XPERIMENTAL EMBRYOLOGY Roberts Rugh EXPERIMENTAL EMBRYOLOGY A Manual of Techniques and Procedures by ROBERTS RUGH Associate Professor of Biology Washington Square College New York University BURGESS PUBLISHING COMPANY 426 South 6ch Street - Minneapolis, Minnesota Copyright 1941-1948 Revised Edition 1948 by ROBERTS RUGH Second Printing 1952 No part of this book may be reproduced without the consent of the author Printed in the U. S. A. MORPHOGENETIC MOVEMENTS DURING GASTRULATION AND NEURULATION (Redrawn from Vogt and Goerttler) TABLE OF CONTENTS Pa^e Frontispiece Introduction - ..Hi Eeference Books for Experimental Embryology — - vi TECHUIQUES IN EXPERIMENTAL MBRYOLOCT Equipment and Procedures in Experimental Embryology 1 Technique for Staining Chromosomes in Tail-fin 26 THE NORMAL DEVELOPMENT OF AMPHIBIA Notes on the Breeding Hahits of Some Common Amphlhla _ 30 The Culture of Amphibian Embryos and Larvae to Metamorphosis - '•8 The Developmental Stages of Amphibian Embryos .._ — _ 56 Eana pi pi ens - 57 Xenopus laevis - — - 90 Hyla regllla _ 92 Ambly stoma punctatum _. 9'*' Trlturus pyrrhogaster — 100 Trlturus torosue ~ 101 Induced Breeding 102 Ovulation and Egg Transport 11** Early Behavior Patterns In the Amphibia 121 EXPERIMENTS ON EARLY DF-^LOPMENT - NON- OPERATIVE Fertilization of the Frog's Egg 126 The Effect of Age of the Egg on Embryonic Development 150 Osmo- regulatl on ' 153 Temperature and Embryonic Development ^ — 137 The Space Factor and the Rate of Growth and Differentiation l't-6 Nutrition and Growth of Amphibian Larvae 152 Mechanical Separation of Growth and Differentiation 157 Chemical Alteration of Growth and Differentiation l62 The Embryo and Narcosis, or The Separation of Form and Function - 166 EXPERIMENTS ON EARLY DEVELOPMENT - OPERATIVE Experiments with the Amphibian Germinal Vesicle - 171 Androgenesi s 179 Artificial Parthenogenesis 1"5 Pressure Effects on Cleavage - 190 The Effect of Centrlfugatlon on Development - 193 The Production of Double Embryos - - - — 198 EXPERIMENTS ON THE EARLY EMBRYO The Behavior of Isolated Embryonic Cells 203 The Organizer and Early Amphibian Development — ~ 210 Morphogenetlc Movements as Determined by Vital Staining 218 The Culture of Isolated Amphibian Anlagen 228 Wound Healing in Embryos - 255 . 2M-0 Parabiosis and Teloblosls Extirpation Experiments on Organ Anlagen •— 214-5 Transplantations — — - - 255 The Origin of Amphibian Pigment ~ — . — ~ 263 I'afie FIELD OPERATIONS Limb Field Operations 27h Eye Field Operations 289 Heart Field Operations — 505 Eegeneration - - — —• 512 ENDOCRINE FACTOBS IN EAKLY DEVELOPMEMT Effect of Thyroid and of Iodine on Amphibian Metamorphosis 522 ThyroideotoncT and Early Amphibian Development — 527 Bypophysectomy and Early Amphibian Development _ 529 CHEMICAL EMBBYOLOGY Cytochemical Tests for Gametes and Embryos of Amphibia ~ 552 CHECMOSOMES AND DEVELOPMENT Heteroploldy Induced by Variations in Temperature 5't-9 Hybridization and Early Development 55° FISH Experimental Embryology of Fish - _ 5^0 The Care and Feeding of Fish - — 56o Fish Diseases - 365 Fish Suitable for Laboratory Experimentation 5^7 Oryzias latipes — 571 Platypoecilus maculatus - 582 Fundulus heteroclitus _ 589 Experimental Procedures with Fish Eggs and Embryos - 595 CHICK Experimental Chick Embryology - '•■19 Procuring and Care of Living Material ~ '+19 Technical and Operating Equipment — - ~ '•■20 The Removal of Chick Blastoderms or Embryos — ■- '••22 Histological Procedures ■■•■- ^21+ Methods for Observing the Development of the Chick Embryo '<-25 Morphogenetic Movements Determined by Vital Staining and Charcoal Particles - '••28 Explanting and Cultivating Early Chick Embryos in Vitro '+52 The Method of Chorlo-allantoic Grafting '+'•■1 Intra- embiyonl c Transplantatl ons - - - '•■'+7 Glossary 1+5'+ " Omne vl vum ex ovo" - -- - ^1 11 INTRODUCTION The material of this "Experimental Emhryology" represents many years of the most Intense research on the part of Innumerable embryologists, from all parts of the world. The author disclaims £iny originality except In those sections relating specifically to his particular investigations. The book is a compendium of data, directions, and ref- erences not generally found in textbooks, but information which is necessary in train- ing the prospective experimental embryologist in the fundamentals of this relatively new and dynamic field of research. There are contained herein some 50 separate experimental procedures, from Andro- genesls to Xenoplastic Grafts, all of which have been tested in the course "Mechanics of Development" given for the past nine years at New York University. The present, completely rewritten book incorporates all the improvements in the various techniques that have come to the author's attention. Each procedure is presented as foundation- al to some basic concept so that the qualified graduate student may be stimulated to pursue further research in the field. The approach is entirely ejcperimental; the subject matter is exclusively the embryo. The organization of each exercise is based upon the general plan of a publish- able scientific manuscript. The usual historical background is omitted, and the discussion (if any) is limited because this is the function of related textbooks. The reference list contains only the most recent and pertinent papers, and certain review articles. Only occasionally there are included papers more than 15 years old, and these because they have been established as classics within the field. It is felt that interested investigators can acquire a complete bibliography through the references given. It is recommended that the student's report include the following, space for which Is generally provided in each exercise: a. Experimental procedure: Any modifications of the procedure as outlined. b. Experimental data: This section must be complete in every detail. c. Discussion: This should be baaed upon "b" above. d. Conclusions: These should be based entirely upon the findings of the student. 9. Eeferences: Only new references which are not included In this exercise. It would be Impossible for any student, under any conditions, to complete the work outlined In this book during a single academic year. There are three solutions to this matter, all of which have been tried in our laboratory and any of which is satisfactory: a. Assign a new procedure for each of the regular weekly laboratory sessions. This is a very heavy assignment and the student would necessarily spend more than the usual four hours per week in the laboratory. The plan has the advantage of making it simpler for the Instructor to anticipate the needs of the entire class, from week to week. He can often schedule the procedures in such a way that they follow in a natural sequence and often conveniently overlap. The major disadvantage is that the student acquires only a passing acquaintance with the various techniques and is apt to as- sume that he is master of all of them. b. Select a logical series of experimental procedures designed to be com- pleted during the first semester, and progressing from the gross to the microscopic, the ci-ude to the refined, the simple to the complex. There is no attempt to cover the entire gamut of techniques. The responsibility of representative selection falls on the instructor, but the student Is quite thoroughly grojxnded in the basic procedures, and is thereby quali- fied later to pursue Independent investigation. This has been the most 111 frequently followed program at New York University during the first aemea- ter. During the second semester the students have been assigned individual and original problems for investigation. c. Assign some of the introductory procedures to the entire class, such pro- cedures as "Induction of Ovulation", "Breeding eind Care of Embryos", and "Temperature and Rate of Development". Then delegate each student to carry out two or three integrated procedures, with the responsibility of thorough work later to be reported in full to the class. This plan de- prives the student of experience in many of the techniques in experi- mental embryology, but it places upon him a responsibility to the entire class which often kindles the research attitude. ^ such a plan most of the exercises can be attempted by an average class of about 15 students. A suggested sequence of exercises, which has been used at New York University, is given below. The assignment is based upon a weekly class session of about k hours, and supplemental time as may be required by the individual student. a. Induction of ovulation and artificial fertilization. b. Normal development: 1. Relation of temperature to early development. 2. Relation of osmotic pressure to early development. 5. The appearance of behavior patterns. c. Experiments with the egg: 1. Germinal vesicle studies. 2. Artificial parthenogenesis. 3. Androgenesis. d. Experiments with the cleavage stages: 1. The effect of unequal pressure on cleavage. 2. The production of double embryos. 5. The behavior of isolated embryonic cells. e. Experiments on the blastula and gastrula: 1. Vital staining and morphogenetic movements. 2. The organizer. f. Experiments with the neurula : 1. Parabiosis. 2. Regeneration. 5- Embryonic inductions in the blastema tissue. g. Experiments with later stages. 1. Wound healing. 2. Hypophysectoiny. 5- Limb or eye transplantations. This program would carry the student through about 1-^ semesters. There would remain about 2 months during which the instructor could direct the students in some of the more difficult techniques with either the fish or the chick embryos. Through the very generous help of Dr. Jane Oppenhelmer and Dr. Nelson T. Spratt, Jr. the sections on fish and on chick embryos have been expanded very considerably. It is believed that the traditional reluctance to use these forms is being broken down by the brilliant work of investigators such as these, and the laboratory of experimental embryology can no longer be limited to amphibian forms but will take in all embryos from the lowest to the highest phyla. It would be ImpoBBlble for the author to properly acknowledge all of the help that he has received in organizing this book. He has enjoyed universal and whole- hearted cooperation, often entailing considerable time and effort on the part of con- temporary Investigators. Where figures, graphs, or photographs have been reproduced, Iv and where certain investigators have constructively helped to organize certain sec- tions, specific acknowledgements are made. It is with pleasure that the author ack- nowledges here the permission granted by The Wistar Institute of Anatoi^T' to reproduce items from papers appearing in their various Journals. It must he emphasized again that this book is made possible by the efforts of many experimental embryologists, many deceased, many active, and an increasing number "presumptive". If biology stu- dents who attempt these various procedures are thereby stimulated to make further con- tributions to the field of experimental embryology, all the effort expended in its compilation will have been justified. Roberts Rugh New York September 191*8 Brae he t, J., 191+ 7 - Child, C. , M., 1915 Child, C. M., 1921 Child, C. M., I92U Child, C. M., I9I+I Cope, E, D., 1889 - Cowdiy, E. V. , 19^3 REFERENCE BOOKS FOR EXPERIMENTAL EMBRYOLOGY Adams, A. S., 19^1 - "Studies in Experimental Zoology." Edwards Bros., Ann Arhor, Mich. Bertalanffy, L. von & J. H. Woodger, 1955 - "Modem Theories of Development." Oxford Univ. Press. Bishop, S. C, I9U5 - "Sandhook of Salamanders." Comstock Publishing Company. Boulenger, G, A., I920 - "A Monograph of the American Frogs of the Genus Rana." Proc. Am. Acad. Arts & Sci. 55:1*^15. Brachet, A., I95I - "L'Oeuf et les Facteurs de l"0ntoge'ne'3e. " G. Doin & Co., Ed. "Emhryologle Chimique." Masson et Cie., Paris. - "Individuality of Organisms." Univ. Chicago Press. - "Origin and Development of the Nervous System." Univ. Chicago Press. - "Physiological Foundations of Behavior." H. Holt and Co., N. Y. - "Patterns and Problems of Development." Univ. Chicago Press. "The Batrachia of North America." Bull. U. S. Natl. Mus. #5'+:l. - "Microscopic technique in Biology and Medicine." Williams & Wilkins, Baltimore. Dalcq, A. M. , 1928 - "Les Bases Physiologlque de la Fecondatlon et de la Parthenogenese. " Les Presses Universitaire de France. Dalcq, A. M. , I958 - "Form and Causality in Development." Cambridge Univ. Press. deBeer, G. E., I926 - "Introduction to Experimental Embryology." Clarendon Press. deBeer, G. K., 1950 - "Embryology and Evolution." Clarendon Press. Detwiler, S. E., I936 - "Neuroembryology. " Macmlllan. Dickerson, M. C, I906 - "The Frog Book," Doubleday Page & Co., N. Y. Driesch, H., I928 - "Science and Philosophy of the Organism." Macmlllan. Durken, B., I928 - "Lehrbuch der Experimentalzoologie. " Verlag. von Gebruder, Borntrager. Durken, B. , I929 - "Grundriss der Entwicklungsmechanik." Gebruder, Borntrager, Berlin. Durken, B., I952 - "Experimental Analysis of Development." Geo. Allen, London. Farris, E. J. (in Press) - "Care and Breeding of Laboratory Animals." John Wiley & Compaiiy. Faure-Fremiet, E., I925 - "Clnetique du Developpement. " Paris. Gray, J., I95I - "A Textbook of Experimental Cytology." Cambridge Univ. Press. Goldschmldt, I925 - "Mechanism of Physiology of Sex Determination." Methuen & Co. ltd. London. Hamburger, V. E., I9U2 - "A Manual of Experimental Embryology." Univ. Chicago Press. Harrison, E. G., - "Cellular Differentiation and Internal Environment." Pub. #-l'4- Am. Ass'n. Adv. Sc. 77:97. Henderson, L. J., I915 - "The Fitness of the Environment." Macmlllan. Herrick, C. J., 19it8 - "The Brain of the Tiger Salamander," Univ. Chicago Press. Huxley, J. S. & G. R, deBeer, 195^ - "The Elements of Experimental Embryology." Cam- bridge Univ. Press. Jenkinson, J. W., I909 - "Experimental Embryology." Clarendon Press. Jennings, H. S., 1925 - "Prometheus." E. P. Duton & Co., N, Y. Just, E. E.,,1929 - "Basic Methods for Experiments on Egga of Marine Animals," Blakiston. Just, E. ;S., 1950 - "The Biology of the Cell Surface." Blakiston. Kellicott, W. E., 1915 - "General Embryology," Holt. Kellicott, W. E., I915 - "Chordate Development." Holt. Kellogg, E,, 1952 - "Mexican Tailless Amphibians in the United States National Museum." Bull. U. S. Nat. Mus. #l60. Korschelt, E., I927 - "Eegeneration und Transplantation." Berlin. Korschelt, E.^ 193 J - "Vergleichende Entwicklungsgeschichte der Tiere." Fischer, Jena. Korschelt, E.'& F. Heider, I900 - "Textbook of Embryology of the Invertebrates," Macmlllan. Lehmann, F. E. , 19'+5 - "EinfiShrung in die Phyeiologleche Embryologie." Birkhauser, Basel, Llllle, F. R., 1919 - "The Problem of Fertilization." Univ, Chicago Press. Llson, L., 1956 - "Histochlmie Animale, Methodes et Problemes." Gauthier-Villars, Paris. Loeb, J., 1915 - "Artificial Parthenogenesis." Univ. Chicago Press, Loeb, J., 1916 - "The Organism as a Whole," Putnam, vl Mangold, 0., I928-I93I - "Das Detennlnationsprotlem. " Ergebn. d. Biol., Vols. 3, 5, 7. Mangold, 0. I95O - "Methodel fur Wlssenschaftliche Biologle." Vol. II. May, R., 194^5 - "La Formation du Systeme Nerveu." Gauthier-Villars, Paris. McBride, E. W. , 191*4- - "Textbook of Embryology." Macmillan (2 vols.). McClung, C. E., 1937 - "Handbook of Microscopic Technique." Hoeber, N. Y. Mertens, E. & L. Miller, I928 - "Liste der Amphibian und Eeptilien Europas." Abhandl. d. Senckenberglschen Naturforschenden Gesellschaft. '+1:3. Meyer, A. W. , I959 - "The Else of Embryology." Stanford Univ. Press. Morgan, T. H., I927 - "Experimental Embryology." Columbia Univ. Press. Morgan, T. H., 193*+ - "Genetics and Embryology." Columbia Univ. Press. Needham, J., I93O - "Chemical Embryology." 3 vols., Macmillan. Needham, J., 19'+2 - "Biochemistry and Morphogenesis." Cambridge Univ. Press. Pantin, C. F. A., 19^+6 - "Notes on Microscopical Technique for Zoologists." Cambridge Univ. Press. Pope, C. H., 19'*^'+ - "Amphibians and Beptiles of the Chicago Area." Chicago Museum Natural History. Przibram, H. , I929 - "Zoonomle, ExperimentEilzoologle." Leipzig und Wien. Eichards, A. N., I93I - "Outline of Comparative Embryology." Wiley. Eitter, W. E. & E. W. Bailey, I928 - "The Organismal Conception." Univ. Calif. Pub, #31. Eobertson, T. B. , 19214- - "Chemical Basis for Growth and Senescence." J. B. Llppincott Company, Philadelphia. Russell, E. S., 1930 - "Interpretation of Development and Heredity." Oxford Univ. Press. Schllep, W., 1929 - "Die Determination der Primitiventvlcklung." Akademische Verlag.- Gesell. Spemann, H., I958 - "Embryonic Development and Induction." Yale Univ. Press. Stejneger, L. & T. Barbour, 19-59 - "A check list of North American An^hlblans and Eeptiles." Cambridge, Harvard Univ. Press (Uth. ed.). Thompson, D'Arcy, I917 - "On Growth and Form." Putnam. Tyler, A., 19^+2 - "Developmental Processes and Energetlce." Qxxart. Rev. Biol. 17:197- ' 353. Waddington, C. H., I956 - "How Animals Develop." W. W. Norton, N. Y. Waddlngton, C. H., 19^0 - "Organizers and Genes." Cambridge Univ. Press. Weed, A. C, 1922 - "New Frogs from Minnesota. " Proc. Biol. Soc, Washington. 35:107. Weismann, A., I893 - "Germplasm. " C. Scribner's Sons, N. Y. Whitman, C. 0., I898 - "Evolution and Epigenesis." Woods Hole Lectures. Wilson, E. B., I925 - "Cell in Development and Inheritance." Macmillan. Weiss, P., 1930 - "Entwicklungsphysiologie der Tiere." Dresden und Leipzig. Weiss, P., 1939 - "Principles of Development." Holt. Wordemann, M. W. & C. P. Eaven, 19*4-6 - "Research in Holland." Elsevier, N. Y. Wright, A. H., 1933 - "Handbook of Frogs and Toads." Comstock Pub. Co., Ithaca. Zwarenstein, H. , N. Sapeikam & H. A. Shapiro, 19'*-6 - "Xenopus laevls, a bibliography." Pub. Univ. Capetown, by The African Bookman. "Embryology is an ancient manuscr ipt with many of the sheets lost, others disp laced, and with spur lous pas sages inter polat ed by a later hand. " Cambridge Natural History, V:79 Vil EQUIPMENT AND PROCEDURES IN EXPERIMENTAL EMBRYOLOGY INTRODUCTION "Biologically Clean." These are the two most frequently emphasized words in any laboratory of experimental embryology, particularly during the initial stages of adjust- ment. Glassware, instruments, solutions, and hands must be "biologically clean" before any experimental results may be considered valid. The term means that, barring any ex- perimental conditions Imposed, there is no possible contamination of living material either by chemical substances, living parasites, or harmful organisms such as bacteria or viruses. The conditions are such that any embryo, introduced into that environment, would be expected to survive. The following precautions, in favor of biological cleanliness, are suggested: 1. Glassware: Eegardless of the source of the glassware to be used, it should be thoroughly scrubbed with hot soap and water, and rinsed in running water for at least 2 hours, rinsed with distilled water and air dried. If the glassware Is cleaned with cleaning fluid {K. Bichromate and Sulphuric acid) it must be thoroughly washed, and rinsed for a longer period (see Richards, 1956) because the cleaning fluid is very adherent to glass. Properly cleaned glassware may be wrapped in clean paper towelling and heat sterilized for ^ hour. As long thereafter as the glassware remains wrapped it may be considered sterile. 2. Hands: A surgeon often spends as much time scrubbing his hands as in operating, and such cleanliness In experimental embryology will lead to more dependable results. The formaldehyde, osmic or hydrochloric acid fumes, adherent to the hands, will contaminate the instruments and ultimately the embryos. 3 . Instrumente : If the Instruments have never before been used, they may be thoroughly washed, rinsed, and sterilized in an autoclave at 15 pounds for 50 minutes. Following this treatment, immersion in 95^ alcohol should be suffici- ent, providing they are never brought into association with any toxic materials. Dissection Instruments from other laboratories cannot ever be used, for the embalming fluids are very tenacious. The student should provide himself with a new set of steel Instruments (not chromium plated) and keep them in a celluloid or plastic tube, or in a cotton filled box, to be reserved for operations on living embryos only. It is often necessary to preserve eggs or embiyos. The student sho\ild have a "contaminated" set of instruments for handling such material, and avoid handling such Instruments (or fixatives) at the same place where the "biologi- cally clean" instruments are kept. k . Embryos : Dead or dying embryos are probably the most common source of con- tamination of cultures. Ailing embryos should be Isolated, and crowding should be avoided at all times. Healthy embryos may be passed through changes of sterile medium to free them of possible adherent bacteria, or some stages may tolerate brief immersion in hypertonic salt which seems to be an effective bac- teriocidal agent. Generally such treatment Is unnecessary if other precautions are taken. There is less likelihood of infection at the lower tolerable tem- peratures, and when the culture dishes are kept covered. The sooner the student learns the significance of the term "biologically clean" the sooner will he enjoy the experience of success In the various procedures outlined in this book. EXPERIMENTAL EQUIPMENT AND PROCEDURES EXPERIMENTAL EQUIPMENT AND PROCEDURES EQUIPMENT AND INSTRUMENTS NEEDED BY EACH STUDENT IN EXPERIMENTAL EMBRYOLOGY OPTICAL EQUIPMENT Blaocular microscope and llshtin^ equipment Objectives 1.7 x 3'5 x most useful, with lOx oculars. The U-shaped base and mirrors should be removable. Microscope lamp: Spencer diaphragm type most suitable. Heat absorbing flask: 250 cc. round bottom Pyrex flask filled with distilled water and supported by screw clamp to vertical stand. GLASSWARE 4 CULTURE DISHES 2 rectangular enamel pans Js x 12 x 2k Inches (for tadpoles) 2 battery Jars with weighted wire covers 2 crystallizing dishes with covers, 12 Inch size 12 Syracuse dishes 12 #1 Stenders with covers 12 #2 Stenders with covers 12 regulation finger bowla 6 pairs Petri dishes 6 shell depression slides i gross microscope slides J ounce #1 cover slips, square or round 2 ft. glass tubing, 5 nmi. diameter (soft glass) 2 ft. glass rod, k mm. diameter (soft glass) h ft. glass rod, 2 mm. diameter (soft glass) 1 hypodermic syringe (2 cc. capacity) with #l8 needles METAL INSTRUMENTS AND EQUIPMENT 2 watchmaker ' s forceps #5 2 adjustable needle holders 1 fine-pointed, sharp scissors (iridectomy scissors are expensive but useful) 1 heavy-duty scissor 2 fine, sharp scalpels, one a lancet type 1 rigid section lifter 1 pair regulation forceps 1 triangle file 1 Bunsen burner with wire gauze. MISCELLANEOUS i lb. Permoplaat, cream colored r- lb. beeswax, white, clear ^ lb. soft melting point paraffin 2 China marking pencils, red and blue 1 diamond or carborundum point pencil 1 wooden needle holder, to hold needles of various sizes 1 celluloid cover for needles and holder 2 ft. rubber tubing 11 or 12 mm. outer diameter 2 ft. rubber tubing 8 or 9 n™- outer diameter THE PREPARATION OF INSTRUMENTS 1. The micro -burner: This will be set-up for class use but it is well that a de- scription be given here for its preparation. The function of a micro-burner is to give a small but intense flame. EXPERIMENTAL EQUIPMENT AND PROCEDURES Secure a 5-lnch piece of 7 nan. soft glass tubing and in a hot flame reduce the diameter of one end to about 1 mm. Then place a right angle in the tube, at ita center (flame tip) and when cool, pass the flamed end through a hole in a large cork. Mount the cork in a burette clamp attached to a ring stand so that the micro-burner is turned upward. To the other end of the tube attach a rubber tubing Joined to the gas outlet. Apply a screw to the rubber tubing. B^ regu- lating the screw clamp a micro-flame of intense heat may be secured. The usual type of micro-burner, where the tube is drawn out to a thin tip of small bore, is not so likely to remain uniform when in use. Hypodermic needles may be used as micro-flame tips. 2. Glass needles : There are two types of handles used for glass needles. The stan- dard type consists of a 7 mm. diameter glass rod cut into 10 cm. lengths by flam- ing longer pieces in the center and drawing them out. In this manner one end of the rod is tapering. The tapering end should be brought into the flame so that it retracts to knob for attachment of the needle, made separately. See that the knob and rod are perfectly straight. Prepare 10 such needle handles. The second type of needle holder consists of the regulation steel needle holder with adjustable screw into which various needles may be inserted and fastened. This type is con- venient and entirely satisfactory. The needles may be made on the electric needle-puller but this is not neces- sary. Secure some h-'^ mm. soft glass rod and in a flame tip draw it out perfectly straight \intil a thickness of about 1 mm. is secured. Break this into about 7-8 cm. lengths and in the micro- flame, put a hook or a bend toward the end of each piece. Hang this "hook" over any support (Metal rod) attached to the ring stand in such a manner that it is directly above a 20 x 60 mm. (or larger) glass vial. In the bottom of the vial place a small amount of cotton. At about 2 cm. from the base of the hanging glass rod apply the micro-flame from the side. It will take practice to apply the correct amount of heat. When the glass at the point of heat application is melted, the weight of the hanging will drop it into the cotton in the glass vial, providing a needle point of micro- scopic dimensions. With practice it may be possible to do this in two steps, the first heating will lengthen and thin out the rod and the second will draw an even finer point. In any case the tapering micro-point should not be long and flexible and will probably have to be trimmed with sharp scissors. Draw out many such needles at one time and mount them temporarily in plasticene, i.e., until ready to attach them to holders or to mount them in a needle board. These needle points may be attached to the glass handles (described above) by bringing them together in a small flame. The needles may be attached at a slight angle which will facili- tate operations. 5. Steel needles: The ordinary steel needles are much too coarse for work with small embryos. However, the finest Insect needles may be secured, cut short, and moiuited in wooden handles and will be extremely useful. k. Hair loops: This device is for handling embryos or isolated tissues. Draw out the end of some 5-6 mm. soft glass tubing so that the total length is about 10 cm. and the smaller end has a diameter of about 1 mm. Close the larger end in a flame. The smaller end should be cut off with a diamond pencil or flamed to make it smooth. Secure some blonde hair from a newborn infant and cut it into 1 inch lengths. With forceps insert one end of the hair into the capillary opening and then the other end Into the same opening. Eegulate the insertion of the hair so that a relatively small loop protrudes. Melt a small amount of soft paraffin on a glass slide and dip the hair- loop into the paraffin, whereupon some of the par- affin will run up into the tube, and harden upon cooling. This will hold the hair loop in place. To remove ariy paraffin adherent to the hair loop Itself, warm a slide in a flame, place on it a small piece of filter paper, and gently touch the hair-loop to the filter paper. Avoid melting the paraffin within the capillary tube. EXPERIMENTAL EQUIPMENT AND PROCEDURES 5. Glaaa 'ball tip: This Instrument la also used for moving embryos about without in- Jury to them. Using 6-7 mm. soft rod of about 18 cm. lengths, draw out the center (flame tip) and break off the excess thread down to within 2-3 cm. of the widening part of the rod. Hold the pointed end of the rod in a flame and a glass ball will form on the end. It can be kept symmetrical by constant spinning of the rod while heating. If it is planned to put a gentle curve in such a rod, this should be done when the rod is drawn out rather than later. Such ball-tips are used also for making depressions in Permoplast, suitable for embryos. It is well, there- fore, to have a small assortment of such ball-tips. 6. Glass pipettes: Three types of pipettes will be used: wide-mouthed, micro- pipette, and micro-pipette with lateral control (see diagrams). Y V \i GLASS TUBING MICRO - BURNER GLASS BALL TIPS // MICRO - NEEDLES \J u HAIR LOOPS @nl FINGER COrfTfiOL OF TERMINAL SUCTION r® •- SAFETY PIN HOLE IN SIDE OF PIP£TTE TXIN RueSER GUARD TRANSFER PIPETTE 2 MM OPENING Ij V PTRALIN • TRANSPARENT SRUCKE BRUCKE TO HOLD TRANSPLANT IN PLACE ( STULTZ '38) The wide-mouthed pipette is used for the transferring of embryos. It Is aimply a wide bore pipette (6-7 mm. diameter) with a curved and amooth-edged tip. Thia should be available at all times. The micro-pipette is made of soft glass with 7-8 mm. outer diameter, pulled out to microscopic dimensions in a manner similar to the making of micro-needles (above). The tip end of such pipettes are often closed but may be trimmed off. These pipettea may be uaed with ordinary rubber nipple or may be uaed with rubber tubing and mouth suction. The micro-pipette with lateral control has special use in the transfer of isolated pieces of embryoa to regions where transplants are desired. It is made in the following manner: Pull out some 7-8 mm. soft glass tubing so that the handle portion has a length of about 10 cm. It is better if the capillary end has a alight curve and tapers rapidly. Close the capillary end by melting it in the Bunaen flame and attach a piece of rubber tubing to the larger open end of the pipette. Bring the aide of the pipette, at about ^-h cm. above the capillary end, RUBBER TUBING OVER SIDE HOLE Spemann Mioropipette for Transplantations EXPERIMENTAL EQUIPMENT AND PROCEDURES Into a small hot flame. If the capillary end is curved, the point of heating should be on the opposite side of the curve, ^^^hen this point is soft, hlow gently into the ruhher tubing and the melted glass will bulge outward. The size of the heated area will determine the size of the bulge. Break off the glass bubble down to the wall of the pipette and smooth off the edges in a micro-burner. V/ith dia- mond point cut off the closed tip of the capillary end so that the aperture will be be about 1 mm. in diameter. Cut a short piece of thin-walled rubber tubing and slip it (from larger end) over the pipette to the point where it covers the lateral hole. Heat the broad end of the pipette until very soft, press down on metal sur- face to give a ridge to hold the rubber nipple. Add rubber nipple to this end, and the pipette is ready for use. This latter pipette is of special value in the transfer of small pieces of tissue under solution. With nipple, suck in solution until the capillary end is filled almost to the lateral hole. Bemove fingers from nipple and place a fore- finger over the rubber- covered lateral hole. With the capillary point under the solution, force out a small amount of the contents of the pipette by gently press- ing on the lateral rubber cover with the forefinger. Then, by releasing the gentle pressure a small amount of fluid, or tissue, may be drawn up into the pipette and held there. This tissue may be oriented at any place by slight pres- sure on this lateral membrane. The capillary end must hold solution (not drip) even when held vertically with the point suspended. Practice the use of this pipette with small objects. 7, Glass bridges : These are small pieces of cover glass, thinnest grade, used to hold transplanted tissues in place for 15+ minutes while they "take" or heal onto the host. Using a diamond point pencil and ruler, cut thin cover glasses into strips of about 2-5 mm. wide and 5-10 mm. long. With forceps run the edges of these glass strips through a micro- flame to make them smooth. To put a slight curve in some of the pieces of cover glass, grasp the edge of a piece with forceps and bring the center of it over a micro-burner with a 1 mm. flame. The weight of the cold end will bend the cover slip slightly when the center is heated. The height of the bridge should be determined by the size of the embryo to be used as host. Frequently flat cover slips will prove to be adequate^ especially when the host is held in a depression. The bridges should be sterilized in 70^ alcohol over cotton and kept thus in a covered stender dish. There is a refinement of this type of bridge (or Briicke) recently described (Schultze, 1958). The cover itself consists of transparent Pyralin of less than 2 mm. thickness cut into rectangular blocks of 1/8 inch by 5/8 inches each. Holes are drilled through each end of the block with #70 or #71 wire gauge drill. The edges of the block are smoothed off with fine file and then dipped in very weak balsam to increase transparency. Take care that balsam does not fill the holes. Small safety pins, (preferably gold plated to prevent rusting) are straightened out and then given three right angle bends as shown in the diagram. The pins are then forced through the holes, to the extent of 5-6 mm. Such a graft cover may be pressed down onto the graft exactly as desired, the pins anchoi-ed in the Permo- plast and the Pyralin allowing constant observation of the graft. In most trans- plantations such an elaborate Briicke is not necessary. OPERATING DISHES Most operations will be carried out in Syracuse dishes or salt cellars. Since em- bryonic tissues often adhere to glass, the base of the Syracuse dish is lined with one or another plastic substance. The fol ''owing are satisfactory: a. Permoplast - American Art "lay Co., Indianapolis, Indiana. b. Beeswax to which has been added lampblack, to take glare off background. 0. Eainbow Wax - American Art Clay Co., Indianapolis, Indiana. d. White refined beeswax. e. Pieces of cellophane or Pyralin. f. Agar, 2% or more concentrated. The Permoplast is probably best for delicate operative procedures, as it is easily molded without iieating. However, under solution it tends to fragment and it may be necessary to compensate for this by melting It with about 20^ low melting point paraffin. EXPERIMENTAL EQUIPMENT AND PROCEDURES For frog work, melted beeswax to which enough lamphlack has heen added to give it a dark gray appearance, will prove to be satisfactoiy. Occasionally, after the dishes are prepared, the wax base will, under water, float off the bottom. To prevent this, place a few pieces of 2 mm. glass rod in the bottom of each dish before adding the melted beeswax. These will add sufficient weight to hold the wax In place. Have at least 10 operating dishes available at all times. Grooves to hold embryos may be made with ball tips when needed. OPERATING SOLUTIONS The operating medium will vary with the age, the condition of the embryo (or lilssue) and the species used. Operations are generally performed in more concentrated solutions, and, after healing, are returned to weaker salt solutions (see exercise on "Wound Heal- ing"). For operations on Urodeles the Urodele Operating Medium is used and the embryos will heal normally if left in these solutions. After the operation wound has healed the Urodele embryo is transferred to Urodele Growing Solution. For the Anura double strength and then Normal Standard Solutions are used. Brief boiling of the solutions to sterilize them may be necessary. If convenient, large volumes could be autoclaved or filtered and stored as stock solutions. Controlled salt solutions are more satisfactory than Spring Water or conditioned tap water because there Is greater uniformity. In all cases remember that surface rather than volume is important. The embryos need be Just covered in the solution and no more, providing evaporation is reduced to a minimum. GLASSWARE Ovulating animals should be kept in fish bowls or small battery Jars, properly covered. If eggs are to be layed in these containers, it will be necessary to provide them with appropriate solutions. The Urodeles generally attach their eggs to vegetation. EXTRA FINGER 80WLS OOOOOOOOQ oooooooo EXTRA OPERATING DISHES HOLTFRETER'S SPRING WATER AUX)HOL © © 95% MICROSCOPE LAMP NEEDLE a HAIR LOOP BOARD EXPERIMENTAL EMBRYOS RING STAND J CONTROL EMBRYOS -» GROUND BOTTOM ^ — ^ )--'"(^ Qoooo / \^__y \ I^OPERATING (SYRACUSE) DISHI c, WtiSJE FINGER BOWL y .<*- >*" .<^^ OPERATIMG BOARD BINOCULAR MICROSCOPE METAL BOX WITH DRAWERS FOR INSTRUMENTS PLAN FOR OPERATING TABLE EXPERIMENTAL EQUIPMENT AND PROCEDURES Tor fertillEation of frog's eggs, finger bowls or Petri dishes are used and all eggs from a single female may be inseminated in a single container providing they are flooded in 15 minutes and separated to lots of about 25 per finger bowl before the first cleavage. Development will continue to hatching in finger bowls but beyond hatching a different pro- cedure will be necessary (see section on "Culturing of Embryos"). Operations are performed in dishes described above. Following the operation, when the transplant has "taken", the embryo may be transferred to a covered stender, the bottom of which has a thin layer of U-5^ agar. This provides a softer base than glass so that there is less danger of injury. Such operated animals should be kept at constant tempera- ture toward the lower levels of tolerance for the particular species under investigation. ASEPSIS The amphibian embryo is relatively immune to infection, or bacterial contamination. Operated embryos are, of course, exposed to such conditions but the wounds heal so rapid- ly that simple aseptic precautions are generally sufficient to protect the embryo. In recent years Detwiler, Copenhaver, and Robinson (19^7) have demonstrated that sodium sulfadiazine (0.5^) in any of the various operating or culture media is perfectly harmless to the embryo, and will reduce the operative casualties on the central nervous system from 95^ to 5^. The sulfadiazine may partially crystallize out, but these crystals are harmless except as they may mechanically pierce the ectoderm of the embryo. At later larval stages, or just after metamorphosis, amphibia may become susceptible to fungus or other skin infections. These can be treated locally with dilute mercuro- chrome, neo-sllvol, or very concentrated salt solutions painted onto the affected area. MEMBRANES The eggs of eJ.1 amphibians are surrounded by secondary (jelly) membranes secreted by the oviduct in addition to the vitelline membrane. These membranes presumably protect the embryo from bacteria and injury during development. Any operations on such embryos re- quires that such membranes be removed. It must be remembered, therefore, that the unpro- tected embryo is rather easily injured and that it is more susceptible to physical and chemical changes in the environment, as well as to bacterial infection. URODELA The Urodele jelly is rather tough and may be peeled off of the eggs with sharp pointed watchmaker's forceps. The vitelline membrane will be resistant to puncturing so that it may be necessary to use glass micro-needles to make the Initial break. Urodele egg capsules should be opened in Urodele Growing Solution. ANURA The Anuran egg is surrounded by loose jelly which is adherent to the vitelline mem- brane. Attempts have been made to remove this jelly by Ultra-violet light, or by dilute KCN, but there is no reliable chemical method of Jelly removal which allows the embryo to survive. Should someone discover a method of fertilizing body cavity eggs (devoid of jelly) this would be a boon to experimental embryology. The Anuran egg jelly can be re- moved to some extent by rolling the egg on filter paper or paper toweling, pushing it along with the flat side of a scalpel. The danger in this method is, of course, excessive mechanical injury to the egg so that the correct rate and amount of absorption (by filter paper) and the exact amount of rolling will have to be determined empirically. With prac- tice it is possible to pierce the jelly with one prong of the forceps, slide a prong of a second pair of forceps along the first, and cut through the jelly with an outward move- ment. Then the split jelly capsule can be shelled off of the egg. The enzyme hyaluronl- dase, so effective In denuding the mammalian egg, may prove of similar value with the amphibian egg. (Personal communication from D. C. Metcalfe and reference to Kurzrok I9I18: Am. Jour, Clin. Pathology, p. '+9I. ) EXPERIMENTAL EQUIPMENT AND PROCEDURES STANDARD LABORATORY EQUIPMENT IN EXPERIMENTAL EMBRYOLOGY* MAJOR EQUIPMENT 1. Water tatle - wood or stone tatle provided with current of water to depth of 1" to be used for holding finger howls, etc., at fairly constant temperatures. 2. Befrigeratlon - electric refrigerators (or constant temperature rooms) with heat- ing units installed so that the temperatures may he regulated. Best temperatures are k°C., lO^C, and 20°C. 3. Incuhators - thermostatically controlled hoxes which can he regulated at tempera- tures above the laboratory temperatures. Best temperatures are 25°C., 29°C., 52°C., and 35°C. Chick incubators regulated at 105°C. for few eggs (Oaks, Chicago) or for several hundred eggs (Buffalo Incubator Company, Buffalo) should be on hand. 1+. Centrifuge - heavy electric centrifuge with large capacity as well has smaller electric and hand centrifuges. 5. Animal cage - a wooden frame with no floor, screened sides and top, should be made to fit into the water table If the table is large enough. The cage may be divided into compartments with the doors opening on top. If the cage is to sit in water, the wood should be treated with some waterproofing. Frogs and salamanders may be kept in such a cage on the water table in healthy condition If they are provided with cool running water. 6. Aquaria - the sizes and shapes of aquaria depend upon the particular animals con- cerned. For Urodele and Anuran tadpoles (larvae) the low) flat tanks with con- siderable surface are best. Ideal dimensions are the 12 x 12 x 1+ enamel (restaur- ant ) pans . For fish, four sizes are desirable: For fry - ^ gallon battery Jars or Woolworth aquaria. For tropicals and breeding pairs - l6^ x 8 l/5 x 8^ inches wide is best ( 5 gallons ) . For larger fish, or large groups of non-belligerent fish - 20^ x lOj x 12^ inches high (about 10 gallon capacity). For Zebra fish which are very active - 2U^ x 6| x 6 Inches wide. All fish tanks should have slate bottoms and glass covers. One corner of the glass cover may be cut off to facilitate removal, emd for feedings. The larger tanks may be sub-divided by cutting a piece of glass to fit and then covering the edges of the glass with split rubber tubing and inserting the glass into the tank. The mbber tubing will hold the plate in position and at the same time block the passage of small fry from one compartment to the other. 7. Microtome - Spencer rotary probably the best. 8. Embedding ovens - Columbia probably the best. 9. Slide warmer - Chicago Apparatus Company, thermostatically controlled electric warmer. 10. Balances - Coarse (200 + gram capacity) and sensitive types. * Note: The following list is rather exhaustive because it Includes equipment needed to carry on all of the complicated procedures outlined. The list is Intended as a guide for the Instructor. Many of the experiments can be conducted with a pair of watchmaker' s forceps, a scalpel, a binocular microscope, and heat absorbed lighting. It is not necessary, therefore, to provide all the items in this list in order to encourage research in experimental embryology. 10 EXPERIMENTAL EQUIPMENT AND PROCEDURES GLASSWARE 1. Battery .lara or aquaria for individual use of students; each with weighted wire screen cover. 2. Crystallizing;^ dishes or 10 inch finger bowls. 5. Finger bowls - regulation size, made to fit Into each other. k. Petri dlahea (10 cm.) with covers. 5. Stender dishes with covers, both #1 and #2. 6. Syracuse dishes - regulation size. 7- Coplin staining .lars and Homeopathic and Shell Vials. 8. Salt cellars for embedding oven. 9- Erlenmeyer flasks - 500 cc. capacity for storing sterile media. 10. Lantern slide covers - used as glass base for operating and for protecting binocu- lar and compound microscope stages when using wet mounts. 11. Graduates - 10 cc. and 100 cc. 12. Beakers - 100 cc. and 600 cc. 15. Slides: Begulation microscope slides. Depression slides, cell type. Ik. Cover slips - glass. 'Best size, 7/8 Inch square and #1. 15. Qlaaa rods - Diameters from 1+ to 7 mm- Soft glass. 16. Glass tubing - Diameters from 4 to 7 mm. Soft glass. SOLUTIONS AND REAGENTS 1. Distilled water - glass distilled preferred. Supply in large carboys with siphon and pinch clamp. 2. Spring water - if possible supply in carboys with siphon and pinch clamp. Great Bear Spring Water (N. Y. City) is excellent. 5- Conditioned tap water - to be stored in carboys after conditioning. This will be necessary where the City water supply la treated ao that embryoa cannot survive in it. This may be tested with sperm or early embryos. Conditioning is achieved by running tap water into large tank in which there will be maximum of surface exposed and in which plant material la abundant. Artificial aeration will help to eliminate chlorine. Paaslng tap water through fine gravel and charcoal la rarely necessary but would aid in conditioning highly toxic water. Three or four days of such conditioning should be sufficient. h. Standard (Holtf reter' a) Solution - this should be available to the students in several concentrations and large volumes. For the convenience of the instructor, the dry salts may be made up in appropriate concentrations and stored in vlala to be added to carboys of glaaa diatilled water when needed. The concentrations needed are 200^, 100^ and 205^. The formula for Holtfreter's solution (J. Holt- f reter, I93I - Arch. f. Ent. mech. 12l*^:Uol*) is: NaCl 0.55 gr. KCl 0,005 gr. CaClp 0.01 gr. NaHCbj 0.02 gr. Water 100.00 cc. (glass distilled preferred) 5. Amphibian Binder's - should be available In concentrated form: NaCl 0.66 gr. KCl 0.015 gr. CaCl^ 0.015 gr. NaHCOj to pH 7-8 Water 100.0 cc. 6. Urodele stock solution: Great Bear Spring Water 10 liters NaCl 70 grams KCl „ 1 gram CaClp 2 grama EXPERIMENTAL EQUIPMENT AND PROCEDURES 11 Urodele Operating Medium : ( hypertoni c ) Stock solution 2 parte Great Bear Spring Water 1 part Urodele Growing Medium: (isotonic) Stock solution 2 parts Great Bear Spring Water k parts 7- Locke's solution - for chick emtryos: NaCl 0.9 gr. ICCl O.OU gr. CaClg 0.021+ gr. (anhydrous) KaHCOj 0.02 gr. Water 100.0 cc. 8. Special variations of the ahove solutions: A. Calcium- free Standard (Holtfreter' s) Solution. B. Sodium-free Standard ( Holtfreter ' s) Solution. C. Potassium- free Standard ( Holtfreter' s) Solution. D. Buffer-free Standard ( Holtfreter' s) Solution. E. Nuclear medium for germinal vesicle studies (Calcium- free Ringer's). NaCl 0.6 gr. KCl 0.01 gr. Glass Distilled Water 100.00 cc. 9- Anesthetics : A. MS-222 (Sandoz Chem. Co., 65 Van Dam St., N.Y.C.) Excellent for embryos. Make up 1/30OO concentration in Spring Water and in Standard Solution of the ahove concentrations. (Tricain Me thane sulfonate) Must be used fresh. B. Chloretone - 0.5^ in Spring Water and in Holtfreter' a. C. Ether - anesthetic form. D. Chloroform. E. Magnesium sulphate (Epsom salts), crystal form. 10. Killing and fixing solutions: A. Smith's fixative - best for yolk-laden aicjihibian eggs. Two solutions to be mixed Just before use. Not to be used if discolored. Solution A: K Bichromate 0.5 gr. Water 87.5 cc. Solution B: Formalin 10 cc. Acetic (glaclal)2.5 cc. B. Bouin's fixative: safest of all fixatives, particularly for late stages. Saturated (aqueous) picric acid 75 cc. Formalin ( oommercial) 25 cc. Glacial acetic acid _ 5 cc. C. Bouin-Dioxan - excellent for yolk embryos and rapid technique. (See Puckett, 1957 - Stain Tech. 12:97). Use Bouin's and Dloxan in equal parts (Dloxan is volatile and toxic). D. Mlchaelis' fluid - For yolk embryos. Cone. HjjClg - aqueous 20 pts. Cone, picric aqueous 20 pts. Glacial acetic acid 1 pt. Water distilled 1+0 pta. E. KLeinberg's pi cro- sulphuric - for polar bodies and spindles (McClungJ. Water 200 vols. Cone. B^SOl^ 2 vols. Picric acid - to saturation F. Chrom- acetic fixative - excellent for cytological studies of amphibian egg and embryos : Chromic acid 10^ 25 pts. Glacial acetic acid 10 pts. Sat. aq. picric acid 100 pts. 12 EXPERIMENTAL EQUIPMENT AND PROCEDURES Killing and fixing aolutlona (Continued) G. Gllaon's fixative - Camoy & Lebrun, l897: La Cellule 12: Nitric acid - 80^. 15 cc. Glacial acetic acid_ k cc. Corrosive sublimate 20 grams Alcohol 80^ 100 cc. Water - distilled 880 cc. H. Gatenby's fluid: K. Bichromate - 2% aqueous 100 cc. Chromic acid 1^ 100 cc. Nitric acid 6 cc. I. Stockard's solution - for fish embryos: Formalin 5 pta. Glacial acetic k pta. Glycerine 6 pta . Water 85 pts . J. Acetic alcohol: Absolute alcohol 90 cc. Glacial acetic 10 cc. K. CoiTosive acetic: HgClg 5 gr . Glacial acetic 10 cc. Water 90 cc. L. Formalin - two concentrations should be available, 10^ and h'^. 11. Bleaching Agents: A. Javelle water - potassium hypochlorite. B. Mayer's chlorine - to be made up Just before use 1. Place few crystals potassium chlorate in vial. 2. Add 2-5 drops HCl. 5. When green chlorine fumes evolve, add 2-10 cc. of 70^ alcohol. h. Transfer specimens from pure 70^ alcohol to this mixture until bleached. C. Amitinnlated alcohol - 2^ NHj^OH in 70^ alcohol to decolorize picric acid stain. CYTOPLASMIC STAINS A. Eoaln - O.J^ in 95^ alcohol. B. Light green (Griibler'a) - 0.5^ in 95^ alcohol. C. Fast green (Nat. Aniline Co., N.G.f.5) 0.5^ in 95^ alcohol. D. Safranln 0 (Nat. Aniline Co., N.S.-IO) 1^ in aniline water. E. Orange G - sat. in clove oil. F. Maason stains (A,B,C.) Excellent for cell types (pituitary). G. Alizarine S - (used in Spaltoholz technique). VITAL DYES A. Nile blue sulphate - 1/200,000 B. Methylene blue - 0.5^ C. Neutral red - 1^ D. Bismarck brown - 1^ E. Janua green - 1^ (aee section on "Vital Staining" etc.) EXPERIMENTAL EQUIPMENT AND PROCEDURES 13 NUCLEAR STAINS A. Iron haematoxylln B. Feulgen, nucleal reaction C. Earris' acid haemalum 12. Miacellaneoua reagents: A. Iodine - saturated iodine In 70^ (to follow Zenker's fixation). B. Ammoniated alcohol - % NBij.OH in IQ^ alcohol for removal of picric. C. Glacial acetic acid - and Normal acetic (60 cc. Glacial/liter). D. I^ydrochlorlc acid - concentrated, 1^ and Normal. E. Sodium hydroxide - concentrated, 1^, Normal and O.OOJN. F. Ammonium hydroxide - concentrated. G. Potassium hydroxide - 1^. H. Bydrogen peroxide - concentrated (can be used as 'bleaching agent). I. Acetone J. Glycerine K. Cleaning fluid (K. Bichromate and sulphuric acid). L. Clearing agents: 1. Xylol (toluene, toluol, benzene (benzol) 2. Cedar oil 3. Aniline oil k. Oil of wlntergreen (methyl salicylate) 5. Oil of cloves 6. Dioxan (volatile and presumably toxic) 7. Chloroform M. Mounting media: N. Embedding media: 1. Canada balsam - dissolved in xylol 2. Gum damar 5. Clarlte k. Egg albumen - best as albumen water (3^) 1. Paraffin: M.P. range from U5OC. to 58°C. 2. Beeswax: pure white ' 5. Bayberry wax (Candle Factory, Falmouth, Mass.) k. Rubber - white rubber to be added to paraffin 0. Vaseline P. pH indicators (LaMotte sets for entire range) MISCELLANEOUS EQUIPMENT A. Operating base 1. Permoplast - American Art Clay Company, Indianapolis, Indiana. 2. Rainbow wax - 3. Beeswax with lampblack to give proper background shade. h. Board with cut-out to fit over binocular base. B. Lampblack C. Agar D. Cellophane, pliofilm or pyralin. E. Lucite - used to conduct light for considerable distances, without heat transmission. Can be used to curve light and point it Into operating dish. F. DeKhotinsky's cement - for mounting razor blade fragments onto glass handles. G. Paper 1. Toweling 2. Lens 5. Filter H. Marking devices 1. China marking pencils: red, blue or black 2. Diamond point 5 . Carborundum mounted on glass rod 1. Cloth 1. Cheese 2. Black velvet (to reduce movement of ciliated larvae) lU EXPERIMENTAL EQUIPMENT AND PROCEDURES J. Meaaurln^ devices 1. Millimeter ruler 2. Glass plate with mm. graded graph paper mounted teneath K. Thermometers - range 0°C. to 110 "-"C. L. Glass blowing equipment - and flame board 1. Buna en burner with tubing 2. Wing tip 5- Micro-burner (metal or glass made with screw clanp regulation of flame )< M. Tripod with wire gauze N. Vial board - made to hold shell, or homeopathic vials of various sizes. 0. Brushes for cleaning vials, etc. P. Slide boxes - cap 25 and 100 slides. Q. File, triangular. E. Pyralin cover for needles and needle board. SOME OF THE EQUIPMENT SUPPLY COMPANIES Baker & Adamson Chemical Company - kO Rector Street, N. Y. Bauch & Lomb Optical Company - Eochester, N. Y. Central Scientific Company - 220 East l4-2nd Street, N. Y. Clay Adams - hk East 25rd Street, N. Y. Eastman Kodak Company - Rochester, N. Y. Elmer & Amend - 655 Greenwich, N. Y. City Fischer Scientific - Pittsburgh, Penn. General BiologicaJ Supply House (Tiu-tox) - Chicago, 111. Harvard Apparatus Company - Dover, Mass. International Equipment Company - 552 Western Avenue, Boston, Mass. Merck & Company - Eahway, N. J. Spencer Lens Company - Buffalo, N. Y. Standard Scientific Supply Company - 3k West l+th. , N. Y. City SOURCES OF ANIMAL MATERIAL FISH; AQUARIUM EQUIPMENT, INCLUDING PLANTS Aquarium Stock Company - 66 West Broadway, New York City, Columbia Tropical Aquarium Company - 61+3 Broadway, New York City. Crescent Fish Farm - I62I+ Mandeville Street, New Orleans, La. Eastern Gardens - Klssena Blv'd. & Rose Avenue, Flushing, L. I., N. Y. Empire Tropical Fish Company - 126 Church Street, New York City. Everglades Aquatic Nurseries - 706 Plaza Place, Tampa, Florida. Grassy Forks Goldfish Hatcheiy - Martinsville, Indiana. Japanese Goldfish Hatchery - North Branch, New Jersey. Metal Frame Aquarium Company - Pine Brook, New Jersey. Nassau Pet Shop - 129 Nassau Street, New York City. Trlcker, William - Brookside Avenue, Saddle River, New Jersey or Rainbow Terrace - Independence, Ohio. U. S. Bureau of Fisheries - Washington, D. C. Wurst, C. - 58U5 Frankford Avenue, Philadelphia, Pa. (dried salmon and ant eggs, and flies) AMPHIBIA (Adults as well as eggs may be secured from most of the following) Carrlbean Biological Laboratories - Blloxi, Mass. Everglades Aquatic Nurseries - 706 Plaza Place, Tampa, Florida, (^yla only) Fletcher, 0. K., Jr., - Biol. Dept., Univ. Georgia, Athens, Georgia. Hazen, J. M., - Al burgh, Vermont. Louisiana Frog Company - Rayne, La. ( Rana catesblana, clamltans, plplens, sphenocephala ) Marine Biological Laboratory - Woods Hole, Mass. EXPERIMENTAL EQUIPMENT AND PROCEDURES 15 HISTOLOGICAL HINTS FOR EGGS AND EMBRYOS INTRODUCTION Satisfactory slides of embryonic material are difficult to ottain. This Is true of toth chorionated and yolk-laden eggs as well as the early stages of development. The es- sential steps are the removal of membranes, proper fixation, incomplete dehydration, and short embedding in wax-paraffin mixtures. ANESTHETICS In general, anesthetics are not necessary when eggs or embryos are to be fixad im- mediately. Occasionally it is desirable to fix an embryo in a certain position, or to reduce body movements during fixation, when anesthesia is in order. a. 1^-222 : This imported poison is the most satiafactoi-y anesthetic available, used in 1/5OOO concentration either in Standard ( Holtfreter' s) Solution, Spring Water, or Locke's solution (for chick embryos). The embryos are immobilized in about 1 minute and, after retiim to normal medium, recover in about 10 minutes without ill effects. Must be used fresh. b. Chloretone : Generally 0.5^ concentration in whatever medium the embryo is accustomed, will give slow but quite satisfactory anesthesia. c. Magnesium sulphate: (Epsom salts) Simply drop a few crystals into small volume of water containing the embryo and await immobilization. d. Cyanide: KCN l/lOOO In salt solutions acts as an anesthetic. e. Ether and chloroform: These volatile anesthetics are for air breathing forma; hence will find little use with embryonic material. f . Chilling: Embryos are rapidly retarded in all of their activities by adding to their media some cracked ice. Such embryos may be operated upon and will recover, upon return to normal temperatures, without ill effects. It may take 10-15 minutes to adequately stupefy the organiama. REMOVAL OF JELLY CAPSULES It is easier and allows better fixation if the Jelly membranes are removed from eggs and embryos prior to the killing process. The Urodele egg is provided with a distinct Jelly capsule which may be punctured with needles or sharp watchmaker's forceps, and pulled off of the egg. If a tear is made by means of a pair of forceps, the embryo will usually "shell out". If the capsule is placed on a piece of paper towelling, filter or blotting paper, to which it will adhere, this operation may be facilitated. The Anuran egg generally has looser but more adherent Jelly capsules. This Jelly may be removed by cutting single eggs away from the mass and placing them- on coarse paper and rolling them along the flat side of a scalpel until the bulk of the Jelly rolls off onto the paper. A better method Is to pierce the Jelly with one prong of the #5 watchmaker's forceps, slide a prong of a second pair of forceps along the first, and, with an outward motion cut through the Jelly. It may then be peeled off. It has been reported that ultra-violet light will dissolve off the Jelly capsules of eggs but it la very likely that the same irradiation will damage the egg or embryo. Chem- ical removal of the Jelly, after fixation, may be achieved by placing the embryo and cap- sule into 10^ sodium hypochlorite or chlorox diluted with 5-6 volumes of water. The Jelly can be shaken off within a few minutes. Javelle water (potassium hypochlorite) diluted 3-1+ times may also be used. Following fixation in Gilson's fluid, the Jelly hardens suf- ficiently so that it may be picked off the embryo which la subsequently hardened in alcohol. l6 EXPERIMENTAL EQUIPMENT AND PROCEDURES KILLING AND FIXING PROCESSES The fixation method of choice depends upon the end results desired. It has recently been discovered that decapsulated amphibian eggs can be briefly boiled to coagulate them, prior to normal chemical fixation. For cytological preparations the corroeive-acetic or chrom-acetic fixations are best; for early embryos with much yolk, Smith's fluid is recom- mended; and for later embryos and tissues in general, Bouin or Bouin-dioxan mixtures are suggested. Fixation may be speeded up by the addition of I'jt Turgitol (Carbide & Carbon Company, N. Y. C.) which reduces the surface tension of the fixative. a. Smith's fluid: This fixative is made up of two solutions which, when brought together, rapidly deteriorate. It is therefore necessary to mix them Just before use and to never use it when it has become discolored (dark). The fixative should be used for 12-2U hours, followed by thorough washing (12-21+ hours) in running water. If the material is discolored, follow bleaching directions below (bichromate bleach). Tissues or embryos fixed in Smith's may be permanently preserved in k$ formalin directly after washing. This fixative is good for yolk-laden eggs and will give a minimum of distortion. b. Bouin' s fluid: The most universally satisfactory fixative known, made up in aqueous or alcoholic solutions. Fixation may be as short as 1 hour (tail tips); 2k hours for whole embryos; or much longer if it is inconvenient to change because Bouin' s is a preservative as well as a fixative. The yellow of the picric acid is best removed by adding about 2']^ NHj^OH to the 705t alcohol when dehydrating, changing the solution every hour until the color is entire- ly gone. Lithium carbonate acts more slowly and may leave crystals, while the ammonia will eventually all evaporate. If chromophlls are to be studied, such tissues must subsequently be properly neutralized by long exposure to pure 70^ alcohol. c. Bouin- Dioxan: This is a rapid and entirely satisfactory method of fixation and dehydration, the proportions being half-ln-half , and the fixation time 12-2lv hours. The mixture prevents shrinkage and hardening that often attends the use of other reagents. Other ratios used are Bouin-2 parts, Dloxan-1 part. If it is necessary to decolorize, transfer directly to ammoniated 70^ alcohol, later to pure dioxan for dehydration. d. Mlchaelis' fluid: Fixation for 8 hours, after which Jelly must be removed be- fore transferring to alcohols or dioxan for dehydration. e. Gatenby's fluid: Used in ratio of about 2 cc. per egg for 12-21+ hours during which time the Jelly capsules will fall off the eggs. f. Gllson's fluid: Short fixation (15-^5 minutes) for cytological studies, recom- mended particularly for oogenesis. g. Chrom-acetic fixative: Excellent for cytological studies of an^hibian egg and early embryos. h. Acetic-alcohol: Fix tissues for 8 hours, transfer directly to absolute alcohol. 1. Formalin fixatives : Gross fixation of embryos or tadpoles which are not to be sectioned may be accomplished in k formalin 8 pte.; 5^ chromic acid 2 pts. as fixative for chromo- some sti-ucture of Anuran eggs. For Urodele larvae he recommends 10^ formalin 7 pts., and 1^ chromic acid 5 pts. J. The following procedure has proven to be very good for amphibian eggs. (See Goldsmith, I929. Trans. Am. Mlcr. Soc. U8:2l6.) 1. Fix in Goldsmith's fluid: Chromic acid 1^ 15 parts K Bichromate 2^ h parte Glacial acetic 1 part (Fix small pieces 2 hours, large pieces 2k hours) EXPERIMENTAL EQUIPMENT AND PROCEDURES 17 2. 3. k. 5. 6, 7. 8, 9. 10. Il- ls. 13. If eggs are left in fixative 2l4-i^8 houra, the Jelly will be removed.. Wash 2k hours in running water. Carry to 70^ alcohol through gradual changes. Change to 1 part aniline oil, and 2 parts 70^ alcohol for 2 to 6 hours. Change to 2 parts aniline oil, and 1 part 95^ alcohol for 2 to 6 hours. Change to pure aniline oil luitil clear - 1 or more hours. Change to 50^ aniline oil plus 50^ toluene for 1 to 6 hoiu-a. Change to 100^ toluene, for 1 to 5 hours. Place in 100^ toluene for 1 to 5 hours. Place in saturated 53° paraffin in toluene for 1 to *+ houra. Place in 53° paraffin for 5 to l^ houra, at 55°C. Embed in 53° paraffin. C. L. The following procedure has been used with considerable success by Dr. Parmenter in studying the cytology of the amphibian egg. 1. Fix for 2k hours in Smith's fluid. 2. Preserve egga in Jelly in 5^ formalin. 3. To remove Jelly: Uae 20^ solution chlorox in distilled water for 3 to k minutes. Watch and atop before the cortex is injured. k. Binse thoroughly in distilled water, several changes. 5. Dehydrate to 70^ alcohol with 5 minute changes in ascending. 6. Dehydrate further: (Leave in this overnight) 8Cff> alcohol 96 cc. Phenol k cc. (See King & Slifer, 1935; Science 78.) 7. Dehydrate further in 95^ alcohol - 2 thirty minute changes. 8. Carboxylol - 1 hour. (Thla may be too drastic for some eggs.) 9. Infiltrate with tisaue mat; '56-58° M.P. paraffin for 1 hour. 10. Imbed, aection and mount. 11. Preliminary to staining, remo.ve paraffin with xylol; rinae in absolute alcohol and dip (or flood with pipette) quickly into a solution of equal parts of absolute alcohol, ether, and 10^ celloid in solution. Place in 70^ alcohol to harden the very thin coating of celloidin on the aections. Thie coating ia a precaution againat loss of an occa- sional section during staining, etc. It does not Interfere with staining and need not be removed. 11. Use any stain deaired. Neither Feulgen nor acid haematoxylin will atain yolk. WASHING Moat tissues in aqueous fixatives should be waahed in running water for 6-21+ hours, the time depending upon the size of the tissue. The function of washing is to remove salts, crystalline substances that may have been added with the fixative, and any extrane- ous materiala within the tissues. The completion of washing cannot be determined by loss of color in the tissues. In Bouln-dioxan fixation, the washing process is generally omitted until the sectioned material is passed down through the alcohols. Alcoholic iodine is used to remove excess corrosive sublimate. BLEACHING Two proceaaea are included under thla title. Firat, the removal of color added in the fixation procees. This may be acconplished by 12 hour changes in saturated aqueous solutions of lithium carbonate when picric acid has been used. A more satisfactory bleaching agent, because it is more rapid and leaves no residue, is a 2^ solution of am- monium hydroxide (NB1).0H) in 70^ alcohol used in half-hour changes until no more of the yellow picric color is visible. The second process has to do with the actual bleaching of tissue pigments, and this may be acconjiliahed with any of the following: a. Mayer's chlorine method: Transfer specimens from 70^ alcohol to freshly made Mayer's solution, cover, and leave for period of from aeveral minutes to several daya, depending upon the degree of bleaching desired. 18 EXPERIMENTAL EQUIPMENT AND PROCEDURES b. Javelle water: Slow tut satisfactory bleaching agent. 0. Bichromate bleach: Following bichromate fixation such as Smith's tissues may be bleached in 10 cc. of 2$ sodium bisulphite to which Is added (just before using) 2-h drops of concentrated HCl. Acts over 6-12 hours. d. gydrop;en peroxide: Used as 2^ solution but will macerate tissues if used for long period. DEHYDRATION The standard method of dehydration la to run the tissues up through a graded series of alcohols (55^ to absolute alcohol) with 15-50 minute stops for tissues and 2-5 minute stops for sections or small eggs. To conserve alcohol, dehydration may be accomplished in small vials by decanting off and changing the alcohols. If 5^ glycerine or triethana- lamine is added to the dehydrants, the tissues will not become so brittle and will be easier to section. It has been found that dioxan is an excellent substitute for alcoholic dehydration. Following the washing (or even without washing) the tissues are put directly into dioxan for 2 two-hour changes. Larger tissues may be left in dioxan for a month without any deleterious effects. If the dioxan Is kept in covered Jars and over copper sulphate, it should last a long time. The dioxan fumes are known to be poisonous to humans. Amphibian eggs and yolk-laden embryos should not be completely dehydrated. It la thought that the dioxan method does not give complete dehydration and that this la one of the reaaona that dioxan givea better reaults with amphibian egga. CLEARING Clearing followe dehydration and must be accomplished before embedding. In any case, the absolute alcohol should be mixed with the clearing agent so that the transition la gradual. Several steps of 15-50 minutes each are best. The dehydrant dioxan acta as its own clearing agent. Xylol - this agent tends to make yolk-laden egga and embryos rather brittle and sec- tions are apt to crack. This tendency can be somewhat compensated by the addition of 5^ lanolin (Sheep fat) to the xylol. Xylol will become cloudy if the embryos were not suf- ficiently dehydrated. Benzole - same aa xylol. Dioxan - this is both a dehydrating and clearing agent, and can be mixed with fixa- tives. The transfer to paraffin is generally made by way of a paraff In-xylol mash. Uaed for both cytological and histological preparations. Chloroform - tissues are transferred from absolute alcohol to a mixture of equal parts of alcohol and chloroform until they sink to the bottom of the container, thence to pure chloroform for final clearing. Keep the vial covered. Wintergreen oil - excellent for yolk masses and glandular tissue, used as chloroform. Tissues become translucent and may thus be photographed. Clove oil - uaed same aa wintergreen oil. Aniline oil - aame aa wintergreen oil except that it will mix with lower alcohols down to 80^. Stained sections are dehydrated to 95^, then to equal parts of 95^ and aniline oil for 10 minutes; finally to 2 ten-minute changes in pure aniline oil. Mount In anillno-balBam. Good for mitotic figures. EXPERIMENTAL EQUIPMENT AND PROCEDURES 19 EMBEDDING This la accompli shed by Infiltration with paraffin, wax, rubber, or mixtiires of these. Whatever clearing agent Is used, the process of embedding shoiild be gradual and at a temperature slightly above the melting point of the mixtures. To the vial contain- ing the clearing fluid and cleared tissue, add shavings of the embedding mixture and bring to about 1+0°C. for several hours. The paraffin will become gradually more concentrated with the evaporation of the clearing agent. Then transfer to embedding substance for 2 half -hour changes. If dloxan Is used for dehydration and clearing, wann a mixture of 25 cc. dloxan, 5 cc. xylol and 20 cc. of 50°C. paraffin and transfer the tissues to this mixture for 50 minutes. Then transfer to pure 50°C. paraffin for 15 minutes and finally to 2 changes of 55-55°2. paraffin. For eggs, a lower melting point paraffin Is better. Prolonged em- bedding tends to make the eggs brittle. Various embedding eubatancea may be used, starting with paraffin of different melting points, the softer paraffin being best for the yolk eggs and glandular tissue and the hsird paraffin for tissues In general. a. Paraffin alone often crystallizes when cooled, or It may even flake, particular- ly If the xylol has not been entirely removed. In order to prevent crystalliza- tion and to facilitate ribbon formation, a mixture has been devised which allows sections down to 5 microns even during the summer, without the use of Ice. Paraffin M.P. l+8°-50°C. - 90 gms. Beeswax, white - 5 gms. (for ribboning) Bayberry wax, pale green - 5 gms. (for hardening) The same mixture can be used for tissues rather than eggs but the higher melt- ing point paraffins would be advised. b. Tissue mat - a commercial mixture probably very similar to the above excellent. c. Rubber - small amount of white rubber may be melted into the paraffin to give better ribbons. Particularly good for large sections or semi-hard (i.e., car- tilage) tissue. The conventional method of embedding Involves paper boxes made in appropriate sizes. Syracuse dishes lined with glycerine or white vaseline may be used for large tissues or large numbers of tissues. Paraffin buttons made by pipetting a small amount of melted paraffin onto a clear slide will prove satisfactory for small tissues. The most satisfactory method is to use Plaster of Paris (see Solberg) embedding boxes. These are made by cutting out several blocks of soft paraffin, cut into the shapes and sizes desired, and making certain that the sides of the blocks all slant outward from the bases. Place these blocks with larger surface down on glazed paper and cover, carefully with wet Plaster of Paris. When dry, tear off the paper and dig out the soft paraffin with a scalpel. Finally shave off the excess Plaster of Paris until a thin-walled embed- ding box is made. To use, first submerge the box in cool water, pour out all of the water; add melted paraffin; add tissue and orient it with hot needle; bring box into ice water but do not submerge it until there is a surface film. When the film entirely covers the paraffin, plunge the whole box beneath the surface of the water and the paraffin block will pop out and come to the surface. SECTIONING Amphibian ceils are among the largest known so that sections should rarely be less than 10 microns. When organ systems are to be studied, sections may be as much as 25 microns. The standard rules to be followed are: to use a clean, sharp knife at a fair angle; to clean the knife blade frequently with xylol; and to section yolk-laden material veiy slowly. Examine the knife under binocular magnification for knlcks. If yolk-eggs are embedded so that the knife cuts from vegetal toward the animal pole, cracks will be eliminated. 20 EXPERIMENTAL EQUIPMENT AND PROCEDURES Some investigators expose the amphibian egg 'by cutting off one or two sections and then soaking the entire block in water, overnight. Such an egg will expand beyond the cut siu'face and several sections will be lost, but frequently very nice serial sections of the remaining portion of the egg can be acquired. A second soaking, half-way through the egg, may be necessary. Apparently a small amount of water invades the paraffin and egg and reduces brittleness. Another modification is to paint each section with a very thin coating of ceiloidln and mastix. Rubber-paraffin mixtures have been used. The early stages of amphibian development are difficult to section satisfactorily. MOUNTING SECTIONS The conventional method is to coat the slide with a thin layer of egg-albumen prior to mounting the sections. Some stains will show up the unevenness of the albumen and it is difficult to control the amount applied. A more satisfactory method is to float the sections on the slide and over albumen water made up of 10 cc. of (boiled) distilled water which has been cooled and to which has been added 1 drop of egg albumen. Enough of this albumen-water should be used to allow complete expansion of the sections over the warming oven, held at 4o°-'+5'-'C. Adherence should be complete In 12 hours. Thick sections and large yolk masses may require additional treatment before they will adhere permanently to the slides. In the hydration process leading to the staining, the mounted sections should be taken through xylol and absolute alcohol and then immersed briefly In a very thin solution of cellodin before going into the lower alcohols. The alcohols and stains will penetrate the ceiloidln satisfactorily. During the hydration process (descent through the alcohols) the yolk-laden sections of amphibian eggs often come loose from the slide, no matter what precautions in albumen- fixation are taken. To avoid this. Just before going into the 95^ alcohol from the 100^ (absolute) alcohol, dip the slides into the following mixture: Ceiloidln 8$ 50 cc. Absolute alcohol _. k^O cc. Ether 1^50 cc. This will provide the sections with a very thin coating of ceiloidln which will hold them In place but will in no way interfere with staining, and subsequent dehydration. HYDRATION OF MOUNTED SECTIONS This may be accomplished with 1-2 minute shifts in the various alcohols after the embedding substance (paraffin) has been completely dissolved off. Dioxan may be used in hydration as well as dehydration. STAINING The choice of stain depends entirely upon the end results desired. Nuclear stains: (Where destaining is necessary, used acidified 70^ alcohol.) a. Delafield's haematoxylin - should be deep wine colored, aged for months, and used In concentrated form for 5-10 minutes. Follow with wash in tap water to blue the stain. Cytoplasmic stains not necessary since Delafield's gives the cytoplasm a slight pink color. b. Harris' haematoxylin - excellent for chromosome studies in tail tips. Use like Delafield's although better to dilute C+x) and stain longer. Destain with 55^ acid alcohol and blue in tap water. c. Harris' acid haemalum - dilute to 25^ with distilled water, stain as with haematoxylin and rinse in tap water. EXPERIMENTAL EQUIPMENT AND PROCEDURES 21 d. Heldenhaln' a Iron haematozylin - still the most reliatle and satisfactory nuclear stain. The alum must be in form of violet crystals when the mordant is made up. Mordant in k'jL for 12 hours, stain in 0.5^ haematoxylin for 3-12 hours (shorter time if 0.1^ Turgitol is used) and deatain in 2']t alum under hinocular magnification. The slide should be rinsed in water when the tissue has become grey and the nuclear constituents first become visible. Elnse thoroughly and dehydrate quickly. There is a modification of the Heidenhain's Iron Haematoxylin method which shortens the staining time and makes the nuclei and chromosomes blue- black instead of intense black, and the cytoplasm retains a slight stain which increases the visibility of the spindle fibres. Two solutions are needed: 1. Haematoxylin: 1% in absolute alcohol Ferric chloride, C. P. 1.2^ HCl _ 0.2'f, Prepare solutions separately. Before ueing, mix equal volumes of 1 and 2; stain about 20 minutes; destaln in a weak ferric chloride (O.l^) under binocular magnification. Intensity of stain relative to concentration of HCl. e. Feulgen stain - this is a chemical test for thymo-nucleic acid and if properly used will give excellent chromosome stain without any trace of the cytoplasm. Polar bodies of the frog's egg will stand out as red or violet in color. The modifications recommended are: 1. Hydrolysis 10-12 minutes at 6o°C. in N-HCl (use 82.5 cc. cone. HCl to 1000 cc. water) 2. Blnae In cold N-HCl. 5. Elnse in d,lstllled water. k. Stain in acld-fuchsin about 80 minutes. Basic fuchsin 1 gm. Distilled water 200 cc. This Is made up as follows: Bring water to boil, add basic fuchsin and stir thoroughly. Cool to 50°C; filter through coarse filter; add 20 cc. of dilute HCl; cool to 25°C, ; add 1 gm. of anhydrous sodium bisulphite. When the solution becomes colorless It la ready for use. Keep in the dark, and do not use if it becomes discolored. 5. Pass sections throi;igh 5 baths of dilute sulfuroua acid Distilled water 200 cc. 10^ aq. solution of anhydrous sodium bisulphite 10 cc. Dilute (N) HCl 10 cc. 6. Binse In distilled water. 7. Counterstaln (if desired) with 0.5^ Grubler's light green in 95^ for 5 to 7 seconds only. 8. Mount in gum damar. f. Mayer's haemalum - very good for sections containing chromosome figures. Cytoplasmic stains: a. Light green - 0.25^ In 95^ alcohol. Good for spindle fibres. Stain 1-2 minutes . b. Eosin - 0.5^ in 95^ alcohol. Merely dip the slides into this quickly. Should not be used when chromosomes are to be studied or photographed. c. Safranin 0 - 1^ in emillne water, wash in tap water. d. Orange G - sat. solution in clove oil, use only 3O-60 seconds. e. Masson stain - differential stain; excellent for pituitary cell types. 22 EXPERIMENTAL EQUIPMENT AND PROCEDURES Solution A Solution B Solution C Acid fuchsln 0.5 gm. This la a 1^ Glacial acetic 2.0 cc. Ponceau de xylidine 0.7 gm. aqueous Distilled water 100.0 cc. Distilled water _ 100. cc. phosphomolyb- Aniline blue to Glacial acetic 1.0 cc. die acid saturation (3- gm. ) Staining procedure: Masson A for 5O-60 seconds: Rinse in distilled water; Masson B for 2-5 minutes; drain, do not rinse; Masson C for 10 minutes to 2 hours, depending on tissue; rinse in distilled water; I'jt acetic acid for 5 minutes to remove excess phosphomolybdi c acid; equal parts of 1^ acetic and absolute alcohol for 1 minute or dropped directly onto the slide; abso- lute alcohol; xylol, etc. PERMANENT MOUNTING While balsam dissolved in xylol is the standard mounting medium, dried balsam will chip and often takes on a yellow tinge with age. Gum damar is preferred. * Remove frog egg Jelly with 10^ Chlorox (see Shumway: I9I+2 Anat. Bee. 85:309). Special staining procedure for skeletons, particularly good for post-metamorphis stages of amphibia and chick embryos beyond the 10th day. a. Fix in 95^ alcohol two weeks to harden. b. Put in 1% KOH for 2l+ hours. c. Put in tap water and pick off as much of fleshy material as possible. d. Put in 95^ alcohol; change once during 6 hour period. e. Put in ether for 1-2 hours to dissolve away any fat; use acetone if there is little or no fat. f. Put in 95^ alcohol for 6 hours; change once. g. Put in 1^ KOH for 6 days. h. Put in Alizarin red "S" for 12 hours. 1. Put in 1^ KOH for 2k hours. J. Put in Moll's solution for 2k hours, k. Store in 100^ glycerine. BECCMMENDED BKFERENCES: Lee : "Vade Mecum" McClung: "Handbook of Mlcrascopical Technique" CLEANING AND BLEACHING OF VERTEBRATE SKELETONS A. Solution of Ammonia - 2%. B. Mixture of 50lt Benzene and 50^ Naptha. C. ^jrdrogen peroxide - 2^. Clean off all soft tissues, picking them away to the bone with scalpel. Place the skeleton in each of the above solutions for about 12 hours each. Skeleton will come out clean and bleached. EXPERIMENTAL EQUIPMENT AND PROCEDURES 23 BEFERENCES : Adama, A. E,, I928 - "Paraffin sections of tissue supra-vitally stained." Sc. 68:305. Bacon, E. L., 1914-7 - "The adaptation of block surface staining of fetuses embedded in ethyl methacrylate." Anat. Eec. 99, Bassett, D. L., 19*+? - "Ethyl methacrylate as a preserving medium for gross anatomical serial sections." Anat. Bee. 99. Becker, E. F., I9I1O - "Experimental analysis of Kuo-Vaseline technique for studying be- havior development in chick embryos." Proc. Soc. Exp. Biol. & Med. ^5:689. Belar, K. , I928 - "Methodik der wlssenschaftlichen Biologie." ed. by T. Peterfi Vol. 1:779. Brachet, J., 19^16 - "La specificlte de la reaction de Feulgen pour la detection de I'acid thymonucleique." Experientia Il/U. Bradbury, F. E. & D. 0. Jordon, I9U2 - "The surface behavior of antl- bacterial substances. I. Sulfanilamide and related substances." Bioch. Jour. 36:287. Bragg, A. N., I958 - "The organization of the early embryo fo Bufo cognatus as revealed especially by the mitotic index." Zeit. f. Zellforsch. u. mikr. Anat. 28:15U. Brown, M. G., 19'+0 - "A direct action micromanipulator especially designed for precise operations in experimental embryology." Anat. Eec. 78:suppl. 123. Bruechner, A. H, , igi^-J - "Sulfanilamide activity as influenced by variation in pH of culture media." Yale Jour. Biol. Med. 15:815. Cleveland, E. & J. M. Wolfe, 1952 - "A differential stain for the anterior lobe of the hypophysis." Anat. Eec. 51:i;09. Child, C. M., I95U - "Differential reduction of Methylene blue by living organisms." Proc. Soc. Exp, Biol. & Med. 52:51;. Cole, E. C, I9U6 - "Improved fixation in vitally stained methylene blue preparations." Stain Tech, 21:l65. Cooper, K. W. & B. H. MacKhight, ' 1957 - "Cooling devie© for the microtome," Stain Tech, 12:25. Davidson, M. H. , I9I+5 - "The preparation of frog embryology slides." Turtox News. 25:53, Dawson, A, B. , 1939 - "Visualization of the vertebrate skeleton In the entire specimen by clearing and selective staining," Am. Biol. Teacher. 1:91. Dawson, A, B,, I959 - "Differential staining of the anterior pituitary of the cat." Stain Tech, Ih:!^^. Dempster, W. T., 1914-1 - "The mechanics of microtome sectioning." Anat. Bee. 79:8uppl. 18. Detwiler, S. B. & G. E. McKennon, 1929 - "Mercurochrome (di-brom oxymercuri-fluorescin) as a fungicidal agent in the growth of amphibian larvae." Anat, Bee. 1+1:205. Detwiler, S. E. & C. 0. Eobinson, I9I+5 - "On the use of sodium sulfadiazine in surgery on amphibian embryos." Proc. Soc. Exp. Biol. & Med. 59:202. Detwiler, S. B., W. M. Copenhaver & C. 0. Eobinson, I9I+7 - "The survival of Amblystoma embryos when treated with sodium sulfadiazine and quinine sulphate." Joui-. Exp. Zool. 106:109. Dickie, M. M., l^kk - "A new differential stain for mouse pituitary." Sc, 100:297. Drury, H. F., I9I+I - "Ancrl acetate as a clearing agent for embryonic material." Stain Tech. l6:21. Fankhauser, G., I952 - "Cytological studies on egg fragments of the Salamander Triton." Jour. Exp. Zool, 62:185. Feulgen, E. , I925 - "Die Nuclealfarbung. " Abderh. Handb. biol. Arbeit. 5:1055- Forbes, J., I9I45 - "Glycerin Jelly mounting medium for frog eggs and early embryos." Trans. Am. Mlcr. Soc. 62:525. Goldsmith, J. B., I929 - "A new fixation of general use." Trans. Am. Mlcr. Soc, l^:2l6. Gray, P., I952 - "Notes on the practice of fixation for animal tissues," Jour, Boy. Mlcr. Soc. 55:13. Gregg, H. E. & W. 0. Puckett, I9I43 - "A corrosive sublimate fixing solution for yolk- laden amphibian eggs." Stain Tech. 18:179. Groat, E. A,, 1959 - "Two new mounting media superior to Canada Balsam and Gum Damar, " Anat , Eec . 7!^^ : suppl . 1 . Harrison, B. G. , I92I - "On relations of symmetry in transplanted limbs." Jour. Exp. Zool. 52:11. Henry, B. J., I9U5 - "The mode of action of sulfonamides." Bact. Bev. 7:175. Henry, B. J. & E. C. Smith, 191+6 - "Use of Sulfuric-Acid-Diehromate mixture in cleaning glassware." Sc. 10l+:l+26. 2h EXPERIMENTAL EQUIPMENT AND PROCEDURES Koneff, A., 1956 - "An iron-haematoxylin-anlllne "blue staining method for routine labora- tory use . " Anat . Eec . 66 : 175 • Kornhauser, S. I., 19'*5 - "A quadrxiple tissue stain for strong color contrasts." Anat. Bee. 85:55- Masson, P., I928 - "Carcinoids and nerve hyperplasia of the appendicular mucosa." Am. Jour. Path. l+ilBl (description of Masson stain). McElroy, W. D., I9UU - "On the specificity of sulfanilamide action." Jour. Cell. & Comp. Physiol. 25:109. McGovern, B. H. & E. Rugh, I9I+I+ - "Efficacy of M-amlno ethyl benzoate as an anesthetic for amphibian embryos." Proc. Soc. Bcp. Biol. & Med. 57:12?. Moore, Betty, 19*4-0 - "Chromosomes of frog eggs and embrj'-os stained by the Feulgen method to avoid excessive staining of yolk granules." Anat. Bee. 78:suppl. 122. Nebel, B. E., 19'4-0 - "Chlorazol Black E as an aceto-carmine auxiliary stain." Stain Tech. 15:69. Needham, J. & E. J. Boell, 1959 - "An Ultramlcro-KJeldahl technique." Bioch. Jour. 55:ll4-9. Nichols, C. W., 19l;0 - "A simple method for mounting embryologi cal material." Stain Tech, 15:5. Patton, P. L., 19i4-5 - "A cool light for dissecting microscopes." Sc. 98:592. Petrunkevitch, A., 1957 - "On differential staining." Anat. Bee. 68:26?. Pickels, E. G., 19'4-2 - "Apparatus for rapid, sterile, removal of chick embryos from eggs." Proc. Soc. Exp. Biol. & Med. 50:22U. Polllster, A. W., I959 - "The structure of the Golgi apparatus in the tissues of the Amphibia." Quart. Jour. Micr. 3c. 81:255. Pollister, A. W., 19'+1 - "Mitochondrial orientations and molecular patterns." Physiol. Zool. li+:268. Price, J. W., 19^5 - "A device for observing living fish embryos at controlled tempera- tures." Ohio JovlT. Sc. ^5:85. Price, J. W. & S. V. Fowler, 19'4-0 - "Eggshell cap method of incubating chick embryos." Sc. 91:271. Puckett, W. C, 1957 - "The dioxan-paraffin technic for sectioning frog eggs." Stain Tech. 12:97- Puckett, W. 0., V^kl - "The Methacrylate plastics as mounting media for biological materials." Anat. Bee. 80:^55 (See ibid, 78:105). Prokofleva, A., 1955 - "On the chromosome morphology of certain Amphibia." Cytologia. 6:lk&. Elehards, A. W. , I956 - "Killing organisms with chromium as from incong)letely washed bichromate sulfurlc-acid cleaned glassware." Physiol. Zool. 9:2^6. Eobertson, 0. H., 19i+2 - "Sterilization with glycol vapors." Harvey Lect. 19^2-^5, p. 227- Schotte, 0. E., 1950 - "Transplantatlonsversuche iiber die Determination der Organ - Anlagen von Anurenkeim." Arch. f. Ent. meeh. 125:179- Schwind, J. L., D. 0. Bemp, & S. Sturgess, 1957 - "A method of measuring the volume of Amphibian embryos." Sc. 86:555. Seaman, G. E., I9U7 - "Penecllln as an agent for sterilization of Protozon cultures." Sc, 101:527. Severlnghaus, A. E., I952 - "A cytological technique for the study of the anterior lobe of the hypophysis." Anat. Bee. 55:1- Shumway, W. , 1942 - "Steiges in the normal development of Rana pipiens." Anat. Eec. 85:509 (Chlorox removal of amphibian jelly). Slater, ,D. W. & E. J. Dornfeld, 1959 - "A Triple stain for amphibian embryos." Stain Tech. U:105. Solberg, A. N., I959 - "The preparation of plaster of Paris embedding boxes." Stain Tech. l!+:27. 3pemann, H., I920 - "Mlkrochlrurglsche Operatlonstechnlk. " Abderhalden, Handb. blol. Arb. V. 5- Stultz, W. A., 1955 - "Devices for experlnents on amphibian embryos." Anat. Eec. 61+: auppl. 1+5. Stultz, W. A., 1958 - "The use of plastic materials for operation on amphibian embryos." Sc, 88:555. Taylor, E. M. & R. J. Chialvo, I9I+2 - "Simplified technic for Inoculating into amniotic eac of chick embryos." Proc. Soc. Exp. Biol. & Med. 51:528. Tyler, A., I9U6 - "Eapld slide-making method for preparations of eggs, Protozoa, etc." The Collecting Net 19 . EXPERIMENTAL EQUIPMENT AND PROCEDURES 25 Twitty, V. C, 1937 - "Experimenta on the phenomenon of paralysis produced ty a toxin occurring in Tri turns embryos." Jour. Exp. Zool. 76:67. Tyler, A. & W. E. Berg, I9I+I - "A new type of micro-respiraometer. " Sc. 9it-:576. Vagtendonk, W. J. van., F. A. Puhrman, E. L. Tatum, & J. Field, 19^+2 - "Triturus toxin: Chemical nature and effects on tissue respiration glycolysis." Biol. Bull. 85:157- Weiss, P. , 1956 - "A convenient retractor for use in operations and dissections of small- sized animals." Sc. 8'+:l61+. Williams, T. W., I9I+I - "Alizarin Red S and Toluidine Blue for differentiating adult or embryonic bone and cartilage." Stain Tech. l6:25. "H/ien it is recalled that the 9,200.000.000 cells m the human cerebral cortex are the nervous e lements of this organ and that they collectively constitute rather less than a cubic inch of protoplasm, it seems almost incred- ible that they should serve us as they do. They are the materials whose activities represent all human states, sen- sations, memories, volitions, emotions, affections, the highest flights of poetry, the most profound thought s of philosophy , the most far-reaching theories of science, and, when their action soes astray, the ravings of insani ty . It is this small amount of protoplasm in each of us that our whole educational system is concerned with training and that serves us through a lifetime in the growth of personal- ity " ^ - G. II. Parker TECHNIQUE FOR STAINING CHROMOSOMES IN TAIL-FIN It Is often desirable to determine the chromosome count in an androgenetic, gynogene- tlc, or parthenogenetic embryo or tadpole. This can be accomplished the better with the Urodela than with the Anura, due in part to the larger ajnount of pigment in the epidermis of most Anura. There are two methods of preparing the material. The one involves fixing the entire larva and subsequently peeling off the entire tail epidermis. This is better with Anuran material. With such a large sheet of cells one can generally find abundant chromosome figures. There is a technical difficulty of keeping the tail epidermis flat- tened through the staining procedure. The second method is suitable for Urodele larvae and allows them to survive, since only the distal l/j or the tail fin is cut off. THE METHOD OF PARMEMTER (for Anura) There is a stage in larval development when the yolk in the tail fin has been reduced to a minimum and yet the cells themselves have not become so small that the chromosomes are difficult to identify. For the frog tadpole this stage is attained in from 15 to 20 days at laboratory temperatures, at the beginning of feeding. The tail fin should be well formed, thin, and transparent. The steps in the process are as follows: a. Fix the entire tadpole in Bouin's or Michaelis' fluid for 2 hours. b. Transfer to 70^ alcohol to which 2% ammonia has been added. This will shortly remove the yellow coloring of the picric acid. c. Transfer through appropriate (alcohol) steps to water; leave for 12-2U hours. This will tend to soften the tissues somewhat. d. Place the head of the tadpole in a depression of a shell-depression slide, with the tall flattened on the slide in a few drops of water. e. With a sharp scalpel trim off the entire margin of the tail fin. Eemove as little of the tissue as possible, but cut to the Junction with the body. f. With sharp scissors make a circular cut around the body of the tadpole just behind the mouth. The cut should not be so deep that it injures internal organs. g. Along the mid-dorsal line cut through the body flap to the junction with the tail. Cut through the mid-ventral line in a similar manner. These cuts will provide lateral flaps of relatively tough body epidermis which is continuous with the lateral tall-fin epidermis. With the tadpole on its side, the tail fin Immersed in water, it will now be possible to grasp the tough body epidermis with forceps and gradually peel off the lateral tail-fin epidermis. This can be done with the aid of a hair loop which can be worked beneath the epidermis as it is raised with the forceps. It is not recommended that a needle be used. If the tail- fin has been proper- ly trimmed, two continuous sheets of epidermis from a single tadpole may be secured, h. If the sheets of epidermis tend to curl, but a few knicks in the edges with a sharp scalpel. Transfer with wide-mouthed pipette. 1. Stain, dehydrate, and clear tail-fins in shell vials or small Stendere. 1. Stain: Heidenhain's Iron Haematoxylin: Mordant 12 hours, stain 2 hours, destain under binocular observation. This is still the best chromosome stain but it has the disadvantage that the destaining must be exact and any persistent yolk material stains black. Harris' Acid Haemalum:* The time and concentration of stain must be determined empirically. It is suggested that 50^ stain be used for 8 to 10 minutes, without destaining. Wash in alkaline tap water. # Mayers Haemalum or Conkllns Haematoxylin are also satisfactory. -26- STAINING TAIL FIN CHROMOSOMES 27 2. Dehydration: This may he accomplished rapidly through the alcohols, vdth 5-iiilnute changes in each grade, or through three 15-iiilnute changes in dioxan. 3. Clear in xylol and mount in clarite. Such large pieces of epidermis may he difficult to handle. If time permits, such pieces can be fastened to slides with thin celloidin and then stained. If permanent mounts are not required, chromosome counts can he made within k hours after fixation hy reducing the hydration time. Tr im off outermost edge of tail fin with sharp scalpel (The tail fin is tr Median ventral be peeled back froff cut this cut edpe of body sl V #^ / i PENTAPLOrD TETRAPLOID TRIPLOID tiC- , « ^'11'^' -•; :^:> ^^ o 00^0 Q ^"^fS Cf^W CHROMOSOMES IN THREE TAILTIPS Small portions of the epidermis of a tetrap- lold, a triploid, and a diploid talltip, each with a mitosis in metapliase. Below, the tliree metaphase plates enlarged. Tlie chromosomes catinot be counted accurately in photomicrographs, but the dimensions and the general appearance of the w)iole chromosome group clearly indicate differences in chro- mosome number. The average size of the non- dividing nuclei is also roughly proportional to the number of chromosomes t)iey contain. Polyploidy In "Eurycea blsllneata" (From FankJiauser 1959 = Joux. Heredity 30:579) RELATIVE SIZES OF NUCLEI AND CELLS FROM THE TAILFINS OF LARVAE OF TRITURUS VIRIDESCENS WITH DIFFERENT NUNfflERS OF CHROMOSOME SETS Above: Surface view of nuclei of epider- mis cells. Nuclear size increases ap- proximately in proportion to chromosome number. Below: Nuclei and cell boun- daries of single gland cells (Leydig cells) . Cell size Increases witli nuclear size. (From Fankhauaer l°ik'j: Quart. Bev. Biol. 20:20) pentaplOid diploid Body cavity of pentaploid distended dist&nded with fluid (ascites). In both larvae the melanophores are "expanded". Tracings of photomicro- graphs. "TrlturuB virldeacena" (From Fankhauaer 19'4-0: Proc. Nat. Acad. Scl. 26:526) NOTES ON THE NATURAL BREEDING HABITS OF SOME COMMON AMPHIBIA . Natural breeding on the part of different species of the Amphibia covers all the sea- sons of the year, in the various latitudes. The one characteristic feature of all but a very few specialized forms is that breeding occurs in or near water regardless of the habitat during the balance of the year. In general, the Amphibia lay their eggs and then desert them. This means that there is a very high mortality and in order to survive, the race must produce a great excess of eggs. It has been estimated (Smith: Science 19'+?, V. 105, P« 619) that the maximum number of eggs layed by any amphibian species is probably in the neighborhood of 55,000. It is interesting that the high numbers are layed by the predominantly aquatic Anura while among the Urodela the number of eggs layed may be less than 100, in certain species. The aquatic environment is apparently the more hazardous when compared with the mildly damp environment where one occasionally finds Urodele eggs. Regarding the insemination of the eggs, the frogs shed their products into the water simultaneously during amplexus; many of the toads similarly shed their gametic products but the eggs are layed In strings and the male Inseminates each egg as it eioerges from the cloaca. Finally, among the Urodela it is necessary for the female to pick up spermato- phores and to take them into her cloaca and genital tract where the eggs are fertilized before being layed. Thus there is considerable variation in the breeding procedure among the various species of Amphibia. There are a number of forms which, because they are relatively common, are likely to be available for use in our laboratories. For this reason there Is presented below a table showing the common name, location, breeding periods, and egg production of these forms. BREEDING HABITS OF SOME COMMON AMPHIBIA Animal Popular Name Locality Breeding # ^gs FBOGS ACRIS GRYLLUS HYLA CRUCIFER HYLA VERSICOLOB PSEUDACEIS NIGBITA RAKA CATESBIANA BANA CLAMITANS EANA PALUSTBIS SANA PIPIENS EANA SYLVATICA Cricket frog Spring peeper Tree frog Swamp tree frog Bullfrog Green frog Pickerel frog Leopard frog Wood frog Central U.S. Eastern seaboard Eastern U.S., Canada All U.S. except N.Eng. East of Bockies Eastern N. America Eastern N. America Entire U.S. Entire U.S. May to July April May and June March and April May to August June to August April and May March to May March to May Few 1,000 50 500 to 1,500 6,000 to 20,000 5,000 2,000 5,000 5,000 TOADS BUFO AMERICANUS BUFO FOWLERI XENOPUS LAEVIS American toad Fowler's toad African clawed-toad Northeastern U.S. Central & East U.S. South Africa April and May April to June April to Sept. 6,000 8,000 15,000 SALAMANDEBS AMBLYSTOMA JEFFERSONIAMJM A. OPACUM A. PUNCTATUM (MACULATUM) Jefferson salamander Marble salamander Spotted salamander East U.S., South Canada East & Middle West Eastern U.S. Early Spring Sept. to Oct. January to May 500 100-250 100-200 -50, BREEDING HABITS OF AMPHIBIA 31 Animal Popular Name Locality Breeding # Eg^s SALAMANDEES ( Continued) A. TIGBINUM Tiger salamander U.S., Canada, Mexi co Dec. to April 100 EUBYCEA BISLINEATA Two-lined salamander Central & East U.S. April to June 20 to 50 HEMIDACTYLIUM Four-toed salamander Eastern U.S. Spring 50 SCUTATUTI PLETHODON CINEREUS Eed-backed sala- mander Entire U.S. June and July 11+ TRITUHJS Japanese fire Japan ? 80 PYEEHOGASTi!;^ salamander TEITURUS Common newt U.S., South Canada April to June 20 to 50 VIEIDESCENS (DIEMYCTYLUS) FROGS ACE IS GEYLLUS, the cricket frog: This frog is found largely In the Central States, from Michigan to Dakota and south to Texas. It ia very small, the head and body never measuring more than l■^• inches. The toes of the hind limbs are webbed. While the color varies there is always a triangular mark between the eyes and a dark ob- lique stripe on the side of the body. The male's throat skin ia grayish-yellow, and its fingers are shorter and body smaller than that of the female. Breeding takes place in shallow, plant-filled water during May to July. The eggs are laid singly axii are attached to stems and twigs. Metamorphosis occurs about the middle of September. HYLA CRUCIFEE, the Spring Peeper. This small frog is found all along the eastern coast and abundantly in Florida. The adults /n™,v-t „ n n d„^ , , ^ -, /n . , , -. ^,X.i T_ jj-uii-i I Courtesy C. a. Pope rarely exceed 1 5/8 inches in length but may be recognized by their j^qi^j^. chica«o Mus high, shrill, clear call so frequently heard in the spring during j^^ Hist ) breeding. There is an oblique cross on the back, the general color being brown of various shades. The digits are not webbed as they are in the genus Eana, but the tips of the toes have discs for climbing on smooth surfaces. The thumb of the male bears a pad on Ita Inner surface, the chin and throat are loose and dark and the males are always smaller than the females. Acrls gryllus, the cricket frOfi,. Hyla crucifer, the spring peeper Tl.e spring peeper's eggs at- taclied to the submerged stem of a plant. After Wri^lit. (Courtesy C. H. Pope l^kk: Chicago Mus. Nat. Hist.) Breeding normally occurs in April when the temperature is about 52°F. The eggs (about 1,000) are layed singly at night while the pair is floating at the surface of the pond. The male inseminates each egg as it emerges from the cloaca of the female. The eggs are small, about 1.0 in diameter, and are generally dark in color. Each egg is surrounded by firm jelly. Hatching occurs in h to l6 days depending upon the water temperature, and temperature tolerance ia from about '+2°F. to 85°F. Metamorphosis is reached in 90 to 100 days, occurring in July. The eggs are excellent for operative procedures. The tadpoles live on diatoms and algae while the adults feed on insects and spiders. Can be fed Droao- phila in the laboratory (preferably the veatigial mutant). 52 BREEDING HABITS OF AMPHIBIA FROGS OVIPOSITION IN HYLA CRUCIFER (Drawings by Mr. Sidney Aberman) a-Normal amplexus of Hyla andersoiili; D-Klrst pliase of ovl- position; c-Second pliase of ovlposltion; d-Tlie back depres- sion release meclianlsin. (From Aronaon 19'+5: Copela '+:256 - Drawings by Mr. Sidney Aberman) BREED ING HAB ITS OF AMPHIBIA 33 HYLA VERSICOLOB, the tree frog. This small (2 to 2^ inches) arboreal species is partial to woodlands and hushy areas but may be found far removed from moist areas, around build- ings, walls, fences. It is found in eastern United States and Canada, The skin la moist and slightly rough; the toes are webbed and their tips expanded into disks for adhering to tree bark; the back may be uniformly colored or blotched, but is never stripped. There ia a white spot under the eye and yellow and brown markings on the groin. These froga can change their body color conaiderably. The male has the usual thumb pad and throat charac- teristics. Breeding occurs in quiet ponds surrounded by high vegetation between the middle of May and the middle of June when the air temperature ia at least 72°F. Egg-laying takea about an hour, the paired animals depositing about 50 egga at a time until one or two thousand are layed. The Jelly, which holds the entire egg mass together, is of loose con- aiatency. The egga are brown at the animal pole and yellow or cream at the vegetal pole. The eggs hatch in U to 5 days into larvae ■^■ inch in length. The tadpole reaches a length of 2 inches and metamorphoses in ^5 to 6o days, never measuring more than 1 inch. The newly metamorphosed froga are green and without characteristic markinga. The length of life ia probably about 9 yeara. These forms are herbivoroua, living on minute algae and diatoms in early life and later living on non-aquatic Insects. PSEUDACRIS NIGRITA. the awamp tree frog. This frog is found widely except in New England) and the diatribut'ed over the United States males rarely exceed l-f inchea and the females 1^ inchea in length. It haa three broad, dark atripes that extend down the back, and the tips of the toea bear small disks. The akin of the chin and the throat of the male is loose and dark. Breeding ia in any small body of water, permanent or temporary, from the middle of March to the middle of April. About 500 to 1,500 eggs are layed in cluatera. Hatching occurs In about 2 weeks, and larval life lasts from 1+0 to 90 days while the tadpole attains a length of l-j- inches. Pseudacris nigrlta, thie swamp tree frog. (Courtesy C. H. Pope 19l).l4-: Chicago Mus. Nat. mat.) BANA CATESBIANA. the bullfrog. This large frog is found East of the Rockies from Mexico to Canada, and is known to have a life span of 15 years. Its typical haunts are small lakes and permanent ponds with much vegetation, generally shadowed by willows and other low trees. The species can be recognized by the fully webbed hind feet, pointed toes, uniformly dull green back (no warts or plicae) and the size of adults ranges from l*- to 8 inches, from snout to anus. The males have a slightly larger tympanic membrane than do the females, a pigmented thumb pad, and yellow throat. The male bullfrog, Rana catesbiana. (Courteay C. H. Pope 191*-'+: Chicago Mua. Nat. Hist.) Ovipositioii of Raiia cates- belana; lateral view. (From Aronson 191+3: Am. Mus. Nov. #1224) (Artist Mr. Eichmond E. Lawler) Its natural food in the larval stage ia diatoms and algae; as young frogs it is in- sects and other small invertebrates; and as adults, any moving object, invertebrate or vertebrate, that can be ingested. This includes fiah, frogs, salamanders, young turtles, moles, mice, and even birds. In the laboratory the bullfrog may be fed smaller frogs. J>^ BREED ING HAB ITS OF AMPH IB lA The breeding season depends upon the latitude, but ranges from June to August. The air temperature must be at least 72°F. and the bottom water temperature at least 66°C. be- fore the eggs are layed. The eggs are small, but as many as 20,000 may be layed by a single female, and the egg Jelly is loosely applied. The eggs will develop between 59°F. and 90°F. , and the hatching span at 68°F. is about l^k hours. There is a long larval (tad- pole) life, the mature tadpole of *+ to 6 Inches total length being ready to metamorphose 2 or 5 years after the egg is layed. Meta- morphosis generally occurs in late July and in August. RANA CLAMITAJfS. the green frog. This frog is found where bullfrogs are found but they prefer permanent, plant-grown aquatic ponds, swamps, meadows, and slow streams. It is a common form in Eastern North America, even at considerable altitudes. The green frog can be recognized by its predominantly green back, with small and widely separated spots, paired ridges of skin from the eyes backward along the back, and webbed and pointed toes as in the bullfrog. It is rarely more than k inches in body length. The tympanum is usually very large, and this is the most readily determined difference between the greenfrog and immature bullfrogs. The tadpoles, which grow slowly, feed on diatoms, and algae; the young frogs and adults eat insects, Crustacea, spiders, snails, earthworms. The food consists largely of non-aquatic forms. Tlie male Raiia clami- tans, tlie green frog, with inflated vocal sacs as seen from above. (Courtesy C. H. Pope 1941+ : Chicago Mus. Nat. Hist.) Typical amplexus of Rana clamitans; lateral view. Egg-laying pos- ture of Rana clamitans just prior to tlie on- set of the ovl- position; dorsal view. I'pstroke of the male Rana clam- itans and the appearance of the first batch of eggs; dorsal view. Downstroke of t)ie male Rana clamitans and tlie formation of the surface film; dorsal view. (From Aronaon 19'^5 : Am. Mus. Nov. #122U. Artist Mr. M. Sorensen) The male has a yellowish throat but also a yellow spot or ring In the center of Its tympanum, and its head is wider than that of the female. Breeding occurs in quiet, shal- low plant -grown ponds from May to August, generally in late June and early July, even in the same latitude and environment. The eggs may number as many as 5,000 and they are ap- proximately the size of those of Eana pipiens, 1.5 mm. in diameter. The eggs and tadpoles can tolerate a low oxygen environment, the tadpoles generally hibernating in mud for one winter and then quickly passing through metamorphosis in early Spring at 570 to UOO days. Low temperatures are disastrous, the embryos being unable to survive lO'-'C. The time lapse from stage #12 to gill circulation at 15°C. ia about 220 hours. SANA PALUSTRIS. the pickerel frog. This is also known as the Spring Leopard Frog, and is found largely in the East, in sphagnum bogs or in cool clear water surrounded by high grass and other vegetation. The adults rarely exceed 5 inches in body length. BREEDING HABITS OF AMPHIBIA 55 The color ia light hrown with numerous squarish dark spots with dark horders, arranged largely in two rows hetween the lateral plicase which run posteriorly from the eyes. The underside of the legs is hright yellow or orange. The thumb pad of the male is un- usually large. These frogs breed in late April and in May, the 2,000 bright yellow eggs measuring ahout 1.6 nan. in diameter. The water tempera- ture is generally between 50° and 65°F., and development is normal between h-6'^T. and 86°F. The eggs hatch in about 2 weeks, the tad- pole reaches a length of about 3 inches and metamorphosis occurs in about 80 days. Under laboratory conditions of 15°C. temperature, it takea 200 hours to go from stage #12 to gill circulation. De- velopment is slightly faster than that of Hana pipiens. nana palustris, the pickerel frog, as seen from above. (Courtesy C. H. Pope I9I+I+: Chicago Mus. Nat. Hist.) It is found in al- BANA PIPIENS, the leopard frog. This is the common leopard frog most frequently used in our laboratories. most all parts of the United States and parts of Canada and Mexico. The adults rarely exceed h- inches in body length (96 mm. ) but females must be 72 mm. or longer before they can be considered sexually mature. The general color is green with light dorsal plicae. The rounded dark spots on the skin have light colored borders. The underside is white. The male is darker and smaller than the female, with firm abdominal muscles, lateral cheek pouches when croaking, and prominent thumb pad. These frogs are omnivorous feeders and difficult to maintain ae adults in the laboratory except under conditions simulating hibernation when they can go for long periods without food. The newly metamorphosed frogs may be fed earthworms cut into 1 inch lengths. These segments of worm will continue to move and thus attract the frogs. Male Rana pipiens (the leopard frog) with vo- cal sacs Inflated, as seen from above. (Courtesy C. H. Pope 19kk: Chicago Mus. Nat. Hist.) These frogs breed from March to May depending upon the latitude in which they are found. They lay about 5,000 eggs (diameter about 1.75 nim. ) which generally reach metamorphosis dui'ing the summer. The span from fertilization to hatching Is about 8 days and to meta- morphosis (in the laboratory at 25°C. ) Is about 75 days. The eggs are normally layed In water at about 15°C. and the upper limit of temperature tolerance la about 31°C. Leopard frogs have been known to live for 5 years. Iholomus End of ovlpositlon. Tlie male, about to release, is sliowing the pre- release movements. Note the female (beneatli) shapiiif!; the eggs into a clump . (From Aronson 1914-2: Bull. Am. Mus. Nat. Hist. 80:127; nucleus N.n tegmentum tiypottialomus { pars ventralls WARNING CROAK [ SRftWNING MOVEMENTS 0 ^ RELEASE olfoctory bulb cerebrol twmispliere preoptic Oreo SWIMMING RESPONSE Diagrammatic sagittal section tlirough tlie brain of Rana pipiens Indicating the regions of tlie brain that were found to be of primary importance for tlie mediation of each of four phases of sexual behavior. (From Aronson l^k-^: Bull. Am. Museum Nat. Hist. 86:89) 36 8REE0ING HABITS OF AMPHIBIA BANA SYLVATICA. the wood frog. Thia frog Is found only in the Northern States and Canada. It rarely exceeds 2-5/U inches in body length, and Is light hrown in color with a dark streak on either side of the head and a dark line from the tip of the snout to the eye. There is also a black patch over the tympanum. The dorsal plicae are prominent but not of a dif- ferent color. The head is pointed. Its feeding habits are like those of Bana piplena. These frogs breed in ponds, in wooded regions where there are dead leaves and mud. They begin breeding in early March in water at about 12°C. The eggs are larger than those of Rana pipiens (about 2.0 mm.) and number about 5,000. Depending upon the tempera- ture, metamorphosis is reached in from ko to 50 days. Development is normal even at U°C., indicating wide temperature tolerance but at a lower level than for Rana pipiens. Stage #12 to gill circulation stage requires only about 95 hours at 15°C. and at 20°C. metamor- phosis la achieved in it-5 days. .laiia sjlvatica, tlie wood frog. (Courtesy C. H. Pope l^kk: Chicago Mus. Nat. Hist.) Exceptional cases of crosa-oviposition Noble and Aronson (19^2) report that when Rana clamitans or Rand sylvatlca males as- sume the amplectlc position with ovulating Rana pipiens females, the grip is lateral and below the axillae rather than ventral, as with pairs of Rana pipiens. This grasp seemed to make it difficult for the female to oviposit and the caudal half of the male's body tended to float away from the female. Even though the cloacae are not approximated (see figure below) the eggs are not generally fertilized or. If fertilized, rarely develop be- yond the gastrula stage in hybrid croasea ( aee aection on ^T-bridization) . Female Rana pipiens ovipositing witli male Rana clamitans (Drawing made by the Illustrators Corps of The American Museum of Natural History. Loaned through courtesy of L. R. Aronson.) TOADS BUFO AMERICANUS, the American toad. This toad, common in gardens as a night prowler, is found in the northeastern United States. The adults vary in size from 2 to U Inches in body length, the males being smaller and possessing black china. The ventral surface of these toads la aand colored and granular while the dorsal surface is warty. The eyes are protruding £ind two large parotid glands extend backward from the eyes. The color is olive green. Food consists primarily of large insects found around gardens. BREEDING HABITS OF AMPHIBIA 37 Breeding oociirs in April and May, the eggs being layed in long spiral tubes of jelly and totalling up to 20,000 in exceptional cases. The average is about 6,000. The time necessary for hatching is 2 to 17 days, depending upon the temperature, and for metamor- phosis in nature is about 60 days and occurs in August. The temperature tolerance is from about 50°F. to 86°F. Bufo fowleri, Fowler's toad . Fowler's toad feigning deat)j. After Dickersoii. Bufo americanus, American toad. tiie Left: Egg cables of American toad twenty-four hours after tliey were laid. Riglit: Same cables tliree days later, s)]Ow- ing newly liatclied larvae. After Dickerson. (Courtesy C. H. Pope 19!+!+: Chicago Mue. Nat. Hist.) BUTO FOWLERI. Fowler's toad. This smaller toad is found along 'beachss, roadsides, and in sandy areas where there may be shallow water in the central, east and northeastern States. The adults measure not more than 5 inches in length. The general color is green with mid- dorsal light stripe. The male throat is black. The warts are generally small, rounded and uniform, and generally at least 5 warts are enclosed in each dark patch. Conspicuous shoulder glands, the parotids, are oval and long and in contact with long ridges behind the eyes. Food the same as Bufo americanus. Breeding occurs from April to June, a little later than for Bufo americanus. About 8.000 eggs are layed in strings, each egg measuring about 1.0 to 1.2 mm. in diameter. The metamorphic span takes about k-0 to 60 days and is generally complete by August. 58 BREEDING HABITS OF AMPHIBIA Bufo amerlcaiiiis pair e\hiDiting the back-arch release mechanism. Oviposition of Bufo americaiius. (From Aronaon I9UI+: Am. Mua. Nov. #1250. Artist Miss P. Hutchinson) XENOPUS LAEVIS. the South African clawed toad. This form has been classified as a toad and as a frog, Its skin texture heing more that of the frogs and its breeding habits more like the toad. It is found in Africa, from the Cape to Abyssinia, and measures about k Inches maximum In the adult stage. It is purely an aquatic form, with clawed toes, no distinct tympanic membrane, tongue- less, single posterior median opening for the Eustachian tubes, dilated sacral vertebrae, carnivorous, and has Sllurold appearing larvae. It Is sluggish, never leaving the water and remaining submerged most of the time. Its greatest activity is seizing food, which it does rapidly. It Is negative to light. It should be fed on alternate days strips of beef, beef heart, liver, earthworms, Tubifex, small newts, tadpoles, Daphnia. It reaches sexual maturity in 2 years. Inguino-amplexus usually in July, thousands of eggs being extruded but often not all fertilized. Egg measure 1.5 mm., with Jelly It is 5.0 mm. Embryos hatch out in 37 hours and Siluroid type transparent tadpoles appear on third day. The tadpoles feed on Chlanj^domonas . Developmental temperature must be at least 22°C. BREEDING HABITS OF AMPHIBIA 39 The adult female: South African clawed toad Xenopus laevis. Photo "by courtesy of Dra. Weis- man and Coates, from "The South African Frog in Preg- nancy and Diagnosis." N. Y. Biologic Besearch Foundation, 19i;4. Dorsal view Ventral view Amplexus (coupling) In Xenopus laevis. (From Dr. H. A. Shapiro 1956: Brit. Jour. Exp. Biol. 15:'+8) AMBLYSTCHA JEFFERSONIANUM, Jefferson's salamander. This salamander is found in southeast- ern Canada and northeastern United States westward to Minnesota and southward to Virginia. It prefers cold climate and the adults measure about 7 inches from tip to tip. It is pri- marily nocturnal, burrowing in loose soil and leaf mold, under logs. It is bluish black or dark brown in color with numerous bluish white flecks concentrated on its sides. The swollen vent of the male protrudes noticeably and its tail is longer and flatter than that of the female. Arablystoma jef fersonianura, Jefferson's salamander. (After Bishop) Hemidactylium scutatum, tlie four-toed salamander found on May 11th. (After Bishop) EGG-LAYING OF SALAMANDERS i Courtesy C. H. Pope. Chicago Mus. Nat. Hist.) 1+0 BREED ING HAB ITS OF AMPHIB lA Breeding occurs in early spring and the exact time i.s dictated by the temperature. The site is any pond, permanent or temporary, and the eggs are layed singly at the rate of about h per minute and are attached to each other and to a twig. Never more than 500 eggs are layed, and the four Jelly capsules often take on a greenish color due to the growth of a unicellular plant. Mating occurs Just before laying, and incubation takes about 6 weeks in nature and about 2 weeks at laboratory temperatures. The larval period lasts from 56 to 125 days, depending upon whether the pond dries up, and at metamorphosis the larva is about 1-7/8 inches long. Sexual maturity is achieved in about 21 months. AKBLYSTCMA OPACUM, the marbled salamander. This 1+ inch salamander Is found in the East and Middle West, but not north of a line from Boston to Chicago. It is quite abundant on Long Island and at Bear Mountain, but is difficult to find except during the breeding sea- son during the latter part of September and October. This is a black salamander with con- spicuous light colored bands across the back. These bands are wider on the sides of the body than on the back. The light colored markings of the male are white and of the female tend to be gray. The male has the characteristically protruding vent. Plethodon cinereus, the red-backed salsunander, found on July 16th. Atnblystoma opacum, thie marbled sala- mander found on October 26tl) in North Carolina. (After Bishop) BROODING OF EGGS BY FEMALE SALAMANDERS (Courtesy C. H. Pope l^kk: Chicago Mus. Nat. Hist.) Breeding occurs in the Fall following an elaborate courtship at the breeding grounds which are on the edge of dry pond beds. Spermatophores are picked up by the female and she proceeds to deposit singly from 100 t* 250 eggs in a nest which is nothing more than a depression under moss or a log. The duration of the pre-hatching stage depends entire- ly upon the rainfall and may not occur until Spring. The adults can drown but the larvae require the Fall rains to hatch. The eggs withstand desiccation but shrink as they dry. They hatch at about 70°F. and mature about 15 months later. AMBLYSTOMA PITNCTATUM, the spotted salamander formerly known as A. maculatum. This is the most common of the large American salamanders and is found in the Eastern United States. There are two irregular rows of bright orange spots along the back. The background color is bluish black. The males have dark bellies and protruding vents. Breeding occurs Immediately after the Spring thaws, and there is a brief courtship on the part of the male before it drops up to ko spermatophores, in shallow water. The water temperature may be as low ae 15°C. The females pick up the spermatophores with their cloacal lips and proceed to lay from 100 to 200 eggs in semi-solid clumps of Jelly about 6 inches below the surface, often attached to a stick or stem. The Jelly is some- times opaque, but the embryos are normal. The Jelly masses lie freely in the water. BREEDING HABITS OF AMPHIBIA 111 Typical egg clusters of spotted salamander as they appeared on April 9. A spermatopliore can be seen on leaf below large clus- ter. (After Bishop) Spermatophores of spotted salamander in situ on April 14. Inset: A single one, greatly enlarged. (After Bishop) (Courtesy C. H. Pope I9UI4.: Chicago Mua. Nat. Hist.) These eggs will tolerate refrigerator temperatures as low as i+°C. and if kept at such low temperatures the time to gaatrulatlon may he extended to ahout 10 days. The optimum temperature for development is between, 12°C. and 15°C., hut the embryos will tolerate 20°C. at the upper limit. At 10°C. the embiyos pass from stage #7 to stage #25 in about 500 hours (13 days). These are excellent eggs and embryos for operative procedures. AMBLYSTOMA TIGBINUM, the tiger salamander. This salamander is found throughout the United States and In Canada and Mexico. There are numerous yellow spots which cover the body but are largely concentrated along the sides of the belly, In contrast with A. punctatum. The background color la deep brown to black and this is the largest of the salamanders except the mudpuppy and siren. It measures about 10 inches in length. The males are the larger sex and their vents protrude. Mature larva of tiger salamander. (Courtesy C. H. Pope I9UU: Chicago Mus. Nat. Hist.) Amblystonia tigrlnum, the tiger salamander. Amblystoraa punctatum, the spotted salaman- der. k2 BREEDING HABITS OF AMPHIBIA Breeding begins in early apring, generally a bit earlier' than for A. punctatum, in February, (January in the Carolinas). The egg clusters (about 100) are attached to tvrigs about a foot below the surface of the water, and are enclosed in a very loose fitting Jelly. The egg is a bit darker than that of A. punctatum but the egg species can be readi- ly distinguished by the consistency of the Jelly. Development is rapid, with hatching in less than 28 days and metamorphosis in about 75 days. Laboratory larvae at 20°C. may not metamorphose for k months, I56 days at 15°C., or 80 days at 25°C, Temperature tolerance Is great, and development from stage #7 to stage #25 takes about 200 hours at 10°C. EUKYCEA BISLINEATA, the two-lined salamander. This form is found in central and eastern States, having a maximum size of h- inches, and is characterized by having five toes on the hind limb and a distinct labio-nasal groove. It is a slender salamander, round like a lead pencil, and is found in streams, springs, and bogs. Sex differences are not clear cut, except that the males upper front teeth are bicuspid. Breeding extends from April to June. After an elaborate courtship, the female picks up the spermatophores and then lays the 20 to 50 eggs, one at a time, on the under aide of atones or other objects. Incubation takes about a month, metamorphosis in about a year and sexual maturity the following Spring. HEMIDACTyLIUM SCUTATUM. the four-toed salamander. This salamander has a wide distribution in the eastern United States, measuring no more than h inches in length. Except for the mudpuppy this is the only common sala- mander with four-toes. The color of the back is orange-brown with a pseudo- reptilian appearance of overlapping plates. The belly surface is spotted white. The male body la smaller but Its tall la longer than that of the female. Mating occurs in late summer and autumn, and breeding is in the spring In open boga, shaded woodland pools, or along quiet streams. The neata are mere cavities in decayed wood, grass or moss. About 50 eggs are layed and Incubation takes from 6 to 8 weeks, depending upon the environmental temperature. The aquatic larval period is about 6 weeks, and sexual maturity in 2-^ years. PLETHODON CIHEREUS, the red- backed sala- mander. Rather widely dlatributed throughout the United States, particu- larly in the middle west, but not over- ly abundant. There are five toes on the hind limbs. It is never more than k Inches in length, la wholly terres- trial, and has two phases; one with black and the other with red back. The male snouta are awollen and they are smaller than the females. Mating occurs in October and again In the spring, with egg-laying in June and July. The eggs are layed in cavi- ties in well -decayed logs or stumps and Courtship of salamanders, a and b: Two-Unert a single cluster may contain only about salamander. c and d: Newt. e and f: Marbled 1I+ eggs. Development is very different salamander (spermatopliore between pair in f ) . ^.j-gm ^j^^t of other Amphibia. (See a, b, e, and f after NoDle and Bradj ; c and d Lynn.) after Bishop. (Courtesy C. H. Pope 19l<^U : Chicago Mua. Nat. Hist.) BREED ING HA3 ITS OF AMPHIBIA »^3 TRITURUS PYRRHOGASTER (From A. Ichikawa 1937: Jour. Fac. Sci., Hokkaido Imp. Univ.^ Sor. VI, Vol. VI, No. 1 TRITURUS TOROSUS Aoove: adult T. torosus female. Middle: adult T. torosus male in breeding condition. The extreme glandular develop- ment during the mating season leaves the skin of Triturus males in a smooth, transparent condition, almost Jelly-like in consistency. The color of the skin during tlils period Is characteristically quite pale in T. torosus, although it may be sometimes considerably darker than in the specimen shown here. Below: male of T. sierrae in breeding condition. (From Twitty l^hQ: Copela, #2) uu BREED ING HAB ITS OF AMPHIBIA TRITURUS GRANULOSUS Above: adult male of T. granulosus from Mendocino County In breeding condition. Kxtreme development of tfie tall fin is characteristic of the breeding males In this species. Adult pigmentation is more variable in this than in other species of Californlan Trituriis, and may be either somewhat darker or considerably lighter tlian in the specimen shown here. The sides of breeding males generally exhibit a characteristic steely-blue coloration (which is, liowever, sometimes approxi- mated also in torosus males). Particularly during terrestrial periods tlie back and sides of both sexes are often ouite dark, almost black, and the belly a yellowisli or pinkish orange. In general the granulosus of Santa Clara County are less darkly pigmented than those farther north (see Bisliop, 1941). Middle and below: lateral and ventral views of an adult T. rivularls female. The band across the cloaca is very cliarac ter ist ic, but not invariable, and may appear also in lesser degree of development in other Californlan species of Triturus. (From Twltty I9I+2: Copela, #2) BREEDING HABITS OF AMPHIBIA U5 TBITUHJS FYKRHOGASTER. the Japanese fire-salamander. This form is very comnon in Japan and is characterized hy a black warty skin and a hright red belly, with scattered black spots. The size is generally about 6 inches, the males being a bit smaller and having large vents and pointed tails. May be fed strips of fresh liver which they will ingest from the water without prompting. Since these forma are imported, they are used in the laboratory and may be caused to ovulate by the injection of frog pituitary glands or extracts of mammalian pituitaries. Generally the best procedure is to inject two female frog pituitaries on alternate dpys for three injections, then place the female in water with Elodea on which she can lay the eggs. Females in colonies may have apermatophores, but if this is not the case the malea can be induced to drop spermatophores by pituitary treatment. The eggs are generally layed after the third injection and a good female will give as many as 80 egga. The fe- males seem to retain their spermatophorea for a long period and the eggs are inseminated as they are layed. TBITUHJS YIBIDESCENS. the common newt, formerly known aa Dlemcytylus vlridescena. These ainall vermilllon spotted newts are found throughout the United States and aouthern Canada, particularly in the East. It lacke the series of vertical grooves on the side of the body, characteristic of salamajiders. The back and sides are olive and the belly la light yellow with moat of the bright vermilllon spots on the sides and belly. The male possesses a row of pits, the hedonlc glands, on the aide of the head and can also be diatinguiahed by Ita protruding cloaca. These forms may be fed strips of liver, or small earthworms, but the food must be moving to be attractive- Breeding occurs in April and May, following an elaborate courtship which is termlnatedr when the female picks up the spermatophores dropped by the male. The egga are layed In sluggish water and are attached singly to stems, leaves, and other submerged objects. Spermatogenesis can be encouraged in the laboratory with temperatures of 55°F. or higher and sperm discharge is achieved by temperatures above 75°F. , even in the winter time. From egg-laying to hatching is about 20-55 days, the larval period lasting about 80 days when the larva transforms into a bright orange, and terrestrial "red-eft". This stage cannot live in the water and possesses a skin which is highly repellent to water. Transformation into an aquatic form occurs after several (2 to h) seasons and breeding then begins. In water. This form responds readily to anterior pituitary induced ovulation in the labora- tory at almost any time of the (academic) year. REFERENCES : Adams, A. E., igl+O - "Sexual conditions in Triturua vlridescena. II. The reproductive cycle of adult aquatic forms of both sexes." Am. Jour. Anat. 66:255- Applington, H. W. , 19i4-2 - "Correlative cyclical changes in the hypophysla and gonads of Necturua maculosus." Am. Jour. Anat. 70. Aronson, L. R., l^kk - "The mating pattern of Bufo americanus, Bufo fowleri, and Bufo terrestris." Am. Mus. Nov. #1250. Aronson, L. R. & G- K- Noble, l^k"^ - "The sexual behavior of Anura- 2. Neural mechanisms controlling mating in the male leopard frog, Sana pipiens." Bull. Am. Mus. Nat. Hist. 86:89. Bellerby, C- W- 8c L- HDgben, I958 - "Experimental studies on sexual cycle of the South African clawed toad ( Xenopus laevis)-" Jour- Exp- Biol. 15:91* Berk, L., I958 - "Studies in the reproduction of Xenopus laevis. 1. The relation of ex- ternal environmental factors to the sexual cycle." S. Afr. Jour. Med. Sol. 5=72. Bishop, S. C, 1925 - "The life history of the red salamander, Pseudotrlton." Nat. Hist. 25:585- Bishop, S. C- & C- Crisp, I955 - "The nests and young of the Allegheny salamander Des- mognathua fuacus-" Copeia- 1955, P- 19^+- Blair, A. P., 191+2 - "Isolating mechanisms in a complex of four species of toads." Biol. Symp. 6:255- Blanchard, F- N-, I925 - "The life history of the four- toed salamander-" Am- Nat- 57:262- Bles, E. J., 1905 - "The life history of Xenopus laevis," Tr. Roy. Soc. Edin. 1+1:789. Bragg, A. N , 1957 - "Observations on Bufo cognatus with special reference to breeding habits and eggs." Am- Midland Nat. 18:275- U6 BREEDING HABITS OF AMPHIBIA Bragg, A. N., 19i)-4 - "Egg laying in leopard frogs." Proc. Oklahoma Acad. Scl. 2k. Branln, M, L., 1955 - "Courtship activities and extra seasonal ovulation in the four- toed salamander." Copeia. 1955, p. 172. Brown, M. G., 1914-2 - "An adaptation in Amhlystoma opacum emtryos to development on land." Am. Nat. 76:222. Conturosvill, P. S., 1956 - "Experiments in changes of sexual cycle in certain tailless amphihia." C. B. (Doklady) Acad. Scl. I'U.R.S.S. 11:125. Coates, C. W. & A. I. Welsman, 19^1+ - "Pregnancy test frogs being hred at will." Jour, Am. Med. Ass'n. 12i*:l+6l. DeAllende, I. L. C, 1958 - "Aparato sexual femenino del Bufo arenarum. " Inst, de Fisiologia Fac. de Cienclas Medicos Cardoba (Argentina). Dwnn, E. E., I926 - "The salamanders of the family Plethodontidae, " Evans, H. M. & K. S. Bishop, I925 - "Existence of hitherto unknown dietary factor essen- tial for reproduction." Jour. Am. Med. Ass'n. 81:889. Flower, S. S., 1925 - "Contributions to our knowledge of the duration of life In verte- brate animals. II. Batraclens." Proc. Zool. Soc. London. 1925:269. Force, E. P., 1955 - "The age of the attainment of sexual maturity of the leopard frog, Eana pipiens In Northern Michigan." Copeia. 5:128. Francis, E. T. B. , 195'*- - "The anatomy of the salamander." Oxford Univ. Press. Galgano, M., 19''-5 - "Trattl fondamentall del ciclo sessuale annuale negll Anfibi del nostri climl," Boll, di Zool. lU:57. Gltlin, G., 19^1 - "Seasonal variations and sexual differences in the fat bodies and other fat deposits of Xenopus laevls." S. Afr. Jour. Med. Sci. 6:156 (see ibid, 7:l6). Glass, F. M. & B. Bugh, l^kk- - "Seasonal study of the normal and pituitary- stimulated frog (Rana pipiens). I. Testes and thumb pad." Jour, Morph. 7'+:'+09. Green, H. T., 1925 - "The egg laying of the purple salamander (Gryinophilus) . " Copeia. Ul:52. Green, N. B., 1958 - "The breeding habits of Pseudacris brachyphona with a description of the eggs and tadpole." Copeia. #2, p. 79, Hlnsche, G. , I926 - "ITber Brunst- und Kopulationarealrtlonen des Bufo vulgaris." Zeitschr. vergl. Physiol, h-.'^dh. Hisaw. F. L. Sb E. 3. Astwood, I9I+2 - "The physiology of reproduction." Ann. Rev. Physiol. 4:505. Hitchcock, H. B., 1959 - "Notes on the newt Trlturus vlrldescens. " Herpetologlca. l:lU9. Ifft, J. D. , X^hl - "The effect of environmental factors on the sperm cycle of Trlturus vlrldescens." Biol. Bull. 85:111. Kauffeld, C. F. , 1957 - "The status of the leopard frogs, Rana brachycephala and Rana pipiens." Herpetologlca, l:81^. Kumpf, K. F., I95U - "The courtship of Amblystoma tigrlnum. " Copeia, 195*+, P- 7- Lantz, L. A., 1950 - "Notes on the breeding habits and larval development of A. opacum." Ann. Am. Mus. Nat. Hist. 5:522. Landgrebe, F. W. & G. L. Purser, I9I+I - "Breeding of Xenopus in the laboratory." Nature. 1U8:115. Liu, C. C, 1951 - "Sexual behavior in the Siberian toad, Bufo raddei and the pond frog, Rana nigromaculata . " Peking Nat. Hist. Bull. 6:1+5. Moore, J. A., V^hk - "Geographical variation in Bana pipiens Schreiber of Eastern North America." Bull. Am. Mus. Nat. Hist. 82:51+9. Noble, G, K,, I95I - "The Biology of the Amphibia," McGraw-Hill, N. Y, Noble, G, K, & L. B, Aronson, I9I+2 - "The sexual behavior of Anura. I. The normal mating pattern of Bana pipiens," Bull, Am, Mus, Nat. Hist. 80:127. Noble, G. K. & M. K. Brody, 1955 - "Observations on the life history of the marbled salamander, A. opacum." Zoologica. 11:#8. O'Donnell, D. J., 1957 - "Natural history of the amblystomld salamanders of Illinois." Am. Midland Nat. 18:1065. Pope, C, H. , I9I+I+ - "Amphibians and Beptiles of the Chicago area." Chicago Museum Nat. Hist. Rostand, J., 1951+ - "Toads and toad life." Methuen & Company, London. Savage, B. W., I95I+ - "The breeding behavior of the common frog Bana temporaria and of the common toad Bufo bufo." Proc. Boy. Soc. London. 1:55. Savage, B. M., I955 - "The Influence of external factors on the spawning date and migra- tion of the common frog, Bana temporaria." Zool. Soc. London. 1955. Schmidt, K. P., I958 - "A geographic variation gradient in frogs." Zool. series of Field Museum. 20:577- BREEDING HABITS OF AMPHIBIA ^1 Shapiro, H. k. , IS'^1 - "The role of distance receptors in the establishment of the mating reflex in Xenopus laevis, The Nares." Jour, Exp. Biol. ll+:38. Smith, B. G., I9II - "Notes on the natural history of Amblystoma Jeffersonianum, A. punc- tatum and A. tigrinum." Bull. Wise. Nat. Hist. Soc. 9:ll4-. Smith, C. L., I956 - "The clasping reflex in frogs and toads and the seasonal development of the brachial musculature." Jour. Exp. Biol. 15:1. Smith. B. E., 19'+'+ - "Mating behavior in Tritunis torosus and related newts." Copeia. t:255. Waring, H., F. W. Landgrebe, & E. M. Nell, 19'+1 - "Ovulation and oviposition in Anura." Jour. Exp. Biol. l8:l. Wilder, I. W., 1921+ - "The relation of growth to metamorphosis in Eurycea bislineata." Jour. Exp. Zool. 1+0:1. Wright, A. H. & A. N. Wright, 1955 - "Handbook of Frogs and Toads." Comstock Publishing Company. "Not only do the body fluids of the lower forms of marine life correspond exactly with sea water in their composition, but there are at least strong indicat ions that the fluids of the highest animals are really descended from sea water . . . . the same substances are present in both cases, and in bnth cases sodium chloride predominates." L. J. Henderson 1913: "Fitness of the Environment" "Of course, I am not forgetting that development and evolution are in the main epigenet ic proces ses by which the more complicated end stages are built upon the less compli- cated earlier ones, but I also refuse to forget that these earlier stages are also complex, that the egg or the Para- mecium are complex organisms and that development is endo- genetic as well as epigenet ic . Both epigenesis and endo- genesis are involved in all deve lopment and evolut ion. " E G. Conklin, 19ii THE CULTURE OF AMPHIBIAN EMBRYOS AND LARVAE TO METAMORPHOSIS Early amphibian embryos possess an abundance of yolk which provides them with all the nutriment necessary for a considerable period of development. Tadpoles of both the Anura and the Urodela can survive for many days after hatching, by utilizing the yolk found be- tween the embryonic gut and the belly ectoderm. The most Important single factor for sur- vival during the earliest stages of development Is the temperature, and second to this is the culture medium. In order that the research worker can reduce to a minimum the environ- mental variables, some suggestions regarding the culturing of common amphibian forms are given here. CULTURE MEDIUM All amphibia lay their eggs In water. The pond water in which the form to be studied is known to breed is the ideal water to use. Since this is not always practical, labora- tory substitutes have been devised, based partly upon a chemical analysis of such pond waters. In general It has been found that slightly hypotonic media are preferred, and development can proceed even in distilled water to some extent (see section on Osmo- Eegulation) . The tap water, in large cities particularly, may be so highly chlorinated that It is toxic to embryos or, in some instances, enough metallic ions escape from the lead, copper, or iron piping that the embryos cannot survive. Tap water which has been run through sand and charcoal, filtered, and allowed to stand for several days with abun- dant plant material in it, will generally prove quite satisfactory. The sand and charcoal take out the debris and dissolved gases, and the living plant material (Elodea, Valeclnerla, Saglttaria, Nltella, etc. ) helps to Increase the oxygen content. It is now quite clear that sodium, calcium, and potassium ions must be present in an approximate ratio of 50:1:1 in order that development of aquatic embryos be normal. Each of these Ions has specific value in cleavage and the developmental process, and If the ratio is maintained the forms can tolerate quite a range in concentration. Solely by the empirical method a number of formulae have been devised, each presumably suitable to par- ticular forms. Some of these formulae follow: STANDARD (HOLTFRETEB'S) SOLUTION: This solution has proven to be the most satisfactory of the synthetic media. The total salt content is 0.585^, which Is hypotonic to adult tissues but seems to be isotonic to the early embryonic stages of the Anura. It is recommended that this solution be made up in double or quadruple strength as a stock solution. The normal concentration follows: NaCl 0.55 grams KCL 0 . 005 " CaClg 0.01 NaHCO, (Buffer) 0.02 Distilled water 100.00 cc. (Note: This solution is satisfactory for T. pyrrhagaster if the NaCl is reduced to half and the buffer is omitted.) AMPHIBIAN RINGEB'S SOLUTION: This solution is hypertonic to Standard Solution and to embryos and embryonic tis- sues, but is satisfactory for adult tissues of the Amphibia. NaCl 0 . 66 grams KCL 0.015 " CaCl2 0.015 " NaHCO^ (Buffer) O.050 " (amount necessary to Distilled water 100.0 cc. regulate pH at 7.8) - ka. CULTURING OF AMPHIBIAN EMBRYOS k9 ORIGINAL FBOG BINGEB'S: (S. NaCl KCl . CaClg Ringer I88O: Jour. Physiol. IJOSo) 0.65 grama O.OII+ 0.012 0,02 NaHCOj .... Na%PQl+,, Glucose Water 0.001 0.20 to 100. SPRING WATER: Great Bear Spring Water. This has proven to te entirely satisfactory for the early development of Anuran embryos particularly. (Available in N. Y. City) URODELE STOCK SOLUTION: The same constituent salts are found in the Urodele media hut in slightly different proportions. This stock solution could be made up in 20 liter carboys and could be diluted for either operating or growing media, using the Great Bear Spring Water for dilu- tions. NaCl 70.0 grama KCl 1.0 " CaClg 2.0 Spring Water 10.0 liters URODELE OPERATING MEDIUM: This medium la slightly hypertonic to the grovlng medium. Embryos may be left in this medium for several hours after transplantations, etc., and then should be transferred to the Growing Solution. Urodele Stock Solution 2 parts Great Bear Spring Water 1 part URODELE GROWING SOLUTION: Urodele larvae grow very satisfactorily in this medium. Urodele Stock Solution 1 part Great Bear Spring Water h parts In making up aynthetic media it is recommended that glass distilled water be used where it is practicable. It has been found that with copper stills, enough copper goes into solution, in some atills, to make the water slightly toxic to embryos. In any crucial experiment the Investigator should pre-test the medium against the embryos to be studied to that he can eliminate this as a possible factor in his experimental results. The data from the Osmo- Regulation experiments will be of value in this regard. FOOD FOR TEE LARVAE: Feeding is not necessary for some days after the mouth of the tadpole (larva) is open. This is because all amphibian larvae are provided with an abundance of reserve food in the form of yolk which is digested and absorbed directly by the tissues. Anura: Feeding is not necessary until stage #25. Most Anuran lai-vae are vegetarians, and the most satisfactory food consists of slightly boiled lettuce or spinach. These greens should be thoroughly washed to rid them of any adherent arsenic or lead which may have been sprayed on them as Insecticides by the gardener. Boiling the greens simply softens the plant tissues. The danger, at least in the beginning, comes from over-feeding. The tanks must be cleaned daily to keep faecal and bacterial accumula- 50 CULTURING OF AMPHIBIAN EMBRYOS latlon at a minimum. For Xenopus larvae cooked, dehydrated, and finely powdered beef liver is an excellent supplement to greens, and algae. Livervorst has been used suc- cessfully with many Anura. Other foods used are powdered egg-yolk; bacto-beef ex- tract mixed with whole wheat flour, dried and pulverized; raw liver, minced; algae and Protozoa. The lettuce feeding seems to produce fewer abnormalities but develop- ment is slower than with spinach ( ^man, 19'+1) • Briggs (19^+2) has shown that a pure spinach diet produces certain minor abnormalities and kidney stones, so that a mixed diet is recommended. On pure lettuce or spinach, or a mixture of the two, tadpoles can be reared through metamorphosis with considerable ease. Anura are essentially vegetarian until after metamorphosis, then they are omnivorous with a leaning toward the carnivorous. Urodela : The Urodela larvae require living, moving food. At first, rich cultures of Protozoa and young Daphnia are fed to the larvae after stage #4-0. The carapace of the older Daphnlas will tear the gut of the larvae. The eyesight of these forms is very poor and their neuro-muscular responses are slow, so that the living food must be active. After a week or two of this diet, when the larvae are more hardy, they can tolerate such foods as the red worm (Tubifex), the white worm (Enchytrea), Daphnia (all sizes, but the young ones are better), mealflies, mealworms, wax moths (Gallerla), Drosophlla (vestigial mutant), plant lice, small ants, and, best of all, amphibian larvae (frog tadpoles of early stages). If not adequately fed the Urodele larvae will tend to nip off each others tails and occasionally larger specimens will devour the smaller ones. Beef liver favors A. tigrinum over A. punctatum while Daphnia and Enchytrea favor A. punctatum over A. tigrinum. Cannibalism la common, and Amblystoma larvae seem to grow best on a diet of Amblystoma. ■)«-***-X--X-#*-X--X-*» If over-feeding is avoided, and any unlngested food is removed daily from the taiaka, it may not be necessary to change the culture medium more than once or twice each week. As the larvae grow, however, there will appear more faecal material in the tanks, and this should be sucked out with a suction bulb and glass tube or the water should be changed more frequently. The water must not be allowed to become turbid with bacteria. FOOD FOB THE POST -MET AMOKPSIC STAGES: After metamorphosis, with correlated changes In the histology of the digestive tract, the food requirements become radically different for all amphibia. Not only must there be more food, but it must represent a greater variety and should contain vitamins. Anura: After metamorphosis the situation becomes reversed in that the Anura require living, moving food. Living earthworms cut into 1-inch pieces, which will continue to move and attract the small frogs, are excellent as food. Blow flies, meal worms, ante, spiders, roaches, caterpillars, grasshoppers, will all be eaten as long as they move. Fish muscle, mammalian liver or muscle, dipped in a thin paste of Brewer's yeast and cod liver oil is an excellent food for recently metamorphosed frogs. Xenopus is a slight exception in that it will eat anything bloody, particularly strips of raw liver. This form shows little activity except in taking of food, after which it settles down in the water again for as long as several days, coming to the surface only occasionally to get air. Some of the potentially larger Anvira (e.g., B. catesbiana, the bullfrog) may grow rapidly and will require more food. These forma may be fed small crayfish, minnows, earthworms, and even small frogs of the same or other species. It is pos- sible but difficult to train Anura to take non-living food. The tree frogs (^la) require a continually humid environment such as an ordi- nary terrarixun and their food consists of small insects. If the terrarium is glass covered, Drosophila (vestigial mutant) may be given to them, but it takes a good many flies to make an adequate meal, ^la will take other living food such as earth- woma. CULTURING OF AMPHIBIAN EMBRYOS 51 Toads require a rather warm and dry environment, live on insects at first and then they will accept worms and even strips of beef, if the food is shaken before their nostrils. Urodela: Salamanders are aquatic or semi -aquatic, and a few are actually terrestrial, hence the food requirements will vary somewhat with the species. In general, the salamanders should be removed to a large crystallizing diah dur- ing feeding. The food at first may consist of cliunps of white worms (Enchytrea), small earthworms or larger earthworms cut into 1-inch lengths suitable for ingestion. As the salamanders grow they can be trained to accept strips of beef or calves liver, if the liver is held in forceps and dangled before their nostrils. These forms act as though they are blind, but their olfactory senses are acute. After the feeding, remove all excess food, rinse off the specimens in fresh water, and return them to their tanks. Uningested or regurgitated food in the tank necessitates frequent com- plete change of medium, and occasional sterilization of the entire tank. Triturus pyrrhogaster, the Japanese fire salamander, should be fed about three times each week, and each adult specimen should receive the equivalent of about 1 inch of liver, the diameter of a small pencil. With this routine they may be kept in healthy condition for many years, producing eggs (luider pituitary stimulation) when desired by the Investigator. SPACE AND OXYGEN The space factor in development has not been adequately recognized but it plays a very important role in the rate of development. In general, the larvae will grow the faster in less crowded conditions, all other factors being equal. It is suggested that a ratio of 1 egg to 2 cc. of medium be used In finger bowls with a maximum of 25 eggs to 50 cc. of medium. As the embryos develop into larvae (tadpoles) this ratio will have to be changed so that at the beginning of feeding there are no more than 10 tadpoles per finger bowl of 50 cc. of medium. At this time it is better to transfer the tadpoles to a larger tank to allow for greater activity (see Rugh, 195'*-)- The amount of water per specimen is not the vital consideration, however. It is the surface area that is Important, so that in a tank measuring 6 x 12 x 2l4- inches one can place 200 tadpoles in water not more than 1 inch in depth. Evaporation from this tank should be compensated for by adding distilled water once each week, but under no condi- tions should tadpoles be placed in deep water. Amphibian embryos can tolerate a wide range of oxygen tension but they are very sen- sitive to anerobic conditions. Artificial aeration is not necessary but it is well to place in the tanks some aquatic plants such as Elodea, Nltella, etc. which will continual- ly add some oxygen to the medium. Anuran larvae seem to require more oxygen than do the Urodele larvae. LIGHT There is no evidence that light is necessary for normal amphibian development (Eugh, 1955) . However, since larvae can derive nourishment from algae and they do require oxygen, it is well to provide normal light so that plant food can grow and can provide some of the necessary oxygen. Direct sunlight Is not advised because of the heat factor. TEMPERATURE The temperature tolerance of various forms is given in the section on Temperature. The range of tolerance for the various forms is about 2l+°C., but the scale is high for some and low for others. The Urodela, for Instance, develop better at the lower tempera- tures while the Anura seem to develop better at the higher temperature levels. 52 CULTURIN6 OF AMPHIBIAN EMBRYOS Within the normal tolerance range it is possible to retard or accelerate the normal rate of development of any of the forms without altering the developmental processes in any way. For Instance, one can keep Eana pipiens eggs at 15°C., 20°C., and 25°C. and have three different stages of development simultaneously, all from the same original hatch of fertilized eggs. The maximum range for all amphibian larvae is 0°C. to it-0°C., with the optimum range between 120C. and 25°C. Most laboratories are kept at between 25°C. and 25°C. which is satisfactory for the Anura but somewhat high for the Urodela. BACTERIA AND PARASITES The most common infection for adult frogs is Eed-Leg. Numerous attempts have been made to control this disease. The best method is to eliminate any infected animals upon receipt; to keep the tanks cool and the animals in running water, and occasionally to treat possibly infected animals with weak KMNOij^ solution. Copper lined tanks will reduce the incidence of Eed-Leg. Saprolegnia is another infection of high mortality and unknown cure. The symptoms include body swelling or bloating. Infected animals should be de- stroyed immediately and the tank sterilized with permanganate. It is a practice in many laboratories to place several copper pennies in the tanks with the frogs, enough copper ions passing into the water to keep down these infections and yet not enough to be toxic for the adults. (In even minute concentrations, copper, lead, zinc, mercury, and bronze are toxic to embryos.) Salamanders are sometimes seriously affected by a fungus, Monilia batrachus, which attacks the lips and causes open sores. Frogs and toada seem to be immune. This growth is contagious but if treated early by painting the lips with 2^ mercurochrome, the disease may be checked. Parasites are often brought in with living food. These include worms, flies, and mites. However, these infected animals normally comprise the food of most amphibia so that there is little or no danger for them. If Tubifex (red worm) is used as food, it la well to keep them in running cold water for several days because they grow in sewage and are apt to bring in an excess of bacteria. Oedema, or swelling of tissues with water, may be due to a malfunctioning of the embryonic kidneys although it is known to occur even before such organs are developed. Early tadpoles often develop an apparent oedema, but the swelling is due to an accumulation of dissolved gases in the digestive tract with some consequent bloating of the body. Generalized oedema can sometimes be relieved by placing the embryos in slightly hypertonic medium. If the oedema is localized, it can be relieved by puncturing with a needle to al- low the escape of the excess fluid. CULTURING OF AMPHIBIAN EMBRYOS 55 BEFERENCES : Adolph, E. F. , 1931 - "Body size as a factor in the metamorphosis of tadpoles." Biol. Bull. 61:376 (see ibid, 6l:550). Anderson, P. L,, 19'+3 - "Ttie normal development of Triturus pyrrhogaater. " Anat. Bee. 86:59. Aronson, L. B., 19*4-1+ - "Breeding Xenopus laevis." Am. Nat. 78:131. Atlas, M., 1935 - "The effect of temperature on the development of Bana pipiens." Phys. Zool. 8. Bellancr, A. W., I922 - "Differential susceptibility as a basis for modification and con- trol of early development in the frog. II. lypea of modification seen in later developmental stages." Am. Jour. Anat. 30:^73- Bilski, F., 1921 - "■QlDer den Einfluss des Lebensrauma auf das Wachstum der Kaulquappen. " Pfluger's Arch. 188:251+. Blea, E. J., I905 - "The life hiatoiy of Xenopua laevis." Tr. Boy. Soc. Edin. 1+1:789. Brachet, J., 19^+0 - "Etude histochimique des Proteines au cours du developpement embryon- naire dea poissons, des Amphiblens, et dea Oiseaux." Arch. d. Biol. 51:l67. Bragg, A. N,, 1939 " "Observations upon amphibian deutoplasm and ita relation to embry- onic and early larval development." Biol. Bull. 77:268. Brigga, B., 19^+6 - "Effect of growth inhibitor hexenolactone on frog embryos. I. Effects on diploid embryos. II. Differential effects on haploid and diploid embryos." Growth, 10:1+5. Briggs, B. & M. Davidson, 19^+2 - "Some effects of aplnach feeding on Bana plplena tad- poles." Jour. Exp. Zool. 90:1+01. Brown, M. G., I9I+2 - "An adaptation in Amblystoma opacum embryoa to development on land.'' Am. Nat. 76:222. Buchanan, J. W. , 1938 - "Developmental acceleration following inhibition." Jour. Exp. Zool. 79:111. Burke, V., 1935 - "Bacteria aa food for vertebrates." Science 78. Burr, H. S. & 3. C. Northrop, I959 - "Evidence for the exlatence of an electro- dynamic field in living organisms." Nat. Acad. Sci. 25:28!+. Burr, H. S., I9I+I - "Field properties of the developing frog's egg." Science, 95:1+1+0. Cameron, J. A., I9I+O - "Effect of flourine on hatching time and hatching stage in Rana pipiens." Ecology. 21:288. Cameron, S. B., I9I+7 - "Successful breeding of Xenopus laevis, the South African clawed Toad-frog." Am. Jour. Med. Technology. May 191+7. Corradetti, A., 1955 - "Variozioni nelle durata della vita iarvale dl Bana esculenta in relazione col colore dell ambiente." Boll. Zool. 1+:17. Daniel, J. F. & E. A. Yarwood, 1959 - "The early embryology of Triturus torosus." Univ. Calif. Pub. Zool. 1+5:521. Dempster, W. T., I955 - "Growth in Amblystoma punctatum during the embryonic and early larval period." Jour. Exp. Zool. 61+:l+95. Dent, J. N., I9I+2 - "The embryonic development of Plethodon clnereus as correlated with the differentiation and functioning of the thyroid gland." Jour, Morph. 71:577. Dickerson, M. C, I906 - "The Frog Book." Doubleday Page, pgs. 253. Dorrla, F., 1955 - "The development of structure and function in the digestive tract of Amblystoma punctatum." Jour, Exp, Zool. 70:1+91. DuShane, G. P. S: C. Hutchinson, I9I+I+ - "Differences in size and developmental rate be- tween Eastern and Midweatem embryoa of Amblystoma maculatum." Ecology. 25:l+ll+. Eakin, B. M., 1959 - "Further studies in regulatory development of Triturus torosus." Univ. Calif. Pub. Zool, l+5:l85, Emerson, H, & C. Norria, I905 - "Bed-leg an infectious disease in frogs." Jour. Exp. Med. 7. Francis, E. T., 195!+ - "The anatoncr of the salamander." Clarendon Press, Oxford. Gilchrist, F. G., I928 - "The time relations of determinations in early amphibian develop- ment." Jour. Exp. Zool. 66:15. Gitlin, D., I9I+I+ - "The development of Eleutherodactylus partorlcenaia, " Copela. 19'+'*-, p. 81, 5U CULTURING OF AMPHIBIAN EMBRYOS Goetsch, W., 1924 - "Lebensraiun und Korpergrosse. " Biol. Zentrlbl. hk:'y2'j). Goodalej H. A., I9II - "The early development of Spelerpes 'bisllneata." Am. Jour. Anat. Grant, M. P., 1951 - "Diagnostic stages of metamorphosis in Amhlystoma Jeffersonlanum eind Amblystoma opacum. " Anat. Bee. 51:1- Griffin, L. E., I93U - "Infection with Amphibian tubercle bacilli connected with a case of sex reversal in a frog." Copela. #5, p. ll**-. Gudernatsch, F. & 0. Hoffman - (See references under each and both in section on "Nutri- tion and Growth".) Hamilton, W. J., 195*+ - "The rate of growth of the toad (B. americanus) under natural conditions." Copela. 19514-, p. 88. Hammett, F. S., 191*6 - "What Is growth?" Scientia, April-June, 19^6. Harrison, E. G. , 19'+5 - "Eelation of symmetry in the developing embryo." Trans. Conn. Acad. Arts & Scl. 56:277. Holtfreter, J., 19^5 - "Differential inhibition of growth and differentiation by mechani- cal emd chemical means." Anat. Bee. 95:59- Howes, N. H. , 1958 - "Anterior pituitary and growth in the Axolotl (Amblystoma tlgrinum) neotonlc form. II. The effect of injection of growth-promoting extracts upon the utilization of food." Jour. Exp. Biol. I'^ikk'J. Himphrey, E. E. , I928 - • "Oviilation in the four- toed salamander Hemldactylium scutatum and the external features of cleavage and gastrulation. " Biol. Bull. 5l*-:507. Hutchinson, C, 1959 - "Some experimental conditions modifying the growth of amphibian larvae." Jour. Exp. Zool. 85:257. Hltchinson, C. & D. Hewitt, 1955 - "A study of larval growth in Amblystoma punctatum and Amblystoma tlgrinum." Jour. Exp. Zool. Il:k6'^. Hyman, L. , V^kl - "Lettuce as a medium for the continuous culture of a variety of small laboratory animals." Trans. Amer. Micr. Soc. 60:365. Janes, B., 1959 - "Studies on the amphibian digestive system. IV. The effect of diet on the small Intestine of Sana sylvatica." Copela, #5;15'+. Krlzenecky, J., I928 - "Studien uber die Funktlon der Im Wasser gelosten Nahrsubatanzen Im Stoffwechsel der Wassertiere. " Zeit. vergl. Physiol. 8. Krogh, A., 1951 - "Dissolved substances as food of aquatic organisms." Biol. Eev. G:klZ. Landgrebe, F. W. & G. L. Purcer, 1914-1 - "Breeding of Xenopus in the laboratory." Nature, llt8:115. Lynn, W. G. , I9I4-2 - "The embryology of Eleutherodactylus nubicola, an anuran which has no tadpole stage." Carnegie Inst. Contrib. Emb. #190, p. 27. (Pub. #51+1). Lynn, W. G. & B. Lutz, I9I4-6 - "The development of Eleutherodactylus Guentheri Stdur." Boletim do Museu. Naclonal Zool., Brazil, 71:1. May, E. M. & M. Coulon, I95I - "Bolte d'elevage pour Jeunes batraciens." Bull. Soc. Zool. de France 56. McClure, C. F. W. , 1925 - "An experimental analysis of oedema in the frog, with special reference to the oedema in red-leg disease." Am. Anat. Memoires. 12:1. McCoy, C. M., 1959 - "Chemical aspects of ageing." (in "Problems of Ageing"). Baltimore. McCurdy, M. B. D., 1959 - "Mitochondria in liver cells of fed and starved salamanders." Jour. Morph. 614- :9. Merwln, E. N. & W. C. Allee, I9I4-I - "The effect of carbon dioxide on the rate of cleavage in frog's eggs." Anat. Eec. 8I: suppl. 126. (See ibid I9I45, Ecology. 2l4-:6l.) Moore, A. E., I915 - "An analysis of experimental edema in the frog." Am. Jour. Anat. 57. Moore, A. E., 1955 - "Is cleavage rate a function of the cytoplasm or of the nucleus?" Jour. Exp. Biol. 10:250. Moore, J. A., 19^0 - "Adaptive differences in the egg membranes of frogs." Am. Nat. 7'+:89. Morgan, A. H. & M. Grierson, I952 - "Winter habits and yearly food consumption of adult spotted newts, Triturus viridescens. " Ecology. 15:5l+' Morgan, T. H., I905 - "The relation between normal and abnormal development of the embryo of the frog. X. A re-examlnatlon of the early stages of normal development from the point of view of results of abnormal development." Arch. f. Ent. mech. 19:588. Morrill, C. V., I925 - "The peculiar reaction of the common newt to a liver diet." Anat. Eec. 26:85. Needham, J., I9I16 - "New advances in the chemistry and biology of organized growth." Proc. Boy. Soc. Med. 29:1577. Nlgrelli, B. & L. W. Maraventano, 191+14 - "Pericarditis in Xenopus laevls caused by Diplostomulum Xenopus, a larval atrlgeld." Jour. Parasitology. I9I+I+. CULTURING OF AMPHIBIAN EMBRYOS 55 Pasteels, J., 1957 - "Sur I'origine de la symetrie tilaterale des aniphlbiens anourea." Arch. Anat. Mlkr. 53:279. Patch, E. M., 1927 - "Blometrlc studies upon development and growth in Amhlystoma puncta- tum and tigrlnum." Proc. Soc. Exp. Biol. & Med. 25:218. Patch, E. M., 19^+1 - "Cataracts in Amblystoma tigrinum larvae fed experimental diets." Proc. Soc. Exp. Biol. & Med. 1+6:205. Peter, K. , 195*+ - "Die Gastrulation von Xenopus laevis." Zetsch. mikr. Anat. Forsch. 55:181. Pike, F. H. , 1923 - "The effect of the environment in the production of malformations." Ecology. l4-:420. Pollister, A. W. & J. A. Moore, 1957 - "Tatles for the normal development of Eana sylvatica." Anat. Bee. 68:1+89. Pratt, E. M., 19^0 - "Effects of vitamin E deficiency on the tadpole of Eana pipiens." Univ. Mich. MS. Thesis. Eaney, E. C. & W. M. Ingram, 19'tl - "Growth of tagged frogs (Eana cateatiana and Rana clamltans) under natural conditions." Am. Midland Nat. 26:201. Eeiman, S. P., I9U7 - "Growth." Ann. Rev. Physiol. 9:1. Eobh, E. C, 1929 - "On the nature of hereditary size limitation. II. The growth of parte in relation to the whole." Jour. Exp. Biol. 6:311. Rose, S. M., 191+6 - "Disease control in frogs." Science. 10l+:550. Eugh, E., 195'*- - "The space factor in the growth rate of tadpoles." Ecology. 15 • Eugh, E., 1955 - "The spectral effect on the growth rate of tadpoles." Physiol. Zool. 8. Savage, E. M., 1957 - "The ecology of young tadpoles, with special reference to the nutrition of the early laevae of Eana tenporaria, Bufo bufo, and Bombinator variegata." Proc. Zool. Soc. London Ser. A 107:21+9 (see ibid 108:l+65). Scott, H. H., 1926 - "The ncrcotic disease of Batrachiens. " Proc. Zool. Soc. London, 685. Shaw, G., 1952 - "The effect of biologically conditioned water upon the rate of growth in fishes and amphibia." Ecology. 15:263. Shumway, W., I9I+0 - "A ciliate protozoon parasitic in the central nervous system of larval amblystoma." Biol. Bull. 78:285. Shumway, W., I9I+O - "Stages in the normal development of Eana pipiens. I. External form." Anat. Eec. 78:159 (see ibid I9I+2, Vol. 85:509). Smith. B. G., I926 - "The embryology of Cryptobranchus alleghenienaia." Jour. Morph. 1+2:197. Stockard, C. E., I92I - "Developmental rate and structural expression." Am. Jour. Anat. 28:115. Swingle, W. W., 1919 - "On the experimental production of edema by nephrectony. " Jour. Gen. Physiol 1. Taylor, A. C. & J. J. Kollros, I9I+6 - "Stages in the normal development of Rana pipiens larvae." Anat. Eec. 9l+:7. 1917 - "Growth and Form." Cambridge Univ. Press. "Correlated genetic and embryological experiments on Triturus." 17l+:259. "The role of genetic differentials In embryonic development of . Symp. 6:291. DeLanney, 1959 " "Size regulation and regeneration In salamander larvae under complete starvation." Jour. Exp. Zool. 81:399' Twitty, V. C. & W. J. van Wagtendonk, I9I+O - "A suggested mechanism for the regulation of proportionate growth, supported by quantitative data on the blood nutrients." Growth l+:5l+9. Tyler, A., I9I+2 - "Developmental processes and energetics." Quart. Rev. Biol. 17:197- Weiaman, A. I. & C. W. Coatea, 19!+!+ - "The South African Frog (Xenopus laevis) In preg- nancy diagnosis." N. Y. Biologic Research Foundation. Weiss, P., 19l^7 - "The problem of specificity in growth and development." Yale Jour. Biol. & Med. 19:255. Weisz, P. B., I9I+5 - "The development and morphology of the larva of the South African clawed toad, Xenopua laevia. II. The hatching and the first and second form tad- poles." Jour. Morph. 77:195. Wilder, I. W. , I92I+ - "The relation of growth to metamorphosis in Eurycea bislineata. " Jour. Exp. Zool. 1+0:1. Wright, A. H., I929 - "Synopsis and description of North American tadpoles." Proc. U.S. Nat. Mus. 7'+:Art. 11:1. Thompson, D. A. W., 1' Twitty, V. C-, 1956 - Jour. Exp. Zool. Twitty, V. C, 19 1+2 - Amphibia." ' Biol Twitty, V. C. & L. E. THE DEVELOPMENTAL STAGES OF AMPHIBIAN EMBRYOS starting with the (unpublished) Harrison series of stages for Amblystoma punctatum, there have appeared more and more descriptions of the early develop- mental stages of various forms. Through the generous cooperation of those who have worked out the normal morphology of a number of different amphibian forms, there follows .all the series and steiges that are now available. It is hoped that this list will be extended, not only for the sake of research usefulness, but in order to determine the extent of any deviations from a standard pattern of development. The author desires to express, here again, his appreciation for the cooperation of those who have made this collection possible. Reference is made to Shumway, Polllster, and Moore, for Rana; to Weisz, Taylor and Kollros for Xenopus; to Eakin for ^yla regilla; to Priscilla Anderson for Triturus pyrrhogaster; to Twitty for Triturus torosus; and to Mrs. Naomi Leavltt who painstakingly drew the Amblystoma series j.rom living and preserved material. Identifying the various stages as in the (unpublished) Harrison series. On the following pages will be found the normal series for: Eana pi pi ens (Shumway) Rana plpiens (Mueller) Rana plpiens, photographs (Rugh) Rana plpiens metamorphosis (Taylor & Kollros) Rana plpiens, photographs of developmental abnormalities (Rugh) Rana sylvatica (Polllster & Moore) Xenopxifl laevia (Weisz) Hyla regilla (Eakln) Amblystoma punctatum (Leavltt) Triturus pyrrhogaster (Anderson) Triturus torosus (Twitty & Bodensteln) -56- STAGING OF AMPHIBIAN EMBRYOS 57 U "1" lO UJ a. Q- < CO Z W U m Z o CD • -H P m c a .-^ © o rH . O -. © *i T-. .2-i d ^ 0 --i ■H © O O O © O c u sS- M a t* © +j ^. CO u . © *-. © -p 3 -p © ■ • ffl t< © © ^ © • © s S l< ti o u © O v-- © p 82 a, Tj © © 4-> ^ w O > V. ^ CO 3 5^ ^ 3 ^ o •rH ffi tH 0 © > -^ 0 u X:j3©-rH©© © ^-. 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Exo-gastrula in which 2/5 of yolk is unincorporated. 22. Gastrulation achieved although dorsal lip has been damaged. 25. Exo-gastrula in which 2/5 of yolk is unincorporated. 2U. Heml-gastrula. 25. - 26. Embryonic areas attempting to develop without incorporated yolk. 27. - 59- Various degrees of exo-gastrulatlon. 1+0. Fairly normal yolk plug but irregular dorsal lip which will condition abnormalities in the embryo. STAGING OF AMPHIBIAN EMBRYOS 81 ABNORMALITIES 30 31 33 34 ( 35 36 37 39 ^— ^0 (RAMA RRENS) 82 STAGING OF AMPHIBIAN EMBRYOS 1+1. Medullary plate forming even with an extruding yolk plug, 1+2. Normal yolk plug but gaetrular surface rough, Indicating early disin- tegration. hj). - k6. Neunilae vrith extruding yolk plugs. If these develop the embryos have severe caudal abnormalities. h^ . - 55' Heml-embryoa, due to incomplete invagination. 5'+. - 55 • Rudimentary abnormal embryos due to failure to incorporate yolk. 56. Hemi-embryo in which there is slight development of the head region. 57- - 59- Anterior end fairly normal in heml-embryos, Internal structures very abnormal. STAGING OF AMPHIBIAN EMBRYOS 85 ABNORMALITIES 41 42 44 (RANA PIPIENS) Qh STAGING OF AMPHIBIAN EMBRYOS 60. - 61. Yolk exposed laterally on tail 'bud. embryo. 62. Persistent yolk plug prevents tail formation in tail bud stage. 65. - 66, Relation of persistent yolk to spina bifida. 67. - 72. Spina bifida In various degrees. Note fair degree of head forma- tion in some cases. STAGING OF AMPHIBIAN EMBRYOS 85 ABNORMALITIES kJX* RANA PIPIENS) 86 STAGING OF-AMPHIBIAN EMBRYOS 75. - 8'+- Types of atmormallties found moBt frequently among parthenogenetlc or androgenetic tadpoles. 85. - 89. Oedema - associated with haploldy as well as osmotic unbalance. 90. - ^h. Haplold tadpoles with reduction of head stnictures. 95. The typical haplold tadpole with microcephaly, doraal-flexion of the tail, telescoping of the entire hody, reduction of gllla etc. STAGING OF AMPHIBIAN EMBRYOS 87 ABNORMALITIES 75 ;rANA RPIENS) 88 STAGING OF AMPHIBIAN EMBRYOS NORMAL STAGES OF RANA SYLVATICA TABLE I TABLE 2 STj^ EXTERNAL -a? rORM 51 Si EXTERNAL -JOfVl FORM st|^' external FORM VesJ>/ o NiVisi-^ Vj^^ EXTERNAL FORM MUSCULAR MOVCMeNT CIIX CincULATION SWIUUrNC- HATCHING TABLE 3 r^^f V. EXTERNAL FORM CORNEA TRANSPARENT ■>AJ TAOWLC rORU TICTM CIMB BUD DESCRIPTION OF STAGES* STAGES I TO 17 (TABLE I) 1. S^g at fertHlzatlon. 2. Eetabllsbment of gray crescent area as first external evidence of development, sharply defined at 1 hour. 5 to 6. Age given la time of appearance of cleavage furrov that eetabllahee the number of cells drawn for the stage. 7 to 9- Later stages In cell multiplication, best determined ty comparison of size of cells at vegetal pole. Appearance of dorsal lip. Blastopore approximately a semicircle. Complete blastopore (yolk-plug) stage. 13 • Silt blastopore or neural plate stage. lU. Neural fold stage. Beginning of closure of neural folds, begin- ning of elongation. Cilia begin to rotate the embryo at about this stage. Closure of neural folds con^Jleted. Beginning of development of tall bud, marlced off from body by ventral notch i^en embryo Is viewed laterally. 10. U. 12. 15 * The stages Illustrated and defined by age are of course essentially an arbitrary series of readily Identifiable points in the continuous process of development. Defined In terms of these points, the total development Is com- prised in a series of periods, each extending from one stage until tile next. In practice the points and the periods are not sharply dlatln- gulshed and It la convenient to describe each period in terms of th^ stage that Initiates It. Thus the development from onset of the heart beat to the beginning of gill circulation would be the period of stage 19* (From A. W. Polllster & J. A. Moore) STAGING OF AMPHIBIAN EMBRYOS 89 NORMAL STAGES OF RANA SYLVATICA STAGES 18 TO 23 (TABLES 2 AND 3) The figures are ventral and lateral views of all but stage 22, which shows dorsal Instead of ventral aspect. 18. Stage begins with development of capacity for muscular movement, i.e., simple uni- lateral flexure In response to mechanical stimulation. This Is verj suddenly ac- quired and is closely correlated with attainment of the external form figured. 19- Time given Indicates onset of heart beat which appears very suddenly and Is accord- ingly a most useful marker for this stage. (Use of strong reflected light is neces- sary for identification of this early pulse.) Tail equals one- third the length of the body. 20. Beginning of circulation of blood corpuscles through a capillary loop of anterior gill is closely correlated with gill morphology, and is the best indication of the beginning of this stage. Shaking will hatch embryos early in this stage; they hatch spontaneously late In 20. Swimming ability is acquired In the latter part of this stage. Tail equals one-half the body length. 21. Cornea becoming transparent so lens is visible as light spot. Body and tail nearly equal in length. 22. Development of posterior bend in gut makes trunk appear asymmetrical from dorsal aspect. A few capillary loops are functional In the tail fin. Epidermis rapidly becoming transparent. 25. Trunk and head have rounded out sind embryo assumes true larval or "tadpole" shape. Horny larval teeth developed. Posterior limb bud identifiable. Opercular fold beginning to develop. Active spontaneous swiimning beglna. INTERNAL ANATOMY (TABLE 5) It has been found useful to have some means of readily Identifying sectioned material in terms of the series of stages described qbove. There are, of course, no difficulties In doing this with embryos up to the time of closure of the neural folds (stage 15). For recognition of stages I6 to 21 table 5 was constructed. The number of somites was counted from frontal or sagittal sections. The development of the eye and ear, as seen in cross sections, are shown by the series of drawings made at a common magnification by a projec- tion method. TABLE 4 Hours from first cleavage required to reach various stages at different temperatures STAGE 10.4°C. 15.*^°C. 18.5°C. 3 0 0 0 1+ 2+ 1.3 1.0 5 5 2.2 2.0 6 3.0+ 5.0 7 11 h.l 3.5 8 2k ll+.O 9.5 9 56 19-5 13.5 10 1^5 2»+.0 16.5 11 60 32.0 21.0 12 72 37.0 25.0 15 96 52.0 33.0 l!+ 112 56.0 37.0 15 12 1^ 63.0 1+2.0 16 lUl 72.0 1+7.0 17 168 83.0 55.0 18 180 90.0 62,0 19 216 108.0 72.0 20 275 130.0 87.0 TABLE 5 16 17 18 M 12 18 EYE EAR 19 20 21 ill 20 EYE ^I EAR (From A. W. Pollister & J. A. Moore) 90 STAGING OF AMPHIBIAK EMBRYOS THE NORMAL STAGES IN THE DEVELOPMENT OF THE SOUTH AFRICAN CLAWED TOAD, XENOPUS LAEVIS 1. Unfertilized egg. Otlique view. Size 0. 9-1.0 mm. diameter. 2. Grey crescent stage. Oblique lateral view. 3. First cleavage completed. k. Second cleavage completed. 5. Third cleavage completed. 6. Early cleavage stage; approximately 52 cells. 7. Mid-cleavage stage. Detenained by easily recognizable, unpigmented rows of macromeres. 8. Late cleavage; blastula stage. Determined by position of lower pigment border. 9. Early gastrula. Oblique ventral view to show semicircular blastoporal rim. 10. Mld-gastrula; yolk plug stage. Oblique ventral view. 11. Late gastrula stage. Heavy pigmentation in place of yolk plug, and loss of pigment in presumptive neural area. Oblique ventral view. 12. Neural plate stage. Very slight lateral compression. Dorsal view. 15. Neural fold stage. Slight elongation. Length 1.2 mm. Ik, Neural folds in contact; ciliary rotation within egg covers. Top, dorsal view. Bottom, frontal view, showing presumptive frontal gland and oral sucker. Length 1.5 mm. 15. Neural tube stage. Top, dorsal view; eye vesicle distinct; tallbud indicated. Bottom, frontal view; ultimate size of frontal gland area as Indicated. Length 1.8 mm. 16. Tall bud stage. Optic, otic and pronephric protuberances distinct. Total length 2 mm. Tallbud 0.2 mm. 17. Beginning of muscular response to mechanical stimulation. Gill plate distinct. Total length 2.8-5.1 mm. Tallbud O.h mm. 18. Gill buds (two pairs) distinct; will hatch if shaken. Total length 5.7-'*.l mm. Tail 0.7 mm. 19. Beginning of heart beat; can be observed under the microscope with strong illumina- tion. Total length 14-.9-5.2 imn. Tail 1.5 nm. 20. Spontaneous hatching; first indications of melanin pigmentation. Total length 5.14-5.7 mm. Head O.9 mm. Body Z.k mm. Tail 2.2 mm. 21. Beginning of first-form tadpole stage. Nasal pits distinct; active spontaneous swimming begins during the phase; epidermis begins to become transparent; mouth open at this time; oral sucker degenerated; cornea transparent. Total length 6.0-6.2 mm. Head I.5 mm. Body 1.5 inm. Tail 5-5 nm. 22. Beginning of second-form tadpole stage. External gills disappeared; respiratory gulping, and feeding begin; thymus gland externally visible; lateral contour of mouth is round. Total length 9 d™' slv. Head 2 mm. Body 1 mm. Tail 6 mm. 25. Beginning of third- form tadpole stage. Hind limb buds indicated; oral tentacle buds indicated; lateral contour of mouth is wedge-shaped. Total length 50 mm. av. Head 7 nm. Body 5 nun. Tail 20 mm. STAGING OF AMPHIBIAN EMBRYOS 91 THE NORMAL STAGES IN THE DEVELOPMENT OF THE SOUTH AFRICAN CLAWED TOAD, XENOPUS LAEVIS STAGE NUMBER AGE-HOUfiS, IB'C Cf5 OWT CMCSCCNT tMI.T Cl.t.T oAM«A.* M'D ' atiTnuia o .arc oMTMuut NCuMk PL'TC STAGE NUMBER AGE-MOURS, I8*C NCUML *OLD m uaat. Twat STAGE NUMBER 20 AGE IN HOURS AT IB* C 39 72 LENGTH IN MILLIMETERS 55 HUSCUL** nci'OHSt ^•i^ ■ '^ '■'"''2^^0^n if " . ,■ f H*TCHC ? 23 910 30 rintT-vonit taopoli • CCONO-roKM TADPOLC TMlND-rOKM TADPOLE Drawings : Courtesy Dr. Paul Welez I9I+5: Anat. Bee. 95 Dorsal, lateral and ventral views of a 35 mm. larva of Xenopus laevls. Photo: Courtesy Dr. Paul Welsz I9I15: Jour. Morph. 77 92 STAGING OF AMPHIBIAN EMBRYOS STAGES IN THE NORMAL DEVELOPMENT OF HYLA REGILLA* StaRe #1^: Length li/h mm,; neural folda high and unfused in cranial part of neurula but in contact with each other in and posterior to region of hindbrain; presump- tive optic vesicle indicated by intensely pigmented depression in ventrolater- al wall of future forebrain. Stage #16: Length 2 mm,; neural tube completely closed; sense and gill plates visible; optic vesicle forming as shallow, thick-walled diverticulum of forebrain. Stage #17 •• Length 2^ mm. ; tallbud s.eparated from body proper by ventral notch; stomodeal depression slight; nonmotile; optic vesicle finger-like outpocketlng. Stage #18: Length 5 nim. ; tail about I/5 length of body; caudal fin appearing; suckers in- dicated by two heavily pigmented areas Joined medially by narrow pigmented band below stomodeum; beginning of muscular response to touch (simple flexure); optic vesicle fully formed and consisting of thick distal wall, relatively thin sides, and narrowing stalk. Stage #19: Length 'j'-l/j nm. ; tail rounded, more than 1/3 length of body, nasal placodes indicated by pigmented shallow depressions; suckers no longer Joined by pig- mented band; coll response to touch; beginning of heart beat; optic vesicle Invaginating to form optic cup; lens placode invaglnatlng into optic cup. Stage #20 : Length 5-l/5 nmi. ; tail less rounded, almost ^ length of body; appearance of external gills, with circulation; melanophores appearing on dorsal part of body; brief and weak swimming movements; optic cup fully formed and consist- ing of thick, inner sensory layer and thin, outer tapetal layer, faintly pig- mented at back of eye; lens vesicle formed but undifferentiated; optic stalk disappearing. Stage #21 : Length 6 mm.; tail pointed, |- length of body; external gills branching; nasal pits deep; sustained and strong swimming movements; lens vesicle faintly visible through cornea; optic nerve beginning to form. Stage #22 : Length 65- mm.; tail longer than body; pigmentation on dorsal aspect of eye visible externally; retina differentiated into relatively narrow inner layer of nuclei {ganglion\ cells) and wide outer layer of nuclei separated by narrow' band of white matteA (inner molecular layer); rods and cones beginning to dif- ferentiate outer parws; lens fibers forming; optic nerve well developed. Stage #23 : Length 7 nun,; proportion of tail to body about h to 5; operculum beginning to form; colon differentiated and bent dorsally; eye completely pigmented; retina further differentiated by formation of outer molecular layer separating nuclei of rods and cones ( outer nuclear layer) from nuclei of bipolar neurones ( inner nuclear layer); outer parts of rods and cones better differentiated, especial- ly ellipsoid. Stage #2U: Length 1^ mm.; operculum covering gills; gut S-shaped; spacious pleuroperl- toneal cavity ventral to colon; dorsal fin bowed; cavity of lens vesicle ob- literated; outer segment, ellipsoid, and perhaps nyoid of rods and cones well formed. * These stages, from #15> to §2h , differ slightly from those of Shumway for Eana plpiens and are here reprinted with the kind permission of Dr. B. M. Eakin (From 19^*7, Univ. Calif. Pub. Zool. 51:2l+5)- STAGING OF AMPHIBIAN EMBRYOS 93 HYLA REGILLA i \ f V V 16 1 20 ^ 33 9U STAGING OF AMPHIBIAN EMBRYOS AMBLYSTOMA PUNCTATUM lOj Naomi Leavltt STAGING OF AMPHIBIAN EMBRYOS 95 AMBLYSTOMA PUNCTATUM AMBLYSTOMA PUNCTATUM 16 Naomi Leavltt 96 STAGING OF AMPHIBIAN EMBRYOS AMBLYSTOMA PUNCTATUM Naomi Leavltt STAGING OF AMPHIBIAN EMBRYOS 97 AMBLYSTOMA PUNCTATUM .;--^ 32 Naomi Leavitt 98 STAGING OF AMPHIBIAN EMBRYOS AMBLYSTOMA PUNCTATUM AMBLYSTOMA V PUNCTATUM 38 /■ -^ 39 Naomi Leavltt STAGING OF AMPHIBIAN EMBRYOS 99 AMBLYSTOMA PUNCTATUM Naomi Leavitt 100 STAGING OF AMPHIBIAN EMBRYOS STAGES OF TRITURUS PYRRHOGASTER* (at 180C.) T, FYKRHOGASTER HARRISON STAGES STAGES AMBLYSTCMA AGE LENGTH REMARKS 1 1 Newly laid 2 2 15| hrs. 2 mm. First cleavage Incomplete 3 20 hrs. Second cleavage Incomplete 1* 5 21-5 A bra. Four cell stage 5 25i hrs. Third cleavage Incomplete 6 k 25 hrs. Eight cell stage 7 26 hra. 2 mm. Fourth cleavage beginning 8 5 27 hrs. Sixteen cell stage 9 6 51 hrs. Thirty-two cells 10 9 2 days Middle hlastula 11 10 1| days Early gastrula, dorsal lip blastopore 22 11* 4 days 2 mm. Middle gastrula, blastopore large 13 12 5 days 2 ram. Late gastrula, yolk plug Ik 13 6 days 2 mm. Blastopore a slit, neural groove 15 15 7 days 2.1 mm. Early neural plate with folds 16 16" 7-7i days 2.1 mm. Neural folds closing, gill plate 17 17 7* days Late neural folds 18 19 8 days Neural folds fusing 19 20 Bi days 2,8 mm. Neural folds fused, 5-6 somites 20 27 10 days 2.9 mm. Optic vesicle, otocyst, gill plate 21 31 15 days 5.9 mm. Olfactory pit, optic cups, gill plate 22 35 15 days 5.5 mm. Balancer, gill plate, rhombocoel 23 37 17 days 7, 5 mm. Forellmb bud, external gills, balancer 2k Uo* 5 weeks 10,5 mm. Balancer, 2 digits on fore- limb. 25 k6- 6 weeks liv.5 mm. Stomodeum, hindlimb bud, coiled gut * From Prlscilla L. Anderson 19i^5 : Anat. Rec. 86:58. The Author presents this series with- out argument for or against the deviation from the Harrison staging used with the Ambly- stoma aeries. (D@)(o)0 (From P. L. Anderson I9U5 : Anat. Rec. 86:58) STAGING OF AMPHIBIAN EMBRYOS 101 TRITURUS TOROSUS the California salamander <^ 3 ^^ 4 ^^^ 5 6 o o o ^ "^ ^ ? ^"f ^ 1! 16 16 17 18 •y,y I 31 32 19 20 21 f 22 25 26 ^i^27 ^^28 •^ 28 30 1^ V .?- J 33 34 35 36 37 38 89 (Courtesy Twltty & Bodensteln) 40 INDUCED BREEDING DEFINITION: The Induction of breeding activity by experimental means and under laboratory conditions, outside of the normal breeding season. PURPOSE : To produce eggs and embryos suitable for laboratory experimentation at specified times. MATERIAIg : Biological: Sana, Bufo, or Xenopus adults (or other Anura). Triturus, Amblystoma, Triton, or Eurycea adults (or other Urodela). Technical: ^TPodermic syringe (2 cc. capacity) and #18 needle Dissecting instruments Battery or aquarium Jars vrith weighted covers METHOD: (General description for Bana plplens; modifications for other forms appended.) Precautions: 1. Animals must be sexually mature. The females should have a body length of at least 7*+ n™- and the males should measure at least 70 mm., from snout to anus. 2. Animals should be well fed and in pre-breedlng condition. Those animals taken directly from hibernation are the beet. 5. Frogs should be kept in running cold water (below l8°C.) in which case they will be satisfactory for several weeks. Feeding is unnecessary during this laboratory confinement, if they were well fed before capture. k. Avoid red-leg contamination. ExEimlne frogs upon receipt. Bed-leg is not to be confused with temporary rash brought on by sudden changes in temperature. The fungus condition known as red-leg is very contagious and tanks with infected animals should be sterilized with permanganate. A copper penny in the frog tank will give off enough metallic ions to keep the red-leg down. 5- The fresh pituitary glands are injected into the abdominal cavity. Avoid injury to the median ventral abdominal vein, the sub-cutaneous veins, and to the intern- al organs. Glands should be used fresh but may be preserved in alcohol, by freezing in water, or by dissection. 6. Female glands are twice as potent per gland as are the male glands. The number of glands necessary to Induce complete ovulation varies with the season. 7. Efegs should be allowed to accumulate in the uteri before stripping any of them. Vigorous stripping is apt to damage the eggs. 8. Pre-check the medium in which the sperm suspension Is made to be certain that the spermatozoa will survive. Spring or pond water are best. Control: It will not be necessary to run controls for this experiment in as much as the major purpose is to secure eggs and developing embryos. However, adequate con- trols for such an experiment would consist of the injection of another equivalent endocrine gland, such as an equivalent number of adrenals or thyroids. Such con- trols have invariably given negative results. Procedure: (Description for Bana plplens; modifications for other forms appended.) 1. Bemoval of the anterior pituitary gland: Insert large, sharp-pointed scissors into the mouth of the donor, at the angle of the Jaw. Cut posteriorly to a point Just behind the tympanic membrane, then across the skull to the other side of the head, and sever the skull from the body. Invert the Jawless head and push aside the oral skin, thereby exposing the cross formed by the parasphenoidal and trans- verse bones. Insert the sharp point of smaller scissors into the cranial cavity, ventral to the exposed medulla, and cut through the floor of the cranium on either side of the brain In an anterior direction. Avoid injury to the brain tis- sue because in doing so the pituitary may be lost. The two parallel cuts should extend well anterior to the transverse bone. With forceps, deflect this flap of bone in a forward direction, thereby exposing the brain. The anterior pituitary gland should be seen lying Just posterior to the optic chiasma and will appear as a pinkish, kidney-shaped body surrounded to some extent by white endolymphatic - 102- INDUCED BREEDING 105 FLOOR OF CRANIUM DEFLECTED FORWARD ENDOLYMPHATIC TISSUE ANTERIOR PITUITARY GLAND OF FROG Diagram showing position of tlie pituitary gland of the frog as it might be seen through the parasphenoUal bone, lying Just beneath the brain. A-Anterior lobe of the pituitary gland. Note its more posterior position. 13-Pars intermedia and pars nervosa. E-Eye ball as seen through oral skin. Lr-Endolymphfitlc tissue adherent to the pituitary. 0-Exoccipital bone. P-Parasphenoidal bone. R-Retractor bulbi muscle. The attachment of this muscle to the parasphenoldal bone must be partially removed. S-Levator anguli scapuli muscle. V-First vertebra. tissue. Occasionally the gland will remain adherent to the floor of the brain case and will have heen deflected forward with the bone. Hemove the gland by grasping the white endolymphatic tissue surrounding it, using sharp forceps. This endolymphatic tissue has no known endocrine function. Place the gleini in 1 to 2 cc. of water in a small stender. In a similar way proceed with the re- moval of as many glands as are necessary. Pituitary donors need not be freshly caught animals but they must be sexually mature and in a pre-breeding condition. In general, the female glands are about twice as potent as those from the male, but there is no qualitative difference. The suggested doaes for Bana pipiens are as follows : September to January January to February March April Male Pituitaries 10 8 5 1^ Female Pltultarlea 5 h 5 2 In.lection of the hormone: For leclplents, carefully select large and obviously healthy females which have been recently received from hibernation. Such females may be kept In the refrigerator at k'-'C. in a small amount of water for a number of weeks but should not be kept at laboratory temperatures for more than a week if they are to be used for ovulation induction. Healthy females received from hibernation in January may thus be kept until June (i.e., at 4°C. ) . At the labo- ratory temperatures the ovarian eggs deteriorate rapidly. Before attaching the needle to the hypodermic syringe, draw up into its bar- rel the requisite number of anterior pituitary glands. It will be best if the lOU INDUCED BREEDING glands are freed from their attached endolymphatic tissue and remain whole as they pass Into the syringe. There will be some loss if the glands are damaged. Apply a large-bore hypodermic needle (#18) to the syringe, and then insert the point of the needle through the skin and abdominal muscles of the female frog, in the lower quadrant of the abdomen. Do not insert the needle far enough to damage any of the vital organs and specifically avoid the ventral abdominal and the sub- cutaneous veins. Inject downward, as the glands are heavier than water. As the glands pass through -the needle into the abdominal cavity they will be broken up into a fine suspension, ready for quick absorption. Absorption is probably ac- complished largely through the numerous ciliated peritoneal funnels on the ventral faces of the kidneys, which funnels open directly into the venous sinuses. The female frog should now be placed In a small amount of water in a wire-covered battery Jar or aquarium. If eggs are required within 2^-1+8 hours, the female should be kept at about 25°C;.; while if the eggs are not needed for i* to 5 days the frog may be kept at 10°C. and the eggs will be Just as good. 5. Test of ovulation: The presence of eggs in the uteri can be determined only by "stripping", or squeezing of eggs from the uteri. It is not necessary to sacri- fice the frogs to get the eggs. Stripping Is accomplished in the following man- ner, without damage either to the frog or its eggs. The legs of the frog are grasped in the left hand so as to prevent body move- ments on the part of the frog. The palm of the right hand is placed over the back of the frog, and the fingers encircle the body Just posterior to the forellmbe. By gentle closure of the right hand in the direction of the cloaca, eggs will be forced from the uteri. The body may be bent at the pelvic region to facilitate removal of the eggs. If Jelly alone or fluid issues from the cloaca the female should be replaced and tested again within 2U hours. It is general practice to remove and discard the first few eggs that emerge because occasionally cloacal fluid allows the swelling of the nearby Jelly on uterine eggs and this renders such eggs rather difficult of insemination. It is well also to dry off the cloacal region of the frog prior to stripping. The fertilization percentage will be higher if the eggs are allowed 2U hours for physiological maturation in the uterus. Each sexually mature female of Bana pipiens should give about 2,000 eggs all in metaphase of the second maturation division, ready to be fertilized. h. Artificial fertilization: Male frogs need not be injected with the pituitary hormone unless it is desired to show amplexus. In this case, inject the male at the same time the female is Injected, with the same dose of pituitary glands, and place them together in the same container. Amplexus, at laboratory temperatures, will be achieved in about 2k hours and fertilized eggs will be layed in the aquar- ium, generally early in the morning. Use water in which embryos are known to develop. From September until April mature, functional spermatozoa can be secured from Sana pipiens males of body length of more than 70 mm. simply by dissecting the testes in Spring or Pond Water, or any medium in which they are known to be viable. Generally two pairs of testes are dissected in about 10 cc. of water and are allowed to stand for 5-10 minutes at laboratory temperatures In order to al- low the spermatozoa to become active. The sperm suspension may be divided between several finger bowls, Petri dishes or large etenders so that in each there will be a thin film of suspension on the bottom of the container. Eggs are stripped directly into this sperm sus- pension in such a manner that all eggs are exposed. If this is not accomplished it will be necessary to pipette some of the suspension onto the eggs. The in- seminated eggs should stand for about 5 minutes and then should be flooded with the same water used to make up the sperm suspension. The eggs should be barely covered with this water. In about 20 minutes pour off this first water and add enough fresh water to again cover the eggs. It is the exposed surface rather than the volume of water that is important for respiration of the eggs. If the eggs are successfully inseminated they should all rotate bo that the animal heml- INDUCED BREEDING 105 THE LEOPARD FROG: RANA PIPIENS 106 INDUCED BREEDING sphere Is uppennost within about an hour and by 2^ hours the eggs should be In the 2 -cell stage at laboratory "temperatures of 23° - 25°C. (a) Egg of Raina plplens at the moment of insemination. (b) Egg of Rana plpiens 29 minutes after Insemination, showing grey crescent. 5. Care of the material: Aa the jelly membranes swell (by Imbibition) the egg mass will expand sind It may become necessary to add some water to cover. The Jelly mass generally sticks to the bottom of the container but may be separated by means of a stiff, clean section lifter. This may be done as soon as 1 hour after in- semination. The Jelly should be allowed to swell to its maximum and then the egg mass should be cut up Into small groups of eggs. This should be done before the first cleavage. The optimum ratio is about 25 eggs per finger bowl of 500 cc. water. The optimum temperature for the normal development of the eggs of Bana plpiens is 18° - 25°C. Development can be slowed down without the production of abnormalities if the eggs are kept at the lower temperatures, but never lower thein 10°C. ?y retarding the developmental rate of some eggs it is possible to have various stages of development available at all times. Eemember, however, that when eggs are removed from a cold to a warm environment, sufficient time must be allowed for the adjustment before the eggs are to be used for experinen- tal procedures. Eggs and embryos should never be transferred suddenly fKjm one temperatiure level to another. INDUCED BREEDING IN OTHER AMPHIBIA There is reason to believe that any amphibian which Is sexually mature and which has not recently undergone its normal breeding reaction, will respond to the pituitary hormone by ovulation (female) or by the release of spermatozoa (male). In general, the Anura will respond to pituitaries from other Amphibia but not from mammalian extracts of the pituitary hormone. Exceptions to this statement are Bufo and Xenopus. The Urodela, in contrast, will respond readily to the pituitary hormone from almost any source. There follows an alphabetical list of the more common Amphibia which have responded to pituitary treatment In the manner described for Sana plpiens. The references given are not complete but represent one of the major sources of other references for the particular form. Amblystoma tigrlnum Bomblnator pachypus Bufo americanus Bufo arenaxnim Bufo calami ta Bufo D'Orblgnyl Bufo fowls rl Bufo vulgaris Desmognathus fuscus Dlscogloseus plctus Eurycea blslineata Grylnophllus porph. ^la aurea Bums & Buyse, 1951 Moskowska, 1955 Wills, Riley, & Stubbs, 1955 Novelli, 1952 Cunningham & Smart, I95U Houssay, 1950 Rugh, 1955 Rostand, 1954 Noble & Evans, 1925 Kehl, 1950 Noble & Richards, 1950 Noble & Richards, 1952 Creaaer & Gorbman, 1955 INDUCED BREEDING 107 Efcrla crucifer - Eugh ( unpublished] Leptodactylus ocellatus - Novelll, 1952 Pseudobranchus striatus - Noble & Richards, 1952 Eana aurora - Creaser & Gorbman , 1955 Eana catesbiana - Eugh, 1955 Eana clamltans - Eugh, 1955 Eana esculenta - Eostand, I93I+ Eana japonica - Osima, 1957 Eana nigricauda - Osima, 1937 Eana palustris - Rugh, 1935 Eana plpl ens - Hugh, 1935 Eana temporaria - Ponse, 1936 Eana vulg ;aris - Adams, I93I Ehyacotriton olympicus - Noble & Eichards, 1932 Stereochilus marginatum - Noble & Elchards, 1932 Triton cri status - Adams, 193 1 Triturus pyrrhogas ter - Earth, I933 Triturus torosus - Witschi, 1937 Triturua viridescena - Stein, I95I+ Xenopua laevia - Aronson, I9I+I+ Size Eange: mm. Number Egg size Breeding Induced Name malea females Eggs (mm) Period Breeding Acrls gryllus 15-30 16-33 250 0.9-1.0 Feb. -Oct. Almost any- time Buf 0 ameri canus 52-86 51-75 5I+-IIO 55-81+ 38-1+8 1+000-8000 1.0-1.1+ 1+000-8000 1.0-1.1+ Laid 1.2-1.1+ Apr.- Apr. May : -Aug. ; -Aug. 15 1-Aug. 1 Oct. to Apr. Buf o fowleri Nov. to Apr. Hyla anderaonll 30-1+1 Oct. to Apr. singly Hyla clnerea e 36-1+8 32-1+8 Small 0.8-1.6 groups Apr. 15 -Aug. 15 Nov. to Mar. Hyla crucifer 18-50 20-3I+ 35-69 800-1000 0.9-1.1 30-1+0 1.1-1.2 in grps. Apr. Apr. 1-June 15 -Aug. 15 Oct. to Mar. Hyla ve rs 1 c olor 32-52 Nov. to Mar. Pseudacris brachyphona. 25-30 27-35 ? ? Mar. -June Sept. to Apr. Paeudacrls nlgrlta f 20-30 22-31+ ? 0.9-1.1 Feb. -May 15 Sept. to Apr. Pseudacris nlgrlta f 21-32 20-38 500-800 0.9-1.2 Mar. 15-May 20 Aug. 20-Feb. Sana cateablana 85-180 90-200 60-100 50-80 5I+-IO2 50-76 6000-8000 1.2-1.7 2000-1+000 1.5 2000-3000 1.6 2000-3000 1.6 ? ? June June Apr. Apr. June -Aug. 1-Aug. 15 15-May 15 1-May 15 15 -Aug. 15 May-Aug. Nov. to July Oct. to Mar. Sana clamltans 52-95 Eana palustrl s 1+6-61+ Bana pi pi ens 52-80 Sept. 1-May 1 Nov. to May Sana septentrionalls 1+8-72 Eana sphenocephala 50-78 53-82 ? 1.6 Feb. -Dec. All year Eana sylvatica 3I+-60 3I+-70 1+1-66 2000-3000 1.8-2.1+ 200-600 1.5-1.8 Mar. Apr. 15-May 1 -Aug. 15 Sept. to Mar. Eana virgatipes ..._ 1+0-63 Nov. to Mar. DATA ON EGG SIZE, EGG PEODUCTION, AND PEEIOD FOE OVULATION INDUCTIO:: MODIFICATIONS OF THE PROCEDURE ANURA BANA: The procedure aa outlined ia satisfactory for Bana pipiens from about September 1 until the normal breeding aeaaon of the species in April. The procedure for other species of Anura is essentially the same except for the season, the dose of the hormone, and the source of the hormone. The above table indicates that it is possible to secure Anuran eggs at any season of the year, providing the appropriate species is chosen. Since there is also considerable over-lapping of susceptible aeasona, material is available for hybridization expertments. 108 INDUCED BREEDING Properly planned, angjhi'bian eggs can be available for experimentation at all times. The genus Rana is available over vride areas. The dose of the hormone varies somewhat, depending both on the donor and the recipi- ent. In general the larger species seem to require more of the glands from the smaller species. In none of the genus Bana has there been uniform success with any source but other an5)hibian glands, the mammalian extracts being generally negative. BOTO: The toad will react not only to the pituitaries from other Anura but also to the pituitariea from other Phyla and to the extracts of mammalian pituitaries and even to the extract of pregnancy urine (antuirtin-S) and Preloban (Xenopus). The response is most reliable, however, when either frog or toad pituitaries are used. The eggs are deposited in a manner quite different from the frog in- that they are layed singly in long strings of Jelly, each egg being inseminated by the male (during amplexus) as it emerges from the cloaca of the female. It is therefore necessary to in- duce amplexus in toads and allow the paired animals to lay their eggs naturally. Amplexus may be induced by injecting the male with doaes of pituitary glands equivalent to those used for the female, and placing the pair together in a small amount of water. Several changes of the water may be necessary before the eggs are layed, to eliminate the faecal and other matter in the water. The water should be appropriate to survival of sperm and eggs. HYLA: The tree toads and other closely related species are difficult to secure out of breed- ing season but if caught during or Just before hibernation they may also be induced to lay their eggs in the laboratory. The nethod is similar to that for Bufo and during amplexus the male may be seen to bring his cloaca down close to the cloaca of the female as each egg emerges, indicating separate insemination of each egg. The Qyla egg is small but excellent for operative procedures. XENOPUS : This is the African clawed-toad which has attained fame through its use in pregnancy tests. It la extremely sensitive to the pituitary hormone, and ovulation can be induced by the pituitary (or gonad stimulating hormones) from any source. The method of inducing the breeding reactions and caring for the early embryos has recently been described in detail by Aronson (19'+'+) Weisman and Coates (19'+'+) and by Cameron (19'+7) whose papers should be consulted. It is likely that the Xenopus egg and embryo will become increasing- ly valuable as a laboratory form for experimentation purposes. URODELA Among the salamanders the eggs are generally inseminated as they pass through the genital tract of the female, after she has picked up the sperm bundles ( spermatophores) dropped in the water by the males. Artificial insemination of the Urodele egg is possible but rather difficult, since the sperm found In the spermatophores are not properly acti- vated. The process of artificial insemination consists of the removal of activated sperm from the genital tract of the female and applying them to oviduccal eggs of a pituitary- stimulated female, not Impregnated. The sacrifice of the several animals la neceasary and only a few eggs are secured. The more satisfactory procedure, therefore, la to inject the pituitary hormone into females known to carry spermatophorea, whereupon they will depoalt fertilized eggs. There are two methoda which may prove of practical value in the production of fertile eggs of the Urodela: Flrat. pituitary atlmulation of the male two days prior to the etimu- latlon of the female thereby causing it to deposit the spennatophores . If this is accom- plished in a proper environment the females may be induced to pick up the spermatophores with their cloacal llpa after which they may be stimulated to ovulate. Second, some suc- ceae has been reported from Europe (Triton) where the paired animals are well fed, then placed In the dark at hibernation temperatures (1+° - 8°C.) for a month or more, and then are broi;ght out into the light at Spring temperatures (l8° - 25°C.), whereupon they will spontaneously produce fertile eggs. INDUCED BREEDING 109 Triturua: (pyrrhogaeter, torosus or vlridescens) These newts can "be kept in the lahoratory for long periods, are fed on liver and earthworms, and can be Induced to ovulate either with amphibian (E. pipiens) pituitaries or with mammalian extracts of the anterior pituitary hormone. Spermatophores are retained for many months and (particularly with T. pyrrhogaster, the Japanese newt) the majority of females will give fertile eggs almost any time. The breeding tanks for Urodeles should be shallow, with facilities for their climbing out onto floating pieces of wood or onto sand. Aq.uatic' plants (Elodea) should be provided not only for aeration but for the attachment of eggs. Efegs will be layed singly, in large Jelly capsules, and will always be attached to plants. Instead of injecting large doses of pituitary it is well to inject about two fe- male (frog) glands per day for several days since Urodele eggs are constantly maturing. If the animals are well fed they may be Induced to lay a few eggs per day over long periods. Ambystoma: (mexicanum, opacum, punctatum, tigrlnum) The'se species apparently do not retain their spermatophores for very long before ovu- lation, hence it is generally more practical to collect the eggs in nature. Ambystoma opacum eggs are available late in September; A. punctatum and tigrlnum eggs from January through May; and A. mexicanum eggs In the spring, time variable. A. punctatum males will drop their spermatophores in slowly running streams or in ponds during the late March rains even when the temperature may be as low as 15°C., several days prior to the migration of the females from the woods. Females captured after they pick up spermatophores, but before ovlposltlon, will lay their eggs in the laboratory spontaneously or under pituitary stimulation. The eggs of A. opacum and A. punctatum are excellent for operative procedures because they develop rather slowly and are very hardy. The eggs of A. tigrlnum are hardy but develop very rapidly, while the axolotl (A- mexicanum) eggs do not stand operative proce- dures very well. OBSEBVATIONS AND TABULATION OF DATA: With each attempted Induction of ovulation the following data should be recorded: 1. Number and source of anterior pituitary glands. 2. Physiological condition of recipient, i.e., size, evidence of sexual maturity, and extent and conditions of laboratory confinement. 5. Temperature at which the injected female was kept. k. Date of injection and date or time of appearance of the first eggs within the uteri . 5. Percentage ovulation, determined by estimate of the volume of eggs remaining in the ovary after the reaction has been completed. DATE SPECIES FEMALE # PITUITABIES PIT. SOURCE TEMP. MEDIUM HRS. OF OVUL. i CLEAVING BC3GS 110 INDUCED BREEDING DISCUSSION: While the relationship of the anterior pituitary hormone to sexual activity haa heen 8\;ispected and then demonstrated for many years, it was In 1929 that Wolf in Wisconsin and Houssay in Brazil almost simultaneously puhlished results of their observations that the An5)hlbia could he induced to ov-ulate by means of anterior pltultaiy Implantations. There- after many investigators, Inspired by the urgent need for more embryologlcal material, have described successfully induced ovulations in a wide variety of forms. Since the mammalian pituitary hormone can induce sexual activity and ovulation in some amphibia, and the amphibian pltultaiy hormone can Induce heightened sex activity and hyper- trophy of the reproductive organs in mammals, it is no longer tenable that there is any species specificity In the gonadotropic activity of the anterior pltuii-ary gland hormones. It is true that frogs have thus far proven to be negative to the mammalian extracts, but toads are most responsive to these same hormones. It is true that ovulation in mammals has not been induced, even with large doses of Anuran pltuitarlea, although the genital system has responded. Indicating that there may be a great difference in threshold and in the quantitative value of the glands. Nevertheless, there is evidence that there Is present a gonadotropic hormone which will stimulate sexual activity in the amphibia in the pitultaries from fish, reptiles, birds, and mammals. That the reverse has not been equal- ly demonstrated may be only an Indication that the gland attains greater gonadotropic potency with the ascendency In the evolutionary scale. The negative reactions of frogs to mammalian extracts may be due to toxic reactions to the extractants, or to some very spe- cific protein sensitivity. No doubt this exception will eventually be explained. It has not yet been determined Just how the axiterior pituitary hormone acts to induce ovulation in the Amphibia. Attempts have been made to eliminate the circulatory and/or the nervous connections. Once the pathway of action is determined It may be possible to explain certain individual or species variations that have thus far eluded investigation. CONCLUSIONS: It is now possible to induce ovulation among the an^hibla so that eggs and embryos of the various species are available at all seasons of the year. BJ!H''i!J t-l 00 .H rH r<^ O p ID •rH In w © N^ O c etJ P. P p O P. od ^ ^1 O Q< in H' ? g* ^ CO •2 ID rH © :3 E E CO +J :3 ;i ^H x c g •s E R ^ o ,Q ,o rH o (D o 1H 1 B o tH «) H •rH tD ■^ •s § m ID (D g^ +} o §> (D m 0) ^ rH rH !-i 3 >> 5 p ID ■p O ^ ^ f-l e ID P (3 o ^ •S 1 ii U CM ^ ID O O 1 ■p CO ^ a^ a a T-l EARLY BEHAVIOR PATTERNS IN THE AMPHIBIA 123 CO (D •H O (D 0( CO ^D -d- J- -* r\i -^ o -4- CO f CO CVI VX1 OJ -=)• w CM C>J 0 OJ m 0) SP 4J C/J -P 1 w 7i m m rH ID ^ (D si 2 JJ 4J rH +3 s C ^ 0 §• 4J § a M 0) rH •H 08 © tA KN 0 8 § a) 0 •rl (D ?. §> rH 1 0 -P 0 cd u 4:> 0 §> ^ ^ >> 0 U CO 03 ■3 rH m (D E p CO JJ :3 g U !~i c E U E E 0 09 ,0 ,J3 f-i (D ID 0 E E 0 ■H s r-l 0 r-^ •H > m ■^ •rH 0 CO «) ^ 5 •H ■^ <4-( +3 ra 5t a 0 0 -p 0 s* m m ed ^ rH rH 0 >> 0) t>v jj g ■P rH ^ 0) rH 0 > (A < z < K of^Jk. \ "^■""Ci^^^^. \ »— ,,.^^6^7\>-v,. ^^V \ ^^^r?--^ii::^ ;:;On. \ ^^^ 0 (T. a^ r~ ^ ■^ •V A (\i 0 '*' u at O ■P 12" 5* A 25* B 100*--; 100* D 3* E 25* F ^wlre screen h" deep (♦Number of animals per compartment) -1U6- SPACE FACTOR AND GROWTH RATE li^7 2. The depth of the culture medium (Spring Water or Standard Solution) must 'be main- tained constant- If there ia evaporation this loss should be replaced with glass distilled water. The water need not be changed until the tadpoles hatch, unless it becomes turbid. If the aquarium is covered with a glass plate and is placed in uniform light and heat, it will require no care until after the beginning of feeding. (Eemove all dead eggs and embiyos, recording the fact,) 5. After the absorption of the external gills, begin to feed the tadpoles on the standard diet of washed and softened spinach. The green leaves should be washed in running water, and softened by par-boiling. Tadpoles must be cleaned and fed daily thereafter. On occasion it may be necessary to remove all tadpoles to marked crystallizing dishes for a few minutes while giving the aquarium a thorough cleaning, to avoid bacterial contamination. OBSERVATIONS AM) EXPEBIMEMTAL DATA: In the beginning the record will consist merely of staging the embryos under the various space conditions. After the tadpoles hatch, it will become necessary to make three records from each of the groups of tadpoles at each reading. It is suggested that the readings be taken at weekly intervals. The three records will be (a) size of the largest tadpole (b) size of the smallest tadpole (c) average of five sizes. Such size readings can be made quickly in Petri dishes over scaled graph paper, and are total length. After about 2^ months (at ordinary laboratory temperatures and adequate feeding) the forelimb emergence will be detected in some specimens. This may be taken as the final step in the experiment, i.e., when ^0% of the tadpoles reach forelimb emergence. Eecord the data in the following manner: (See record on following page. ) DISCUSSION: During the early cleavage stages there may be no detectable difference in the rate of development under the various conditions of reduced crowding. As soon as there is freedom of movement the tadpoles must seek out their food and, in doing so, encounter each other and they are thereby stimulated to further activity, and we begin to find differences in growth rate. Even in the same clutch of eggs there may be genetic differences which might explain differences in growth rate, hence it is Important to compare the averages and the composite averages from the compartments of the same number. The groups are so arranged to minimize any differences relative to the accumulation of metabolites and faecal waste. The single variable in this experiment is supposed to be the available space per tadpole, all other factors of oxygen, food, light, temperature, etc. being equal for all specimens. The surface area is identical for all compartments, and presumably the dissolved oxygen is the same, particularly if the activity of the tadpoles causes suffici- ent agitation of the medium to circulate all dissolved gases to an homogeneous condition. Lynn and Edelman (I956) have carried this experiment to metamorphosis and find that crowd- ing not only affects the rate of development but the success of achieving metamorphosis. The optimum conditions for development, at least in the pre-feeding stages, is a ratio of 1 tadpole per 2 cc. of medium in a total of 50 cc. per finger bowl. After feeding begins, this ratio would be considered as definitely crowding the tadpoles and development woilld be consequently retarded. 1U8 SPACE FACTOR AND GROWTH RATE M3 ^ ICN r-l .-t ^ ^^^ rH ^ H rH O S3 ^ 1 o\ s H w CO H M r- MD m -=t KA CM H - s i * IT IP lO IP IP UA < E-" (-^ g 4- +j -p jj +3 4- pH ^ 4-^ 0 a +J tr a: ■p tr I 4J a a; 4J ro a 4J 0 a Ph ro (T 1 s 3 m (C a s m ID o H S ) ID a) 6 0 -H a 3 tc a) a S' ^ H t ) - ^^ tjD r- u M r- !h M r- Sh bD -H »H 6 ) ^ %< m 1- a a U g a / \^ ^ V a; irt E a: U ^ (S a) B > ^^ a- t^ CO M CO ^ < / / ^^ < ^ , ^ t5 cr > < / / w V V fe R O W c s a o ^ H f-^ H ^ s ** =*= M •• S@ r, 0 w ^ E t> y (^ m ^ o g § of A. trigrlnum larvae living 2 months, but this effect was counteracted by adding 1.25^ of the total protein of cystine. ) •152- NUTRITION AND GROWTH OF LARVAE 155 Amino aclda for growth and differentiation of Anura: (Gudematach & Hoffman, I956)* Glycine "] Alanine > for maintenance Leucine J Aspartlc acid 4. j i. j. _i. j j. _, . . ., toxic, tut may support maintenance Glutamic acid ' Arglnlne~ lysine [> for grovrth Cystine Phenylalanine"] Tyrosine > for differentiation Tryptophane J _ .. toxic hut may support differentiation Proline (* Note: The hest acid of each group Is In the lowest, or most advanced position. Most of these acids are expensive, and their use in experimental analysis of protein nutrition is prohititlve, hut the paper hy Gudematsch and Hoffman should be studied for its J~emarkahle findings.) METHOD: Precautions : 1. Any food, particularly those which become acidic, will be a source of bacterial contamination. It will be necessary therefore to change the culture medium, and add fresh food on alternate days at first, and finally, every day. 2. Overfeeding is not possible, but maximal feeding will be achieved only by pro- viding excess food. Experience alone will determine for any group of tadpoles the amount of food which is Just in excess of maximal. 5. Green vegetables must be washed to free them of arsenic used by gardeners to get rid of Insects. Control: A variety of foods should be tried, and that which provides the most rapid and normal growth may be considered the control diet. All other conditions of space, medium, light, teniperature, etc. must be the same for all embryos. Procedure : 1. Provide uniform containers, each with a measured volume of the culture medium. Begulation finger bowls holding 5 tadpoles or Urodele larvae and 50 cc. of medium will prove satisfactory. The large (12 inch) crystallizing dishes may be used with 50 tadpoles and 100 cc. of medium. Mark the containers adequately, and place them in a uniform environment. The temperature should be about l4°-l8°C. for Urodeles and about 5°C. higher for the Anura. 2. Select three distinctly different diets, such as carbohydrate, protein, and some synthetic mixture for comparison. Set up at least 5 finger bowls (or 2 crystal- lizing dishes) for each diet offered, using a minimum of 25 tadpoles (or larvae) for each diet. 5. The larvae may be carried to and through metamorphosis (Anura) which takes from 2^ months to longer, depending upon temperature, space, and food. In this ex- periment, with temperature and space controlled, the emergence of the forelimb can be regarded as the beginning of metamorphosis and a terminal point for the nutrition experiment. OBSERVATIONS AND EXPERIMENTAL DA^A: Growth measurements should be taken at weekly Intervals to determine the relative value at different stages of the different nutritional offerings. These readings may be taken rapidly by placing a piece of calibrated graph paper beneath a Petri dish, axid the tadpoles transferred to this dish for direct size readings. l^k NUTRITION AND GROWTH OF LARVAE DIET INITIAL SIZE Species :_ 1 2 BODY LENGTH - MM. (Snout to end of tail) WEEKLY MEASUREMENTS (AVEEAGE OF 5 SPECIMENS) 5 6 7 8 9 10 11 12 13 li+ 15 l6 1. 2. 3. DISCUSSION: Feeding of anphlbian larvae is not necessary for several days after the mouth opens as there is at that time still some reserve yolk which can be utilized. For the Anura feeding may begin at stage #95 and for the Urodela at stage #'+0. A varied diet is gener- ally considered the best, and the Anura are thought of as herbivores and the Urodelee as carnivores, although they may both be omnivores. Certainly the frog tadpoles, when they are short on rations, will eat dead tadpoles, worms, and other organisms. In fact, there is some evidence of cannibalism among the frog tadpoles. The elongated intestine of the Anuran tadpole is associated with Its vegetarian diet and those forms raised entirely on a protein or non- vegetarian diet tend to have shorter intestines. The Anura do better on food with a green color while the Urodela do better on living, moving food such as Daphnla, worms, and small tadpoles. When crowded and underfed, the Urodela will snip off each others tails, and growing appendages. TABLE I Incidence of kidney stone in tadpoles reared on spinach and non-spinach diets. See +ext for more detailed explanation. Kl'MBERS or TADPOLES WITH DIETS Very numeroas kidney stooes "Nortnal" kidneys n2 small stones or less ) NomiBl kidneys (no stones) Spinach and liver food Spinach Liver food Lettuc* and liver food Lettuce 306 (9270 ) 27 (8%) 13 (93%) 1 (7%) 0 (0%) 0 (0%) 0 (0%) 32(10%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 13 (100%) 277 (90%) 15 (100%) From Briggs & Davidson, 191*2: Jour. Exp. Zool. 90:Uoi NUTRITION AND GROWTH OF LARVAE 155 16 Q 14 cn g 12 CL a: O 10 < t- a CD 3 — liver food spinoch a liver food _L 80 100 120 TIME — DAYS 140 Effect of diet on time of metamorphosis. The number of completely metamorphosed tadpoles Is plotted against time in days after fertilization. From Brlgge & Davidson I9I+2: Jour. Exp. Zool. 90:i^01. The kidney atones produced from oxalic acid of spinach and found coimnonly among the frog tadpoles at metamorphosia (Brigga & Davidson, I9I+2) are not seriously damaging, and spinach seems to carry the forms to metamorphosis more rapidly than any other diet. Daphnla and then Enchytrea have proven to he the best for Urodele larvae, with small strips of liver being offered after metamorphosis. iiqrlnwn 10 60 90 100 Growth curves for tigrinum and punctatum larvae raised under similar environmental conditions out on differ- ent diets. Vertical lines indicate the beginning of feeding. From Hutchinson & Hewitt 1955: Jour. Exp. Zool. ll:kG'^. 156 NUTRITION AND GROWTH OF LARVAE BEFKRENCES: Allee, W. C, E. B. Oeatlng, W. H. Hoaklns, I936 - "Is food the effective growth- promoting factor In homotypi cally conditioned water?" Physiol. Zool. 9- Borland, E., 19^+5 - "The production of experimental goitre in Sana pipiens tadpoles hy cabhage feeding and methyl cyanide." Jour. Exp. Zool. 9'+:115> Briggs, E. & M. Davidson, 19^2 - "Some effects of spinach-feeding on Eana pipiens tadpoles." Jour. Sxp. Zool. 90:14-01. Dorris, F., 1955 - "The development of structure and function in the digestive tract of Amhlyatoma punctatum. " Jour. Exp. Zool. lOik^l. Evans, H. M, & K. S. Bishop, 1922 - "On the existence of a hitherto unrecognized dietaiy factor essential for reproduction." Science 56:560 (also Jour. Am. Med. Aas'n. 81:889). Gudematsch, F. & 0. Hoffman, I956 - "A study of the physiological value of a-amlno acids during the early periods of growth and differentiation." Arch. f. Ent. mech. 155:156. Hoffman, 0. & F. Gudematsch, 1955 - "Further studies on amino acids in development. VIII. On the physiological value of the amino acids of glutathione and of some pro- teins in amphibian development." Proc. Am. Physiol. Soc. U7th An. Meeting. Howes, N. H., 1958 - "Anterior pituitary and growth in the axolotl (Amblystoma tigrinum) the neotonic form. II, The effect of injection of growth- promoting extracts upon the utilization of food," Jour. Sxp. Bio. 15-14-14-7. Hutchinson, C, 1959 - "Some experimental conditions modifying the growth of amphibian larvae." Jour. Exp. Zool. 82. Hutchinson, C. & D. Hewitt, 1935 - "A study of larval growth in Amblystoma punctatum and Amblystoma tigrinum." Jour. Exp. Zool. 71:1+65. Hyman, L. H., I9I+I - "Lettuce as t. medium for the continuous culture of a variety of small laboratory animals." Am. Microscopical Soc. 60:365. Janes, E. G., I959 - "Studies on the amphibian digestive system. IV. The effect of diet on the small intestine of Eana aylvatica." Copela. 5:15'*-" McCurdy, M. B, D. , I959 - "Mitochondria in liver cells of fed and starved salamanders." Jour. Morph, 6k ■.^. Morgan, Ann H. & M. C, Grlerson, 1932 - "Winter habits and yearly food consumption of adult spotted newts, Triturus virideacena, " Ecology, 13:5^- Morrill, C. V., I925 - "The peculiar reaction of the common newt to a liver diet." Anat. Eec. 26:85. Patch, E. M., I9HI - "Cataracta in Amblystoma tigrinum larvae fed experimental diets." Proc. Soc. Exp. Biol. & Med. 1+6:205. Pratt, E. M., 191+0 - "The effects of vitamin E deficiency on the tadpole of Eana pipiens." Univ. Illinois Pub. Zoology. Twitty, V. C. & L. E. DeLanney, 1959 - "Size-regulation and regeneration in salamander larvae under complete starvation.'' Jour. Exp. Zool, 81:599. Twitty, V, C. & W. J. van Wagtendonk, I9I+O - "A suggested mechanism for the regulation of proportionate growth, supported by quantitative data on the blood nutrients," Growth. l+:5l+9. "Maintenance, growth, and differentiation are the three visual expressions of the metabolic processes which go on in the developing organism." "In development, we are dealing with several concurrent processes which, though advancing synchronously, nevertheless are independent of each other. " J. F. Gudematsch 193i MECHANICAL SEPARATION OF GROWTH AND DIFFERENTIATION PUBPOSE : To inhibit growth (increase in mass) hy limitation of the physical environment and the determination of the effect of such limitation upon differentiation. MAT^IAIS: Biological: Eggs and early emhryos of Anura and Urodela. Technical: Agar, 1^ to 55^ concentrations made up in appropriate culture media for the forms used. Petri dishes and #2 Stenders. METHOD: Precautions: 1. Bacterial contamination may appreciably shorten the duration of this experiment. It might be effective to use 0.5^ sodium sulfadiazine in the agar mixture to cut down on the incidence of certain bacteria. This drug is non-toxic to embryos. 2. Avoid drying up of the agar through unnecessary exposure to warm or dry air. The containers should be covered during the experiment, and the agar should be sub- merged in the appropriate culture medium. Control: This consists of similar stage and age embryos kept under unrestricted con- ditions in the normal culture medliim, in similar containers. Procedure : (Best results will be achieved with Amblystoma) 1. Divest the eggs or early embryos of their membranes and place them In sterile, slightly hypertonic culture medium. This will partially dehydrate them before the Immersion in agar. 2. Prepare several Petri dishes or #2 Stenders with the agar mixture (above), using only enough agar to Just cover the embryos to be studied. When the temperature reaches a tolerable level for the embryos and when the agar begins to gel, trans- fer an embryo to the agar by means of a wide-mouthed pipette, and a minimum of sterile medium. Gently press the embryo (with hair loop) into the agar until It is submerged. 5. As soon as the agar is Jelled, cover it with about ^ inch of the sterile culture medium used to make up the agar mixture. This can also contain 0.5^ sodium sulfa- diazine. k. With older embryos (I.e., neurula or tail bud) place the specimen so that the gill, limb, or eye anlagen are uppermost and near the surface of the agar. When Jelled, and submerged in culture medium, examine under a dissection microscope and scrape away the agar immediately covering one of these organ anlage thereby releasing it from mechanical restriction but retaining the balance of the embryo under restriction. This should allow differential growth of the exposed area. ************* 5. If the agar is sufficiently concentrated, it may be cut Into rectangular blocks, each containing an embryo, parts of an embryo, or even Isolated cells. These blocks may be transferred to larger volumes of the culture medium and are very convenient to handle in that they may be turned over and the embryo be examined from various aspects. With the tail bud or later stages the partial dehydration by pre-treatment with hypertonic medium seems not to be so essential. OBSERVATIONS AJTD EXPERIMENTAL DATA: Record in the space on following page, by a series of drawings and/or photographs, the changes that seem to occur during each day following the embedding of the embryo in the agar. If free of bacteria some of these embryos may survive for 10-12 days, particu- larly at temperatures slightly below those of the laboratory. -157- 158 SEPARATION OF GROWTH AND DIFFERENTIATION DRAWINGS OF GBOWTH- INHIBITED EMBRYOS DISCUSSION: Whltaker and Berg (19't'+) first suggested this method of separating the processes of determlnati-on, growth, and differentiation, using the Fucus egg. Recently (I9U5) Holt- freter has made a similar study on the amphibia with very instructive results. He says: "Embryonic development is brought about by the integrated cooperation of various chains of biological processes, such as cell multiplication, morphogenetic movements, histological differentiation, differential changes of size and form of the embryo, etc., each of which has been given ample attention in analytical research while information on the relation- ship of these processes with each other is scarce and has been the by-product rather than the aspired aim of experimental work." He says, further, that "Instances have been re- corded where cytological differentiation occurred without cleavage, growth without differ- entiation, tissue formation without morphogenetic movements, metabolism without growth or differentiation." The agar medium provides an isotonic but restrictive environment where there may be relatively free exchange of respiratory gases but which prevents expansive growth or the acquisition of body cavities (neurocoel, archenteron, coelom) . Such pressure as is ex- erted is due to the forces of expansive growth of the embryo, for it is first exactly fitted into a relatively fluid agar medium. Such uniform restriction does not impede the formation of neural folds, or of other invaginations such as the stomodeum, proctodeum, olfactory pits, etc. The total volume of the egg (or embryo) remains almost static. The evaglnationa, such as the optic vesicles, gills, balancers, and limb-buda are apt to be restricted. Intra-cellular imbibition are limited and concern particularly the mesenchyme. It is not yet known whether mitosis is in any way inhibited, but certainly morphogenetic movements are not. While the general shape and size (growth factors) of the embryo re- mains much as they were at the time of embedding, there is not present a parallel restric- tion of differentiation. SEPARATION OF GROWTH AND DIFFERENTIATION 159 Figs. 1-4. Embryos of Amblystoma punctatum of equal age, which have been erabedded for 10 days In agar. According to whether they were Included completely or partially, they exhibit total or local growth inhibitions respectively. Cytologlcal differentiation has not been affected. Figure 4 shows the agar matrix still present. From Holtfreter, 1914-5: Anat. Bee. 93:59. REFERENCES: Adblph, E. F. , 1951 - "Body size as a factor In the metamorphosis of tadpoles." Biol. Bull. 61:576, Barnes, M. R., 19^11+ - "The metabolism of the developing Rana plplens as revealed hy specific Inhibitors." Jour, Exp. Zool. 95:399- Berrlll, N. J., 1935 - "Cell division and differentiation In asexnial and sexual develop- ment." Jour. Morph. 57:353- Bliss, A. E,, 1914-0 - "The effect of trypsin on development of Rana plplens," Proc, Soc. Exp. Biol. & Med. 1+5:769. Blount, R., 1955 - "Size relationships as influenced by plttdtaiT- rudiment Ingilantatlon and extirpation In the urodele embryo," Jour. Exp. Zool. 70:1. Bodensteln, D., I9I4-7 - "The effects of nitrogen mustard on embryonic amphlblsin develop- ment. I. Ectodermal effects." Jour. Exp. Zool. 10l4-:511. Boerema, I., I929 - "Die Dynamic des Medullarrohrschlusses. " Arch. f. Ent. mech. 115:601. Bragg, A. N., I958 - "The organization of the early embryo of Buto cognatus as revealed especially by the mitotic Index." Z. Zellf. mirk. Anat. Berlin. 28:15l4- (see also 1959; Bio. Bull. 77:268). Brlggs, E., I9I+6 - "Effects of the growth Inhibitor, hexenolactone, on frog, embryos." Growth.- 10:1+5- Brlggs, J, B. & R. W, Brlggs, I9I+5 - "The effects of water-soluble carcinogen on early frog development," Cancer Research. 5:1- Brown, L. A., I927 - "On the nature of the equation for growth processes," Jour, G«n. Physiol. 11:57, Brown, M. G. & V. Hamburger, 191+1 - "Density studies on amphibian embryos with special reference to the mechanism of organizer action." Jour. Exp. Zool. 88:555- l60 SEPARATION OF-GROWTH AND DIFFERENTIATION Buchanan, J. W., I938 - "Differential acceleration following inhibition." Jour. Exp. Zool. 79:111 (see ibid, 19^+0: vol, 85:255). Burt, A. S., 19'^-5 - "Neurulatlon in mechanically and chemically inhibited Ambly stoma. " Biol. Bull. 85:105. Clements, D. I. & N. H. Howes, 1958 - "Anterior pituitary and growth in the axolotl (Amblystoma tigrinum) neotonic form." Jour. Exp. Zool. 15:5^1. Cohen, A. &. N. J. Berrill, I956 - "Cell division and differentiation in growth of specialized vertebrate tissues." Jour. Morph. 60:2it-5. Copenhaver, W. M. & S. B. Detwiler, 19'+1 - "Developmental behavior of Amblystoma eggs sub- jected to indolebutyric acid." Anat. Bee. f9:2kT. Crozier, W. J., I926 - "On curves of growth, especially in relation to temperature." Jour. Gen. Physiol. 10:55- Dalcq, A. & J. Pasteels, 1957 - "Une conception nouvelle des bases physiologiques de la morphogenese . " Arch, de Biol. 148:669. Dawson, A. B., I958 - "Effects of 2,U-dlnltrophenol on the early development of the frog. Bana plpiens." Jour. Exp. Zool. 78. Dempster, W. J,, 1955 - "Growth in Amblystoma punctatum during the embryonic and early larval period." Jour. Exp. Zool. 6k:k<9^. Dent, J. N., 19^2 - "The embryonic development of Plethodon cinereus as correlated with the differentiation and functioning of the thyroid gland." Jour. Morph. 71:577. Detwiler, S. B. & W. M. Copenhaver, I9UI - "Further experiments upon the production of developmental abnormalities in Amblystoma." Jour. Exp. Zool. 88:1. Eakln, E. M., 1959 - "Further studies in regulatory development of Trlturus torosus." Univ. Calif. Pub. Zool. 45:185- Gilchrist, F. G., I928 - "The time relations of determinations In early amphibian develop- ment." Jour. Exp. Zool. 66:15. Hamilton, W. J., 195^ - "The rate of growth of the toad Bufo americanus under natural con- ditions." Copela. p. 88. Hammett, F. S., 19*^6 - "What is growth?" Scientla April-June, I9I4-6. Holtfreter, J., I9I+5 - "Differential inhibition of growth and differentiation by mechanical and chemical means." Anat. Bee. 93:59- Hutchinson, C, 1959 - "Some experimental conditions modifying the growth of amphibian larvae." Jour. Exp. Zool. 82. McCay, C. M., 1959 - "Chemical aspects of ageing." in "Problems of Ageing." Baltimore- Needham, J., 1957 - "Oii the dissociability of the fundamental processes in ontogenesis-" Biol. Eev. 8:l80. Patch, E. M., 1927 - "Biometric studies upon development and growth in Amblystoma puncta- tum and tigrinum." Proc. Soc. Exp. Biol. & Med. 25- Pike, F. H. , 1925 - "The effect of the environment in the production of malformations." Ecology. U:i*.20. Baney, S. C. & W. M. Ingvar, I9I+I - "Growth of tagged frogs (B. catesbiana and E. clami- tans) vmder natural conditions." Am. Midland Nat. 26:20. Beiman, S. P., I9U7 - "Growth." Anr Eev. Physiol. 9:1. Eobb, E. C, 1929 - "On the nature of hereditary size limitations. II. Tne growth of parts in relation to the whole." Br. Jour. Exp. Biol. 6:511- Shaw, G., 1952 - "The effect of biologically conditioned water upon the growth in fishes and amphibia." Ecology. 15:265. Stockard, C. E., I92I - "Developmental rate tind structural expression." Am. Jour. Anat. 28:115. Thompson, D. A. W., I9I7 - "Growth and Form." Cambridge Univ. Press. Twitty, V. C, 19*42 - "The role of genetic differentiation in the embryonic development of Amphibia." Biol. Symposium. 6:291. Tyler, A., 19'47 - "An auto-antibody concept of cell structure, growth, and differentia- tion." Growth Symposium. 7-19- welsa, P., 19*40 "The problem of cell individuality In development." Am. Nat. 6*4:514. Weiss, P., I9I+7 - ''The problem of specificity in growth and development." Yale J. Biol. & Med. 19:255. Whl taker, D. M. & W. E. Berg, I9I414 - "The development of Fuous eggs in concentration gradients. A new method of establishing steep gradients across living cells." Biol. Bull. 86:125- SEPARATION OF GROWTH AND DIFFERENTIATION 161 Wltschl E. 1950 - "Experimentally produced neoplasms In the frog." Proc. Soc. Exp. Biol. & Med.' 2^■.k^^. Youngstrom K. A., I958 - "On the relationship hetween choline esterase and the develop- ment of behavior in Amphihia." Jour. Neurophysiology. 1:557. Yung, E., 1885 - "De 1' influence des variations due milieu phy3icopchemlq.ue sur le developpement des animaux." Arch, des Sci. Phyg. et. Nat. 14:502. Fig. 1. - Fig. 2. - Fig. 3. - Figs. 4, 5, Embryo embedded In the late gastrula stage exhibits, 8 days later, the original egg shape but has developed stunted external organs. The ventral portion of an embedded neurula has grown through a hole In the agar investment, while the upper portion lias not Increased In volume but continued differentiating. The embryo has for 12 days retained the general shape existing at the time of embedding. Left nasal placode and right gill region form abnormal exten- sions into holes cut into the a^ar matrix. 6. - Differential Inhibition of development in blastulae of Rana fusca that have been kept for 3 days closely packed In between other eggs. The frontal portion which alone had free access to the Immersion fluid Is least inhibited showing a medullary liead fold and a sucker (Figs. 4-5). In the absence of exogastrulatlon such eggs develop Into microcephalic larvae (Fig. 6) . ( From floltf reter, I9U5: Anat. Bee. 95:59) CHEMICAL ALTERATION OF GROWTH AND DIFFERENTIATION FUBPCSE: The study of the effect of a specific chemical, Lithium Chloride, on early amphibian development. MATEEIALS: Biological: Early gaatrula stages of Anura (stage #9) and Urodela (stage #10). Technical: Various solutions as follows:* 1. Balanced lithium solution (Hall, 1914-2): Lithium chloride 1.12 gr. Lithium sulphate 1.5 gr. Calcium chloride 0.I5 gr. Potassium bl phosphate .... O.Qlt- gr. Sodium bicarbonate 0.5 gr. Glass dist. water 1000.0 cc. 2. Unbalanced lithium solution (Hall, 1942): Lithium chloride I.09 gr. Lithium sulphate 1.5 gr. Sodium bicarbonate 0.11 gr. Glass dist. wBter 1000.0 cc. 3. Unbalanced sodium solution (Hall, 19'+2): Sodium chloride 1.5 gr. Sodium sulphate 1-97 gr. Sodium bicarbonate 0.25 gr. Glass dist. water 1000.0 cc. h. Pure lithium chloride solutions: (a) 1.9 grams LlCl in 1,000 cc. of glass distilled water. (b) 1.9 grams of LiCl in 250 cc. of glass distilled water. 5. Control solutions: For Anura: Standard ( Holtfreter' s) solution For Urodela: Urodele Growing Solution METHOD: Precautions : 1. Glass distilled water should be usedj if possible, to make up tne experimental solutions to avoid even the minutest traces of metallic or other ions. 2. In returning the exposed (experimental) gastrulae to the nonnal medium, it is wise to pass them through several changes to remove all adherent Lithium. Control: This consists of similar gastrula, similarly denuded of their membranes, but kept in control media. Conditions of temperature, light, etc. must be identiceil. Procedure : 1. Prepare 12 finger bowls, each with 50 cc. of solution as follows: Two finger bowls (a and b) each, of solutions 1, 2, 5, U, and tne appropriate control 5. Cover them and iceep them at a cool temperature. (About 18°C. for Anura or lit-°C. for Urodela. ) 2. Strip eggs (Anura or Urodela) of their Jelly membranes. Do not attempt to re- move the vitelline membranes, particularly of the Anura. The stages should be the same and in early gastrula (Anura #9 and Urodela #10). A total of at least 500 undamaged eggs should be quickly prepared. (Since it takes 5 hours to reach stage #10 in the Anura, at least this much time should be allowed for the denud- ing.) Note: Effects similar to those produced by Lithium Chloride may be produced by un- balanced solutions of NaCl, NaOH, ethyl alcohol, NHj^OH, MgCl^, Mg(N0j)2, ether, chloroform, and by x-raye. -162- CHEMICAL EFFECT ON GROWTH 4 DIFFERENTIATION 165 DEAWINGS AMD PHOTOGRAPHS OF CHSMICAL EFFECTS l6it CHEMICAL EFFECT ON 'GROWTH 4 DIFFERENTIATION 5. Place 25 gastrulae in each of the finger bowls, and record the exact time and stage. k. After 2k hours, pour off all experimental media and wash twice with control medium. Finally place the embryos in 50 cc. of the control medium. 5. Allow the embryos to develop under controlled conditions to Anura stage #22 or Urodela stage #^+0 (swimming), examine, and fix in 10^ formaldehyde. (Various degrees of exo-gastrulae, monorhyny and cyclopia will be found.) ************* 6. The long exposure of 2k hours may prove to be too drastic for the species used, as evidenced by the mortality and teratoligies of the experimental embryos. For those solutions which produce such drastic effects, reduce the exposure to 6 or even to 2 hours. DISCUSSION: Experiments in animalization and vegetalization have been standardized for the Echinodermata but Adelmann, Lehmann, and Hall have each demonstrated that Lithium Chloride will effect the medium strip of organizer material of Amphibia resulting in exo-gastrulae, which have faulty inductions (in consequence). A large variety of substances interfere with gastrulation, but Lithium Chloride seems most satisfactory in the uniformity and reproducibility of its effects. Hall (19^+2) has shown that even with Lithium Chloride the results will depend upon a number of factors such as (a) concentration of Lithium (b) duration of exposure to the salt (c) phase of development of the embryo (d) temperature of medium (e) presence of other salts. The effect on differentiation is due to upsetting the delicately balanced developmental factors that are so important at the time of gastrulation, and the manifes- tations relate largely to the head and the tail organizers. By applying the Lithium Chloride for 6 hour exposures at different stages of development, Lehmann has determined a shift in susceptibility with progressive development. The effects are lessened at the lower temperatures, and in the presence of calcium and the salts of the control medium. Histological examination is desirable but not necessaiy to identify the various types of developmental abnormalities. Material may be fixed in Bouin's for sectioning, or in 10^ formaldehyde for dissection. -N_-t) CYCLOPIA IN AMBLYSTOMA PUNCTATUM EMBRYOS Figs. la. embryo ol' 32 somites; lb - embryo of 8 mm.: Ic - embryo (In Mg) of about 12 mm. Figs. 2a. eraorj'o of 31 somites; 2b - embryo of 5.5 mm.; and 2c - embryo of about 9.5 mm. An but Ic from lithium treatment. The "1" group ex- liiblt cyclopia complete and the "2" group exhibit cyclopia incomplete. From Adelmann 195'^: Jour. Exp. Zool. 67:21? CHEMICAL EFFECT ON GROWTH & DIFFERENTIATION 165 There is a current interest in mitotic Inhltitora such as colchicine and the nitrogen mustards, which indirectly affect development. It is expected that within the next few years the chemical separation of ontogenetic processes will he extended to great lengths by the use of such and related chemical suhstances- on the amphihian emhryo. BEFESMCES : Adelmann, H. B., I956 - "The problem of cyclopia." Quart. Eev. Biol. Il:l6l & 2Qk. Bellamy, A. W. , I919 - "Differential susceptibility aa a basis for modification and con- trol of early development in the frog." Biol. Bull. 57:312. Burt, A., 19ii3 - "Neurulation in mechanically and chemically inhibited Amblyatoma." Biol. Bull. 85:103. Child, C. M,, I9I1O - "Lithium and echinoderm exo-gastrulation; with a review of the physiological gradient concept." Physiol. Zool. l^:k. Cohen, A., 1938 - "Myotome fusion in the embryo of Amblystoma punctatum after treatment with lithium and other agents." Jour. Exp. Zool. 79:'+6l. Gurwitsch, A., I895 - "Ueber die Einwirkung des Llthlonchlorids auf die Entwicklung der Frosch- und Kroteneier (E. fusca und Bufo vulgaris)." Anat. Anz. 11B;65 (See also 1896 Arch. f. Ent, mech. 3-) Hall, T. S., 19'j-2 - "The mode of action of lithium salts in amphibian development." Jour. Exp. Zool. 89:1. HenJ.ey, C, 191+6 - "The effects of lithium chloride on the fertilized eggs of Nereis Umbata." Biol. Bull. 90:188. Herbst, C, I9U5 - "Die Bedeutnungen der Salzversuche fur die Frage nach der Wlrkungsart der Gene. Zusammenschau elner Hydrationsbzw. Mikrolaboratorien theprie der Genwirkung." Arch. f. Ent. mech. 1^2:319 (See ibid I896, 2:1+55). Holtfreter, J., I9I+3 - "A study of the mechanics of gastrulation. " Jour. Exp. Zool. 9l+:26l. Lehmann, F. E., I937 - "Eegionale Verschiedenkeiten des Organlsators von Triton, insbe- sondere in der vorderen und kinteren Kopfregion, nachgewiesen durch phasenspezifische Erzeugung von Lithlum-bedingten und operativ bewirkten Regionaldefekten- " Arch. f. Ent. mech. 138:106. Lindahl, P. S., 191+0 - "Neue Beitrage zur phyaiologiachen Grundlage der Vegetativisierung des Seeigelkeimes durch Llthiumionen. " Arch. f. Ent. mech. Il+0:l68. Pasteels, J., 19^2 - "Lea effets du LiCl sur le developpement de Eana fusca." Bull, de la Classe de Scl., Belgique. 27:605 (See also 19^+5, Arch. Biol. 56:105). Eanzi, S., 19^+2 - " Naturwiss. 30:329 (See also Pubbl. Staz. Napoli. Zool. 9:8l.) Baven, C. P., I9I+2 - "The influence of lithium upon the development of the pond snail, Limnaea stagnalis." Proc. Nederl. Akad. von Wetena. 1+5:856. Euunstrom, J., 1935 - "An analysis of the action of lithium on sea urchin development." Biol. Bull. 68:378. Spiegelman, S. & F. Moog, I9I+5 - "A comparison of the effects of cyanide and ozide on the development of the frog's egg." Biol. Bull. 89:122. Stockard, C. B., 1921 - "Devel5pmental rate and structural expression; an experimental study of twins, double monsters and aingle deformities, and the interaction among embryonic organs during their origin and development." Am. Jour. Anat. 28:115- Tondury, G., I938 - "Weitere Beitrage zur Frage der Kopfentwlcklung bel Urodelen. ±1. Erzeugung von Mikrokephalle durch Einwickung von Lithium chlorid auf die Gastrula von Triton alpestris." Arch. f. Ent. mech. 157:510. von Ubisch, L., I929 - "Ueber die Determination der larvalen Organe und der Imaginanlage bei Seeigeln. " Arch. f. Ent. mech. 117:80. Waterman, A. J., 1957 - "Effects of salts of heavy metalos on development of the sea urchin, Arbacia punctula." Biol. BuJl. 75:1+01. Wolsky, A., M. A. Tazelaar, J. S. Huxley, I956 - "Differential acceleration in frog development." Physiol. Zool. 9:265. 'Tr easure your except ions .'" - Bateson THE EMBRYO AND NARCOSIS, OR THE SEPARATION OF FORM AND FUNCTION PURPOSE: To determine the efficiency of various depressants as emtryonic narcotics, and to utilize such narcotics in an attempt to separate the development of form from func- tion. MATERIALS : Biolopjlcal: Early Anuran or Urodele emhryos through stages of (muscular) motility, (Anura stage #17, Urodela stage #27). Technical : Various known depressants: (None of these except freezing affect the cilia) Acetanilid - l/l,000 (lethal at 1/200) Bapid recovery Alypin Anyl alcohol - 0.15^ Barhital sodium - l/lOO (lethal at l/l5) Slow recovery Borocaine Butyl alcohol - 0.5/^ Chloral hydrate - 0.1^ or I/5OO. Bapid recovery * Chloretone - 0.2 to 0.9^ or l/20,000 (lethal at 1/20OO) Bapid recovery Chloroform - 0.05^ Bapid recovery CC^ gas Cocain Diethyl ether - l.U^ * Ethyl alcohol - k-.S^o or I/50 Bapid recovery Ethyl chloride - 0.046 moles/liter * Ethyl urethane - 2.(yfi (Not suitable for prolonged exposure unless in weaker concentrations, i.e., 1.0^.) Freezing Methyl alcohol - 6.1|^ * MS 222 (M-Amino-Ethyl-Benzoate) - 1/5OOO. Can he autoclaved.** Nembutal - 1/750 Slow recovery Novocaine Panthesine Paraldehyde - lAoO (lethal at l/lOO) Propyl alcohol - 1.5^ Stovaine Tutocain METHOD: Precautions : 1. To function as an anesthetic, a drug must interfere with some physiological process such as respiration or enzyme activity. The action of untested drugs must not he prolonged beyond the stage of total anesthesia unless testing for its persistent effect or lethality. 2. All depressants are Immediately lethal in high concentrations. In very low con- centrations, and repeated immersion, the embryo may become increasingly resistant to narcosis. Controls : The controls consist of untreated embryos of the same age and stage, kept under identical conditions except for the drug being tested on the experimentals. ♦Suitable for these experiments. ** Available through M. Sandoz 8e Co., 68 Charlton St., New York City. -166- SEPARATION OF FORM AND FUNCTION 167 PEOCEDUEE: EFFECT ON THE SPERM AND FERTILIZATION 1. Prepare an ovulating female, Sana pipiena. 2. Prepare two sperm suapenaiona (Eana pipiena), aa followa: a. Control - 2 paira of teates in 10 cc, of Spring water. b. Experimental - 2 paira of teatea in 10 cc. of Spring water containing 1/3,000 MS 222. Examine a drop of this auapenaion Just iDefore atripping the eggs into it to determine whether the sperm are motile. 5. Fertilize eggs from the same female in the two aperm suapensiona Flood in I5 minutes with more of the aame solutions used to make the suapensiona. k. After 1, 5, and 5 hours remove eggs from the Experimental mass and place them in pure Spring Water. Lahel the container, and make a further change in Spring Water in 5 minutea. Allow these eggs to develop under the same conditions aa the con- trola. Observe frequently. 5. Obaerve the development of the eggs remaining in the MS 222. RECORD OF FERTILIZATION DATA (SpeciesL CONDITION TOTAL # EGGS i CLEAVE i BLASTULA % GASTRULA % NEURULA ■jt HATCH Control MS 222-1 hr. MS 222-5 hrs. MS 222-5 hra. MS 222- hra. EFFECT ON EARLY MOTILE STAGES Anura St. #17-#S5: Urodela St. #27-#35 1. Select a large number of embryos of the same age, atage, and species. There ahould be evidence of motility. Anura stagea #21-25, Urodela Stages #31-#55 are best. 2. Prepare finger bowls or Petri dishes of MS 222 in 1/5,000 concentration. 5. Place the embryos, 5 at a time, in a finger bowl of anesthetic and, with a stop watch and 2-3econd Interval stimulations, determine the time of the first and the last of the 5 embryos to lose their responsiveness to tactile stimvilation. It is best to use a hair loop and stimulate the same region, i.e., the side of the body. Mark each finger bowl with the exact time of anesthesia. k. At intervals of 1, 6, 2k, 56, and 1*8 hours (or approximately similar houra) transfer the anesthetized embryos from one of the finger bowla to pure Spring Water. With a atop walch and 2-second interval stimulations (as above) determine whether snort or long anesthesia has any effect on the period of recovery. Note, Incidentally, whether there haa been any injury to the experimental embryos as evidenced by ab- normal movements . 168 SEPARATION OF FORM AND FUNCTION RECORD OF ANESTHESIA TIME IN MS 222 Time In aeconda for total anesthesia EMBRfOS (Species A B C D E AVERAGE TIME Group 1 Group 2 Group 5 Group *+ Group 5 RECORD OF RECOVERY TIME FROM MS 222 Time In second or minutes for total recovery EMBRfOS (Species A B C D E AVERAGE TIME Group 1 Group 2 Group 5 Group h Group 5 TOTAL TIME UNDER ANESTHESIA DEVELOPMENT OF FORM WITHOUT FUNCTION Anesthetics knock out the normal functioning of the nervous and/or the muscular sys- tem but seem to have no effect on ciliary motion. It Is Instructive to determine to what extent the muscular and the nervous systems can develop (normally) although the embryo la under the continued Influence of an anesthetic. Select a group of embryos of the same species and at an age and stage prior to the Initiation of muscular movement (see exercise on "Behavior Patterns"). In fact, the best stage to begin narcosis for thla observation is #1^*- for the Anura and #17 for the Urodela, when there are as yet no somites. Place them, five to a finger bowl of 50 cc. total vol- ume, In l/lO,000 MS 222 freshly made up in Spring Water for the Anura and Urodele Growing Medium for the Urodela. Keep controls under the same conditions except for the anesthetic, and allow the embryos to remain undisturbed for periods ranging from 1 day to 2 weeks, at appropriate temperatures for the species considered. The Anura will do well at laboratory temperatures but the Urodela should be kept at temperatures below 18°C. For the extended obaervatlons, replace the experimental medium with fresh experimental medium every k-^ days, since there is some loss of potency of the MS 222 in solution. Upon returning the embryoa from prolonged narcosis to normal medium, determine the speed of recovery of response but more particularly compare the stage of development with that of the controls. Note any morphological and behavior variations when compared with the untreated contiX5ls. Have the muscles developed normally In spite of total anesthesia? SEPARATION OF FORM AMD FUNCTION I69 BECOBD OF EFFECT OF PBOLONGED NAECOSIS DISCUSSION: There are three major theories regarding the mechanism of narcosis. They are the permeability theory of Lillle and Winterstein; the adsorption theory of Warhurg; and the lipoid theory of Meyer-Overton. According to Henderson (1950) the Meyer's theory is the moat plausible but "No theory of anesthesia' says Henderson "will prove acceptable which is based on a proof of a depression of the resting oxidation of the cell." Moog (19^'t-) found a smooth rise in the normal respiration of Rana plpiens eggs from fertilization to the heart-beat stage (#19) and that chloretone, from 0.051^ to 0.09^, had a small but increasing effect on that respiration through late gastrula. The gastrula seemed to be resistent to chloretone. After neunilatlon the effect was more pronounced, weak chloretone effects being reversible, stronger ones producing various permanent ab- normaiitiee, and still stronger ones causing disintegration and cytolysis. On the basis of differential reaction at different stages, Moog postulates two separate chloretone- sensltive respiratory systems, one related to "activity" and the other to "maintenance". Karczmar and Koppanyi (V^kT) In brief notes list a large group of anesthetics which were used with larval salamanders. They classify the drugs as to the rapidity with which they bring on Immobilization and from which the larvae recover. The precise mechanism of narcosis has not been determined, and therefore it is unlikely that the action of these various depressants can be directly compared. However, from the point of view of prac- ticality, a reliable, non-toxic, non-injurious anesthetic is necessary for many of the pro- cedures in experimental embryology. Thus far MS 222 has proven to be the most satisfactory of all. It is nevertheless recommended that the student test the value of other depres- sants, particularly chloretone, chloroform, chloral hydrate, ethyl alcohol, and freezing. This exercise throws light on the relation of the development of structure in rela- tion to function, since the larvae are immobilized during the development of the muscula- ture. 170 SEPARATION OF FORM AND FUNCTION REFERENCES: Anderson, B. G. & G. J. Jacobs - "The apparent thresholds of narcosis and toxicity for some normal aliphatic alcohols and their isomers." Anat. Rec. 99= suppl. k'}. Bancroft, W. D. & G. E. Rochter, I95I - "The chemistry of anesthesia." Jour. -Phys. Cham. 55:215. Clark, A. J., 1957 - "The action of narcotics on enzymes and cells." Trans. Faraday Sic. 55:1057. Fisher, K. C, I9U2 - "Narcosis." Canadian Med. Ass'n. Jour. kT:klk. Fox, D. L., 1955 - "Carbon dioxide narcosis." Jour. Cell. Comp. Physiol. 5:75. Greig, M. E. , I9I+6 - "The site of action of narcotics on brain metabolism." Jour. Pharm. Exp. Therap. 87:185. Henderson, V. E., 1950 - "The present status of the theories of narcosis." Physiol. Bev. 10:171. Hiller, St., I92U - "Influence de I'alcool ethylique sur le developpement de Rana fusca." Bull, de I'Acad. Polonaise des Sc. et des Lettres. B. 625. Johnson, F. A., D. Brown, D. A. Marsland, I9U2 - "A basic mechanism in the biological ef- fects of ten5)erature, pressure, and narcotics." Science. 95:200. Jowett, M., 1958 - "The action of narcotics on brain respiration." Jour. Physiol. 92:522 Karczmar, A. G. & T. Koppanyi 19^+7 - "The effect of stimulant drugs on overt behavior." Anat. Rec. 99: suppl. 64. Koppanyi, T. & A. G. Karczmar 19'<-7 - "The effect of depressant drugs on overt behavior." Anat. Rec. 99: suppl. 6^+. Mathews, S. A. & S. R. Detwiler, I926 - "The reactions of Amblystoma embryos following prolonged treatment with chloretone." Jour. Exp. Zool. 1+5:279. McElroy, W. D., I9I+7 - "The mechanism of inhibition of cellular activity by narcotics." Quart. Rev. Biol. 22:25. McGovem, B. H. 8e R. Rugh, l^kk - "Efficacy of M-Amino-Benzoate as an anesthetic for amphibian embryos." Proc. Soc. Exp. Biol. & Med. 57:127. Michaelis, M. & J. H. Quastel, 19*^1 - "The site of action of narcotics and respiratory processes." Bloch. Jour. 55:518. Moog, F. , 19'*'+ - "The chloretone sensitivity of frog's eggs in relation to respiration and development." Jour. Cell. & Comp. Physiol. 25:151. Narins, S. A., I95O - "A quantitative study of the effect of chloroform on oxygen consiimp- tion of the tadpole (Bana clamitans)." Physiol. Zool. 5:519- Parker, G. H., I959 - "General anesthesia by cooling." Proc. Soc. Exp. Biol. & Med. U2:186, Pamas, J. K., & S. Krasinska, 1921 - "Uber den Stoffwechsel der Amphlblen larven. " Blochem. Zeitschr. Il6:108. Eideal, E. K., I9I+5 - "Surface chemistry in relation to biology." Endeavor. U:85. Roblin, R. 0., Jr., I9I+6 - "Metabolite antagonists." Chem. Rev. 58:255. Rothlln, E., 1952 - "MS 222 (loslichee anaesthesin) ein Narkotikum fur Koltbluter." Schweiz. und. Wchnschr. 62:10U2. Twltty, V. C, 1955 - "Nature of paralysis produced in Amblystoma by Triturus transplants." Proc, Soc. Exp. Biol. & Med. 52:1285. Valko, E. I., I9U6 - "Surface active agents in biology and medicine." Ann. N. Y. Acad. Scl. 46:1+51. "The deternmat ion or cheno-dif ferent iation of any given part (i.e., the decision as to what any cell or group of cells shall develop into) takes place inv isibly some time prior to the process itself. " Spenann "No concre te argument can be advanced to separate form and function in their essence." Dalcq EXPERIMENTS WITH THE AMPHIBIAN GERMINAL VESICLE* PURPOSE : To study the living but Isolated nucleus of the amphihlan egg, with special emphasis on the chromosoine structure and the effects on the chromoaomes of various en- vironmental variables. MATERIALS : Biological: Ovaries of any Anuran or Urodele. Technical: Stenders, Syracuse dishes, depression slides, medicine droppers, electrical apparatus (diagram on p. 175), and various solutions: Phenol red as pH indicator. Nuclear medium (N-medium) which is Ca-free Singers solution made up with glass distilled water. Fixatives: Bouin, HgClg sat. aq.., h% formalin, aceto-orcein. Stains: Mayer's Eaemalum, 1^ crystal violet in N-medium; aceto-orcein (1+5^ glacial acetic acid and 0.55^ orcein), 1^ methyl green in 1^ acetic acid. General: 0.1 N-NaHPOi^ 0.001 N, 0.005 N, and M/iOO HCl 0.001 N, 0.003 N, and m/100 NaOH METHOD: Precautions: 1. All glassware Is to be thoroughly washed and rinsed at least once with glass dis- tilled water to remove all traces of metallic ions. 2. Glass distilled water must be used to make up all solutions, particularly N-medlum. Ordinary distilled water often contains traces of (Jopper which are deleterious. 5- Calcium and all heavy metals must be avoided. (N-medium is calcium- free) . h. Reduce to a minimum the amount of light, carbon dioxide, and bacteria. 5. Keep the isolated geirminal vesicle beneath the surface of the solution at all times to avoid contact with air, air bubbles, surface films, and dust particles. Controls : 1. The standard control is the fixed and stained germinal vesicle. 2. Where environmental variables are used the environment of the nuclear (N) medium is to be considered as the control environment. 5. Where the ~0H ions are used, the +H Ions may be iised for contrast; where an elec- tric current is used, it may be reversed. Procedure: Where possible, use the eggs of some Urodele for in these the germinal vesicles are relatively larger and the chromosomes are the more easily studied than in the eggs of other amphibia. However, since the frog (Anura) is more readily available, the following description will be concerned with the germinal vesicles of Rana pipiens. A. REMOVAL OF THE GERMINAL VESICLE Remove the ovary of a sexually mature and hibernating female frog and place It in Amphibian Ringer's solution in a finger bowl. Wash free any adherent blood. This ovary will remain healthy for about 2k hours at 20°C. or for about h days at 10°C. Cut off a small portion of the ovary (20 to 50 eggs) and transfer to a #2 Stender containing Nuclear Medium. Pour off this solution after a few minutes and replace with * This exercise has been organized with the generous aid of Dr. W. R. Duryee. -171- 172 THE AMPHIBIAN GERMINAL VESICLE fresh N-medium. Cut this piece of ovary Into smaller pieces each containing 3 to U eggs and transfer one such cluster of eggs to a Syracuse diah containing N-medium. DISPERSED OERMIKAl fOLK VESICLE GERHlmi VESICLE •NIHAL POLE FOLLICLE WALL FOLLICLE CELLS- FOLLICLE CELL FOLLICLE WALL. FORCEPS REMOVAL OF THE AMPHIBIAN GERMINAL VESICLE With very sharp forceps and under the low magnification of the microscope^ make a email tear at a point in the follicle sac and egg wall in the region of the animal hemi- sphere but close to the margin of the vegetal material. The germinal vesicle is located in the center of the animal hemisphere. The tear should not be larger than 1/5 the diameter of the egg. The egg substance will Immediately flow out, carrying with it the rather large and spherical germinal vesicle. When working with the smaller, immature oocytes, a needle puncture may suffice to liberate the vesicle. Using a clean medicine dropper, force a gentle flow of N-medlum over the Isolated germinal vesicle to wash away the yolk globules which are adherent to it. This will be- come increasingly difficult with time so that the cleaning procedure should Immediately follow the liberation of the vesicle. The clean germinal vesicle should appear 9s a clear spherical sac without visible contents. The chromosomes cannot be seen at low magnification. Transfer the germinal vesicle to fresh N-medium in another Syracuse dish for further cleaning. The transfer can be made by sucking up a small amount of fluid into the medicine dropper and then drawing up the vesicle, followed by more of the same fluid. The transfer must be made in nuclear fluid to a position beneath the surface of the fluid in the new container. The vesicle must not come into contact with air. Re-examine the vesicle and clean it further if necessary. The yolk actions tend to coagulate the vesicle and must be removed entirely. A medicine drop- per with diameter slightly greater than the diameter of the vesicle can be used to suck the vesicle in and out (gently), the edges of the dropper thus scraping off the adherent yolk. B. EXAMINATION OF THE GERMINAL VESICLE Place a small amount of Permoplast on each of the four corners of a small, square coverslip (a quarter size coverslip is satisfactory). Add equal amounts of Permoplast to each of the comers but only enough to elevate the coverslip a bit more than the diameter of the vesicle. Now transfer the germinal vesicle, in a drop or two of N-medium, to a cleeua slide and gently cover with the coverslip. Add more N-medium from the side of neces- sary. The advantage of a small coverslip is that various reagents may be added readily from the side. OBSERVATIONS AND EXPEBIMENTS: NORMAL APPEARANCE OF THE GERMINAL VESICLE 1. The isolated germinal veaicle should appear exactly as it does in the egg except for a very alight swelling. THE AMPHIBIAN GERMINAL VESICLE 175 GERMINAL VESICAL OVARIAN EGG OF ANURAN S^' *^A* : : » • 'V'^'''? -'^Jfe- '^r* • ..v^^i I X CHROMOSOME CORE IN CENTER: SACS ON SURFACE^ COLLOID GROUND SUBSTANCE •A„ ^T: GERMINAL VESICLE v . j^-' • STUDIES *^ ' (COURTESY OR. W.R. DURYEE) . v^>: '^L*.^^^^' 1^. • . ' '^ *<•- SIDE VIEW VESICLE SACS 8 MEMBRANE SURFACE VIEW OF VESICLE SACS If TEMPORARIA CHROMOSOME IN 0 1 % NACL R PIPIENS CHROMOSOMES IN GERMINAL VESICLE *%:;,fjil OUTWARD FLOW OF SHRINKAGE {70%) "iv^S-^—^ COLLOID UPON TEARING DURING FIXATION OF ■ '■".■" NUCLEAR MEMBRANE : OF T PYRRHOCASTER GERMINAL VESIClE NUCLEAR SUBSTANCE A GEL. nh THE AMPHIBIAN GERMINAL VESICLE 2. The hyaline condition of the ground substance should persist for several hours . 5. The nucleoli and the hyaline chromoaomes should maintain their relative central positions In respect to each other. k. Fixation pictures are distinctly different. To see the chromoaomes within the central chromosome core it will be neceasary to add a drop of 0.1-N NaHPOj; or a small amount of calcium (as in normal Elnger's solution). The chromosomes may then be stained with 1^ Crystal Violet or with Aceto-orceln and studied under high power magnification. With Aceto-orceln, which is a combination fixative and stain, the vesicle is permanently fixed. The germinal vesicles of large, medium, and small ovarian eggs are structurally dif- ferent and should be studied in detail before applying any of the experimental procedures. The smaller eggs are relatively more transparent, due to the lack of yolk or pigment. The general characteristics of the three types are as follows: Large egg veaicle (nucleus) 1. Nuclear membrane has outside sac-like bulges (see photographs). 2. Nucleoli clusters appear in the center. 3. The chromosome frame appears in the center of the ring of nucleoli. This Is a gel structure which gives rise to the first maturation spindle. h. Very small contracted chromoaomes appear on the framework. The diploid num- bers of a few of the common forms are as follows : Bufo (various species) - 22 chromosomes Sana esculanta - 2k Sana plpiens, E. fusca - 26 Eana catesbiana - 28 (26?) Hyla arborea - 214- Triturus (various species) - 2k T. vlrldescena - 22 Deamognathus - 2k Salamandra - 2k Amblystoma tigrlnum - 28 (axolotl 16, 2k, 28, 30) Plethodon - 2k Half or medium- si zed egg veaicle (nucleus) 1. Nuclear aacs small. 2. Nucleoli are next to nuclear wall. 5. Chromosome frame fills the entire nucleus. k. Chromosomes are spread out to their maximum extension, and possess large lateral loops . Small-sized egg vesicle (nucleus) 1. Nuclear aacs barely visible. 2. Nucleoli peripheral. 3- Chromosome frame fills the entire nucleus. '+. Chromosomes are much smaller than in the larger nuclei. B. PERMANENT FIXATION AND STAINING OF THE GERMINAL VESICLE The student is again cautioned about the use of fixatives in the laboratory where living material is also to be kept. Further, fixation artefacts are most readily apparent in germinal vesicle fixation, hence this section should be treated as a further study of the germinal vesicle and its reactions to environmental factors. This in addition to ac- quaintance with methods of providing permanent preparations. A rBther new fixative-stain is recommended ( aee La Cour, 19'+l) In which aceto-orceln Is applied directly to the Isolated vesicle. This stain consists of k'^'fi acetic (glacial) acid and 0.5']^ orcein into which the vesicle la placed for 3O-60 seconds. It is then run up rapidly through the alcohols (in which some of the dye will dissolve out) and into an alcohol-free mounting medium. A stain of 1^ Methyl Green (acidified) acts in much the same manner. Other permanent mounts may be made with Bouln's fixation followed by Mayer's fixation followed by Mayer's haemalum stain. THE AMPHIBIAN GERMINAL VESICLE 175 A study of the action of fixatives on the vesicle Is very profitable. Isolate 20 to 30 full-sized vesicles in N-medlum and, while observing them under low- magnification of the microscope (under elevated coverslips) add singly such fixatives as Bouin's, i^^ formalin, satizrated HgClp in water, etc. Immediate changes in size and consistency of the vesicle should be noted. Since Bouin's contains an acid, follow the Bouln- fixation with some alkaline treatment to attempt to counteract the acid factor in this fixation. A study of the fixed germinal vesicle raises the legitimate question as to how far we may assume that fixed material accurately represents the structures of the living germinal vesicle. Probably the more accurate picture is a composite one, arrived at by the study of both fresh and fixed material. C. IONIC EFFECTS A visible Iso-electric point can be demonstrated passing through the germinal vesicle by the addition, to one side of the coverslip, of a small drop of dilute BCl. If a slow reaction is desired, use O.OOIN-HCI; if a fast reaction is desired, use O.OOJN-HCl. The germinal vesicle is negatively charged. Note the Brownlan movement of granules. The dif- fusion of weak acids through the germinal vesicle gives an effect known as the "Eing Phenomenon". As proteins within the vesicle reach the iso-electric point (I.E. P., here- after) they become insoluble and appear as floccules. The size of the floccule Is relative to the speed of the acid penetration. If penetration is fast, the floccules will be small; if penetration is slow, they will aggregate and be large. Observe the fusion of micelles to build up the so-called "llnln reticulum". The chromosomes will appear and become distinct -only when the solution Immediately aroimd reaches the I.E. P. of the chromosomes. Watch the I.E. P. passing to the center of the germinal vesicle and the subsequent swelling of the outer colloidal area, which be- comes positively charged and reverses Its reaction. >- t- - (O 6 I— ^IWwaI 4^ RESISTANCE COIL in VOLTMETER SWITCH -^ ELECTRODE / REVERSING SWITCH •lujy ELECTRODE NUCLEAR (N) MEDIUM SLIDE BY - PASS SWITCH '^' MILU - AMMETER Sq^^H COVER SUP ELEVATED BY PLASTOCENE ON CORNERS . VESICLE IN N - MEDIUM TAP KEY ELECTRICAL STIMULATING - EQUIPMENT FOR GERMINAL VESICLES 176 THE AMPHIBIAN GERMINAL VESICLE When the germinal vesicle in weak acid clears^ add a similar amount of weak alkali ( NaOH) and the reactions will he reversed. In NaOH alone the germinal vesicle will hurst. If a very dilute haae is used^ this swelling can he compensated hy acid shrinking. The reactions can he made to go hack and forth under experimental control. D. ELECTBICAL CHARGE AND THE EEACTION OF THE GERMINAL VESICLE An electrical set-up has heen devised hy means of which a known current may he sent through the germinal vesicle and then reversed. A hrief description of the apparatus is given helow. The equipment consists of a '+5-volt dry cell, a reversing switch, key, platinum electrodes mounted in glass, with voltmeter and milli-ammeter in the circuit as shown in the accompanying diagram on the preceding page. Study the electrical set-up hefore attempting to use it, making particular note of the various switches. The milll-simmeter and the voltmeter must he out of circuit at all times and are to he used only hy the instructor to check the apparatus. The student should use only the reversing switch and the tap key. The reversing switch will reverse the direc- tion of the current and the tap key will complete the circuit hetween the points of the platinum electrodes providing there intervenes a conducting (salt) medium. Genninal vesicles should he placed on slides in such a manner as to allow a small amount of fluid to flow heyond each side of the Permoplast supported coverslip. The platinum electrode must he immersed in the fluid on each side of the coverslip, and con- sequently on each side of the germinal vesicle. When the ohject is in focus heneath the compound microscope, press the tap key down and hold it as long as you want the current to pass through the solution. To reverse the current simply throw the reversing switch. (See Flndlay: "Practical Chemistry" p. 155 for comparahle set-up.) 1. Sign of Nuclear Charge; Use the germinal vesicle of half-sized eggs in which the chromosomes are relatively large. When the vesicle is in position, press the tap key for two-second contact and ohserve the suhstance of the vesicle piling up on the positive (+) side, indicating a negative (-) charge. Watch the migration of the nuclear suhstance and the effect of the release of the current. 2. Beactions of Chromosomes: If copper (Cu) electrodes are used, which give off hy- drogen ions, sustain the current and note that the chromosomes will pile up at the posi- tive (+) pole while the wall of the germinal vesicle will hurst toward the negative (-) pole, due to the release in that direction of hydroxyl (OH) ions. It would he well to add a deop of phenol red to this solution prior to initiating the current, in order to detect these changes in pH. (It is Important that after each experiment the copper electrodes he thoroiighly cleaned with cotton and acid alcohol, followed hy distilled water, since they will corrode. ) 5. Comhined Acid and Electric Current: Use the set-up as in #2 ahove hut hefore pass- ing the current through the solution containing the germinal vesicle, add to the N-medium, at a point opposite yourself, a small drop of O.OOIN-HCI. This will hring in the hydrogen ion effect at right angles to the direction of the current. As the I.E. P. passes over the germinal vesicle, apply the current in short shocks. A continuous current will produce an Irreversihle coagulum. Movement Inside the vesicle will he toward the anode will he toward the cathode (-). There will he no movement of the I.E. P. THE AMPHIBIAN GERMINAL VESICLE 177 SKETCHES OB PHOTOGRAPBS OF ISOLATED GERMINAL VESICLE SKETCHES OF AMPHIBIAN CHROIOSCMES VTETHIN VESICLE 178 THE AMPHIBIAN GERMINAL VESICLE Rii;i''KKENCES: Brachet, J., 1959 " "Quelques proprieties chlmlquea de la veslcule germlnatlve Isolee." Arch. f. exper. Zellforsch. 22:51+1. Diiryee, W. E., 1938 - "A microdissection study of amphibian chromosomes." Biol. Bull. 75:5^^5. La Coiir, L., 19^1 - "Aceto-orceln: a new stain- fixative for chromosomes." Stain Techno. 16:169. Nehel, B. E., I959 - "Chromosome structure." Bot. Eev. 5:565. Oguma, K. & S. Makino, 1957 - "A new list of the chromosome numhers in Vertehrata." Jour. Fac. Scl. Hakkaido Imp. Univ. Japan. 5:297. Strauh, J., 19^+5 - "Chromosomenstructur." Naturwiss. 51=97. Waddington, G. H. , 1959 - "The physico-chemical structure of the chromosomes and the gene." Am. Nat. 75:500. "Liquid crystals , it is to be noted, are not important for biology and embryology, because they manifest certain properties which can be regarded as analogous to those which living systems manifest (models), but because living systems ac tually are liquid crystals , or, it would be more accurate to say, the par acrys tall ine state undoubtedly exists in liv ing cells . " J. Needham 1936: "Order and Life" "The species is contained in the egg of the hen as completely as in the hen, and the hen's egg differs from the frog's egg as the hen from the frog. "The adult organi zat ion is ident ical in its individual- ity with that of the egg." C. 0. Whitman "Nature is never more perfect than in small things. Pliny ANDROCENESIS * PURPOSE : To study the variations in early development of the embiyo under the influence of the haploid set of chromosomes from the sperm nucleus alone. MATERIAIS : Biological: Recently inseminated eggs of any Amphibian, preferably Triturus or Sana. Technical: Glass needles and needle holder; micro-pipette (O.16 mm. in diameter) with attached rubber tubing; #2 Stenders with covers. METHOD: Precautions: 1. Since the polar bodies are very small and not distinctly colored, it is impor- tant that maxlimim spot-lighting be achieved. The heat of the light must be absorbed, preferably by water-filled Florence flask. The overhead and other lights should be off to reduce extraneous sources of light. 2. The time of oviposition must be known because the egg nucleus is to be removed as it comes to the surface of the egg to give off the second polar body. In Eana pipiens this occurs from I5 to 55 minutes after insemination. 5. A minimum of cytoplasm and yolk is to be removed with the egg nucleus. k. Haploid eggs and embryos are less viable than controls, and must be given special post operative care. Controls : 1. Control eggs should be puntured in a manner identical with the experimentals, but at a point well removed from the position of the maturation spindle of the egg nucleus. The same amount of yolk should be removed. 2 . Some untreated eggs should be allowed to develop to determine whether they are otherwise entirely normal. Procedure: Provide yourself with optimum lighting conditions. This Includes a spot light directed at the eggs from a ^5° angle in order to shine on the upper surface of each egg and to cast a shadow from the first polar body and, by contrast, to reveal the polar body pit. Low power magnification will be adequate after the polar bodies and pits are recognized. It is necessary here to give a brief description of the amphibian egg nucleus at the time of oviposition, and during the few minutes after insemination. The nucleus of the ovarian egg of the hibernating and non-ovulating amphibian is in the germinal vesicle stage, prior to any maturation divisions. This germinal vesicle breakg down at the time of ovulation (liberation from the ovary) so that coelomic eggs show neither a vesicle nor chromosome figures. As the egg enters the oviduct (within 2 hours) the metaphase figure of the first maturation division appears, and as the egg progresses through the upper third of the oviduct it extrudes the first of two polar bodies. The egg nucleus remains near the periphery and about the time the egg reaches the uterus, the metaphase figure of the second maturation division will appear. The egg remains in this condition until it is fertilized (or dies). The procedure described below takes advantage of the peripheral position of the egg nucleus, removing it before it has a chance to fuse with the spemi nucleus entering the egg at another point. REMOVAL OF THE FEMALE NUCLEAR ELEMENTS An ovulating female frog Is secured and concentrated frog sperm suspensions are pre- pared as thin films In h Syracuse dishes. When the optical equipment is adjusted, strip * The author acknowledges, with appreciation, the help of Dr. K. E. Porter In organizing this exercise. -179- 180 ANDKOGENESIS Fertilization of the egg and the removal of the female nucleus (androgenesls) in Trlturus vlrldescens. P^C' I DnmBg of id tgg ki ■howinf two dark ipiinn marki in its trairt tba MroDd malDril Iti upaul* about 12 miautM altn dapMiUOB, nd th* lifbll.T pifDi«ttl«it poUr ara* conUIDlnt on (pindk, markn) b; a small plgai(int*d apal. PifC- i Andra^Dftic cmbrjo 7 daii old (b), lbs diploid coalrol of lb« mem ttft annlrol of tka auw ago (e), and jmiagrr diploid larra (hi, of about Ih* aamr alaff of JoTvlopmMt and from Ih* aamr frmalp a* th« andrograrlic Urra. Tbr dlfffrvncro In the aia* af Ibo irllb aa *rll a* Ihr miw of the flKmtnl rrlU of thr Kaploid lad Ibe two diploid larva* arc tvr; notinabk- Tba banloid larta u «]» ibortrf than bolb diploid lartar. Fig. 1. Drawing of an egg In Its capsule about dark sperm marks and the lightly plgmen the second maturation spindle, marked b Fig. 2. Diagram of the pipette, needle, and an Fig. 3. Androgenetlc embryo 7 days old (b) , the the same female (c) , and a younger dlpl development as the androgenetlc embryo. genetic embryo are smaller than those o Fig. 4. Androgenetlc larva 12 days old (a) , the and younger diploid larva (b) , of about the same female as the androgenetlc lar gills as well as the size of the plgmen lold larvae are very noticeable. The h diploid larvae. 12 minutes after deposition, showing two ted polar area containing In Its center y a small pigmented spot. egg In position for puncturing. diploid control of the seune age and from old embryo (a) , of about the same stage of The head and eye vesicles of the andro- f either one of the two diploid embryos. diploid control of the same age of (c) , the same stage of development and from va. The differences In the size of the t cells of the haploid and the two dip- aplold larva l6 also shorter than both (Kaylor, 195?: Jour. Exp. Zool. 76:375) ANDROGENESIS l8l a few of the egga from the female into one of the Syracuse dishes. At 10 minute Intervals strip additional eggs into other Syracuse dishes, marking the exact time of insemination on each dish. Within 3 to 5 minutes after insemination, flood each dish with Spring Water (or Standard Solution) so that the eggs are completely covered. The other dishes are to be similarly flooded at comparable intervals thereafter. The water on the eggs may be changed to clear it of excess spermatozoa. The nucleus is to be pulled out by means of a glass needle. This needle should be made of soft glass but must have a sharp and rigid point. A large number of needles should be made available with an appropriate needle holder. When the egg is inseminated the first polar body is already located between the egg and the vitelline membrane. This small gray bead representing an extruded nucleus may sometimes be located on the animal pole surface, near its center. One must focus very sharply onto this animal pole surface, and use light coming onto the egg from an angle, in order to see the very small polar body and its shadow. From 7 to 10 minutes after insemination there will appear a small depigmented area near the center of the animal pole, and within this area will develop a pin-point depres- sion. This is caused by a temporary retraction of the surface coating just above the form- ing second maturation spindle. Insert the tip end of a glaea needle Just below this polar body depression at such an angle that it will extend below the spindle and with a very slight withdrawing and upward motion, bring the spindle out with an exudation mass of yolk. This mass will necessarily be somewhat larger than the size of the first polar body, but with practice its size may be reduced and yet include the whole second spindle. Since there are a total of k Syra- cuse dishes of eggs which were inseminated at 10 minute intervals, the second dish will be ready about the time the first dish of eggs has been experimentally treated. ANDROGENESIS IH THE UROCELE EGG The eggs of Triton, Triturxis pyrrhogaster and T. virldescens have been used success- fully in androgenesis. The salamander egg is generally fertilized as it passes through the genital tract of the female where spermatophores are stored for variable periods. Ovulation can be induced by anterior pituitary injection (see section on Induced Breed- ing). Since the eggs are fertilized shortly before they are deposited by the female, it is Important (when using Urodele eggs) to note the exact time of ovlposition of each egg. The removal of the maturation spindle must occur within 50 minutes after ovlposition. Urodele eggs are normally polyspermic and the multiple sperm entrance points can be identified by dark spots caused representing the accumulation of pigment. Toward the center of the animal hemisphere will be seen a clear area, considerably larger than a sperm entrance spot, marking the position of the metaphase spindle. With watchmaker's forceps remove the several layers of jelly but avoid the vitelline membrane. With a wide- mouthed pipette transfer the egg to a Syracuse (operating) dish in which there is a wax depression appropriately molded to fit the egg. Use Urodele Growing Solutior\ or Spring Water as the medium. The Urodele maturation spindle may be removed by the needle method, as described above. Another method, developed by Xaylor (1957), Involves sucking out the nuclear ele- ments with a mlcro-plpette. The egg must be oriented with the animal hemisphere dorsal in position. Then, with a fine glass needle (1 to 2 ji in thickness) rupture the vitelline membrane, but avoid injury to the egg cortex, at several points directly above the posi- tion of the spindle. Attach a micro-pipette to a small bore rubber tubing, the tapered end of the pipette having a diameter of not more than 0.l6 mm. Place the end of the rub- ber tubing in your mouth, hold the pipette firmly in one hand, and with the other hand adjust the low power microscope and the operating dish. Bring the open end of the micro- pipette down directly onto the vitelline membrane just above the region of the spindle and with negative pressure (gentle suction) draw the entire spindle, out of the egg. A 3^32 ANDROGENESIS DWAWTWOR AND PHOTOGRAPHS OF AWDBOGENETIC LABVAE ANDROGENESIS I83 small amount of cytoplasm and yolk will he included, and this must be kept at a minimum. Transfer the operated egg to a covered #2 Stender with fresh Urodele Growing Solution or Spring Water and keep it at a constant temperature in the vicinity of 15° to 18°C. CARE OF MATERIAL: Operated eggs should he examined within several hours to determine whether they are cleaving. Such eggs as seem to he developing, and show operation exudates, should he isolated in #2 Stenders with the appropriate medium and should he kept at the cooler tem- peratures within the normal range. Controls must be kept at the same temperatures and under the same conditions of medium and space. OBSERVATIONS AND TABULATION OF DATA: There are two criteria for successful androgenetic operations. a. Delay in cleavage and early development. The first cleavage may be delsiyed as much as k'^ minutes (at 22°C.) and development through neurulation will tend to be delayed. After hatching, the embryo will manifest those characteristics normally associated with experimentally induced haploidy such as in artificial parthenogenesis. These include stunting, dorso-ventral thickening, dorsal flexion of the head and tail, oedema, reduction of the gills, etc. (see photo- graphs of abnormalities in development and haploid larva in section on Arti- ficial Parthenogenesis). Make drawings of androgenetic embryos and tadpoles. b. Chromosome count which should be haploid. This can be determined in the neuru- la stage either by sectioning and staining the material, or by making cover- glass smears of neural crest cells (see Culturing Isolated Embryonic Cells) and staining them with Harris' haematoxylin. Those tadpoles which survive for 10 days or more can have their tails clipped and examined (see Tall Tip Chromo- some Technique) for chromosome figures, without killing the tadpole. 181* ANDROGENES IS ■ 0 '»♦ mn -.0 J^ ^ <^ POLAR BODY FORMATION: RANA PIPIENS (From Porter, 1959: Biol. Bull. 77:235) Figs. 1-4. Semi-diagrammatic representations of four sta^^es in second polar body forma- tion of R. plpiens eggs. Drawings were made with camera lucida and give ex- act distribution of pigment granules, yolk platelets and cliromosomes, only part of which are shown. Selected from considerable material sectioned at 10 )i. (Eggs inseminated and kept at 12°C.) 1125 X. Fig. 1. Division spindle as in egg at time of insemination. Fig. 2. Anaphase of maturation division. Stage at which spindle can be seen from ex- terior of egg as small black dot. Egg fixed 35 minutes after insemination. Fig. 3. Early telophase. Egg fixed 50 minutes after insemination. Fig. 4. Polar body just forming. Egg fixed 56 minutes after insemination. BEFERENCES : Baitzer, F. & V. de Eoche, I956 - "Uber die Entwlcklungsfahigkelt haploiden Triton al- peatria." Eev. Suisse de Zool. k^-.k'^'^. Eaat, E. M., 19514- - "The nucleus-plasma problem," Am. Nat. 68:289 & k02. Fankhauaer, G. & C. Moore, 19^1 - "Cytological and experimental studies of polyspermy in the newt, Triturus viridescens. II. The "behavior of the sperm nuclei in androgene- tic egga (in the absence of the egg nucleus)." Jour. Morph. 68:387. Kaylor, C. T., 19'*-1 - "Studies in experimental haploidy in salamander larvae. II. Cyto- logical studies on androgenetic eggs of Triturus viridescens." Biol. Bull. 81:14-02. Porter, K. R., I9UI - "Diploid and androgenetic haploid hybridization between two forms of Bana pipiens Schreiber." Biol. Bull. 80:238. "How it chanced that a man who reasoned upon his premises so ably should assume his premises so foolishly is one of the great mysteries of human nature. " MacAul ey to Dr. Johnson "It IS this t hought- transmi t t in^ prepotency of the human species, more than any other, that gives it a superlative lead over all the creatures of the globe." A. J. I.otka 1925 ARTIFICIAL PARTHENOGENESIS* PURPOSE : To repeat the earlier experiments^ using modern methods of inducing ovulation and of equipment, in an attempt to initiate the development of the amphibian egg hy artificial means (i.e., without benefit of spermatozoa). MATERIAIS : Biological: Uterine eggs from an ovulating anuran: Eana or Bufo. Blood fron a second non-ovulating female anuran, same species. Technical: Slides, Petri dishes, finger howls, #2 Stenders, moist chamber, section lifter, sharp-pointed (5 to 9 p) glass or platiniim (20 to JO n) needles, and china marking pencil. METHOD: Precautions: 1. All articles and female frogs must be kept sperm-sterile. The instruments and glassware may be boiled for 5 minutes or immersed in 70^ alcohol and air dried. 2. The female frog which produces the eggs for the experiment should be isolated from all males for several days prior to the experiment and should be washed off with tap water and dried before stripping. Controls : Two types of controls are necessary for this experiment. 1. Some eggs are to remain untreated, but should be placed side-by-side with the eggs experimentally treated. This provides identical environmental conditions for the experlmentals and the controls, with but a single variable. 2. The eggs should be tested to determine whether they are in fertilizable condi- tion. Following the conclusion of the experiment, some of the uterine eggs should be normally inseminated by frog spermatozoa, of the same species, in £Ui- other laboratory. There must be no possible contamination of the experimental eggs with spermatozoa. Procedure: 1. Adjust a low-power microscope so that the heat-absorbed light will strike the eggs from a ^+5° angle from above. A lantern slide cover glass might be used to protect the microscope stage from water. 2. Place 10 clean microscope slides, a slight distance apart, on clean paper towel- ling. On the upper left hand comer of each mark "C" (for controls) and below on the lower left hand corner of each slide mark "X" (for experlmentals), using a china marking pencil. Number the slides in sequence. 3. Strip a single row of eggs from the uteri of an ovulating female, placing them along the length of the slide opposite "C" and then another opposite "X". Try to strip eggs in a single row so that they will not lie over each other, and will adhere to the slide. Place all 10 slides of eggs in a moist chamber where they may remain for an hour or more without deleterious effects. (The chamber should have stood for at least an hour so that the contained air is completely saturated with water vapor.) Such eggs will lose their CC^ and thereby facilitate their physiological maturation (Bataillon &. Tchou-Su, 1930). k . Pith a non-ovulating female frog; lay it on some paper towelling; cut through the leg muscles to prevent further reflex movements; open the abdomen and expose the heart. With the frog on its back cut off the tip end of its ventricle and allow the blood to flow freely into the body cavity, mixing there with the coelomlc fluid. Keep the abdomen closed until ready to use the blood. 5. Remove one slide from the moist chamber. Take a small strip of abdominal muscle from the non-ovulating female, draw it through the mixture of blood and coelomlc fluid, and gently pass it over each of the two rows of eggs. Avoid any pressure on the eggs but see that each egg is provided with a partial coating of blood and coelomlc fluid. * This laboratory procedure has been organized with the very generous help of Dr. C. Parmenter. -l85- 186 ARTIF IC lAL PARTHENOGENES IS Cortical stimulation. Gently but firmly prick each egg with a sharp point of a glass or platinum needle. The puncture should he applied within the animal hemisphere but not in its exact center where the second maturation spindle la likely to be located. Leave the eggs in row "C" untouched as controls. Immediately after pricking the experimental row of eggs, immerse the slide in Spring Water or Standard Solution in which normal development la known to occur. It is best to use Petri dishes and only about 2 cm. depth of medium to cover. Eepeat the above procedure with h other slides from the moist chamber. Follow the above procedure with the remaining 5 slides from the moist chamber but limit the pricking to the vegetal hemisphere of the egg. Mark these slides to Indicate location of stimulation. ************ If time permits, the same procedure should be followed with single variations which might increase the incidence of successful stimulation. Such variables are as follows: h. Allow the eggs to remain within the uterus at IQOC. for 5 days before stripping (Zorzoll and Rugh, 19^1). Such aged eggs must be allowed to come to the labora- tory temperature before stimulation. Keep the female at refrigerator (l4-°C-) temperature, and in the moist chamber provided with ice cubes, to determine whether a lower temperature alone would in- crease the sensitivity of the egg to artificial stimulation. Omit the use of blood or serum (Guyer, 1907; Bataillon, I9II and I919). Vary the depth of cortical Injury, deep or shallow pricking. Allow the eggs to dry (partially) on the slide before pricking. Allow the jelly to swell in water to various degrees, before stimulating. The cortical pricking will be a bit more difficult through swollen jelly. Follow the artificial stimulation of the egg by immersion in media of various osmotic conditions. Determine the role of the presence and the absence of calcium (using oxalates and citrates) in the response to parthenogenetic stimulation. OBSERVATIONS AND TABULATION OF DATA: Record the data from your experiment in tabular form as follows: CONDITION NUMBER & PERCENTAGE Total Controls Total Experimentala Pseudo- cleavages ^ Cleavages ^ Blastula i Gastrula i Animal Pole Vegetal Pole Aged Eggs Frozen Eggs Deep Injury Dried Eggs Eggs with Swollen Jelljr Total experimentala should include all eggs stimulated. The paeudo- cleavages in- clude irregular cleavages and superficial indications of attempts at cleavage. Along with this data, include a statement regarding the exact method of stimulation. Instrument used. ARTIFICIAL PARTHENOGENESIS 187 DRAWINGS OB PHOTOGBAPHS OF PABTHEHOGEHETIC EMBEYOS 188 ARTIFICIAL PARTHENOGENESIS and any variations in the prescribed technique. If, perchance, you achieve an unusually high percentage of cleavages, you will want to be able to repeat the procedure in every detail. There are qualitative aspects of the problem which should be recorded under: 1. Pattern of cleavage when it is not normal. Is the injury point In any way related to the position of the cleavage furrow or the position of the grey crescent? 2. Bate of cleavage. This observation will have value only if the temperature for the experimentals and controls Is identical. 5. Analysis of haploid characteri sties of the tadpoles which develop. These in- clude microcephaly (due to sluggish or Incomplete gastrulatlon) ; dorsal flex- ion of head and tall; increased number of cells per unit area (except the notochord) ; and frequent oedema which Is thought to be due to the malfunction- ing of the excretory system of haplold tadpoles. Oedema alone is not an ade- quate criterion for there are many environmental factors which will cause this condition in normally diploid tadpoles. h. Fix and stain tail tips of parthenogenetlc tadpoles 9 to 10 days old to make chromosome counts. (See under Tail Tip Technique.) Many of the parthenogeneti cally activated eggs will proceed to early stages of development and then cytolyze. It would be instructive to make a rapid preparation of a healthy neurula stage to determine whether the cells are truly haplold, using the method of Tyler (l9'+6). This simply Involves placing the neurula on a coverslip; separating or teasing apart its cells In a minimum amount of culture medium, possibly with the aid of 0.1^ KOH; inverting the coverslip over a second coverslip on which is placed a large drop of Bouin's fixative. The edges of the upper coverslip should cross the corners of the lower coverslip so that they can be separated the more easily after fixation. If the coverslipa are moved over each other slightly, this will separate the cells of the neurula and they will become fixed and moat of them will become attached to one of the coverellps. After 5 minutes, place the paired coverallps in a Syracuse dish of Bouin's fluid and gent- ly tease them apart with needles. Allow the fixative to act another 5 minutes. From this point on the coverslipa may be treated as any mounted cytological preparation, and may be stained for chromosomes. It should be possible to locate some mitotic figures in the neural crest cells which will answer the question relative to ploidy. In general the frog's egg lenda itself admirably to this type of experiment. The results should give from 0^ to 18^ cleavages, with the average about 6^. The eggs which show relatively normal cleavages should be isolated and given apeclal care In the hope that aome may develop into tadpoles. BEFERENCES : Bataillon, E., 1929 - "Analyse de la fecondation par la pathenogenese experlmentale." Arch. f. Ent. mech. 115:711. Dalcq, A., I928 - "Les Bases Physiologlquea de la Fecondation et de la Parthenogenese." Les Problemes Blologlques, Paris. Harvey, E. B. , 19^+0 - "Development of half-eggs of Arbacia punctulata obtained by cen- trlfuging after fertilization, with special reference to parthenogenetlc merogony." Biol, Bull, 78:'+12. Just, E. E,, 1959 - "The Biology of the Cell Surface." Blakiston, Lillle, B. S., 19'*-1 - "Further experiments on artificial parthenogenesis in starfish eggs with a review," Physiol. Zool, l'+:259. Loeb, J., 1921 - "Further observations on the production of parthenogenetlc frogs." Jour. Gen. Physiol. 5:529. Parmenter, C. L., I9U0 - "Chromosome numbers in Sana fusca parthenogeneti cally developed from eggs with known polar body and cleavage histories." Jour. Morph. 66:2l4-l. Pincus, G. & H. Shapiro, I9U0 - "The comparative behavior of mammalian eggs in vivo and in vitro. VII. Further studies on the activation of Babbit eggs." Proc. Am. Phil. Soc. 85:651. Boatand, J., 1958 - "La Parthenogenese des Vertebres," Actualities Sclentifiques er Industrielles. 651:5 (Herman er Ci, Paris). ARTIFICIAL PARTHENOGENESIS 189 Tchou-Su, M., & Chen, Chao-Hsi, 19'+0 - "The technique of Professor Bataillon and some three year old parthenogenetlc frogs." Chinese Jour. Exp. Biol. 1:305. Tyler, A., I9I+I - "Artificial Parthenogenesis." Biol. Bev. l6:291, Tyler, A., 19'+6 - "Hapid slide making method for preparation of eggs, Protozoa, etc." The Collecting Net I9. Zorzoli, A. & B. Bugh, 19*+! - "Parthenogenetic stimulation of aged anuran eggs." Proc. Soc. Exp, Biol. & Med. U7:l66,, PARTHOGENETIC CONTROL RANA PIPIENS "It is deserving of emphasis that the function of imagin- ation IS not merely the conception of mythical creations, bat also, and quite particularly, the presentation, to the mind, of realities. Hence imaginat ion plays an important role in the exact Sc ience s . " A. J. Lotka 1925 "I am unwilling to accept the defeatism of the vitalist, so long as means of investigation by experiment are avail- able. " fi. G. Harrison 19^5: Trans. Conn. Acad. Arts & Sc i . 36:311 PRESSURE EFFECTS ON CLEAVAGE PURPC6E: To determine the effect of altering the yolk- cy top laemlc axis on cleavage and on the subsequent development of the embryo. Specifically, to attempt to shift the third cleavage from the equatorial to the meridional plane by the application of un- equal pressure. MATER lAIB : Biological: Fertilized eggs of any Amphibian. Technical: Petri dishes, glass tubing 2.0 mm. in diameter. METHOD: Precautions : a. Do not crowd the eggs; allow sufficient medium for appropriate aeration. b. Separate and remove the eggs whose cleavage plane has been altered, placing them in #2 Stenders where they may be given special care. Controls : Eggs fertilized at the same time, from the same female, but not subjected to any pressure. Procedure : A. Strip some uterine eggs into a sperm suspension in an Inverted cover of a Petri diah. Gently shake them so that they spread out into a single layer of eggs. Flood with Spring Water or Standard Solution in 5 minutes. Mark the time of insemination on the dish. Ey 2^ hours after insemination these eggs should be in the 2-cell stage, and 1 hour later (Ja hours after insemination) most of them should be in the four cell stage. The first two cleavages are normally vertical (meridional) and generally bisect each other in the center of the animal pole. The third cleav- age is horizontal but slightly above the true equator of the egg, at right angles to both the first and the second cleavages. As soon as most of the eggs are in the l+-cell stage, place the bottom of the same Petri dish over the eggs and, while observing them under low power magnification, add water to the upper dish until pressure is exerted on the eggs to such an extent that they are definitely distorted but not ruptured. The bottom of the Petri dish partially filled with water provides the pressure, and this pressure can be controlled by adding or removing water. The dish also acts as a pseudo-lens so that the eggs bene&th can be observed directly and at all times. The pressure must be maintained from before the initiation of and through the time of the third cleavage of both the experlmentals and controls. B. A second method of applying pressure is to draw up the eggs with their Jelly in- to glass tubing which has a d-lameter slightly less than that of both the egg and its Jelly- This will be about 2.0 mm. for Sana pipiens eggs. The eggs should be drawn up by suction at the l+-cell stage, and observed through the side of the tubing, under water. The eggs will be considerably distorted and sketches should be made while the eggs are under pressure and Immediately thereafter. OBSERVATIONS AND TABULATION OF DATA: The observations here are purely qualitative and a series of sketches or photographs should be made of several eggs whose cleavage planes have been altered. Then the eggs, properly identified with their sketches, should be isolated In #2 Stenders and allowed to develop as far as they will normally. Any variations from the controls should be Indi- cated by parallel sketches of experlmentals and controls. .190- PRESSURE EFFECTS ON CLEAVAGE 191 SKETCBES OF PBESSUBE EFFECTS UPON CLEAVAGE 192 PRESSURE EFFECTS ON CLEAVAGE DISCUSSION; Pfluger (188^) was protably the first to discover that vertically applied pressure will alter the normally horizontal third cleavage plans of the frog's egg and will make it vertical, as are the first two cleavages. This means that in the 8-cell stage, all eight cells extend from the animal to the vegetal pole. Driesch (1892) applied pressure to the cleaving sea-urchin egg in an attempt to alter its cleavage plane. Born (1895) and 0. Hertwig ( l895 ) repeated and refined the work on the frog's egg. None of these investiga- tors left series-sketches to indicate whether the grey crescent was involved, and the ef- fect on suhsequent cleavages. Their concern was primarily with whether succeeding develop- ment would be normal. The first cleavage in the frog's egg occurs about 2^ hours after insemination, the second about 1 hour later and the third about ^ hour after the second. There is an ac- celeration of cleavages. Each of the cleavages is at right angles to the preceding cleavage, and the spindle axis lies in the longest protoplasmic axis (see the laws of Hertwig, Sachs, and Balfour in the Glossary). The third cleavage is normally horizontal (equatorial), and the fourth is again meridional (vertical). If the protoplasmic axis of the blastomeres is shifted at the i^-cell stage, the cleav- age plane will be shifted. If the third cleavage, under pressure, is shifted to the verti- cal, and the egg is then released from pressure, the next cleavage (normally vertical) will tend to be horizontal. Generally eggs which have been subjected to this type of unequal external pressure will survive perfectly well and will develop quite normally providing the pressure is not maintained too long and it does not rupture the surface coating of the egg. Such a shift in cleavage pattern as generally occurs means a shift in the distribution of nuclei. Since abnormal embryos are not generally produced by altering one of the cleavage planes, it must be assumed that there is no qualitative distribution of the nuclear material in these early blastomeres. BEFERENCES: Born, G., l89^ - "Ueber neue Compressionversuche an Froscheiern. " Jahreabericht der Schlesischen Gesellschaft fur waterlandische Cultu. Zool. Bot. Driesch, H. , I892 - "Zur Verlagerung der Blastomeren des Echinodeneies. " Anat. Anz. StJ*^. Hertwig, 0., 1895 - "Ueber den Werth der ersten Furchungszellen fur die Organbildung des Embryo." Arch. Mikr. Anat. 42. Morgan, T. H. , I9IO - "The effects of altering the position of the cleavage planes in eggs with precocious specification." Arch. f. Ent. mech. 29. (See also "Experimental Embryology" 1927.) Pfluger, E., I88U - "Ueber die Einwirkung der Schwerkraft und anderer Bedingungen auf die Rlchtung der Zelltheilung. " Arch. Ges. Physiol. 5U, Roux, W., 1895 - "Gesammelte Abhandlungen uber Entwicklungamechanik der Organisman." Leipzig. THE EFFECT OF CENTRIFUCATION ON DEVELOPMENT PURPOSE : To determine the auaceptibility of various stages from the unfertilized egg to the neurula stage to centrifugal force, and the types of ahnonnalities produced by a shifting of the egg or embryo contents. MATERIAI£ : Biological: Ovulating female frogs (Sana pipiens), sexually mature males (Rana pipiens) and early developmental stages of Amblystoma . Technical: Centrifuge with large tubes, controlled speed, and brake. Nujol. METHOD: ^''recautlons : a. Calculate centrifugal force in terms of gravity, using the formula _, mv where F is the gravitational force; r is the radius from the center of the cen- trifuge to the rotational position of the biological material; m is the total mass in grams; and v is the velocity as determined by 2 tt x revolutions per minute. In general the B.P.M. figure is somewhat greater than the force times gravity. In every Instance, record exactly the value for r and m and the number of revolutions per minute so that computations can be checked if necessary. If the same apparatus is used throughout, the relative values of R.P.M. will be ade- quate. (See Costello, Science, May 2, 19*+?, p. '+?'+ for a criticism of centrl- fugation experiments.) b. Crowding must be limited to the duration of centrifugatlon, azii the controls should be similarly crowded for a corresponding period. As soon as possible after treatment, the eggs or embryos should be given optimum conditions of volume, and temperature . Control: Eggs from the same source, untreated by centrifugatlon but otherwise kept under conditions Identical with the conditions of the experlmentala.- Procedure: A. CONST ITUEtlTS OF THE AMPHIBIAN EGG 1. Apply a very thin coat of albumen-water to several microscope slides. Open a sexually mature female Eana pipiens and excise several ovarian eggs. Place a single egg (within its capsule) on each of the five slides, and rupture it with watchmaker's forceps, allowing the contents to flow freely over the dried albumen- water. With the edge of a coverslip, the egg contents may be spread evenly and thinly over the egg-albumen base. a. Examine one of the egg smears under the microscope before It has dried. Note the yolk granules of various sizes. Can you identify any other formed structures? b. Expose two of the slides (after they have become dried) to formaldehyde or osmic vapors to fix the egg contents, and then stain with an alcoholic solution of Sudan III. This dye Is specific for fat. c. When thoroughly dried, apply the plaamal teat to the other two alides (see section on Chemistry of the Embryo). 2. Dissect the ovaries from a sexually mature female frog and crush them in a mor- tar, (in an ice bath If available). The crushing may be accomplished the batter with a small amount of clean sand. a. To half the egg brel add 10 volumes of cold phosphate buffer (m/200 at pH 7), mix well, and centrifuge for 10 minutes at 5,000 R.P.M. The am- -195- 19*^ EFFECT OF CENTR I F UGAT I ON ON DEVELOPMENT phlbian egg contains fat, translucent protoplasm, heavy yolk, pigment granules, and a germinal vesicle- The pigment will be found at the centri- fugal pole, and the so-called microsome layer vd.ll he found between the fat and the pigment, as a cloudy layer. The translucent protoplasm comprises the middle layer and the centripetal pole will have the whitish, opaque cap of hyaloplasm. With micropipette, remove material from each of these layers and examine Immediately under high magnification of the microscope. (Do not expect to find an Intact germinal vesicle.) b. To the other half of the egg brei add 10 volumes of cold phosphate buffer (M/200 at pH 7), mix well, and centrifuge for 10 minutes at 5,000 B.P.M. Before there has been any opportunity for mixing of the various layers, remove each with micropipette into separate homopathlc vials. Biochemical tests should be applied to these isolated egg constituents, particularly to the microsome layer which can be Identified as the cloudy layer between the fats and pigment. Place the microsome layer in a centrifuge tube £ind centrifuge for 20 minutes at 12,500 E.P.M. (ultracentrifuge) . Note the supernatant fluid and the pellets. To the latter apply the following tests: indophenol- oxidase; peroxidase; -SH; and plasmal. (See section on Biochemistry of the Embryo . ) ANALYSIS OF EGG CONSTITUENTS EFFECT OF CENTR I F UGAT 1 ON ON DEVELOPMENT 195 B. RESISTANCE OF EMBRYONIC STAGES TO CENTR IFUGATION DAMAGE There are two aspects of this study: (A) The ability of various stages to survive centrifugation damage and (B) The variety of ahnormallties produced hy standard centrifu- gation at different stages of development. The stages that are to he used are: Uterine eggs, recently fertilized ("but uncleaved) eggs; hlastulae; and gaatrulae. With the large International Centrifuge the approximate speed to he used should range from about 1500 to 3000 R.P.M., but the data should be recorded in terms of the value times gravity (see formula on preceding page). The dura- tion should be from 1 to 10 minutes, the shorter interval at the higher speeds. (For ex- ample, a speed of 180 times gravity for 10 minutes to 1800 times gravity for 1 minute might be the extremes tested. ) If but one speed and time are used, the lower force for the longer interval is recommended for all stages. It has been suggested (Brachet) that if the eggs are centrifuged immediately after fertilization, the eggs that do not develop fail because all of the ribonucleic acid is carried to one pole, opposite that of the yolk. Centrifugation at later stages (e.g., blastula) may produce trlploid embryos because of the excessive concentration of ribo- nucleic acid in specific areas. The effect of fertilization can be tested very simply by stripping several hundred eggs from an ovulating female Into a concentrated sperm suspension. The female is then to be opened and the uteri tied off above and below, eind removed as a double sack full of eggs. The two uterine sacks may then be separated and placed directly into a centrifuge tube, previously coated (internally) with Nujol (paraffin oil). In a balancing tube, place the fertilized eggs, and centrifuge simultaneously. All eggs will be from the same female and the only difference will be that one group are fertilized. The unfertilized eggs should be fertilized Immediately upon removal from the centrifuge, by cutting open the uteri and stripping the eggs into a concentrated sperm suspension. The later stages of development, such as the blastula and gastrula, are to be cen- trifuged within their Jelly membranes and in the Standard Solution. Eecord the data in the following tables: TABLE I: CENTRIFUGATION. _X GRAVITY # EGGS i DEVELOPED AiTEB CENTBIFUGATION i NOBMAL TYPES OF ABNOEMALITIES Unfertilized 1 Fertilized Blastiila Early Gastrula (Crescent Lip) Late Gastrula (Yolk Plug) 196 EFFECT OF C E NTR I F UGAT I ON ON DEVELOPMENT TABLE II: CENTRIFUGATION_ .X GRAVITY # EGGS ^ DEVTILOPED AFTER CENTEIFUGATION ^ NORMAL TYPES OF ABNORMALITIES Unfertilized Fertilized Blastula Early Gastrula (Crescent Lip) Late Gastrula (Yolk Plug) Some of the types of abnormalities produced "by centrifugation are: depigmentation, permanent blastulae; inhibition of gastrulation, anaxlal and hypaxlal conditions, doubl- ing of embryos, spina bifida, head defects, and accessory appendages. These should be photographed or sketched below, always with a record of the stage and conditions of cen- trifugation. DRAWINGS AND PHOTOGRAPBS OF ABNORMALITIES FOLLOWING CENTEIFUGATION EFFECT OF CENTR I F UGAT I ON ON DEVELOPMENT 197 BEFEBENCES : Banta, A. M. & E. A. Gortner, I915 - "Accessory appendages and other abnormalities pro- duced in amphibian larvae through the action of centrifugal force." Jour. Exp. Zool. I8:li37. Beams, B. W. & B. L. King, I938 - "Pigmentation changes in tadpoles of Bana pipiena following centrifugation during the early gaatrula." Jour. Morph. 6j> -.hTJ . Brachet, J., 19^7 - "Localisation de I'acide ribonucleique et des proteines dans I'avalre de Grenoullle normal et centrifuge." Experientia III/8. Costello, D. P., 1914-0 - "The fertilizability of nucleated and non-nucleated fragments of centrifuged egga." Jour. Morph. 66:99. Barvey, E. N., I95I+ - "The air turbine for high speed centrifuging of biological material, together with some observations on centrifuged eggs." Biol. Bull. 66:i4-8. Beilbrunn, L. V., I927 - "The viscosity of protoplasm." Quart. Bev. Biol. 2:250. Jeener, E., 1914-6 - "Essai de fractionnement dea proteines du noyau cellulalre par ultra- centrlfugation." Compt. rendu. Soc. Biol. Ili0:1103. Jenlcinson, J. W., I915 - "On the relation between the structure and the development of the centrifuged egg of the frog." Quart. Jour. Micr. Sci. 60:6l. Kostoff, D,, 1937 - "Chromosome alterations by centrifuging." Science. 86:101. Luyet, B. J., 1955 - "Behavior of the spindle fibres in centrifuged cells." Proc. Soc. Exp. Biol. & Med. 55:l65. Pasteels, J., 191+0 - "Eecherches aur lea facteura initiaux de la morphogenese chez lea amphibiena anoures. IV. Centrifugation axiale de I'oeuf feconde et Insegmente." Arch, de Biol. 51:535- Schechtman, A. M., 1957 - "Mechanism of anomaly induction in frogs egga by neana of the centrifuge." Proc. Soc. Exp. Biol. & Med. 57- Tchou-Su, M., 1956 - "Embryona double obtenua par la centrifugation d'oeufs d'Anoures recemment fecondes. Origine des localisations germinales." Comp. rendu. Soc. Biol. 2lt:10ll5. Whi taker, D. M., I957 - "Determination of polarity by centrifuging eggs of Ricua furcatua." Biol. Bull. 75:21+9. "An axiate pattern, a three -dimens ional co-ordinate system, a symmetrical plan, exists in all eggs and embryos, made up of poles and gradient s between poles, recogni zable by means of quantitative differences along them." Child "If arithmetic, mensuration, and weighing be taken away from any art, that which remains will not be much. Plato "Theory without fact is fantasy , but fact without theory is chaos. Divorced, both are useless: united, they are equally essential and fruitful. C. 0. Whitmao THE PRODUCTION OF DOUBLE EMBRYOS A. THE EXPERIMENT: DOUBLE EMBRYOS BY INVERSION PURPOSE : To produce double monsters ty Inverting the egg In a gravitational field at the two-cell stage, shifting the egg deutoplaam and thereby affecting subsequent gastrula- tlon. MATERIALS : Biological: Ovulating Rana pipiens and adult males of the same species; Urodele eggs In the 2-Gell stage. Technical: Standard equipment. METHOD: Precautions: a. Avoid excess handling if eggs and embryos. b. Avoid desiccation of eggs, crowding, and heat. c. Practice adhering eggs to filter paper and glazed paper before experimenting. Controls : Eggs from the same source as the experl mentals, in the same stage of develop- ment, adhered to the same kind of paper and in the same manner but without suffici- ent tension to prevent rotation of the egg within its membranes. These controls may be inverted along with the experimentals, but they must be able to rotate within their membranes. Experimental Procedure: Ovulate a female Rana pipiens and fertilize the eggs about 2 hours before the time of the experiment. Cut some clean paper (both filter paper and white, smooth paper) into 1 inch squares and practice adhering eggs with their Jelly capsules to this paper. Place one egg only on each piece of paper. The egg is transferred to the square of paper with a minimum of water. Then, using a scalpel, spread the egg Jelly down onto the paper in all directions in such a manner that the drying Jelly will hold the egg firmly to the paper. Allow the Jelly to dry slightly, in air. Test by inverting the paper and the attached egg over a finger bowl of culture medium for 2 minutes and then re-examine to determine whether the egg has rotated or has been held firmly in the inverted position. Remember that there must be some tension to hold the egg sufficiently to prevent rotation. Prepare several finger bowls of culture medium and quickly adhere 2-cell stages to the single pieces of paper in the manner described. In all cases orient the egg so that the animal pole is uppermost. After making certain that there Is sufficient tension to prevent rotation. Invert the paper, with adherent egg, in the finger bowl of culture medium and leave it undisturbed through at least the two subsequent cleavage as determined by parallel-developing control eggs. If eggs are mounted separately they may be examined briefly after the completion of the second cleavage, and those which do not remain inverted should be so marked or discarded. The pieces of paper float and the eggs are adequately submerged in the medium. After the 8-cell stages has been achieved by the controls (about h hours after insemination) carefully remove all of the experimental eggs from their paper squares and place them separately in #2 Stenders. If particular eggs did not remain in- verted, or were distorted by the Jelly-tension, It would be well to make a sketch record In order to have a possible explanation of later developmental monstrosities. The original method of placing the eggs between glass plates (glass slides) and compressing them sufficiently to prevent rotation when the plates are inverted, cein be attempted. The objection to this method is simply that the pressure factor is not uniform and should be taken into consideration. -198- PRODUCTION OF DOUBLE EMBRYOS I99 The first cleavage normally occurs 2^ hours after the eggs are inseminated, and the second cleavage follows within 1 hour. It is important that the eggs be inverted immediately after the congiletion of the first cleavage and not later. It would be well, therefore, to segregate eggs inverted at various stages of the first cleavage development to determine the effect of this variable on double monster pro- duction. DRAWINGS OF DOUBLE EMBRYOS PBODUCED BY INVERSION 200 PRODUCTION OF DOUBLE EMBRYOS B. THE EXPERIMENT: DOUBLE EMBRYOS BY CONSTRICTION PURPOSE: To determine the ability of single blastomeres of the 2-call stage to develop complete embryos, follovlng accentuation of the first cleavage furrow. MATER IAI£: Biolof^ical: Urodele eggs in the 2-cell stage. Amblystoma eggs may be collected in nature (see section on Breeding Habits) or Triturus egga may be layed in the laboratory as a result of anterior pituitary stimulation (see Induced Ovulation) . Technical: Standard Solution for Anura and Growing Medium for Urodela. 0.1^ KOH in appropriate medium. Hair loops, silk fibers, operating glass needles. METHOD: Precautions: 1. Avoid over-exposure to the KOH solution (see section on Isolation of Embryonic Cells). 2. After separating the blastomeres, keep specimen in adequate medium and at a cool temperature. Controls : These will consist simply of eggs from the same clutch, kept under identical conditions except for the separation of the blastomeres. Procedure: Prepare simple loops of fine (blonde) baby's hair so that each loop is slightly greater than the diameter of the egg and its jelly capsule. Prepare several Syracuse dishes with Permoplast base and depressions calculated to hold the 2-cell stage and its Jelly capsule. Fill with appropriate medium and then select eggs in the two cell stage for constriction. :^y placing these eggs for a brief period in 0.1^ KOH (made up in the same cul- ture medli i) the surface coat will be weakened and the cleavage furrow will be ac- centuated. Eemove the egg and pass it through three changes of culture medium before the furrow has progressed very far. This should be practiced, for it may not be easy to stop the KOH action as abruptly as desired. Constriction of an amplilDiaii egg, wiDiin Its jelly capsule, at the beginning of the 2-cell stage, by means of a hair loop. Wlien blas- tomeres are separated, two embryos develop; when the furrow is merely deepened, double embryos result. Remove the 2-cell stage to the Syracuse dish with Permoplast depression and press the hair loop into the bottom of the depression (two ends above the depres- sion) and maneuver the egg into the depression and loop so that the cleavage furrow lies directly parallel to the loop. With practice one can determine whether it is beet to anchor one end of the loop in the nearly Permoplast, leaving but a single loose end to tighten as the egg is held In position by forceps. It may also help to build up the Permoplast about the egg to hold it the better, '/^en secure, use watchmaker's forceps and tighten the loop so that it constricts the 2-cell stage be- tween the blastomeres, through the Jelly capsule and all. It may be necessary to PRODUCTION OF DOUBLE EMBRYOS 201 re-orlent the egg after the hair loop has attained a grip on the capsule. The blastomerea can he separated without rupturing the fertilization (vitelline) mem- brane . The loop should hold as a result of friction and the egg can he removed to an appropriate #2 Stender for continued development and observation. However, very- fine hair or individual fibers of silk can sometimes be looped twice, resulting in ever better (friction) holding. The degree of constriction can be controlled with practice. A second method but one which aims at complete separation of the blastomeres is to remove the jelly capsule and separate the blastomeres (through the fertilization membrane) by means of a cutting movement of the side of a glass needle. This can be done without rupture of the membrane. Controls for this consist simply of eggs deprived of their Jelly. OBSERVATIONS AMD TAHJLATION OF DATA: 1. Make sketches of any changes in the superficial pigmentation of the inverted eggs, as compared with the controls. Determine whether these changes are carried over to the period of gastrulation. Sketch at periodic intervals. 2. During neuLulation it should be possible to select those embryos which will, in all probability, develop into double monsters. Keep accurate and periodic records of developmental changes in specific eggs of this category. It is most Important that any sequence of sketches represent the changes in a single egg. BEFfeRKNCES : Hadorn, E., 1957 " "Die entwicklungsphysiologische Auswirkung der disharmonischen Kern- Plasma-Kombination beim Bastard-Merogon Triton palmatus x T. cristatus." Arch. f. Ent. mech. 156:14-00. Harvey, E. B. , 19i4-0 - "A new method of producing twins, triplets, and quadruplets in Arbacia punctulata, and their development." Biol. Bull. 78:202. Hinrichs, M. A. & I. J. Genther, 1951 - "Ultra-violet radiation and the production of twins and double monsters." Physiol. Zool. l+:'+6l. Lynn, W. G. , I958 - "Conjoined twins and triplets in trout." Anat. Bee. 70:597- Mangold, 0., I92I - "Situs inversus bel Triton." Arch. f. Ent. mech. 1*8:505. Pasteels, J., 1955 - "Eecherches aur les facteurs Inltlaux de la morphogenese chez lea amphibienes anoures. I. Kesultates de 1' experience de Schltze et leur interpreta- tion." Arch. Biol. !+9:629. Penners, A. & W. Schleip, I928 - "Die Entwlcklung des Schultzeschen Doppelbildungen aus dem Ei von Eana fusca." Zeit. f. Wiss, 150:506. (Also ibid. 151:1.) Schwind, J. L., 19l*2 - "Spontaneous twinning in the Amphibia." Am. Jour. Anat. 71:117' Spemann, H. & H. Falkanberg, I9I9 - "Ueber asymmetrische Entwlcklung und Situs Inversus viscerum bel Zwillingen und Doppelbilungen. " Arch. f. Ent. mech. 14-5:571. Weasel, E;, I926 - "Experimentell erzeugte duplicltas cruclata bei Triton." Arch. f. Ent. mech. 107:1+81, TAIL HEAD CHIMEflA - fusion of anterior ends of Raiia plpiens embryos. HEAD 202 PRODUCTION OF DOUBLE EMBRYOS DRAWINGS OF DOUBLE EMBBYOS PBODUCED BY INVEBSION BEHAVIOR OF ISOLATED EMBRYONIC CELLS* PUBPOSE : To determine the structure and the hehavior of embryonic cells isolated from each other, with particular emphasis on their motility, adhesiveness, phagocytosis, and differentiation. MATEBIAI5 : Biological: Urodele emhryos from cleavage to neurula. used hut are not as satisfactory. Anuran eggs and embryos can be Technical: Operating needles, slides, coversllps, depression slides. Carbon and carmine particles, finely divided. Nile blue sulphate: l/750,000 in Standard Solution. Solutions : Standard Solution - both hypo- and hypertonic concentrations. Standard Solution pl'ia 1^ KOH, freshly made up and adjusted to pH. 9.0 - 11.0. Standard Solution made up vrithout the CaClg (Ca-free Standard). Potassium oxalate (O.^i-^) and sodium citrate (0.1^^), used to oppose the solidifying action of calcium. KCN: m/1+O to m/614-0 made up in Standard Solution. METHOD: Precautions: Use reasonably aseptic conditions, particularly in the differentiation observations. Sterilization is not generally necessary. Controls : None are possible in this type of qualitative experiment. Procedure: MOTILITY Remove the bulk of the Jelly from a blastula or gastrula stage, leaving the fer- tilization (vitelline) membrane Intact. Place the embryo In If) KOH in Standard Solution and observe continually under the low power magnification. When the cellular mass has become disarranged, remove it (within the membrane) to fresh Standard Solution (without KOH). After a few minutes change again to fresh Stand- ard Solution. Now rupture the fertilization membrane with sharp watchmaker's forceps. This will liberate the cells which may then be picked up with a fine pipette and transferred to a microscopic slide for examination beneath a cover- slip elevated by two hairs or glass slivers. Study under both low and high mag- nification and note internal Brownian movement, pseudopodial formation, emd general activity. (Compare with accompanying figures from Holtfreter's paper.) Stain an entire embryo at any early stage, using Nile blue sulphate. Allow the embryo to remain in the vital dye until its surface is distinctly blue in color. Now follow liirections under "a" above. The peripherally exposed parts of cells will be stained the more heavily and motility can be studied in relation to the original polarity or axis of the cell. c. Repeat either or "b" but place the disarranged cells in calcium-free Stand- ard Solution and note the effect on amoeboid movement as well as cellular aggre- gation. d. Cells may be separated from each other mechanically, with fine glass needles. This should be attempted, particularly with the later (neurula) stages where the germ layers can be distinguished. * The author acknowledges with pleasure the suggestions made by Dr. Hbltfreter in organiz- ing this exercise. -203- 201+ BEHAVIOR OF ISOLATED CELLS Fig. 1. Isolated amp)ilbian gastrula cell. Fig. 2. Ectoplasmic movements in isolated KmDryoiiic Cell. Rotating movements in the absence of (Fig. 3), and in the presence of endoplasmic sol-gel formation (Fig. 4) . Fig. 5. Fission of a cell Into unequal halves. Fig. 6. Unfertilized frog egg budding off spherical frag- ments. Holtfreter, I9U6: Jour. Morphology. 79:27. BEHAVIOR OF ISOLATED CELLS 205 e. Separate the cells of a neurula within its external membranes by means of the KOH Standard Solution, This should require from 10 to 60 minutes. When the cells are fully separated, return the neurula to Standard Solution and ohserve at inter- vals over a period of 2 or 5 days. Frequently there will be complete re-organl- zatlon of the neurula and development will be normal. f . Other solutions to be tested against neurulae to determine their ability to sepa- rate cells in a manner similar to KOH. Such solutions as K oxalate, Na citrate, Ca-free Standard, and m/61+ KCN might be tried. FRAGMENTATION Isolated embryonic cells can be caused to fragment or pinch off knobs of protoplasm or form blister-like protrusions by a variety of means. a. Observe an amoeboid embryonic neural plate cell which shows a passing wave of constriction along its main axis. Gently handle this cell with a glass needle and often the wave-like constriction will cut the cell into two. b. Chemical fragmentation of cells may be accomplished by means of hypertonic (standard) solutions; alkaline media; pure sodium chloride solutions (isotonic); and a variety of agents such as cysteine and alloxan. Such fragments should be returned to Standard Solution and observed for the duration of activity, which may be as long as 7 days. The best results will be achieved by treating the cells with KOH in Standard Solution where the pH is raised to 10 or 11. ADHESIVENESS Embryonic cells are most adhesive immediately after their Isolation or separation from each other. This adhesiveness is gradually lost even in Standard Solution. (See the Glossary under such terms as cytotaxis, cytolisthesis, cytotropism. ) The developmental stage of the cell and its histological type will also affect the degree of adhesiveness. Following the above procedure of isolating embryonic cells, place the isolated cells in each of the following media to determine the effect of the medium on the tendency of cells to stick together. a. Calcium- rich Standard Solution. b. Calcium- free Standard Solution. c. 105^ Standard Solution (hypotonic). d. Alkaline Standard Solution with pH above 9* 6. e. Neutral Standard Solution with pH at 7.0 to 9-0. PHAGOCYTOSIS This observation is rather difficult, but can be observed if the student has abundant patience and can concentrate on endoderm, mesenchyme, endothelial cells and neuroblasts. The cells of a neurula should be isolated with 0.1^ KOH in Standard Solution and then transferred to fresh Standard Solution (without KOH) to which some carbon or carmine par- ticles have been added. Occasionally one will see the amoeboid-type of ingestion of the foreign particles, a process similar to normal phagocytosis. DIFFERENTIATION The neurula-stage isolated embryonic cells in Standard Solution may be placed in a culture dish or a depression slide and sealed with a rim of vaseline around the cover or coversllp. All conditions must be aseptic. The cells will often survive from 2 to 7 days and many will differentiate. 206 BEHAV lOR OF ISOLATED CELLS / HIGRATIOH ccr:; S ^<:^ c d MIGRATION AHO AOHESiON 10 ATTRACTION WITHOUT ADHESION Q"& PHAOOCnoSIS Fig. 7. Migrating vermiform cells, Isolated from the medullary plate, failing to aggregate. Fig. 8. Cells from the medullary plate becoming adhesive to each other while chang- ing from a cylindrical into a spherical shape, the Intervals between each picture being about 5 minutes. Fig. 9. Successive phases of kinetic relations between a sessile and a migrating neuroblast. Fig. 10. Three ectoplasmic cell fragments exhibiting reciprocal attraction, but no adhesion. Fig. 11. Ectodermal cells in tlie process of aggregating, tlie wliole process taking about 20 minutes. Fig. 12. Aggregations comprlsln;^ various numbers of cells, some of whicli are at the seune time spreading on glass. Fig. 13. Spreading embryonic cell containing 2 particles of carbon. Fig. 14. Cylindrical cell having Ingested a drop of paraffin oil. Fig. 15. Neuroblast attempting but failing to incorporate a droplet of paraffin oil. Fig. Ifi. Neuroblasts leaving an embryo wliicli lias been exposed to a hypertonic salt solution. Holtfreter, 19l^7: Jour. Morphology. 80:57. BEHAVIOR OF ISOLATED CELLS 20? DRAWINGS AND PHOTOaEAPHS OF ISOLATED CELLS 208 BEHAVIOR OF ISOLATED CELLS If one can select a mesectoderm cell for particular atudy, the differentiation ia most graphic. These mesectoderm cells can he isolated from the closed neural tube stage .by temporary immersion in hypertonic solutions. (See figures from Hbltfreter's paper.) The chemical Isolation of neurula-stage cells may require several hours of exposure. The Isolation of blastula stage cells takes from 3 to 5 minutes. A rapid cytological examination of normal but Isolated blastula, gastrula, or neurula cells and of isolated cells of the neurula which have differentiated, can be achieved by the technical procedure recommended by lyier (l9'*-6). The isolated cells are placed on the center of a coverslip and inverted (in the hanging drop) over another coverelip on which there is a drop of Bouin's fixative. If the edges of the upper coverslip are placed across the corners of the lower coverslip, they can later be separated the more easily. The Bouin's fixative should be allowed to act for 5 minutes, and then the coverslips are to- gether immersed In a Syracuse dish of Bouin's fixative and the upper coverslip is gently separated from the lower one by means of a needle. Allow the Bouin's fixative to act on the cell smears for another 5 minutes, then transfer to ]Cf^ alcohol in a Columbia staining dish made for coverslips. From this point on the usual cytological procedures can be fol- lowed, staining the smears with Feulgen for thymonucleic acid; Harris' haematoxylln for gross chromosome structure; pyronln for ribosnucleic acid, etc. DISCUSSION: This exercise has been organized from a series of investigations by Hbltfreter (l9'*-5- I9I4.7) which represent a new approach to the problems relating to morphogenetic movements. Holtfreter has shown that up to a certain stage, any Isolated cell of the embryo is ready to unite with any other similar cell, provided the cells face each other with their un- coated surfaces. Such isolated cells show an inherent tendency to movement due to the autonomous activity of the cell membrane and not to any activity of the endoplasmic core. There are wave-like contractions of the plasmalemma and an internal shifting of a clear fluid mass which often results in the formation of lobopodia. Aggregation of cells re- sults in the reduction of exposed surface tension. The general cytology of the amphibian cell is remarkably like that of the Amoeba. There are four major parts: 1. Central core of seml-llquid endoplasm (plaamosol) which contains the nucleus, yolk, lipo-proteln granules, melanin granules, and cytoplasmic ground sub- stance. 2. Caps\LLar wall of endoplasm, the plasmogel. 5. Outer shell of fluid ectoplasm which contains smaller particles. This is generally mlscible with water, and is rather thick. h. Thin refractive surface membrane, the plasmalemma, which forma irregular sur- face bulges. This is semi-solid. Movements are initiated and executed by forces localized in this layer or membrane but "they may be associated with local solatlon and re-gelation of that portion of the endoplasm which under- lies a fully developed ectoplasmic bulge." (Holtfreter) The adhesiveness of isolated embryonic cells is associated with the fluid environment, the developmental stage of the cells, and the cytological type of cell involved. Cells in isolation tend to lose their adhesiveness, and the hyaline bulges of the moving cells are less adhesive. Adhesion is definitely toward other cells rather than toward the sub- stratum such as glass. Cytolizlng cells become non-adhesive and are generally expelled from an aggregation of cells. Any living cell which exhibits amoeboid movement, forming lobopodia, would be expected to phagocytize particles from the environment. Some of these embryonic cells are more ef- ficient than others, the difference being the more apparent in cells from the neurula stage. Holtfreter has been able to keep isolated embryonic cells of the Amphibia alive and active for weeks. There is around each cell an elastic surface coat whose strength in- creases during development (differentiation) and whose existence is important in the be- havior, survival, and differentiation of that cell. As long as this surface membrane is Intact the cell is protected. BEHAVIOR OF ISOLATED CELLS 209 BEFERENCES : Butschli, 0.^ 1892 - "Untersuchungen uter mlkroscopische Schaume und daa Protoplasma. " Leipzig. Chambers, E., 19*^-3 - "Electrolytic solutions compatiTDle with the maintenance of proto- plasmic structure." Biol. Symp. 10:91. Conklin, E. G., 1955 - "Development of isolated and partially separated hlastomeres of Amphioxus." Jour. Exp. Zool. 64:505. Holtfreter, J., 19^5 - "Properties and functions of the surface coat in amphibian embryos." Jour. Morph. 95:251. Holtfreter, J., 19^6 - "Structure, motility, and locomotion in isolated embryonic amphibi- an cells." Jour. Morph. 79=27. Holtfreter, J., l^k^ - "Observations on the migration, aggregation, and phagocytosis of embryonic cells." Jour. Morph. 80:25 and 57- Holtfreter, J., ISkf - "Changes of structure and the kinetics of differentiating embryonic cells." Jour. Morph. 80:57. Lewis, W. H., 1959 - "Some contributions of tissue culture of development and growth." Growth Symposium. 1959- Eoux, W., 1894 - "Uber das Cytotropismua der Furchungszellen des Grasf roaches (Eand fusca)." Arch. f. Ent. mech. I:l6l. Selfriz, W,, 1914-5 - "The physical properties of protoplasm." Ann. Eev. Physiol. 7:55' Tyler, A., I9I+6 - "Bapld slide making method for preparation of eggs. Protozoa, etc." The Collecting Net 19 . (See also exercise on Culture of Isolated Anlagen.) "The stab ilizat ion of our institutions rests ultimate ly upon our ability to know and to test assumptions, and upon willingness to revise them without par t i zanship , or bitter- ness, or distress. " Simpson: Am. Math. Monthly 1921 "Man is the only animal who in any considerable measure bequeathed to his descendants the accumulated wisdom of past genera t ions . " A. J. Lotka 1925 "Truth comes out of error more readily than out of confusion . Bacon THE ORGANIZER AND EARLY AMPHIBIAN DEVELOPMENT FUBPOSE: To test the organizing potencies of the dorsal lip of the blastopore when trans- planted, or when Introduced into the hlastocoel of another embryo. Also, to teat the Inductive capacities of other regions, and other (non-living) substances. MATEBIALS : Biological: Urodele* gaatrula, stage #10. Technical: Syracuse dishes with agar bases and standard operating equipment. METHOD: Precautions: a. Blastula and early gaatrula stagea are very delicate and must be handled with extreme caution. A soft agar base is best. Avoid shaking or Jarring after the membranes have been removed. b. The membranes may be removed in lOjd Standard Solution, or even more hypotonic media, but the operation is to be performed in full-strength Standard Solution. After the wound has healed and the transplant haa become incorporated in the host, gradually return the Urodele embryo to the Urodele Growing Medium. c. Use sterile instruments throughout. Control : The control for dorsal-lip transplantation or implantation is the use of a comparable sized piece of tissue from the same donor, but from a region other than that of the dorsal lip. Procedure : The student should be thoroughly acquainted with the morphology and with the morphogenetic movements of the late blastula and the early gastrula before attempt- ing these delicate operations. To do this it will be necessary to dissect the vari- ous stages, to locate and identify the blastocoel, the early gastrocoel, and the surrounding yolk and cell layers. It will further familiarize the student with the conditions of the early gastrula if he removes the membranes and hardens some of the specimens in 10^ formalin for 2l4- hours and then dissects some and cuts others in sagittal and other planes. (Avoid contamination of usual dissecting instruments with fixative.) (See the section on "Morphogenetic Movements and Vital Staining.") The Jelly and vitelline membranes are to be removed from the early gastrula while it is in 10^ Standard Solution over the agar base in a Syracuse dish. Do not use as hosts any embryos which have been injured unintentionally. Place the denuded embryo in a depression in the agar, and orient it with a hair loop in anticipation of one of the following operational procedures. (Remember that the embryo is alive and that during delayed preparations the embryo may progress from stage #10 to stage #11.) A. INJURY EFFECTS AND REGENERATION OF THE DOBSAL LIP With sharp pointed glass needles and a hair loop, remove a rectangular group of cells from the mid-dorsal region. Just anterior to the dorsal lip. Vary the size and the shape of the exciaions in different gastrulae, but sketch each embryo Immediately after the operation and put it aside (carefully) in a separate #2 Stender with sterile medium and allow it to regenerate. (See section on "Wound Healing") If there is Incomplete regenera- tion there should be incomplete or abnormal induction of parts of the medullary plate, determined within about 5 days at laboratory temperatures. (If there are abundant embryos, remove sections of the lateral marginal zone and observe for regeneration and effect on nexurulation. ) The Anuran embryos are not as satisfactory as the Urodele embryos for these experiments. -210- THE ORGANIZER 211 PRESUMPTIVE MEDULLARY PLATE DORSAL LIP BLASTOPORE ANURA STAGE I I SHOWING REGIONS TO BE EXCISED OR TRANSPLANTED ORGANIZING IMPLANT HOLTFRETERS SOLUTION BLASTOCOELE VENTRAL LIP GASTRULAR SLIT DORSAL LIP STAGE 10 ORGANIZING IMPLANT VENTRAL LIP HOLTFRETERS' SOLUTION BLASTOCOELE DORSAL UP NOTOCHORD MEDULLARY PLATE STAGE 12 212 THE ORGANIZER RECOED AND DRAWINGS OF DOBSAL LIP OPERATIONS THE ORGANIZER 213 B. IMPLAJfTATION OF TEE DORSAL LIP MATERIAL Having 1)80011)6 acquainted with the size, location, and extent of the hlastocoel of the Urodele, the student will now attempt to place a dorsal-lip (from stage #11) within the hlastocoel of an otherwise complete blastula of ahout stage #7 or #8. This can be accomplished best by making the transfer in a small-bore pipette (see figures) and allow- ing gravity to carry the cells through the roof of the blastula into the blastocoel. The terminal bore of the pipette should be Just large enough to hold the group of dorsal lip cells to be implanted. Spemann's pipette with a side hole covered with a thin rubber tubing, with pressure controlled by gentle thumb pressure over the covered hole, has proven to be very satisfactory. The cells tend to fall apart and the "organizer" region becomes highly disorganized when the implantation is attempted with forceps or needles. It must be remembered that the blastocoel is filled with a fluid and that any pres- sure exerted on the fluid or contents of the pipette will tend to "blow up" the entire blastula. When the donor cell area has been excised, suck up a small amount of medium into the transfer pipette, then pick up the dorsal lip cells, and before the transfer is made (under water at all times) it will be noted that the dorsal lip is pulled by gravity to the tip of the pipette. It will therefore be necessary only to penetrate the roof of the blastocoel and the cells to be implanted will drop in. Slowly and carefully withdraw the pipette, aided (if necessary) by a hair loop. Allow the wound to heal and then do not disturb for 3 days or more. If the student becomes proficient in the above, it is suggested that he coagulate several gastrulae in hot water, excise the dorsal lip and make a similar In^jlantation to determine the relative "organizer" and "inductor" e'ffects of the dorsal lip areas. C. KXPLANTATION OF TEE DOBSAL-LIP MATERIAL When the belly ectoderm of the neurula (stage #15) is peeled off as a sheet of cells, it will normally round up in the form of a tube. It is possible to take advantage of this fact by prior excision of the dorsal-lip material and placing it on the Inside of such a sheet of cells so that the "organizer" will become wrapped up within indifferent ectoderm. The whole may then be treated as the above operated gastrulae and observed for inductions during 5 to 1+ days. D. TRANSPLANTATION OF THE DORSAL- LIP Select two early gastrulae (stage #10) and place in Syracuse operating dish over agar and in 10^ Standard Solution. After removing the membranes, select the best specimen to be the host. From the prospective host remove a small rectangular piece of ectoderm from the presumptive flank or belly region. From the donor quickly excise a similarly sized pi«ce including the dorsal-lip, and transfer it on the point of a needle, \mder water, to the wound on the host. This is a difficult procedure because the host must be oriented and kept In position within the agar depression, and also because mitosis is so rapid, the cells are so large, and cell movements are so extensive that the transplants are often pushed out of the wound before they have a chance of becoming adherent. It may be neces- sary to use a glass bridge or Briicke to hold the transplant in position for 30 to '+5 minutes during the healing process. Such a cover cannot be used longer because it interferes with respirations. Observe the healing process and re-examine during 5 days. E. TRANSPLANTATION TO THE DORSAL- LIP REGION As in "D" above, select two embryos at stage #10 and remove the membranes. The transplantation is to be made from the presumptive flank region of the donor to a position Just anterior to the dorsal lip of the host. Since the dorsal lip cells move rapidly it Is necesaaiy to: a. Make the excision from the donor first. b. Make the host wound with the donor material nearby, and complete the transfer as quickly as possible. The host wound must be sufficiently anterior to the forming dorsal-lip so that the transplant will "take" well before it reaches the level of involution. 211^ THE ORGANIZER RECORD OF TRAMSPLAMTATIONS THE ORGANIZER 215 A variation on this procedure is reconmiended for those students who prove to he pro- ficient in the first part. The donor may he previously stained with Nile hlue sulphate (1 part in 500,000) so that the transplant will he identifiahle. After it has become at- tached to its new location (i.e., the dorsal lip region of the host) for ahout k hours, remove it (without bothering to protect the host), brush away all host cells with a hair loop, and then implant the stained cells into the blastocoel of another embryo at stage #7- Examine during 3 days for evidence of "organizer" activity acquired by the usually Indifferent flank ectoderm temporarily transplanted and located in the dorsal lip environ- ment. F. INDUCTIVE CAPACITY OF THE NOTOCHOBD OB ABCHEHTERIC ROOF Dissect living embryos at stages #15 and #1^+ to locate the notochordal tissue direct- ly ventral to the neural folds. Bemove, and clean strips of notochord by means of a hair loop and watchmaker's forceps. Such notochordal tissue may be Implanted into the blas- tocoel ("B") or explanted ("C") to determine organizer or inductive capacity. The notochord is derived from cells involuting over the dorsal-lip and it is of interest to determine how long the notochordal cells will maintain their influential activity. If possible, determine the portion of the notochord used, whether anterior or posterior. Similarly, the archenteric roof may be identified (generally grayish cells) and parts of it may be implanted and explanted to test the duration of inductive capacity. The origin- al experiments of this nature led to the concept of "individuation". (See glossary.) G. EVOCATION BY INOBGANIC SUBSTANCES This portion of the exercise constitutes essentially the control experilments for "B" above, the implantation of the living dorsal lip material. Obtain the smallest particles of silicon (Okada, 1958) or pieces of cellophane pre- viously soaked in l/lO,000 methylene blue (Waddington, et al 1956) and dried. Insert these small inorganic masses into the blastocoel of stages #7 or #8 and observe during 5 days for evidence of Inductions. Sterols, saponins, glycogen, cephalin, oestrogenic and carcinogenic substances, dead tissues from a variety of animal sources, and tissue extracts from worms to mammals have been used to successfully cause significant changes in contiguous but otherwise indiffer- ent ectoderm. (See Waddington, 19'+0.1 *************** OBSEBVATIONS AND EXPEBIMENTAL DATA: The post-operative care of the embryos generally includes returning them to their normal growing medium after the healing of the wounds ?n the operating medium. Specimens should be kept in separate #2 Stenders or finger bowls, properly marked for identifica- tion, and placed at cool temperatures to reduce bacterial growth. The maximum duration of observations for these experiments is about k days after the operation. Sketches and photographs at the time of the operation, with similar records at appropriate intervals, and finally, histological confirmation of the macroscopic ef- fects are recommended. The results are qualitative in that no two experiments could pos- sibly be alike, hence complete and accurate records of each specimen are most important. (The student should study the Glossary to learn the distinction between organizer. inductor, and evocator as illustrated the above experiments.) 2i6 THE ORGANIZER EECORD OF INDUCTIONS AND EVOCATIONS THE ORGANIZER 21? BEFERENCES: Barth, L. G. & S. Graff, 19'i^3 - "Effect of protein extracts of neural plate plus chorda- mesoderm on presumptive epidermis." Proc. Soc. Exp. Biol. & Med. 5'+:ll8. Bautzmann, H., 1932 - "Experimentelle analyse des organiaatorischen Geachehens in der Primitiventwicklung von Amphibian: Determinationszustand und Aufgabenverteilung der Eandzonenanlagen Im Organosationsprozees. " Verb, der Anat. Gesell. 75=221. Brachet, J., 1939 - "Etude du metaboliame de I'oeuf de grenoiaille (Eana fusca) au cours du developpement. V. Le metabolisme proteique et hydrocarbone de I'oeuf en relation avec le probleme de I'organiaateur. " Arch. Biol. 50:233- Child, C. M., 19I+6 - "Organizers in development and the organizer concept." Physiol. Zool. 19:89. Goerttler, K., 1931 - "Desorganization durch Einwirkrung von Organi satoren auf organiaier- endes Materiel." Verb. Anat. Ges. 37:152 (Also Anat. Anz. 71:132) Harrison, H. G., 19'+5 - "Belationa of symmetry in the developing embryo." Conn. Acad. Arta & Scl. 36:277- Holtfreter, J., 19^5 - "Neuralization and epldermization of gaetrula ectoderm." Jour. Exp, Zool. 98:161. Jaeger, L., 19^+5 - "Glycogen utilization by the amphibian gaatrula in relation to in- vagination and induction." Jour. Cell. & Comp. Physiol. 25:97. Lehmann, F. E., 1938 - "Eegionale Verscheidenhelten des Organlsatora von Triton." Arch. f. Ent. mech., I38. Needham, J., 19'<-0 - "Biochemical aapects of organizer phenomena." Growth Suppl. p. h'^. Oppehheimer, J., I936 - "Structures developed in amphibiana by implantation of living fish organizer." Proc. Soc. Exp. Biol. & Med. 5lt:U6l. Shen, S. C, 1939 " "A quantitative study of amphibian neural tube induction with a water- soluble liydrocarbon." Jour. Exp. Zool. 16:1^3. Spemann, H., I938 - "Embryonic development and induction." Yale Univ. Press. . Spemann, H. & H. Mangold, 192^* - "Uber Induction von Embryonalanlagen durch Implantation artfremder Organi satoren. " Arch. f. mikr. Anat. u. Ent. mech. 100:599- Vlntemberger, P., I938 - "Sur lea reaultata de la transplantation d' organi sateurs d'entenduea differentes chez Rana fusca, dans la region blastoporale ventrale." Compt. rendu. Soc. Biol. 127:'+36. Vogt, W. , 1929 - "Gestaltungsanalyse am Amphlblenkelm mltortllcher Vitalfarbung. " Arch. f. Ent. mech. 120. Waddington, C. H., 19*^0 - "Organizers and genes." Cambridge Univ. Press. Weiss, P., 1935 - "The so-called organizer and the problem of organization in Amphibian development." Physiol. Hev. 15:639- Woerdeman, M. W., I938 - "Embryonale Induction und Organization." Inst. Internat. Emb., London, Aug . I938 . "Between vitalism and mechani sm there is a middle ground which may be called 'Organizationism' or 'Emergence', which holds that life, differential sensitivity and reactiv- ity, fitness and psychic phenomena, are results of increas- ing organization, these properties 'emerging' as it were, by a process of creative synthe s is . E. G. Conklin 19ii MORPHOCENETIC MOVEMENTS AS DETERMINED BY VITAL STAINING PURPOSE: To stain the early embiyo with vital dyes hy means of which movements of various cell areas can be followed to their final location in organogenesis. MATERIALS : Biological: Blastula stages of Anura and Urodela. Technical: Powdered or crystalline agar, cellophane, sind vital dyes (Nile blue sul- phate and neutral red, preferably Gruebler's). METHOD: Precautions : a. These vital dyes are water-soluble. It is therefore wise to soak the pieces of stained agar or cellophane in distilled water briefly before using them for stain- ing embryos, to remove excess dye. b. The smallest pieces of stained medium should be used. These can be prepared in the dry state by cutting into small beads beneath a dissection microscope. c. While being stained the embryo should be as dry as is compatible with the main- tenance of normal conditions. d. All membranes except the vitelline membrane must be removed. The vitelline membrane can be punctured if it is otherwise difficult to hold the embryo in position. e. The Permoplast or soft paraffin base must be rigid enough to hold the egg in place for k'^ to 6o minutes without applying abnormal pressure. Control : This is an exploratory and qualitative type of experiment so that controls are not possible. Localized injury of cell areas could be used to impede such cell movements as seem evident from the vital staining observations. Procedure : PREPARATION OF STAINING MEDIUM Bring 100 cc. of distilled water to a boil in each of 2 Erlenmeyer flasks. To each, add 2 grams of pure powdered or shredded agar, and dissolve congjletely by further boiling. Avoid burning by constantly stirring with a glass rod. To one flask add 1 gram of Nile blue sulphate (Gruebler's) and to the other add 1 gram of Neutral Red (Gruebler's). Heat gently until the solutions are homogeneous. Tilt some clean lantern slide covers (or other glass plates) slightly on paper towel- ling, and pour the warm and stained agar mixture onto the plates so that there is a thin and even layer. Allow the agar to dry thoroughly in a dust- free environment, and then wrap the plates in white typewriter paper and label for future use. (See Vogt, 1925 •) Generally the thin layer of stained and dried agar can be chipped off of the glass plate with a scalpel, but if this proves difficult, simply add a drop of distilled water to the edge of the agar film and allow it to swell, after which it is possible to cut out a small strip of stained agar. This can be further subdivided with sharp scissors. A recent modification of this original procedure is to use the thinnest sheets of cellophane or pliofilm which take up the stain and can be cut Into small pellets. Such pieces of stained cellophane can be kept in envelopes until needed. The staining dishes are generally Syracuse dishes provided with Permoplast or soft paraffin bases. Permoplast is softer and easier to mould than paraffin, but it Is apt to crumble when left in water for aqy length of time. It is well to prepare 10 to 12 dishes well in advance of these experiments, each provided with depressions of various sizes in anticipation of various sized embryos. Depressions cein be made easily in a paraffin base by means of a warmed ball-tip, while the dish Is partially filled with water. -ei8- VITAL STAINING AND MORPHOGENET I C MOVEMENTS 219 STAINING PROCEDURE* It is not alwaya possible to stain an exact area with a particular dye. The usual procedure is to place various small vitally stained pellets within the wall of a depres- sion in the Permoplast (or paraffin) and in the appropriate medium and then to fit the egg or embiyo into the depression, moulding the material to hold the embryo firmly in place. It is not particularly important to use any special configuration as long as a record is made, immediately after staining, of the exact distribution of the stained areas on the egg or embryo. Bemove the Jelly membranes from the egg, or embryo, and place it in the depression. Gravity will orient the egg so that the vegetal pole takes most of the stain in any de- pression. This position can be varied by holding the egg in position with a hair loop while building up a closely confining cover of the Permoplast (or paraffin) with a ball tip. If the colored pellets are properly alternated within the depression, and properly spaced, the transferred marks on the embryo will not become confluent. Cover the egg or embryo with Standard Medium and leave undisturbed for h^ to 6o minutes at the laboratory temperature. Gently uncover the embryo and shake it out of the depression. STAGES AND AREAS TO BE STAINED 1. Grey Crescent: Within 20 to 30 minutes after insemination a grey crescent will appear between the animal and the vegetal hemispheres of the frog's egg, the more pronounced the longer the eggs are aged in the uterus. Using but a single (Neu- tral Bed) pellet, attempt to orient the grey crescent region adjacent to the dye and allow it to stain for half an hour. A second method of staining -the grey crescent is to utilize the Jelly of the egg by spreading it gently onto a small square of filter paper Just enough to hold the egg firmly in place. Take a stiff piece of dry and colored agar pellet held with forceps and, using it as a pencil, mark the region of the grey crescent by applying and holding the dye against the egg for as long a period as possible. (Do not overstain. ) Study the movement of the stained grey crescent, and determine Its relation to the first cleavage furrow and to the subsequent position of the initial involu- tion of gastrulation. 2. Blastula Stage: At about the 614--128 cell stage (stage #8 Sana) apply h stain marks of two colors around the germ ring, alternating the colors. These spots should appear circumferentlally placed. On other blastulae of similar stage, ap- ply a line of alternating colors from the germ ring of one side, through the dorsal hemisphere, to the germ ring of the other side. These stained areas should not only move but should change shape, depending upon their location in relation to the morphogenetlc movements of gastrulation. (See Goerttler, I925 and Vogt, 1925.) 3. Gastrula Stage: At the first indication of gastrulation (stage #10) mark the dor- sal, the lateral, and the (presumptive) ventral lip regions of the future blasto- pore. If you are successful in this work, attempt to repeat Goerttler' 3 work of staining a line of spots both dorsal and ventral to the Initial involution of the blastopore. h. Yolk Plug Stage: (stage #11 Sana or Amblyatoma) a. Presumptive notochord: Carefully apply a stain to the medium upper lip of the early blastopore. When this embryo has reached the tall-bud stage (Bana, stage #1? or #l8) dissect it with needles to locate the position of the iftvaginated colored cells. * Either Anuran or Urodele eggs (or embryos) may be used, but the latter are preferred, because of the reduced natural pigmentation. 220 VITAL STAINING AND MOR PHOGE NET I C MOVEMENTS VITAL STAINING AND MOR PHOGE NET I C MOVEMENTS 221 SUCKER LENS NEURAL FOLD EAR LIMIT OF HEAD REGION LIMIT OF INTURNED MATERIAL VISCERAL POUCHES ANTERIOR LIMB BUD LIP OF BL ASTOPORE LIP OF BLASTOPORE POSTERIOR- DORSAL VIEW RIGHT SIDE VIEW PRESUMPTIVE REGIONS OF ANURAN BLASTULA (ADAPTED FROM VOGT 1929) DORSAL MARGIN OF INM«31NATI0N SPLANCHNIC MESODERM ECTODERM REGION OF DORSAL UP VENTRAL POSTERIOR (BLASTOPORIC) VIEW RIGHT SIDE VIEW PRESUMPTIVE REQONS OF URODELE BLASTULA (ADAPTED FROM VOGT ^ 1929) 222 VITAL STAINING AND MOR PHOGENET I C MOVEMENTS GASTRULATION IN THE FROG ORGAN PRIMORDIA PRESUMPTIVE NEURAL PLATE DORSAL LATERAL ) LIPS *** ^ fi^' REMOVE ECTODERM PARABIOSIS STAGE 17 TELOBIOSIS 242 PARAB lOS IS AND TELOBIOS IS Parabiosis means lateral fusion. This may be side-to-side; back-to-back; belly-to- belly; or a Combination of these. The slde-to-slde fusion is generally the best because the larvae are permitted to move about and to feed in quite a normal manner. The lateral fusion of three embryos has been achieved. TELOBIOS IS The technique of terminal fusion is identical with that above except that the Permo- plast depression is long and narrow, and the injured areas are terminal rather than lateral. Simply cut off the tip end of the heads, tails, or combinations, using sharp and sterile scalpel or scissors. Bring the cut surfaces together and hold them approximated until healed. The relations may be head to head; tall to tail; or head to tail. Also, one of the embryos can be Inverted. OBSERVATIONS MP TABULATION OF DATA: a. Determine the growth rate of paired embryos as compared with the controls. b. Is there any evidence of dominance of one member of the pair over the other? c. How large a discrepancy in stages can be used for successful parabiosis? Dis- crepancy in species? (See section on "Transplantations".) The data should be recorded in the form of photographs or drawings, and preserved specimens which survive to the later stages. FUSED AT STAGP. 16 DIED AND PHOTOGRAPHED AT 73 DAYS OF AGE Raiin plpiens tadpoles in parabiosis, fused at stage 16 photographed at stage 24 (Railey) , PARABIOSIS IN METAMORPHOSED FROGS (RANA PIPIENS) Tullpan & Schrelber, 191^2 PARABIOSIS AND TELOBIOSIS 2k3 DRAWINGS AND PHOTOGRAPHS OF PARABIOTIC UNIONS 2hk PARABIOSIS AND TELOBIOSIS REFERENCES : Baltzer, F. , 19^1 - "Untersuchungen an Chlmaren von Urodelen und ^la." Rev. Suisse de Zool, l+8:iH5, Burns, E. K, Jr., 1955 - "The process of sex transformation in parabiotic Amblystoma. III. Conversion of testis to ovary in heteroplastic pairs of A. tigrinum and A. punc- tatum" Anat. Rec. 65: Fele, E., I929 - "Experimentelle Studlen an Parahiose-Tieren uher Physiologie und Biologle der Sexualhormone. " Arch. f. Gynakologie. 158:l6. Humphrey, R. E., 1956 - "Studies on sex reversal in Ambly stoma. IX. Reversal of ovaries to testesin parabiotic A. tigrinum." Jour. Exp. Zool. 75:1. Humphrey, E. E. & E. K. Burna, Jr., 1959 " "An incompatabllity manifested in heteroplas- tic parabiosis or grafting in Amblystoma due- to a toxin of cutajneous origin." Jour. Exp. Zool. 81:1. Kaylor, C. T. , 19'*-0 - "Experiments on the parabiotic union of haploid and diploid larvae of Triturus pyrrhigaster." Anat. Bee. 78:52 (suppl.). Smith, P. E., 1921 - "Some modifications induced by parabiotic union of the hypophysec- tomized to the normal tadpole." Anat. Eec. 21:85 (abs.). Stone, L. S., 195^+ - "Production and metamorphosis of chimeras in anurans and urodeles." Proc. Soc. Exp. Biol. & Med. 51:108^*-. Witschl, E., 1957 -"studies on sex differentiation and sex determination in amphibians. IX. Quantitative relationships in the induction of sex differentiation, and the prob- lem of sex reversal in parabiotic salamanders." Jour. Exp. Zool. 75:515. Witachi, E. & H. M. M. McCurdy, 19^5 - "Sex differentiation in heterogenous parabiotic twins (Amblystoma x Triturus)." Essays in Bio., Univ. of Calif. Press. "Chez tous les etres vivants le milieu inter ieur qui est un produit de I'organisme, conserve les rapporte neces- saires d'echange avec le milieu exterieur; mais a mesure que I'organisme devient plus parfait, le milieu organique se specific et s'isole en quelque sorte de plus en plus du milieu amb iant . Claude Bernard 1885 "A graduate from one of our larger universities, when asked why he changed from Biology to Philosophy said: 'Well. I found that there was so much. to be learned in Biology that I had no time to think, so I took up Philosophy, where there is nothing to be learned and J had all my time to think.'" E. M. East EXTIRPATION EXPERIMENTS ON ORGAN ANLACEN PUEP^E: To remove organ anlagen and to determine the ability of the embryo to adjust to the loss. MATERIALS : Biological: Various stages of Amblystoma as designated for each experiment. Technical: Standard equipment for operating on and caring for embiyos. METHOD: Precautions: a. The post operative care of embryos is Important, for they are naturally suscep- tible to bacterial infection. This can be reduced somewhat by operating and healing in 0.1^ sodium sulphadiazine, and keeping them in cool media. b. Bemove only the areas and cell layers indicated, at the designated stage. Organ fields become contracted with succeeding stages. c. Study section on "Wound Healing". Controls : The controls for extirpation experiments consist of the same operation but without the actual removal of the organ anlage. Procedure: Operate in 10^ Standard Solution and transfer to full strength Standard Solution when the wound is fully healed. THE UROCELE BALANCERS The balancers are slender rod-like appendages which project from the side of the head, slightly posterior and ventral to the eyes, and which serve as supports to hold the head off the bottom and maintain balance in many Urodeles (see Amblystoma series stage #^0). They are present in most species of Triturus, in Amblystoma punctatum (maculatum) Jeffer- sonlanum, mlcrostomum and opacum but are absent in A. tigrinum. The club-shaped ends of the fully formed balancers are sticky, and this condition may be taken as a criterion of normal development. Select Amblystoma punctatum or opacum at stages #28 to #52 and extirpate the balancer anlage on the righL side, leaving the left side as the control. Eemove both the ectoderm and the underlying mesoderm from a circular area posterior and slightly ventral to the eye, on the mandibular arch. The dorsal margin of the balancer area is on a direct line from the dorsal border of the eye, and the area extends to a level below that of the eye. The area also extends from the posterior margin of the eye to the first gill slit. (See sketch) Sketch (photograph) immediately Sketches (photographs) of after extirpation subsequent development -21+5- 2U6 EXTIRPATION OF ORGAN ANLAGEN THE EXTERNAL GILLS Select Anuran emtryos at stage #1? or #l8 or Amtlystoma embiyos (any species) at stage #25 or #26 and locate the gill swellings. If avail- able study Anura stage #23 and Amblystoma stage #U0 to see the position of the filamentous external gills which normally develop from the anlagen. Since all three germ layers contribute to the forma- tion of the external gills, it will be necessary to extir- pate an adequate area of the ectoderm and a considerable amount of the underlying mesoderm of these early stages in order to make the operation complete. This may be done with glass needles, and deep excavation by means of a hair loop. Ihe phaiyngael cavity should be exposed. The gill position of the balancer an- anlage on the right side may be removed and the lett siae ^^^^ (stippled) and posterior left untouched for control comparison. to it the gill swelling: AraDlystoraa stage #28. Sketch (photograph) Immediately after extirpation Sketches (photographs) of subsequent development EXTIRPATION OF ORGAN ANLAGEN 2hj THE EYE FIELD OF THE NEURULA Manchot (I929), Adelmann (1950) and Mangold (1951) have shown that the two eye fields are located close together near the median line of the anterior medullary plate of stage #1*^ or #15 of the Urodela or stage #1^ of the Anura. The eye field extends beyond the ultimately realized eye area, and extirpations and transplantations often result in pro- ducing accessory eyes. Attempt to extirpate the eye forming area of the right eye alone. With glass needles out deeply along lines indicated on the accompanying diagram, exposing the archenteron beneath. Bemove the ectoderm and the substrate of this area while the embryo is in 10^ Standard Solution and allow it to heal completely before gradually returning it to the full strength Standard Solution. It will be necessary to operate on a number of embryos because survival ia low. Variations of this extirpation should be in the direction of widening the area toward the left aide; moving the area toward the mid-line; removing the epidermis with and with- out the substrate. (See section on Eye Field Operations.) Sketch (photograph) immediately Sketches (photographs) of after extirpation subsequent development 21*8 EXTIRPATION OF ORGAN ANLAGEN THE OPTIC VESICLE The optic vesicles of toth the Anura and the Urodela are capable of regulation. This means that if the entire eye field is not removed, there may be a degree of restoration or regeneration of the excised parts. No regeneration occurs when the entire optic vesicle is removed, indicating that the eye-forming properties do not extend beyond the limits of the prospective eye forming area. The above statements can be verified by removing only the outer retinal portion of the optic vesicle In some embryos and the entire optic vesicle in others, examining by dissection and by histological (sectioning) technique after a minimum of a week of recovery and development at laboratory temperatures. The Anuran embryos at stage #17 (R> sylvatlca and palustris preferable to plplens) or the Urodele embryos at stage #25 or #26 may be used. Eemove the membranes and place the embryo on its left side in a Permoplast or agar depression in a Syra- cuse dish. Locate the optic bulge and with a glass needle and hair loop cut a rectangular piece of epidermis directly over the eye, leaving the upper margin of the rectangle uncut (a hinge). Lift the flap of epidermis and locate the under- lying optic vesicle. A. In some embryos, take the hair loop and cut off the most lateral (outer portion) of the optic vesicle, that portion known to give rise to the retina. Extirpation of the retina and the entire optic vesicle Rana: stage #17. B. In other embryos, excavate the entire optic vealcle, clipping it off at its base. If the flap of epidermis is still intact, replace it over the wound. If it has been damaged, simply leave the embryo in 10^ Standard Solution until the wound has closed com- pletely by the spreading of adjacent epidermis, and then transfer to full strength Standard Solution. The extirpation of the optic vesicle does not do away with the eye muscles entirely but they are reducea In size and are atypical. Sketch (photograph) immediately after extirpation Sketches (photographs) c subsequent development EXTIRPATION OF ORGAN ANLAGEN 2k9 THE HEART ANLAGE The partial or even the complete extirpation of the heart forming mesoderm of Ambly- stoma ia generally followed hy regeneration, which may mean the entire heart. The heart field extends well teyond the limits of the ultimate heart forming area so that even the extirpation of an area equivalent only to the ultimate heart may result in the formation of a small heart or hearts. With these facts in mind, derived from the work of Copenhaver, attempt to extirpate parts of the heart as follows: Amblystoma stage #15 or Anuran stage #1*+ should be used, for the heart mesoderm is derived from two separate lateral primordla representing the free ventral margins of the two hypomeres. These fuse in the mid-line, ventral to the pharynx and Just posterior to the thyroid anlage to give rise to a single tubular heart. The two prlmordia meet ventrally at about stage #27 in the Urodeles and about stage #1? in the Anura . Eemove the membranes and orient the medullary plate stage with the right ventro-lateral aide uppermost, in a depression in agar or Permoplast. The excision and healing should be in lO'jt Standard Solution. With sharp needles outline the heart area on the right side of the mid-ventral line and cut out a rectangular area deep enougn to include all three germ layers (see figure). It may be necessary to excavate cells with a hair loop, and this should be done extensively on the right side. Keep the embryo in 10^ Stan- dard Solution until the wound is healed over and then gradually return to full strength Standard Solution. Medullary plate stage of an amphibian embryo showing the presumptive heart area to be extir- pated (stippled) . Urodele embryos may be allowed to develop as long as possible because the heart development can be viewed through the thin covering ventral epidermis. The Anuran embryos should be dissected, or fixed, stained, and sectioned within about a week following the operation to determine the extent of damage or recovery of the above extirpation. (See section on Heart Field Operations.) Sketch (photograph) immediately after extirpation Sketches (photographs) or subsequent development 250 EXTIRPATION Of ORGAN ANLAGEN THE BRAIN Amblyatonia punctatum (or tigrinum) embryos should be freed from their Jelly membranes at stage flh to #l6 and allowed to grow at about l8°C. In Urodele Growing Medium until they reach stage #52- At this time the primary brain vesicles have been developed and can be outlined from the exterior of the embryo. Embryos of this stage show some movement and can be quieted in 1/5OOO M.S. 222 fresh- ly made up in Growing Medium. When fully narcotized, transfer to a Syracuse dish with bottom of soft paraffin or Permoplast and filled with Growing Medium saturated with sodium sulfadiazine. With a sharp scalpel make a single cut Just anterior to the gill anlagen and the first somite, Just posterior to the auditory vesicle. The head will be removed by such a cut, but particularly the entire brain. A sharp scalpel will bring the cut surfaces together and within l8-2l+ hours the wound should be entirely healed over. The purpose of such an extirpation (or ablation) is to determine the degree of further differentiation without benefit of the brain and the three seta of sense organs. (See Detwiler's papers) Obviously such an embryo cannot feed so that its life spand is deter- mined by the amount of yolk available. Bbwever, the anlagen in the vicinity of the cut (gill and balancer fields) will be the most likely to show variations in the direction of size and number of parts. Such embryos will generally show full development of the heart and circulatory system; will respond to stimuli; but will be somewhat more difficult to anesthetize than the controls (Anagnostis, 19^*8 unpublished). (Note: Extirpation of limb anlagen will be accomplished in connection with limb operations. ) Sketch (photograph) immediately Sketches (photographs) or after extirpation subsequent development EXTIRPATION OF ORGAN ANLAGEN 251 OBSERVATIONS AJIP EXPEBIMENTAL DATA: Under each sut-heading for Extirpations, follow the development of each emhryo with a series of sketches. The first drawing should be made Immediately after the excision, and the last one after the excised area has heen completely reformed, whether or not It Is regenerated. The time elapsed (days) and, the medium and temperature should all he noted. (See section on "Wound Healing".) TAIL BUD \ HYPOPHYSIS RANA PIPIENS STAGE 16 CLOSED NEURAL TUBE ORGAN FIELDS NEURAL TUBE PRONEPHROS . * FORE LIMB EAR HIND LIME YOLK M ASSj_ _' BALANCER AMBLYSTOMA PUNCTATUM STAGE 31 \ HYPOPHYSIS 252 EXTIRPATION OF ORGAN ANLAGEN BEFERENCKS : Adelmann, H. B., 1937 - "The effect of the partial and complete exclelon of the prechordal substrate on the development of the eyes of Amhlystoma punctatum. " Jour. Exp. Zool. 75:199. Bodenatein, D., 19'+3 - "An analysis of halancer development in Tri turns torosus." Physiol. Zool. I6:l+U. Copenhaver, W. M., 1939 " "Initiation of the beat and intrinsic contraction rates in the different paits of the Amhlystoma heart." Jour. Exp. Zool. 80:195' Detwller, S. B., 19^6 - "A quantitative study of locomotion in larval Amblystoma follow- ing either midbrain or forebraln excision." Jour. Exp. Zool. 102:321. Ekman, G. , I929 - "Experimentelle Untersuchungen uber die fruheste. Herzentwicklung bel Eana fuaca." Arch. f. Ent. mech. Il6:327. Harrison^ E. G., I92I+ - "The development of the balancer in Amblystoma, studied by the method of transplantation and its relation to the connective tissue problem." Jour. Exp. Zool. l4-l:3'4-9. Kollros, J. J., I9U0 - "The disappearance of the balancer in Amblystoma larvae." Jour. Exp. Zool. 85:33. Petersen, H,, I925 - "Berichte uber Entwicklungsmeckanlk. I." Ergebn. d. Anat. u. Entw'gesch. 2it-:327. Eotmann, E. , 1935 - "Der Anteil von Induktor und reagierendem Geweb an der Entwicklung der Kiemen und Ihrer Gefasso." Arch. f. Ent. mech. 133:225. Schotte, 0. E., & M. V. Edds, 19'4-0 - "Xenoplastic Induction of Eana pipiena adhesive diaca on balancer site of Amblystoma punctatum." Jour. Exp. Zool. 8^:199. Severlnghaus. A. E. , I93O - "Gill development in Amblystoma punctatum." Jour. Exp. Zool. 56:1. Stohr, P., 1925 - "Entstehung der Herzf orm. " Arch. f. Ent. mech. 106:l4-09. Stohr, P., 1929 - "Zur embryonalen Herztranaplantatlon. " Arch. f. Ent. mech. 109:300. 'Vitalistic hypotheses assume some sort of intelligence, or will, or psychic principle in organisms themselves that act as guiding or directing causes of adaptation - a sort of deus rn machina. Among these hypothe se s are the ' per fee ting pr inc iple ' of Ar istotle and Nageli, the 'indwelling soul' of Plato and Bruno, the 'active te leological pr inciple ' of Kant, the 'will' of Schopenhaur , the 'desire, need, and appetency' of Erasmus Darwin and Lamarck, the 'unconscious purpose' of Hartmann, the 'vital force' of Bunge, Wolff, and Virchow, the 'elan vital' of Bergson, the 'ente lechy' of Driesch, and the 'psychism' of Pauly, Boveri, and Spemann. The chief objec- tion to these hypothese s is that they are mere names, ghosts without substance , not open to exper iment or analys is . There- fore they tend to prevent further inquiry and are a hindrance to re sear ch, unless they are recogni zed as s ignpos ts point ing to the unexplored . " E. G. Conhlin 19^^ in "End as Well as Means in Life and Evolut ion" TRANSPLANTATIONS PURPOSE: To determine the ability of various organ anlagen to adjust to and differentiate within a new environment. MATEBIALS : Biological: Early embryos of the various amphibia, Anura and Urodela. Technical: Standard equipment. METHOD: Precautions : 1. Eemove and transplant only the area and the cells prescribed. This is very im- portant because the areas may vary with the age of the donor, and the various germ layers may have different developmental relations to the organ, 2. Adequate excavation of the host site must be made, especially since there is very rapid healing of any embryonic wound. 5. The transplant must "take" (become firmly attached) before the host is moved to a new environment, or changed to a different medium. k. Twitty (1937) and others, have found that the tissues of certain amphibia produce toxins which paralyze or kill the host (or transplant) in xenoplastic combina- tions. Triturus tissues are particxilarly potent when combined with Amblystoma. Therefore, In making xenoplastic transplants one must keep in mind the possibility of tissue in compatibility. Controls: Since most of the organ anlagen that will be used are bilateral, the organ of the unoperated side of the host may be considered as the control organ. Procedure : The following directions will be specific for each of the various organs. It is recommended that the student consult the Chapter in this Manual pertaining to the organ under study. Where natural pigmentations can be used to identify and trace a transplant (graft) in the host environment one need not add any further marking. In homoplas- tic transplants it will be necessary to pre-stain the donor tissue (graft) in Nile Blue Sulphate or Neutral Bed, in order to identify and follow the fate of the graft. Operated embryos should be allowed to recover In #2 Stender dishes with agar bases. LIMBS The f orelimbs of Amblystoma punctatum ( texanum, Jef fersonianum, opacum and Triturus torosus) are smaller and slower growing than those of A. tigrinum and A. mexicanum (axolotl). However, the A. punctatum anlage appear early (Stage #57) and develop digits shortly thereafter (Stage #1+1), while the forelimb buds of the A. tigrinum do not appear until about the beginning of the larval period (when the yolk is resorbed). Detwiler (1958) states that the prospective limb material is determined as early as the late yolk-plug stage, and Swett has shown that the two axes of the limbs are laid down consecutively. (See exercise on Limb Fields.) i. Eemove the vitelline membranes of 10 specimens of Amblystoma (Stage #28) and place them in sterile 10^ Standard Solution. If necessary, narcotize the embryos in 1/5000 MS 222. If the transplants are to be within the same species, pre-stain the donors in Nile Blue Sulphate. 2. Select a pair of embryos of similar developmental stage and place them side- by-side in an operating dish over soft paraffin or Permoplast. Mould a depression for the host, and place it in the depression with the right side uppermost. 5. Locate the forelimb anlage. This will be found ventral to somites #5-#5, Just posterior to the gill swelling and includes a portion of the ventral slope of the pronephric bulge. -255- 25lt TRANSPLANTATIONS RECORD OF LIMB TRANSPLANTATI ONS TRANSPLANTATIONS 255 h. Prepare the host by cutting a square hole with a glass needle (or a lancet) about 5 somites In diameter at the level of the pronephric bulge but just beneath the somites #8 to #10. The excavation nnist be deep enough to include some under- lying mesoderm. This may be done with a hair loop. 5. Prepare the donor forelimb anlage by excising the limb area, including the underlying mesoderm. This can be done by passing a glass needle from ventral to dorsal beneath the body ectoderm Just posterior to the gill buds, and dorsal to (and including) the pronephric bulge. By bringing the needle upwards, the ectoderm will be cleanly cut. In a similar manner, make a parallel vertical cut beneath somite #5 at the posterior limit of the pronephric bulge. Then cut the third side of the square between the two ventral points of needle Insertion. This will provide a flap of ectoderm which can be worked away from the neighboring tissues but with the under- lying mesoderm attached. Finally, make the dorsal cut to free the graft and quickly transfer It (always under water) on the tip of a glass needle, Into the previously made hole In the host. Note the orientation of the graft with respect to its origi- nal axes. It may be necessary, to further enlarge the hole of the host, due to heal- ing movements while preparing the graft. Gently press the graft Into place with the hair loop and cover it with a piece of (chipped) cover slip or glass bridge. If the depression is properly made, the cover slip edges will rest on the Permoplast and its center will continually press the graft into place. Do not distort the host by excessive pressure. 6. After about haJf an hour gently remove the cover slip bridge and allow the embryo to adjust to the new situation. If the graft has not taken, replace the bridge. After another half hour gently shake the embryo from Its depression, and transfer It to a #2 Stender (preferably with agar base) for further growth. If there are loose cells about the wound, clean them away with the hair loop. Variations 'In the above procedure would include transplanting the limb ectoderm above or exchanging the limb ectoderm indifferent belly ectoderm before transplant- ing the whole anlage, to determine the place of limb mesoderm In limb determination. Eecord by drawings or photographs the condition of the graft at the time of the transplantation and during subsequent weeks. Xenoplastlc transplants between A. punctatum and A. tlgrinum are very instructive. (See section on Limb Field Opera- tions for further details.) THE GILLS The external gills of the Anura appear as anlagen at stage #l8 and in the Urodela at stage #26. In the Anura they develop as branched, and filamentous outgrowths by stage #22 and in the Urodela by stage #^^1 they are fully formed. Before operating it is well to become fully ac- quainted with the normal morphology and development of the external gills. All three germ layers contribute to the formation of the external gills and Harrison (1921) has shown that the gills of Amblystoma punctatum are determined by stage #21, (see also Severinghaus, 1950 and Eotmann, 1935). Trans- plants can be varied to check the relative place of at least the ectoderm and the endoderm, It being rather dif- ficult to Isolate the intermediate mesoderm for such an experlpiental test, although it can be done. 1. Select two embryos, Anura or Urodela, of the same age and size and place them in Standard Solution in a Permoplast or paraffin operating Syracuse dish. Amblystoma tlgrinum gill anlage transplanted to the belly region of A. punctatum. Prepare shallow depreesiona to hold thnm right side uppermost. Eemove all the membranes. 256 TRANSPLANTATIONS EECOBD OF GILL TBMSPLANTATIONS TRANSPLANTATIONS 257 2. Prepare- the host site hy removing a piece of ectoderm and underlying mesoderm from different sites on different emhryos, e.g.^ Just posterior to the normal position of the gill anlage; heneath somites 9 to 12; just posterior to the anterior limb hud; Just anterior to the posterior limb hud; or in the position of the eye. Ex- cavate deeply enough to provide adequate room for a transplant with considerahle thickness. 5. The gill swelling will be found in a line with the eye but beneath the first several somites. Its anterior extremity will be Just posterior to the otic vesi- cle. With operating glass needle and hair loop cut out the entire gill swelling, and Include mesoderm and some of the pharyngeal endoderm. Keeping the piece in- tact, and properly oriented, transfer it on a needle to the prepared site on the host. Further excavate the host site to hold the transplant, and then hold it in place for JO minutes by means of a cover-slip bridge. Eemove the bridge carefully, and clean away any sloughed off cells around the margin of the wound by means of a hair loop. After complete healing transfer to the growing medium. Variations in the above procedure not only Include a different site on the host, but isolation of the germ- layer constituents of the anlage to determine (if any) their sepa- rate ability to develop into gills; rotation of the anlage; and xenoplastic transplants between the slow developing species (A. punctatum) and the rapidly developing species (A. tigrinum) . Make drawing or photographic records of individual transplants at appropriate inter- vals. THE EYE The eye is a composite organ made up from brain (neural) ectoderm and head ectoderm (lens). Transplantations may be made at various stages, and of constituent parts, but it is the purpose of this exercise merely to determine to what extent the entire optic vesi- cle and overlying ectoderm can adjust to a new site on the host, a site devoid of the normal second cranial nerve. 1. Secure embryos ( Anura stage #17 or Urodela stage #25) and place them in the oper- ating Syracuse dish over Permoplast and in depressions, in Standard Solution. The optic vesicle protuberance can be easily located on the right side of fhe head- Prepare one embryo as the host by excavating a hole in the ectoderm and underlying mesoderm in the lateral body wall; Just anterior to the -position of the hind-limb bud; or in the tail bud. 2. Eemove the entire optic vesicle on the right side of the donor, including a good portion of the diencephalon. With the overlying ectoderm Intact, transfer the en- tire transplant on a needle to the host site and pack it into place with a hair loop, and hold it for 50 minutes with a piece of cover slip. The eye anlage is a rather compact unit and since there is less yolk and mesoderm around it than around other anlagen, it may not adhere so readily to the host site. For this reason the excavated site should be somewhat deeper than the thicknes& of the transplant, and the surrounding ectoderm should be allowed to partially close over the transplant. This procedure can be varied in a number of ways. Extra eyes may be transplanted close to the site of the host eye to determine the degree of fusion or interfet-ence; an older anlage may be transplanted to a younger host (the reverse is likely to give negative results); the eye may be so oriented that the optic vesicle faces inward (in which case an extra covering of ectoderm must be provided); and xenoplastic transplants should be attempted, particularly between species of Amblystoma. (See Hewitt, 1955, Jour. Exp. Zool. 69:255 for a study of xenoplastic transplants of eye rudiments between various Anura and Amblystoma.) (Some ingenious transplantations, rotations, and regenerations of older, larval eyes, have been made by Stone and Zaur, I9I+O.) (See exercise on 'Eye Field Opera- tions and on Wolffian Begeneration. ) As in all transplant experiments, make drawings or photographs immediately after the operation and at appropriate intervals thereafter. Sectioned material at a later stage will answer the question relative to innervation. 258 TRANSPLANTATIONS SECORD OF EYE TRANSPLANTATIONS TRANSPLANTATIONS 259 THE BALANCERS Balancers are paired, alender, rod-like appendages which project from the side of the head slightly behind and helow the level of the eyes. They are found in Triturus and in Amhlystoma, (except for A. tigrinum), hut never in Bana. They serve as supports which hold the head off the bottom, preventing the larva from losing its balance before the develop- ment of functional forelimbs. They consist of an epithelial membrane whose glands secrete a mucoid substance at the tip and a mesodermal central core with a nerve and blood vessels (see Bodenstein, 19it-5). They normally develop at about stage #^h but the anlage appear as early as stage #21-22 (Bodenstein, I9U5). 1. Select two Urodele embryos of about stage #28 and shell them out of their mem- branes. Place them in a Syracuse operating dish with Permoplast base and partial- ly filled with Urodele Operating Medium. Prepare depressions with ball tip, side- by-side and Just adequate for the embryos. 2. Prepare the host embryo. Choose the site for the transplantation on one of the embryos. The most satisfactory positions are Just posterior to the pronephros and in line with it, or Just posterior to the otocyst. With glass operating needles cut out a rectangular piece of ectoderm about the size of two somites. This may be done by piercing the ectoderm with the point of the needle; pushing the needle forvard, beneath and pai-allel to the ectoderm; allowing the needle to emerge at the upper level of the pre-choeen area, and then (if the needle is rigid) lifting it upward and thereby making a clean cut. If this seems impractical, rub a hair loop against the needle until the ectoderm is cut. Bepeat this procedure along the parallel line of the pre-chosen area, then across the ventral and finally the dorgal edge. Lift out this ectoderm and discard. Excavate some underlying meso- derm. 3. Preparation of transplant. The balancer site is on the mandibular arch. Just pos- terior to and slightly ventral to the eye. The dorsal limit of the balancer an- lage is on a level with the dorsal limit of the eye and the ventral limit on a line below the ventral limit of the eye. Anteriorly the balancer area touches that of the eye and posteriorly it is below the second gill slit. In a manner similar to that of host ectoderm removal (above) remove the balancer ectoderm from the area indicated and on the tip of a needle (or hair loop) transfer it to the prepared (excavated) site on the host. Try to retain the same orientation of the transplant in the new, host environment, in respect to the original antero-posterior, dorso-ventral axes. (Do not rotate the transplant.) If the host implantation site has closed over in the Interim, remove some mesenchyme cells with the hair loop and widen the hole to fit the transplant. Work as quickly as possible since the transplant is apt to fall apart. As soon as the transplant is in position, press it gently into place with the hair loop and lay over it a piece of coverslip (which will act as a bridge). The embryo should be left undisturbed for at least 30 minutes, in a cool, dark place. After about 30 minutes if it seems that the transplant is not "taking", or has been moved, scratch the host site with a needle, replace the transplant and bridge, and await further healing. When the transplant is definitely attached, gently remove the bridge and allow the embryo to adjust to the new situation for a few minutes. Work the embryo out of its depression, by means of hair loops, shake it free, and (after about 1 hour or more) remove it with a large-mouthed, clean pipette to a #2 covered Stender provided with a sterile agar base. Place at a cool ( l8°C. ) temperature. It will be instructive to make the transplant between individuals of different ages, and also to rotate the transplant 90° or 180*^ In the host site to determine the degree of determination of the axes at the time of transplantation. Since a balancer does not nor- mally develop on A. trigrinum, an heteroplastic transplant from siny other species to A. tigrinum should be atten^Jted. Both donor and host may be kept in the same Stender, and the balancer site and anlage of the other (bilateral) side of each may be considered as control. In the space on the following page make sketches of each (donor and host) immediately after this operation and 260 TRANSPLANTATIONS RECORD OF BALANCER TRAMSPLMTATIONS TRANSPLANTATIONS 26l at intervals during the next two weeks. Balancers are not permanent structures. (See papers by Bodenstein I9I4-3; Harrison, I92I+; Kollros, iglf-O; Nicholas, 1924; l^otmann, 1935; Schotte and Edds, 19^0.) Make drawing or photographic records of individuals at appropriate intervals. OBSEBVATIONS AMD EXPERIMENTAL DATA: Each experiment will be peculiar to itself in that it is impossible from a technical point of view, to exactly repeat any operation. For this reason it is important that the student make numerous records, drawings, or photographs, to indicate (a) the donor area (b) the depth of the donor tissue transplanted (c) the site on the host and the depth of the excavation (d) the orientation of the donor transplant in the host field (e) and periodic drawings or photographs of the success of the transplant. Only when a transplant might Involve the central nervous system (e.g., the eye) should the material be sectioned to determine the nenroua connections established. Space is provided with each section for such records. DISCUSSION: The value of these experiments is the establishment of the stage in development when various organ anlagen are determined, and the degree of determination. Detailed experi- ments on the limb and eye are provided elsewhere, but it is instructive for the student to determine for himself the fact that certain anlagen are exchangeable and that certain transplantations are perfectly viable while others (see Twitty, 1937, Humphrey and Burns, 1959, and Eakln and Harris, 191+5) are not. There are, of course, numerous other anlagen that can be used such as the sense plate (stage #1^+), the olfactory placode (stage #15), the lens (stage #l6), the ear, parts of the nervous system (Detwller, 19^+2), thyroid (Allen, I9I8), pituitary (Blount, 1933), gonads (Burns, I928, Humphrey, 19'+'+), and heart (Copenhaver, 19^+5). The basic procedure can be established with the above four organ anlagen. (See sections on Eye, Heart, Limb.) Heteroplastic grafting (graft location different from the graft source) is instruc- tive in relation to growth and differentiation, and may give data on rate of growth, early and ultimate size factors, pigmentation, inhibitions found to exist in the normal environ- ment but non-existent In the transplant environment, mutual compatibility, etc. The stu- dent should consult the Glossary for the distinction between the various types of trans- plants such as homoplastic, heteroplastic, heterotopic, xenoplastlc, and the extreme case of the chimera (Baltzer, I9I+I) . EEFEEENCES Allen, B. M., I918 - "The results of thyroid removal in the larvae of Sana plplens." Jour. Exp. Zool. 2l+:l+99. Atwell, W. J. & J. W. Taft, I9I+O - "Functional transplants of the epithelial hypopfiy^ts in three species of Amblystoma." Proc. Boo. Exp. Biol. & Med. l+l+:53. Baltzer, F., I9U1 - "Untersuchungen an chlmaren von Urodelen und Hyla." Eev. sulsse de Zool. k^iklk. Blount, B. F., 1939 - "Heteroplastic transplantation of hypophysis between different species of Amblystoma." Proc. Soc. Exp. Biol. & Med. 1+0:212. Bodenstein, D. , I9I+3 - "An analysis of balancer development In Triturus torosus." Physiol. Zool. 16 :!+!+. Bom, G. 1897 - "Uber Verwachsungsversuche mit Amphiblenlarven. " Arch. f. Ent. mech. l+:349. Bums, E. K. , Jr., I928 - "The transplantation of larval gonads in Urodele amphibians." Anat. Eec. 39:177- Copenhaver, W. M., I9I+5 - "Heteroplastic transplantation of the sinus venosus between two species of Amblystoma." Jour. Exp. Zool. 100:203. Detwller, S. B., I938 - "Heteroplastic transplantation of somites." Jour. Exp. Zool. 79:361. Detwller, S. E., I9I+2 - "Neuroembryology." MacMlllan. 262 TRANSPLANTATIONS Eakln, B. M. & M. Harris, .19^5 - "IncompatllDillty between amphibian boats and xenoplastic grafts as related to host age." Jour. Exp. Zool. 98-55. Emerson, H. S., 19'+'+ - "Embryonic grafts in regenerating tissue. II. The behavior of the transplants during metamorphosis in Eana pipiens." Jour. Exp. Zool. 95:6l. Geinitz, B., 1925 - "Embryonale treinsplantation zwischen Urodelen und Anuren." Arch. f. Ent. mech. 106:557. Harrison, E. G., 1955 - "Heteroplastic grafting in embryology." Harvey Lectures, p. Il6. Hertwlg, G., 192? - "Beitrage zum Determinationa-und-Begenerationaproblem mittels den transplantation Haploidkemigen Zellen." Arch. f. Ent. mech. 111:292. Hewitt, D. C, 1955 - "Xenoplastic transplantation of amphibian eye rudiments." Jour. Exp. Zool. 69:255, Humphrey, E. E., 19'+'+ - "The functional capacities of heteroplastic gonadal grafts in the Mexican axolotl and some hybrid offspring of grafted animals." Am. Jour. Ant. 75:265. Hungjhrey, E. E. & E. K. Bums, Jr., 1959 " "An incompatibility in heteroplastic parabiosis or grafting in Amblystoma due to a toxin of cutaneous origin." Jour. Exp. Zool. 8l:l. Kollros, J. J., 19'*-0 - "The disappearance of the balancer in Amblystoma larvae." Jour. Exp. Zool. 285:55. Korschelt, E., I927 - "Eegeneration und Transplantation." Berlin. Loeb, L. , 1950 - "Transplantation and individuality." Physiol. Bev, 10:5'+7. Oppenheimer, J., 1959 - "The capacity for differentiation of fish embryonic tissues im- planted into amphibian embryos." Jour. Exp. Zool. 80. Piatt, Jean, 19^1 - "Grafting of limbs in place of the eye in Amblystoma." Jour. Exp. Zool. -86:77. Eichardson, Dorothy, 1952 - "Some effects of heteroplastic transplantation of the ear vesicle in Amblystoma." Jour. Exp. Zool. 65:'+15. Eotmann, E., 1955 - "Der Anteil von Induktor und reagierendem Gewebe an der Entwicklung der Kiemen ihrer Gefasse." Arch. f. Ent. mech. 155:225. Schotte, 0. E. & M. V. Edds, 19'4-0 - "Xenoplastic induction of Eana pipiens adhesive discs on balancer site of Amblystoma piinctatum. " Jour. Exp. Zool. 8'+:199' Schwind, J. L., 1957 - "Tissue reactions after homoplastic and heteroplastic transplanta- tions of eyes In the anuran amphibia." Jour. Exp. Zool. 77:87. Severinghaus, A. E., 1950 - "Gill development in Amblystoma punctatum. " Jour. Exp. Zool. 56:1. Stone, L. S., 195'*- - "Production and metamorphosis of chimeras in anurans eind urodeles." Proc. Soc. Exp. Biol. & Med. 51:108'+. Stone, L. S. & I. S. Zaur, 19'+0 - "Eeimplantation and transplantation of adult eyes in a salamander (Triturus viridescens) with return of vision." Jour. Exp. Zool. 85:2'+5. Tvitty, V. C, 195'+ - "Growth correlations in amphibia studied by the method of transplan- tations." Cold Spring Harbor Symposium Biology. 2:1148. Twitty, V. C, 1957 - "Experiments on the phenomenon of paralysis produced by a toxin occurring in Triturus embryos." Jour. Exp. Zool. 76:67. von Ubisch, L., I95I - "Keimblattchlmaren. " Verhandl. du Deutsch Zool. Gesell. I78. "Living things are analyzed into organs, tissues, cells, chromosomes, genes, and their functions into tropisms, re- flexes, and forced movements , uihile the synthes is of all of these elements into the broader aspects of the organism and its re lation to env ironment are too much ne gleet ed . E. G. Conklin 19ii THE ORIGIN OF AMPHIBIAN PIGMENT PURPOSE : To experlmentaHy test the thesis, by excision, explanation, homoplastic and heteroplastic transplantation, that the amphibian pigment is derived from the neural crests. MATEBIALS : Biological: Urodele larvae stages #13 to #3!+ Anuran larvae stages #14 to #l8 Technical: Standard operating equipment. Glass tubing measuring 0.25 to 0.14-5 mm. in diameter. Petri dishes, depression slides, cover slips. METHOD: Precautions: 1. Sterile precautions will Insure greater success, although amphibian tissues are veiy hardy. This Is particularly true of the isolation cultures. The media should be boiled or autoclaved; the Instruments sterilized either by boiling or in alcohol; and the denuded embryos should be put through several changes of sterile medium to remove surface bacteria. Controls : 1. For the excision experiments the control consists of excision of any region other than the neural crest. 2. For isolation experiments of neural crest anlage, the control likewise consists of the isolation of any region other than the neural crest, and the tissue may be taien from the same donor. 3. For transplantation experiments the control consists of the transplantation of regions other than the neural crest to the same locality in the host as the experimental transplant. Procedure: The following descriptions will apply specifically to Amblystoma but may be followed with comparable stages of Anuraji material. EXCISION OF THE NEURAL CREST The neural crest may be easily excised at the neural fold stage, before the folds have come together, at stage #15 for the Urodele embryo (or stage #14- for Anura). 1. Eemove the membranes from Urodele lai-vae, stage #l'+-#15 In Operating Mediiun. 2. Place the embryo in a shallow depression in Permoplast or paraffin and, using a double-edged lancet or needles, excise the neural fold on the right side as indicated in the accompanying diagrams. Include the dorsal ectoderm. 3. Leave the embryo in Operating Medium until the wound closes over, then transfer it to Growing Medium at about 15°-l8°C. and allow it to develop. A single speci- men in a #2 Stender or finger bowl is best. This operation will have the more graphic results in the more highly pigmented Amblystoma tlgrinum or T. pyrrhogaster. -263- 26U ORIGIN OF AMPHIBIAN PIGMENT BECORD OF NEUEAL CREBT EXCISION EXPERIMENTS HOMOPLASTIC NEURAL CREST TRANSPLANTATIONS Removal of neural crest material from the normal to a strange site should similarly alter the final pigment pattern of the em'bryo. It is generally too drastic to make the transplant on the same embryo, but comparable stages should be used or the donor material may be taken from an embryo slightly older than the host. STAGE 15 STAGE 16 STAGE 17 STAGE 18 STAGE 18 STAGE STAGE 9 STAGE 32 OR IGIN OF AMPHIBIAN PIGMENT 265 1. In Operating Medium, prepare a transplant site on the host emtryo (stage #15) some- where in the lateral helly region, far removed from the host neural folds. The emhryo should lie on its side in a Permoplast or paraffin depression deep enough so that the helly ectoderm is flush with the surface of the Permoplast. 2. Excise the neural fold of the right side from a similar emhryo of stage #15 or older, and place it in the host wound, making the transfer on a needle point or by means of the hair loop. Hold it in place with a piece of cover slip (Briicke) for 30 minutes without di sturhance . 5. Transfer the embryo by means of wide-mouthed pipette to Growing Medium at iS'-'C. or less for recovery and development. Eecently (19^5) Twitty rotated the neural crest and adjacent somite material with interesting results (see accompanying figures). This operation should be attempted but might be varied in respect to position on the host. If the somites and crest are re- versed, it would be significant to make the l80° rotation at the level of the hind- or forelimb to determine not only the alteration in pigment pattern but also in the muscular control of the adjacent appendage. Fig. 2. Dorsal view of Trlturus torosus larva showing tMe transverse strands which connect the paired bands of melanophores located on the spinal cord. Fig. 3. (a) Extirpation of neural (trunk) folds. (b) Closure of "crestless" neural tube. (c) Block of tissue excised and grafted upside down In substitution of the somites of another embryo (d & e) . (d) Diagram of transplant In host (d.m.s. - dorsal margin of myotome) (e) Inverted somites and neural crest in host. (f) Modified distribution of melanoDhores in host. V. C. Twitty, I9k^: Jour. Exp. Zool. 100:li^l. 266 ORIGIN OF AMPHIBIAN PIGMENT RECOED OF HOMOPLASTIC TEANSPLANTATIONS HETEROPLASTIC NEURAL CREST TRANSPLANTATIONS If Amblystoma punctatim and A. tlgrlnum larvae of stages #25 to #28 are available, heteroplastic transplantations resulting In varied pigment patterns should he attempted. If the white axolotl is available, the results are even more graphic. (See section on "Transplantations" to determine the viable combinations.) When Urodeles of different developmental rates are used, this factor must be considered. In general, the graft tis- sue should be from a donor slightly older than the host. Heteroplastic transplantations are best made to the normal site, transferring dorsal ectoderm and ganglion crest, along with some of the nerve cord, to insure presence of crest. Some suggested combinations: Amblystoma tlgrlnum crest stage #2k to A. punctatum stage #25- Amblystoma punctatum crest stage #28 to A. tlgrlnum stage #25- The treinspiant should be treated as in the homoplastic experiments, the operation being carried out in Urodele Operating Medium and after the graft has taken, transfer the host to Urodele Growing Medium for further development. ORIGIN OF AMPHIBIAN PIGMENT 267 EECOED OF HETEROPLASTIC TRANSPLANTATIONS ISOLATION CULTURE OF THE NEURAL CREST Piecee of embryonic neural tube (Urodele stages #23 to #26) are stripped of their dorsal ectoderm and then cut into small fragments, each with some neural crest, Jid are cultured In isolation (Twitty & Bodensteln, 1959, Twitty, 19^5) to derive chromatophores. There are two methods, and several culture media. The sterile culture media are: (1) Standard ( Holtfreter'e) Solution, in which the bicarbonate is dissolved separately from the other salts and the two voliines are mixed after boiling and cooling. (2) Coelomic Fluid which consists of fluid from the coelomic cavity of the adult of the same species, removed under sterile conditions by means of a sterile syringe. Twitty {19'+5) has found that females during the spawning season provide the most abundant coelomic fluid. The culture methods are: (1) In a depression slide sealed over with a vaseline ringed, coverslip. (2) On a coverslip inverted over a depression slide, the culture be- ing in a hanging drop. (5) On a microscopic slide under coverslip slightly elevated by a ring of soft paraffin. In each instance, the Isolated neural crest is in a llqiild en- vironment, protected against evaporation and extrinsic change, and provided with facili- ties for growth and expansion. The coelomic fluid is generally the best, for it provides more than Just the isotonic salt requirements for maintenance and growth. Twitty and Bodensteln (1959) found that boiling did not destroy the effectiveness of peritoneal fluid in stimulating pigment development, but did reduce the risk of infection. The most successful Isolation cultures will probably be achieved if sterile coelomic fluid is used and the neural crest Is Isolated Into this medium on a clean circular cover- slip which is ringed with white vaseline. If the sterile Inverted depression slide is brought down over the isolated tissue, pressed against the vaseline, and left in this position for 2.k hours before re-inversion, the explant will become adherent to the cover- slip so that when it (and the depression slide) is turned over, the neural crest and any 268 OR IGIN OF AMPHIBIAN PIGMENT derived cells will be merely the thickness of the coverslip away from microscopic examina- tion. Such cultures should last for 8-10 days, or longer at lower temperatures. Recently (19^*^) Twltty cultured pigment cells from neural crests within capillary tuhes measuring 0.25 to 0.14-5 in diameter. The neural crests were drawn into the tuhes by oral suction, while submerged in sterile Standard ( Holtfreter's) Solution, and the tubes were tilted slightly (within the Standard Solution) and the end of each tube farthest away from the end containing the explant was embedded in non-toxic vaseline. This vaseline acted as a plug, and at the same time held the tube in place. The tilting was achieved by a mid-way ridge of vaseline, but this could be accomplished equally well with a knotched paraffin ridge. The free end, i.e., where the explant Is located, was open to the culture medium. During examination, the curved sides of the capillary tube acts somewhat as a lens, magnifying the contained neural crest and pigment cells. Twltty found that pigment formation in the capillary tubes was dependent upon substances diffusing from the nervous tissue of the explant, and that the first cells to become pigmented were those found deepest within the tube. He attributed this to regional differences in the concentration of oxygen and pigment precursors or oxidases essential to melanin formation. Place in Petri dishes filled with Holtfreter's solution, the tubes containing the explants are held In position by partly imbedding them in a row ridge of vaseline. The end near which the explant lies is left open; the opposite end is plugged with vaseline to pre- vent any flow of fluid through tlie tube which might De created by tipping or disturbance of the dish. Semi-diagramraatlc drawing. A semi-diagrammatic drawing to repre- sent the differential onset of melati- Izatlon within cultures of embryonic pigment cells developing in capillary tubes . From Twltty, l^kk: Jour. Exp. Zool. 95=259. The record for this experiment will consist of a series of daily drawings of the explants and the cells (pigmented and otherwise) which are derived therefrom. ORIGIN OF AMPHIBIAN PIGMENT 269 EECOEDOF ISOLATION CULTUEE EXPERIMENTS 270 ORIGIN OF AMPHIBIAN PIGMENT SELECTIVE STAINING OF IN VIVO NEURAL CREST DERIVATIVES In transplantation and isolation experiments there is always the question as to whether the derivatives of the excised anlage are the sajne as they would have been in the original site of the donor. Stone (1952) refined a method of preserving the vital dye Nile Blue Sulphate in the fixed emhryo so that it could be located in sectioned material. It is therefore in the nature of a confirmatory experiment that the following procedure 1b given. 1. Select Urodele embryos of stage #25, and remove all of their membranes in Growing Medium. Place them in an operating dish with a Permoplast or soft paraffin base, and mould depressions to hold them with the neural folds uppermost. 2. Cut a piece of Nile Blue dyed agar (0.1 gr. Nile Blue Sulphate in 2^ agar in 100 cc. of distilled water, dissolved by heating-, and poured, while hot, onto glass plates covered with a very thin layer of glycerine. When dried, the agar can be pealed off of the plate In thin sheets of any size or shape (e.g., the shape and size of the neural fold). Place this minute piece of agar flat on a piece of coverslip and pass the coverslip through a flame. This will cause the agar to melt slightly, and become adherent to the coverslip chip. With practice one can provide a marker of the exact shape and size of the neural fold. 5. After the neurula stage (#25) is firmly placed within the depression. In Standard Solution, bring the (Inverted) coverslip chip into position so that the Nile Blue Agar will make precise contact with one of the neural folds. Press it against the embryo, and anchor the edges of the glass chip in the surrounding Permoplast. Hold the neurula in this position for 20-50 minutes, while the dye is being trans- ferred to the neural fold, and then gently remove it without tearing away any of the cells of the embryo. k. Transfer the embryo to Urodele Growing Medium for development until stage #28 or later. The dye will remain for a long time and spread with the cells It has In- vaded, so that It may be found even after the pigment has begun to appear. 5. (a) Fix the embryo in Zenker-acetic for two hours; wash in running tap water 1 hour. (b) Place in 1^ aqueous solution of phosphomolybdlc acid for 2 hours (Lehmann, 1929). (c) Transfer for half hour periods through the ascending alcohols to each of which has been added 0.1^ phosphomolybdlc acid. This acid preserves the dye. (d) Transfer for one-half hour to a mixture of equal parts of 100^ alcohol, (con- taining 0.1^ phosphomolybdlc acid) and cedar oil. Then place in pure cedar oil until clear (overnight). (e) Embed in three changes of paraffin-Bayberry-beeswax mixture (90 - 5 - 5) and, when hardened, section at 10 microns. The sections may be treated in the usual way (with xylol) and mounted under clarite. If the tissues are not passed through any water they will retain the Nile Blue Sulphate dye in the cells which were originally stained, and with appropriate lighting the demarcation between stained eind unstained areas can be made out easily. Becord on following page by drawings or photographs the results of in vivo staining of the neural crest anlage. OBSERVATIONS AND TABULATION OF DATA: The data for these experiments are qualitative rather than quantitative, and space is provided with each section for appropriate records. Histological sections, when possible, will confirm the macroscopic analyses. (See DuShane, 1955= Jour. Exp. Zool. 72:1 for cytologlcal procedures relative to the various types of chromatophores. ) ORIGIN OF AMPHIBIAN PIGMENT 271 RECOED OF IN VIVO STAINING EXPEBIMENTS DISCUSSION: The neural crest arises as a strip of cells lying between the neural plate and the dorsal ectoderm, this plate being separated from both of these structures during the closure of the neural folds. It is now established that the neural crest is the principal and probably the sole source of all pigment cells (except the tapetum) in all of the vertebrates that have been studied (DuShane, 19^3)' Melanophores are found in the epi- dermis and dermis, meninges, visceral mesenteries, peritoneum, and in close association with the blood vessels throughout the body. This means that from the original source of such pigment cells there has been very extensive migration. The forces involved in this migration are illustrated in the capillary- tube isolation experiments of Twitty {19^k). But the pre-migratoiy neural crest cells cannot be distinguished from their neighbors because they show no pigment differentiation until about the time they reach their normal destination. Such pigment cell differentiation includes: Melanophores - wide distribution, cells with brown to black melanin. Lipophores - dermis and epidermis, having diffuse yellow pigment (lipochrome) in solution. Guanophores - pericardium and most lateral line organs, highly refractive, granular, golden yellow guanin crystals with metallic lustre. The pigment pattern may be used to identify different species of Amblystoma or Tri- turus and frequently in heteroplastic transplantations there is evidence that both gene- tic and environmental factors (such as humoral or contiguous cell influences) may be 272 ORIGIN OF AMPHIBIAN PIGMENT Important In directing ultimate cellular differentiation. In Amblystoma punctatum there la even distribution of melanophores while the llpophores are fused to give a continuous sheet within the dermis. In A. tlgrlnum the melanophores are large and dark and are ar- ranged In groups, as are also the llpophores. The time of melanophore appearance Is species specific. In A. punctatum the first melanophores appear at stage #5^ lateral to the medulla, beneath the epidermis, and by stage #56 they have reached the level of the pronephros. The llpophores begin to appear at stage #28. Potential melanophores from various species manifest different abilities to develop in Isolation, and even in the normal environment there are melanophores which are dependent upon the presence of pig- mented epideimls to produce melanin. Other derivatives of the neural crest may include chromaffin tissue, mesenchyme, sheath cells, visceral cartilages, spinal ganglion (neuro- blast) cells, sympathetic ganglia, and adrenal medulla. Twitty and Bodensteln (1959) found that pigment appears in the isolated chromatophorea when cultured in Standard ( Holtfreter' s) Solution later than in peritoneal fluid, they darken less rapidly, and the melanophores become more widely dispersed. The active migra- tion of melahoblasts in vitro is confined to the period before the pigmentation is estab- lished, in both species of Triturus studied, although migration does not preclude the possibility of further melanlzation. They found, in general, that when the volume of the culture medium was kept low that pigment development was the better, and small drops of peritoneal fluid were the best. Xanthophores appear more frequently in cultures of Amblystoma than in cultures of Triturus crests. Szepsenwol (19^+5) found that the eyes of larvae (Amblystoma and Eana) effect the body color either by a humorsl substance (early in development) or reflexly (later In develop- ment), when the nervous control of the melanophores dominates the humeral. These observa- tions resulted from parabiotic union studies in which the eyes of one or both members of the pair were excised, and subsequent pigment pattern studies were made. BEFERENCES : Adler, A., 1959 - "Melanin pigment in the central nervous system of vertebrates." Jour. Conip. Neur. 70. Atwell, W. J., & E. Holley, 1956 - "Extirpation of the pars intermedia of the hypophysis in the young an^jhibisin with subsequent silvery condition and metamorphosis." Jour. Exp. Zool. 75:25. Baltzer, F., 19'j-l - "Untersuchungen an chlmaren von Urodelen und ^yla. I. Die Pigmen- tlerung chlmarischer Molch-und Axolotllarven mlt Byla-Gangllenlelste." Eev. Suisse de Zool. hSihl^. Barden, R. B., 19*^2 - "The origin and development of the chromatophores of the amphibian eye." Jour. Exp. Zool. 90:U79 (See ibid. 92:171). Beams, E. W. & E. L. King, I958 - "Pigmentation changes in tadpoles of Eana plplens follow- ing cent rifugat ion during the early gastrula." Jour. Morph. 65. Bytlnaki-Salz, H. , 1959 - "Chromatophorenstudien II. Struktur xmi Determination des adepldermalen Melanophorennetzea bei Bombina." Arch. f. Zellforsch. 22:125. Collin, E. & P. Florentin, 1958 - "Absence de pigment melanique chez un trlton." Bull. Mens. Soc. Sci. Nancy. J):lhj,, Dalton, H. C, I9U6 - "The role of nucleus and cytoplasm in development of pigment pat- terns In Triturus." Jour. Exp. Zool. 105:169. Dawes, Ben, I9I+I - "The melanin content of the skin of Eana temporaria under normal condi- tions and after prolonged light and dark adaptations." Jour. Exp. Biol. 18:26. DeLanney, L. E., I9UI - "The role of the ectoderm in pigment production, studied by trana- plantation and hybridization." Jour. Exp. Zool. 87:525« Detwiler, S. E., 1957 - "Observations upon the migration of neural crest cells, and upon the development of the spinal ganglia and vertebral archea in Amblystoma." Am. Jour. Anat . 61 : 65 . Detwiler, S. E., & W. M. Copenhaver, 191+0 - "The growth and pigmentary reaponses of eye- less Amblystoma embryos reared in light and in darkness." Anat. See. 76:21+1. Dorrls, Frances, I9U0 - "Behavior of pigment cells from cultures of neural creat when grafted back into the embryo." Proc. Soc. Exp. Biol. & Med. Ul+:286. Drager, G. A. , & B. F. Blount, I9U1 - "The time of the appearance of melanophore expand- ing hormone in the development of Amblystoma maculatum." Anat. Bee. 8I: auppl. 95- ORIGIN OF AMPHIBIAN PIGMENT 275 DuShane, G. P., I943 - "The embryology of vertebrate pigment cells. I. Amphibia." Quart. Rev. Biol. 18:109. Eaatllck, H. L,, 19i^0 - "The localization of pigment- forming areas in the chick blastoderm at the primitive streak stage." Physiol. Zool. 13:202. Elias, H., 191+3 - "Cause for blue, green, and red colour in Anura." Aaat. Sec. 87:12. Etkln, v., 191+1 - "On the control of growth and activity of the pars intermedia of the pituitary by the hypothalamus in the tadpole." Jour. Exp. Zool. 86:113. Fostredt, G., 1939 - "Preliminary in vitro studies of melanophore-principle activity of the pituitary gland." Proc. Soc. Exp. Biol. & Med. Ii0:302. Harrison, E. G., I955 - "Heteroplastic grafting in embryology." The Harvey Lectures, p. 116. Humm, F. D. , I9I+2 - "The growth and migration of cultured melanophor^s from the neural crest when grafted into the embryo." Jour. Exp. Zool. 90:101. Lehmann, F. E., I929 - "Die Entwicklung des Anlagenmusters im Ektoderm der Tritongastrula. " Arch. f. Ent. mech. II7. Lynn, W. G, & Sr. A. de Marie, 19^+6 - "The effect of thiouracil upon pigmentation in the tadpole." Science, 10li:31. Marsland, D. M. , 191+5 - "The mechanism of pigment displacement in unicellular chromato- phores." Biol. Bull. 87:252. Niu, M. C, 19l*-7 - "The axial organization of the neural crest, studied with particular reference to its pigmentary component." Jour. Exp. Zool. 105:79. Parker, G. H. , I9U3 - "Animal color changes and their neurohumors . " Quart. Eev. Biol. 18:205. Eaven, Chr. P., I936 - "Zur Entwicklung der Ganglienlelste. V. Uber die Dlfferenzlerung lea RuBipfganglienleistemnaterials." Arch. f. Ent. mech. l^k:122. Bosln, S., 191*^3 - "Experimente zur Entwlcklungsphysiologie der Pigmentierung bei Amphibien." Eev. Suisse de Zool. 50:1+85. Sawyer, C. H. , I9I+7 - "Cholinergic stimulation of the release of melanophore hormone by the hypophysis in the salamander." Jour. Exp. Zool.. I06. Serra, J. A., I9I+3 - "Sur la nature des melanlnes et la melanogenese. " Genetlca, 23:300. Stone, L. S., 1952 - "Selective staining of the neural crest and its preservation for microscopic study." Anat. Rec. 51^267. Stone, L. S., 1933 - "The development of the lateral-line sense organs in Aniphibiana in living and srital-stained preparations." Jour. Comp. Neur. 57:507. Szepsenwol, J., I9I+5 - "The influence of the eyes on the melanophores in Amphibia." Anat. Rec. 93:185. Twitty, V. C, I9I+5 - "The developmental analysis of specific pigment pattern." Jour. Exp. Zool. 100:11+1. Twitty, V. C. & D. Bodensteln, I9I+I+ - "The effect of temporal and regional differentials on the development of grafted chroma tophores . " Jour. Exp. Zool. 95:213. Waring, H., I9I+I - "The co-ordination of vertebrate melanophore responses." Biol. Eev. 17:120. Watterson, E. L., 191+2 - "The morphogenesis of dovm feathers with special reference to the developmental histoiy of melanophores." Physiol. Zool. 15:23l+. Weiss, P., I9I+I - "Melanin formation by deplanted fragments of thalBmus In Amphibian larvae." Proc. Soc. Exp. Biol. & Med. l*8:3'+5. Willler, B. H. , 191+1 - "An analysis of feather color pattern produced by grafting melano- phores during embryonic development." Am. Nat. 75:156. "There is fundamentally a natural corrective for our inclination to allow likes and dislikes to influence our reason. This corrective is found in the instinct of curi- osity, the faculty that impels men to seek the truth, even if it be unpalatab le . A. J. Lotka 1925 LIMB FIELD OPERATIONS [anlage) of the PURPOSE : :^y the application of experimental methods to the limb field Urodele embryo, to determine the a. Powers of regeneration following partial and total extirpation of the anlage. b. The effect of rotating the limb field axis. c. The effect of splitting the limb field. d. The relative f mictions of the ectoderm and the mesoderm in limb formation. e. The differentiating capacity of heterotopic transplants. f. The relation of host and donor in xenoplastic transplants. g. The genetic factors relating to limb formation, by means of 1. Heterogenic transplants (i.e., between species with different rates of development. 2. HJsteroplastic transplants involving pigmentary differences. MATERIALS : Biological: Urodele larvae of stages #29 to #55. Slow developing forma Amblystoma punctatum ( maculatum) Amblystoma texanum (mlcrostomum) Amblystoma Jefferaonianum Amblystoma opacum Triturus rivularls Tritunis slmulana Trdturus torosus Bapid developing forma Amblystoma tlgrlnum Amblystoma mexicanum (axolotl) Technical: Standard Operating Equipment. Vital dyes and MS 222 freshly made up cone. l/5,000. METHOD: Precautions: a. Use 0.1^ sodium sulfadiazine if bacterial infection is encountered. b. Be reluctant to examine the transplant. It should remain undisturbed for 50 minutes or more and any necessary transferring should be by wide-mouthed pipette. c. Temperatures lower than that of the laboratory are best for Urodele recovery and survival. Some species do best at 10°C. d. Operations are performed in Operating Medium; embryoa are later transferred and raised in Growing Medium. Feeding should begin when the balancers disappear. e. Muscular activity begins at about Stage #3**-. If operations are attempted at this stage it will be neceasary to use freshly made MS 222 in operating medium l/5,000. Control: The controla for each of the following experimental procedures will, of neces- sity, be different. Procedure: Each of the following exercises deals with the limb field but since each illustrates a different principle, the procedures will naturally differ somewhat. REGENERATION FOLLOWING EXTIRPATION OF THE ANLAGE** Secure embryos at stage #29 and remove all membranes. Place the embryos in a Petri dish with an agar base. Select those which are exactly at stage #29 and compare with the accompanying diagram on following page. * The author acknowledges, with appreciation, the help of the late Dr. F. H. Swett in organizing this exercise. ** Limb extirpation la included in the exercise on "Extirpations" but the procedure here Is considerably more detailed. -21k- LIMB F lELD OPERATIONS 275 A - P = 4NTER0 POSTERIOR MIS A ■ P'-- EMSRYONIC axis' D - V ! OORSO VENTRAL AXIS GILLAIyLASEN LIMB AND EMBRYONIC AXES AMBLTSTOMA STAGE 29 IREORAVm FROM SWETT '371 FORE - LIMB FIELD OPERATIONS AMBLVSTOMA STAGE 29 SPLITTING THE UMB - FIELD AMBLYSTOMA STAGE 29 276 LIMB F lELO OPERATIONS Place the emtryo (stage #29) on its left aide In a Permoplast or paraffin depression In an operating (Syracuse) dlah. With a douhle-edged lancet or glass needles make cuts as Indicated below and In the acconipanying diagram. The circular cut can be made most easily by rotating the operating dish while cutting with iridectony scissors in an immobile hand. The ectoderm is pigmented, the underlying mesoderm is white and the deeper endoderm is grayish-tan (having alight pigmentation). The purpose of the following procedures is to determine the areal extent and the depth of the limb field as of stage #29. The limb area is ventral to ncrotomes #5-#5, Just posterior to the gill anlagen. a. Eemove ectoderm alone from area marked "A" (see accompanying diagram). b. Remove ectoderm and all underlying mesoderm from area "A". Be certain to clean out all the white mesodermal cells beneath the excised ectoderm. c. Bemove ectoderm from the larger area "B", leave the mesoderm undisturbed. d. Hemove the ectoderm and mesoderm from the larger area "B". Again scoop out all of the white mesoderm cells by means of a hair loop. e. Make a semi-circular cut (as indicated by arrows) peripheral to the margin of area "B", deflect this flap of ectoderm anteriorly, and clean off and scoop out all mesoderm cells from beneath the flap of ectoderm. Should the excavation be so extensive that the flap of ectoderm does not cover, fill the cavity with yolk cells from another embryo of the same age. Apply a bridge (Briicke) for 50 minutes to hold the ectodermal flap in place until it heals. The unoperated side of the embryo will function as the normal or control for compari- son with the operated side in respect to limb development. Maintain in the same environ- ment, however, some other unoperated embryos of the same age and stage as supplementary controls. The best controls would be operated embryos but with the tissue left in place, particularly since this is an attempt to determine the effect of extirpations. KECOHD OF LIMB EXTIRPATION EXPERIMENTS LtM3 F lELD OPERATIONS 2?? HETEROTOPIC TRANSPLANTS The orthotopic (homotopic) position is the normal position, the heterotopic one "being a different or strange position for the anlage. It is the purpose of this experiment to determine the differentiating capacity of the limb anlage at other than the normal regions. If several donors are available, supernumerary limbs may be provided. Eemoving the ecto- derm and/ or the mesoderm from the donor, make the following transplantations: a. Ectoderm alone from the area "A" of donor to region "X" of host. b. Ectoderm and mesoderm from area "A" to region "X" of host. c. Ectoderm and mesoderm from area "B" to region "X" of host. d. Ectoderm and mesoderm from area "A" to region "Y" of host (on hand). In all cases it is well to place the donor and the host side by side and to locate the regions concerned in both embryos before the operation Is begun. Use MS 222 (1/3,000 cone.) if necessary, to quiet any muscular activity. The transplant must be made quickly and the transfer may be made on the point of a needle or on a small hair loop. With a ball tip gently pat the transplant into place and hold it there by means of a chipped coverslip (Briicke) for at least 50 minutes. If the glass cover tends to adhere to the ectoderm, try a small piece of lens paper whose ends may be fastened to the Permoplast base. In the case of the transplant to region "Y" on the host it will be necessary to place the host in a Permoplast depression with the head protruding. Provide an adequate wound area in the host, for healing is rapid. EECOED OF HETEROTOPIC TEANSPLANTATIONS 278 LIMB F lELD OPERAT-IONS ROTATION OF THE LIMB AXES In these experlmenta it is necessary to mark the donor material in some way so that the axes may be identified when the transplant is oriented in the host site. Generally this may he accomplished in the normal course of excision where identifying marginal nicks may- be used as identifying markers. Both anterior-posterior (A-P) and dorso-ventral (D-V) axes are concerned. Use Urodele embryos of stage #29 or younger. a. Carefully cut out the ectoderm and the mesoderm of area "B" so that the bulk of the limb area remains intact. Clean out the wound with a small hair loop, removing all loose (white) mesodermal cells, and replace the excised mass of tissue in the same wound area after making a l80° rotation of the graft, as in- dicated by arrows in the accompanying diagrams (Fig. F) . Both axes will, in this instance, be reversed. b. Remove ectoderm and mesoderm of area "B" from the right side of the donor and orient it with the D-V axis still dorso-ventral but in the host wound area at the region of the left limb anlage'. In this instance the A-P axis will be re- versed. (Fig. A) c. Without disturbing the normal field of the host, transplant from other embryos both areas listed above, similarly rotated (i.e., as in "a" and "b") but to the locality of "X" on the hosts. In this manner a direct comparison may be made with the hosts normal limb field. (See diagrams for various other possible rotations of the two primary axes,) d. Bepeat the above rotational transplantations with older embryos, using Urodele stages #55-#56, in order to determine whether there has been any further deter- mination of axes within the limb field. These transplantations should be followed as long as the larvae can be maintained so that the axial relations of the well developed limbs may be determined, and also to study the degree of innervation of the transplanted limb. The control for these experlmenta consists of excision and replacement without rotation of the limb field. EECORD OF BOTATION OF LIMB FIELD AXES LIMB FIELD OPERATIONS 279 280 LIMB FIELD OPERATIONS EFFECT OF SPLITTING THE LIMB FIELD Surgical interference vith the limb field will often result in duplication of limbs, the frequency varying with the species. Amblystoma punctatum is much more favorable than la A. tigrinum. Transplants often result in duplications, due to disturbances to the limb field. Locate the limits of the limb field on Amblystoma stage #29 and then cut away a thin strip of ectoderm along the dorso-ventral axis of the field, splitting the field into two semi-circles (see figure below). With a small hair loop and needles, clean out the meso- derm lying in the exposed region (beneath the removed strip of ectoderm). From another embryo of the same age or slightly older, remove a strip of mid-dorsal ectoderm with underlying neural tube material, trimming the strip down until it will fit into the excavated region of the limb field. See that all loose mesoderm is removed, and then place this strip of ectoderm (epithelial and neural) into the excavated region, hold- ing it in place with a coversllp chip until it "takes" hold. Such a foreign strip of tis- sue will block the Integration of the anterior and the posterior halves of the limb field. Similarly split the limb field into dorsal and ventral halves. Notochord with over- lying ectoderm provides an efficient block, but the notochord should be taken from later stages. BECOED OF SPLIT LIMB FIELDS LIMB F lELD OPERATIONS 28l RELATION OF ECTODERM AN[) MESODERM IN LIM3 FORMATION The experimenta on regeneration (atove) throw some light on the relationship of limb hud ectoderm and mesoderm to normal limh development. Since mesoderm alone cannot he grafted, it is necessary first to transplant ectoderm to which the foreign mesoderm at- taches, and then to make a second transplantation, carrying along together the normally unrelated ectoderm and mesoderm, a. Transplant the ectoderm alone from area "B" (see diagram) to the region "X" on a second embryo. The transplant should he exclusively ectoderm. I.e., all original mesoderm must he cleaned away with a hair loop. Within 2k hours the graft should he completely healed. Prepare another host by removing the entire limh anlage', ecto- and mesoderm of area "X" of the previously operated emhryo. This consists of original limh ectoderm plus flank mesoderm. Note carefully the exact stage of development of all embryos used; the size of the donor tissue; size of the host area; and the extent of the excision of mesoderm. Should this operation produce a perfectly normal limh It would indicate that the limb disc ectoderm is of prime and exclusive importance, in the development of the limb. b. Place flank ectoderm over limb-disc mesoderm, making certain that all flank mesoderm has been cleaned off of the transplant. Should a normal limb develop from this operation, it would suggest the importance of the limb disc mesoderm and the lability of the flank ectoderm. c. Transplant flank ectoderm only over limb disc mesoderm for 2k hours, until thoroughly healed. Then excise and transplant the entire disc material (ecto- and mesoderm) to the flank region of another host. In this case the limb disc mesoderm alone is being transplanted to a foreign region. Should a normal limb develop, this would confirm the importance of the mesoderm in limb development. These heterotopic transplants are more graphic if they are made between A. punctatum and A. tlgrinum (Harrison, 1955) and at stage #29 and #55- EECORD THE RESULTS OF THESE TRANSPLANTATIONS 282 LIMB FIELD OPERATIONS GENETIC FACTORS RELATING TO LIMB FORMATION A, BATES OF GBOWTH - HETEROGONIC: The following experiments are deviaed to demonstrate the growth rate differences in- herent in transplants, especially when made between fast and slow growing species. Up to the beginning of the larval period, when the yolk has been absorbed, the more slowly de- veloping Amblystoma punctatum has a considerable forearm with two digits while at the cor- responding stage of development of Amblystoma tlgrinum (fast developer) and A. mexlcaniun larvae have only mesenchymatous nodules. Shortly after this stage, however, the A. ti- grinum limbs reach and surpass the size of those found in A. punctatum. To demonstrate that the transplanted limb generally maintains its inherent (genetic) rate of growth (Harrison, 19214- ) the following experiments are designed: a. Amblystoma tigrinum limb-bud is completely replaced by A. punctatum limb bud at stage #35. b. A. punctatum limb bud completely replaced by A. tlgrinum limb bud at stage #55 • c. Heterotopic transplants made between A. tigrinum and A. punctatum, with host limb fields untouched. This gives an excellent basis for comparisons. Such transpleintations as these. Involving mesoderm alone, would demonstrate most con- clusively the relative functions of ectoderm and mesoderm in limb formation, outlined in the previous set of experiments. The mesoderm of the limb bud seems to control the form, the rate of growth, and the ultimate size of the urodele limb. And in turn, the limb af- fects the number of ganglion cells in the adult spinal ganglia (Schwind, 1952). DORSAL VIEW VENTRAL VIEW HETEROPLASTIC TRANSPLANTATION OF LIMB FROM A. TIGRINUM TO RIGHT SIDE OF A. PUNCTATUM B. PIGMENTARY DIFFEEENCES: This experiment Is best demonstrated with the white axolotl, A. mexlcanum, in limb transplantations with the highly pigmented A. tigrinum. There are three main types of pig- ment cells; Melanophores, with darker granular pigment; Xanthophores, with yellow lipoid LIMB FIELD OPERATIONS 283 pigment; and Guanophores, with the metallic, gold or silver, guanin. The pigmentation of a transplanted limb always resembles that of the host, except in transplants involving the white axolotl (Harrison, 1935). There are microscopic differences not only in color of pigment cells, as suggested ahove, hut in their size and shape. Incidental to part "A" of this experiment, the effect of the host on the pigment of the donor limh can be determined in the A. tigrinum and A. punctatum transplants. If the rare white axolotl embryos are available, this experiment would most graphic and significant. The pigment cells are derived from the ganglion crest cells which migrate toward the limbs in the earliest motile stage of the embiyos (DuShane, 193'+). A few xanthophores and melanophores are developed when ectoderm from a normally pigmented embryo is grafted to the limb bud of a white form. This suggests that the white axolotl has the melanophores but that the ectoderm of the white axolotl lacks some of the activating principle which is found in the ectoderm of the pigmented species. DuShane suggests that the ganglion crest gives rise to cells which normally develop pigment and that a second factor (ecto- or mesodermal) is necessary to activate the process. Periclinal and sectorial chimeras often appear. (See section on "Neural Crest Origin of Pigment".) EECOED OF GENETIC FACTOES IN LIMB DEVELOPMENT 281+ LIMB F lELD OPERATIONS XENOPLASTIC LIMB TRANSPLANTATIONS These are transplantations 'between different genera and even more distantly related ^ or separated) species. Using Amblystoma donors, such transplants may te attempted to Bufo, Hyla, and Sana. In using Triturus donors, the student should first consult the work of Twitty (1957), and others, which indicates that Triturus embryos produce a toxin (from yolk) that paralyzes emhryos of other genera. Depending upon the availability of material, the following are suggested: a. Anuran belly ectoderm transplanted over the limb-field of a Urodele. b. Urodele limb-field ( ecto- and mesoderm) transplanted to the post-gill region an Anuran embryo in the tail- bud stage. c. Anuran belly ectoderm (stage #l6-#17) transplanted for 2k hours over Urodele limb-field mesoderm; then re-transplanted along with the urodele mesoderm, to the flank region of Urodele embryos. This would clearly demonstrate the rela- tion of foreign (xenoplastic) ectoderm and limb mesoderm in the formation of an heterotopic limb. BECOED OF XENOPLASTIC LIMB TEANSPLANTATIONS LIMB F lELD OPERATIONS 285 REGENERATION OF THE UROOELE LIMB Newly developing limbs of Urodele larvae have remarkatile powers of regeneration. Amblystoma or Tri turns larvae with limbs (stage #38 or older) should be anesthetized in 1/5,000 MS 222 and the digits and limbs cut at various levels and angles, and be allowed to regenerate. The following factors should be considered: a. Whether regenerative potencies can be eliminated by repeated extirpations. b. Whether the regenerated portion is structurally identical with that extirpated. c. Whether the level of the cut is the controlling factor in degree or perfection of regeneration. d. Whether regeneration is achieved equally in limbs previously transplanted to orthotopic or heterotopic positions. e. Whether limb regeneration is controlled by the associated girdle or nerve ele- ments. In order to answer some of the above questions, it will be necessary to acquaint your- self with the normal development and morphology of the limb and digits of the salamander. ng.2 ANTERIOR LIMB AND GIRDLE OF AMBLYSTOMA PUNCTATUM Fig. 1. Mpdlal view of tlie skeleton of the left anterior limb and girdle of Amblystoma punctatum, showing the areas of attachment of the muscles (length 44 mm.) . Fig. 2. Lateral view of the skeleton of the left anterior limb and girdle of A. punctatum, showing the areas of attachment of the muscles (length 44 mm.). ABBREVIATIONS USED IN FIGURES 1 AND 2 C, centrale Ce, carpale Co, coracoid H, humerus I, intermediujn Lat. Ep. , lateral epicondyle M, metacarpale Med. Ep., medial Epicondyle PCo, procoracold Ph, phalanges Proc. Lat., lateral process R, radius Re, radlale Sc, scapula SSc, suprascapula U, ulna Ue, ulnar e From I. W. H. Blount 1955: Jour. Exp. Zool. G^-.kO'J 286 LIMB F lELD OPERATIONS Spalteholtz preparations of llmta and entire larvae should be made. Then, make the follow- ing cuts on appropriate larvae and study the regeneration potencies. a. Cut off the right forelimh at the level of the wrist. b. Cut off the right forelimb halfway between the wrist and girdle, above the bend of the elbow. c. Eepeat "a" and "b" but make the cuts at the greatest possible angles. d. I4ake transverse cuts at the levels of "a" and "b" but extend the cut only half- way through the limb (or wrist), leaving all parts attached. In each case, make a sketch of the limb and the extent of the cut. When regeneration is complete, dissect out the parts to demonstrate the details of regeneration or clear the larva by the Spalteholtz method. This method will show cartilage and bone development but little information relative to nerve and muscle regeneration. BECORD OF LIMB REGENERATION EXPERIMENTS OBSERVATIONS AND TABULATION OF DATA: Drawings, photographs, and preserved specimens constitute the record of the above ex- periments on the limb field. It is most important that detailed records be kept, particu- larly regarding the age and stage of the donor and of the host, the ex\;ent (a-eal and depth) of transplants, the angles of cuts, etc. Confirmatory histological analysis is ex- cellent but Is not always necessary. It is more Important that the student carries out thoroughly one of the above procedures rather than to attempt parts of the entire exercise. LIMB F lELD OPERATIONS 287 DISCUSSION: The time at which the antero-posterior axis of the limt field has been irreversibly polarized has pushed hacl!: to the slit blastopore sta^e (Detwiler, 1955). The dorso-ventral axis undergoes permanent polarization during stages #55 and #5^1. The relationship of the ectoderm and the mesoderm in partial transplants suggests that the specific form of the limb is probably contained within the mesodermal portion of the limb-field while the ecto- dermal covering maintains a passive relation to the growth pattern of the underlying meso- derm. The fusion of transplants, splitting and twinning of limbs, altering of axeSj and the relation of the girdles all Indicate that the fundamental limb forming material is primarily mesodermal. BEF.ERENCES : Balinsky, B. I., I927 - "Xenoplastische Ohrblaschentransplantation zur Frage der Indulrtion einer Extremltatenanlage." Arch. f. Ent. mech. 116:65. Blount, I. W. H., 1955 - "The anatomy of normal and reduplicated limbs in amphibia, with special reference to musculature and vascularization." Jour. Exp. Zool. 69:1+07. Brandt, W., 19^0 - "Experimental production of functioning reduplications - a triple and functioning (juintuple hindlimb in the frog." Jour. Exp. Biol. 17:596. Butler, E. G. & 0. E. Schotte", 19^1 - "Histological alterations in denervated non- regenerating limbs of urodele larvae." Jour. Exp. Zool. 88:507. Detwiler, S. E. , I9I7 - "On the use of Nile Blue Sulphate in embryonic tissue transplan- tation." Anat. Bee. lit-. 14-95. Detwiler, S. E. , 1935 - "On the time of determination of the anterior-posterior axis of the forellmb of Amblystoma." Jour. Exp. Zool. ^k:kO^. Detwiler, S. B. & B. L. Maclean, I9I+O - "Substitution of limbs for brachial somites." Jour. Exp. Zool. 85 •1+1+$. DuShane, G. P., I95I+ - "The origin of pigment cells in amphibia." Science. 80:620. Filatow, D., 1932 - "Entwicklungsbeschleunigung in Abhangigkeit von einer Kunstlichen Vergrosserung der Anlage. Yersuche an Amphibienaugen und extremitaten." Zool. Jahrb. 51:589. Glick, B. , 1951 - "The induction of supernumerary limbs in Amblystoma." Anat. Bee. 1+8:1+07. Graper, L., I927 - "Entwicklungsmechanik der Wirbeltierextremitaten. " Argebn. d. Anat. u. Entwicklungsgesch. 27:639- Hamburger, V., 1958 - "Morphogenetic and axial self-differentiation of transplanted limb primordia of 2-day chick embryos." Jour. Exp. Zool. 77:579- Harrison, B. G., 1955 - "Heteroplastic grafting in embryology." Harvey Lectures, p. II6. Hollinshead, W. H., 1955 - "Determination of the dorso-ventral axis of the forelimb in Amblystoma tigrinum. " Jour. Exp. Zool. 75=185- Kraczmar, A. G. , I9I+6 - "The role of amputation and nerve resection in the regressing limbs of Urodele larvae." Jour. Exp. Zool. 105:''t-01. Korschelt, E., I927 - "Begeneration und Transplantation." Berlin. Nicholas, J. S., 1955 " "The correlation of movement and nerve supply in transplanted limbs of Amblystoma." Jour. Comp. Neur. 57:255. Piatt, J., I9I+2 - "Transplantation of aneurogenic forellmbs in Amblystoma punctatum. " Jour. Exp. Zool. 91:79- Puckett, W. 0., 1956 - "The effects of x-irradiation on limb development and regeneration in Amblystoma." Jour. Morph. 59:175- Bobb B. C, 1929 - "On the nature of hereditary size limitation. II. The growth of parts in relation to the whole." Brit. Jour. Exp. Biol. 6:511. Schotte', 0. E. & E. G. Butler, I9I+I - "Morphological effects of denervation and amputation of limbs in urodele larvae." Jour. Exp. Zool. 87:279. Schwind, J. L., I932 - "Further experiments on limbs containing tissue of two species." Jour. Exp. Zool. 65:565. Stone, L. S., I926 - "Further experiments on the extirpation and transplantation of meso- ectoderm in Amblystoma punctatum." Jour. Exp. Zool. 1+1+ :95. Stultz W. A., 1956 - "Belations of symmetry in the hind limb of Amblystoma punctatum." Jour. Exp. Zool. 72:317- Swett, F. H., 1937 - "Determination of limb-axes." Quart. Bev. Biol. 12:322. Swett F. H. , I9I+5 - "The role of the peribrachial area in the control of reduplication in Amblystoma." Jour. Exp. Zool. 100:67. 288 LIMB FIELD OPERATIONS Taylor, A. C, i9'+5 - "Development of the innervation pattern in the limb bud of the frog." Anat. Bee, 87:579- Thornton, C. S., 1958 - "The histogenesis of the regenerating forellmb of larval Amblystoma after exartlculation of the hiunerus." Jour. Morph. 62:219. Tvrltty, V. C, 1957 - "Experiments on the phenomenon of paralysis produced by a toxin oc- curring in Triturus embiyos." Jour. Exp. Zool. 76:67. Twltty, V. C. & J. L. Schwind, 1951 - "The growth of eyes and limbs transplanted hetero- plastically between two species of Amblystoma." Jour. Exp. Zool. 59=61. Waddington, C. H. , I955 - "Heterogony and the chemical ground-plan of animal growth." Nature. 151:15'+- Warren, A. E., 1959 - "Observations on limb development in Sana sylvatica following uni- lateral eye extirpation." Arch. f. Ent. mech. 159:50. Weiss, P., 1955 - "Homologous function of supernumerary limbs after elimination of sensory control." Proc. Soc. Exp. Biol. & Med. 55=50. Weiss, P. & E. Lltwiller, 1957 - "Quantitative studies on nerve regeneration in Amphibia. II. Innervation of regenerated limbs." Proc. Soc. Exp. Biol. & Med. Weiss, P., 1959 - "The epigenetic factors in limb regeneration of Amphibia." Current Science, August 1959- ". . . we shall do well to give up all the old arguments about form and matter, rep lac ing them by two factors more in accordance with what we know of the universe , that is to say. Organization and Energy. " J. Needham 19i2 "Continuity of organization is indeed a species of preformation. Genes are not produced e pigenet ically but by division of pre-existing forms." C. 0. Whitman "However great the difference between an infusor ian and a highly organized animal, it cannot be a qua 1 1 tat ive one . " C. 0. H/iitman EYE FIELD OPERATIONS PURPOSE ■• To determine the anlage for the optlco-ocular apparatus; the limits of regenera- tion of parte of the eye field; the time and extent of self-differentiation; the induc- tive relations; and the extent of experimentally induced cyclopia. MATERIALS : Biolopjical: Anuran emhryos stages #7 to #17; Urodele embryos stages #11 to #50. For heteroplastic transplajitations, use: Eana: cateshiana, paluatris, pipiens, and sylvatica. Amblystoma: punctatum and tigrinum. Technical: Standard operating equipment. METHOD: Precautions: 1. It is Important that the student hecome thoroughly acquainted with the normal development of the eye. This may be accomplished by dissecting living and pre- served embryos at the stages listed above. 2. The usual precautions must be observed for operations (see section on "Wound Healing" ) . 5. The optimum temperature at which to rear the operated embryos differs for the Anura and Urodela. For the Anura the range is 18°C. to 25 C. and for the Urodela it is about 12°C. to 18°C., depending upon the species. Control For the excision, cauterizing, transplantation, and regeneration experiments the controls will be different. In many instances the untouched (bilateral) aide may be considered as the control. Procedure: PRELIMINARY STUDIES ON THE EYE FIELD It is necessary to give here a brief description of the topography of the eye-forming materials. This is made possible largely by the work of Vogt (I929) in his classical paper on vital staining and mapping of anlagen, from Woerdeman (I929), Manchot (I929), Petersen (I925) and Fischel (I92I). OpKc Stalk- Course optic n«i ittc 51alk 'lapetum Fig. Fig. Fig. Fig. 1. 2. Sc)iema of the topographical relations of the eye anlagen ( ' Augenplatten' ) in the early neural plate of an axolotl egg. (Copied from Woerdeman, 1929.) Schema of a young urodelan neurula: the cross-hatched territory represents tfie material for the optic vesicles, optic stalks, and the recessus opticus, the broken line outlines the territory which the same material will occupy m the stage when the neural folds have elevated. (Copied from Manchot, 1929.) Schema of the topography of the optic anlage, according to Peterson, 1923. Schema of the topography of the optic anlagen, according to Fischel, 1921. From Adelmann 1950: Jour. Exp. Zool. 57:225. -289- 290 EYE FIELD OPERATIONS OPTIC VESICLES In the Anura the eye forming materials have been localised in the late blastula as lying about 1+0° to 50° above the equator (see section on "Vital Staining and Morphogene- tic Movements"). In the early neunila when the boundaries of the medullary plate are bare- ly discernible, the eye anlage occupies a circular region with a diameter of about l/j the greatest breadth of the neural plate, at its antero-lateral boundary (see diagrajns). Dur- ing the elevation of the neural folds there is a ventro-lateral evaglnation of the newly formed brain cavity to form the optic -FOREBRAH vesicles. A narrow strip of median material separates the two /^f\ eye anlage. This median strip forms the chiasma and a portion ""■"" of the lamina terminalis. Actually, therefore, the two eye anlagen are not completely separated. Mangold (1S;28) found that when the presumptive eye field had been underlain with archenterlc roof, as early as the medium sized yolk-plug stage (Anura stage #11 or Urodele stage #15), the field had eye forming potencies when introduced into a younger blastocoele. Mangold (1951) discussed the possible re- lation of the whole organism, the anlage itself, and the immedi- ate environment of the anlage in the segregation of the eye form- ing potencies. It is one of the purposes of this exercise that the student determine the exact time and extent of the deter- mination and segregation of the eye-forming potencies. There are, however, species differences. (See Glossary for definitions of "double assurance" in relation to Bana esculenta and dependent differentiation.) (Redrawn from Spemann, 1958) BEEOLE POIMT Exploratory dissections: 1. Using Anura stages #l6 to #18 (or Urodela stages #20 to #50), dissect away the ectoderm of the head region to expose the optic vesicles and the central ner- vous system. This may be done with living material, or with formalin-hardened specimens. 2. Anesthetize Anuran stages #21 and older (or corresponding Urodele embryos) in 1/5,000 MS 222 (or 0.0^^ chloretone) and dissect out the entire optico-ocular apparatus. Note the position and state of development of the lens. The larger tadpoles should be pinned down to a Permoplast base and covered with lens paper (except for the head). 5 • Eemove the lens of later stages as follows. Pierce the skin in front of and ventral to the eye with a sharp glass needle, push the needle backward or up- ward between the eye and the cornea parallel to the cornea. Pierce again at the posterior or upper margin of the eye. Cut the cornea by inserting the needle beneath it and rubbing a scalpel against it. Take two small but unequal sized hair loops and pass them over the lens, from opposite sides, and then pull them apart. In this manner the lens will be cleanly removed (as with scissors) with minimum of damage to the other parts of the eye. U. In a somewhat similar GUSS HICRO-MEEOLE METHOD OF CUTTING THE CORNEA TO REMOVE THE LENS FROM THE AAIPHIBIAN EYE TO TEST FOR WOLFFIAN (LENS) REGENERATION (Needle is inserted tlirough cornea on one side of the iris, passed between the cornea and the lens to emerge tlirough the cornea on the far side of the Iris. The needle Is lifted against the cornea, and a sharp scalpel is then scraped against the needle, providing a cutting edge to , ,, cut a silt In the cornea.) remove the entire eye ball of adult frogs or salamanders. Such hemorrhage as occurs is generally not of any serious consequence (see diagram of adult aye). manner, but with scissors. EYE FIELD OPERATIONS 29 1 RANA PIPIENS LENS ANLAGE 5 ..■•■ REGIONS OF OPTIC VESICLE ANLAGE INDICATED BEFORE CLOSURE OF FOREBRAIN STAGE 13-14 OPTIC PROTUBERANCE A. STAGE 17 (34 HRS. LATER) DEFECT EXPERIMENTS EXCISIONS:* (See papers by Adelmann) 1. Anura (stage #lU) or Urodela (stage #15) vr^^mrr fhn a. Study the accompanying schematized diagram of a neurula showing the limits of the eye field. Eemove the ectoderm only from either area "A" or area "B". -*% ..^' ^^.„ Camera-luclda drawing of a section through the prechordal re'^ion of an Amblystoraa neurula (Harrison stage #lo) . ine mesoderm has Just begun to separate from the lateral por- tion of the roof of the archenteron. This gives an idea as to the distribution of the (mesentoderm) substrate referred to In the excision experiments. y Drawing from Adelmann 1950: Jour. Kxp. Zool. 57:223. * See section on "Wound Healing" for details of post-operative care. 292 EYE FIELD OPERATIONS EYE FIELD OPERATIONS NEURAL FOLD jd ■. > ; cW ANURAH STAGE 13-14 NEURAL OR MEDULLARY PLATE NEURAL FOLD OPTIC VESICLE FIELD LENS FIELD MESENCHYME SECTION THROUGH PRESUMPTIVE FOREBRAIN VESICLE FIELD -LENS FIELD HOT NEEDLE HOT NEEDLE PRIMORDIA OF OPTICO - OCULAR APPARATUS (REDRAWN FROM SPEMANN '38) TRANSPLANT ■ ARCHENTERIC ROOF STAGE 12 STAGE 10 INDUCTION OF EYE FIELD (Eedrawn from Spemann 1958) EYE FIELD OPERATIONS 295 b. Bemove ectoderm and autatrate ( mesentoderm) from the same area ("A" or "B") of another neurula. c. Bemove ectoderm only from area "C". d. Bemove ectoderm and auhstrate { meaentoderm) from area "C". e. Bemove the entire area "A - C - B", ectoderm only. f. Bemove the entire area "A - C - B", ectoderm and suhstrate (meaento- derm) . Anura* (atage #l6 or #1?) or Urodela (stage #21 to #25) a. Bemove the ectoderm only from over the right optic vealcle. h. Bemove the ectoderm and the optic vesicle from the right side. Diagram, showing In stipple the area which it Is necessary to remove in order to prevent complete- ly the formation of an eye on the operated side. From Adelmann I929 : Jour. Exp. Zool. 5l+:2i<-9 c. Insert a sharp operating (glass) needle, beneath the ectoderm and, with an up-lifting movement, cut the ectoderm along the dorsal, pos- terior, and ventral margins of a rectangular .area which includes the entire eye field. This will provide a flap of ectoderm, with an anterior hing?. Deflect this flap forward, and with a stiff hair loop, scoop out the entire optic vesicle from beneath. Beplace the • ectodermal flap and allow it to heal in place. If the excavation is so extensive that there is no base upon which the ectoderm can lie, fill in the hole with yolk from another embryo. B. CAUTEBIZATION: Using a heated and slightly bent needle, attempt to cauterize the entire optic-ocular prlmordia on. one side, as indicated in the accompanying diagrams. It may be necessary to dip the needle into glycerine to prevent the hot needle from drawing out some of the cellular contents of the neurula. The entire anlage may be found both within and without the medullary plate area. This is a delicate operation and there will be high mortality, but the results (if the operation is properly executed) will be very significant. When the embiyos have reached the external gill atage, fix them in 10^ formaldehyde and diasect out the optico-ocular apparatus to compare It with the controls. During the healing and early development of the excised areas, macroscopic examinations and records should be made. (See Glossary on "Double Assurance". This does not hold for Bana aylvatica, Bana palustris, Bana catesblana or Amblystoma maculatum. ) SELF-DIFFERENTIATION OF THE EYE ANLAGE Consult the exercise on "The Culturlng of Isolated Amphibian Anlage" for the descrip- tion of the basic procedure necessaiy for this part of the exercise. Isolate and attempt to culture the same areas used in the "Defect Experiments" ( above) . The degree of self -differentiation is indicated by the degree of independent develop- ment in isolation culture. This part of the exercise should Inform the student as to Just when the lens and the vesicle (retina, etc.) are "self-differentiated". * Bana aylvatica or Bana palustria are better for this than is Bana pipiens. 291+ EYE FIELD OPERATIONS EYE INDUCTIONS This is an extremely delicate operation (Mangold, 1951) and ahould be attempted by only those students who have proven their skill In operative procedures. Gastnilae (Anura stages #10 and #12) are decapsulated. The donor (stage #12) Is dis- sected so as to expose the most anterior portion of the archenteric roof. This material la excised In one piece and la quickly Inserted through the hlastocoellc roof of the younger (stage #10) gastrula (see diagrams). If the normal "Inductive" (see Gloasaiy) In- fluences are exerted on the overlying ectoderm of the blastocoellc roof, accessory optic structures will be formed. (See exercise on "The Organizer" for procedures.) TRANSPLANTATIONS Under this heading will be Included the simpler transplantations of optic cup and/or lens primordlum In order to determine their interrelationship in the normal development of the eye as a whole. There will also be Included the homoplastic, heteroplastic, and xeno- plastic transplantations of larval eyes. LENS INDUCTION: (See Stone & Dinnean 1914-0 and Liedke I9U2) 1. Remove the presumptive lens ectoderm from over the optic vesicle or cup of Anuran stage #l6 or #17, (Urodela stages #21 to #26)* without injuring the underlying structures. Quickly excise a slightly larger piece of belly ecto- derm from another embryo, of the same stage previously stained with Nile Blue Sulphate. Place it over the exposed optic vesicle. Gently pat it into place and, if necessary, hold It in place with a Briicke, lens paper, or piece of coveralip. It should heal within 20 to 30 minutes. (See section on "Wound Healing" . ) 2. Remove the ectoderm from over the optic vesicle of Anuran stage #17 (Urodela stages #21 to #26)*. Prepare a host embiyo (of the same stage) by making a deep pit ventral to the somites at about the mid-body region, leaving the flap of ectoderm over the pit Intact. (See A-2-c \inder "Defect Experiments" on the preceding page.) Quickly cut out the optic vesicle of the donor and transfer it to the excavated pit of the host. Orient the vesicle in the same position as in the donor, i.e., with the bulbous part of the vesicle facing outward. Replace the ectodermal flap over the transplanted optic vesicle, and hold it in place with Briicke until healed. These two experiments are reciprocally related. Part 1 will indicate whether foreign (non-presumptive) ectoderm will respond to inductive Influences from the Intact optic vesicle and Part 2 will indicate whether the optic vesicle, transplanted to a foreign site, can there induce a lens in the over- lying (foreign) ectoderm. All such embryos should be allowed to progress to the external gill stage before dissection analysis. TRANSPLANT ATIONS : TRANSPLANTING THE ANLAGE When the anlage is transplanted to a slightly older host, the conditions for the expression of any self-differentiation are somewhat different from those of an isola- tion culture. However, the nutritive factors are generally the more favorable, and eucial relations can be determined. These operations are best on Urodele embryos.** 1. Decapsulate some Urodele embryos in stage #11+, and locate the eye field at the anterior limit of the medullary plate (see figure on following page). Stain the entire donor In l/500,000 Nile Blue Sulphate. * After Urodele stage #26 (Anura stage #17) the potential lens forming ectoderm becomes ad- herent to the underlying optic cup, and cannot be completely removed. The older embryos can be anesthetized In MS 222. **Follow the standard operative procedures described elsewhere. See Schwind (1957) for results with the Anura. EYE FIELD OPERATIONS 295 2. Prepare host larvae 'by excising the ectoderm, mesoderm and some of the under- lying yolk on the lateral belly region of stage ^h or #25 ( see figures below) , The prepared wound should "be slightly larger than the prospective graft. 5. On the point of a needle transfer from the stained donor a. The right optic anlage b. The median transverse neural fold and medullary plate c. The entire (anterior) optic anlage (A-C-B in figxire above) on the prepared host sites. Hold in place with Briicke, or by hand, for a few minutes until the graft becomes adherent. The vital dye will indicate the limits of the graft. Scliema of a typical experiment, showing the re- lations of the transplanted median and left lateral strips of neural plate and their orien- tation in the hosts. R, right; L, left. From Adelmann 1950: Jour. Exp, Zool. 57:225. By recording the exact shape of the graft at the time of excision, it will be possible to record the axial relations of the graft in the host site. DRAWINGS OF ANLAGE TRANSPLANTATIONS 296 EYE FIELD OPERATIONS TRANSPLANTING THE OPTIC VESICLE AND LENS ECTODERM The most complete study of heteroplastic eye transplantations has been made by Harri- son (1929) and recently Eakln and Harris (19^5) have determined the degree and onset of tissue incompatibility in xenoplastic transplants using the eye as the test object. As with other organ anlagen, eye transplants between the various species of Amblystoma are most successful and instructive. Since there are intrinsic differences in growth rate, these become exaggerated in such heteroplastic transplants. Harrison (I929) pointed out that homotoplc transplantations (in order to produce func- tional eyes) will succeed best at stages #27 to #29 at which time the vesicle is well marked off from the stalk and mortality is consequently lower. If, however, it is desired that the mesodermal and mesectodermal tissues be eliminated from the study, he suggests using stage #21, Just after the closure of the neural folds. In any case, after making a circular incision around the eye region the optic vesicle at stage #21 (or cup at stage #28) is readily separated from its surroundings and the optic stalk may be cut close to its origin from the brain. The whole may then be reioved without disturbing any of the adjacent mesodermal (mandibular arch) or mesectodermal (ganglion crest) structures. When Eyes transplanted from Amblystoma tigrinum to the belly region of Amblystoma puiictatum at stage #29. Amblystoma eye transplanted to the region of the ear. (From Detwller 1914-5: Jour, Comp. Neur. 82:ll+5) reciprocal transplants are made it must be remembered that the eye vesicle of the A. punc- tatum embryo, at comparable early stages, is much larger than that of A. tigrinum. In consequence it is more difficult to place the punctatum graft in the A. tigrinum host than vice versa. The A. tigrinum optic vesicle, at a comparable stage, is smaller than that of A. punctatum, but it grows more rapidly and soon surpasses the A. punctatum eye in size (see illustrations from Harrison's paper). Procedure:* (Transplants between A. punctatum and A. tigrinum embryos or from A. mexicanum to A. punctatum stages #27 to #29.) A. First attempt to transplant the entire optic vesicle with overlying ectoderm from one species (Amblystoma) to another Into an heterotopic region, i.e., to the lateral belly region. (See photographs.) * Follow the usual operative procedures described elsewhere. EYE F lELD OPERATIONS 297 has been accomplished, transplant the entire optic vesicle and over- lying ectoderm of another donor to the excavated optic region of another host (homotopic and heteroplastic transplemt) . It would he hest to make the first attempts from A. tigrlnum to A. punctatum. Make the reciprocal transplant indicated in "B", i.e., from A. pianctatum to A. tigrinum, including the entire optic vesicle and overlying lens ectoderm. Make a I'arge "U" shaped Incision through the ectoderm covering the optic vesicle of A. punctatum at stage #27, with the open end of the "U" (uncut mar- gin) toward the anterior, remaining as a hinge. Deflect this flap of ectoderm forward, and scoop out the optic vesicle, cutting it at the origin from the hrain. Quickly remove the overlying ectoderm from a donor embryo optic vesicle (same stage hut either A. tigrinum or A. mexicanum) and cut it out without damage. Place it in the previously prepared host region, beneath the flap of host ectoderm. So far as possible attempt to orient the donor optic vesicle in the a&me position as the original host vesicle. DRAWINGS AND BECOED OF HETEROPLASTIC- HOMOTOPIC TRANSPLANTATIONS 298 EYE FIELD OPERATIONS GRAFTED EYES IN COMPARISON WITH THE NORMAL RIGHT EYES INTERCHANGED BETWEEN A. PUNCTATUM AND A. TIGRINUM y<- '.\*?*' 10 &^ I ' ;' ''» f ■ r\ i 10 Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. Fig. 10, Punctatum larva from above. Same from right, showing large grafted A. tigrlimra eye. Same from left, showing normal eye. A. tlgrlrium larva from aoove. Same from right, showing small grafted A. punctatum eye. Same from left, sliowing normal eye. A. punctatum, in metamorphosis, showing grafted A. tigrinum eye on the right side. Same, left side, showing normal (control) eye. Control A. tigrinum larva. Larva of Arablystoma tigrinum with grafted optic vesicle from A. punctatum which has developed Into a smaller eye, shown on the right. Specimen 79 mm. In length, 96 days after operation. From Harrison 1929: Arch. f. Ent. moch. 120:1 EYE FIELD OPERATIONS 299 WOLFFIAN REGENERATION This term Is derived from the work of- Wolff who, in l895, found that when he removed the lens from the eye of the European Triton that a new lens would regenerate. Such re- generation is presumed to occur from the dorsal rim of the iris by a hudding process and has been demonstrated for a large group of Urodeles, with but few exceptions. Its occur- rence among the Anura is questioned. Among the Urodeles it has been described for Triturus taeniatus, T. crlstatus. Salaimandra and the Axolotl (Wachs, 191*+); for Triturus viridescens (Stone and Chace, 19^1); for Triturus torosus (Dinnean, 19*<-2); for Amblystoma tigrlnum up to stage #i*-5 (Ballard, I956). It has also been seen in Amblystoma Jefferson- lanum, A. mlcrostomum, and A. opacum as well as in the Japanese fire salamander, Triturus pyrrhogaster. In fact, Triturus (of all species) seems the most reliable, Amblystoma un- dependable, and the Anura generally negative. 1. Select larvae of A. tigrlnum (up to stage #'+5), A. opacum, or any species of Triturus. The operation should be performed on a minimum of 10 specimens, all of the same age and stage. 2. Anesthetize the larva in l/5,000 MS 222 (or 0.04^ chloretone) and place it in a Pennoplast depression made to fit so that the larva lies on its side. 5. Fasten the larval body into position with strips of lens paper held to the Permoplast with insect pins. h. To remove the lens from the right eye use a sharply- tapered glass needle (strong and pointed) and pierce the cornea at one side and run the needle beneath the cornea, across the lens, and out through the far side of the cornea. Avoid injury to the underlying lens. Gently scrape a sharp scalpel against the cornea, over the needle, thereby using the two Instruments to cut through the cornea. 5. The lens is generally glass-clear. After separating the lens from the Iris, with the glass needle, pick the lena out with #5 'watchmaker' s forceps. If this proves difficult, it may be possible to reach under the lens with the glass needle and lift it out. There should be no hemorrhage. Specimens in which the retina is Injured should be discarded. The slit in the cornea will close and heal by itself. Healing is most rapid in bicarbonate- free Standard or Growing Medium. Examine the removed lens under the microscope. Keep the operated larvae in separate, properly marked, finger bowls at controlled tem- peratures. 6. At weekly intervals anesthetize the operated larvae and examine the eye for signs of regeneration. Note changes in the pupil immediately after the opera- tion and at regular Intervals of 3 to U days thereafter. These changes are indicative of changes in respect to the lens within. 7. After one month, during which the larvae are maximally fed, they should be fixed in 10^ formaldehyde and the eyes dissected. The unoperated eye may be considered as the control. Estimate the ratio of the diameter of the lens against the diameter of the eye, for both the control and the operated sides, to determine the degree of regeneration. 8. If there are abundant larvae, the progress of Wolffian regeneration can be best studied by sectioning the eyes at 1+ to 5 day intervals after the operation. There has been considerable discussion, in recent years, about the possibility and the method of lens regeneration in later larval stages (Schotte and Hummel, 1959 and Stone and Sapir, 1914-0). The latter authors investigated Urodeles, Anura, and Fish, all of which were at least 25 and many as much as 80 mm. In length, and they came to the conclusion that at these advanced stages the rim of the iris does not possess the power to regenerate a new lens. In fact, there is evidence of species variation at the earlier larval stages for Stone and Dinnean (1914-0) found no Wolffian regeneration in A. punctatum at any time. Stone and Sapir (191+0) state: "Among the Triturus this unique phenomenon has been proven beyond all doubt. It opens up an interesting field of study to determine more clearly what factors inhibit and release the regeneration of a lens from the rim of the Iris in this group of salamanders." 500 EYE FIELD OPERATIONS ENUCLEATION AND PIGMENTARY RESPONSES Scharrer (1932) in reference to salamander larvae, stated that "• . . .In addition to sight and smell, the lateral line sense organs may play a role in obtaining food" and Nicholas (1922) claimed that in the absence of the eyes, the sense of smell became para- mount in the feeding reaction as evidenced by the animal's positive response to substances diffusing in the water. Detwller and Copenhaver (19'+0) state: "We wish to emphasize the fact that in the absence of both the eyes and the nasal placodes the larvae feed as well as do the normal animals." Utilizing these facts, it ia possible to enucleate (i.e., re- move the eyes) Amblystoma larvae (stages #25 to #27) and rear them on Enchytrea (white worms) in light, darkness, and even under various concentrations of monochromatic light- ing, to determine the relation of the eyes to both growth and pigmentary responses of the skin. 1. Enucleate Amblystoma larvae (stages #25 to #27-) and keep them in adequate aquaria for 5 to U days in order to select those individuals which survived the operation most satisfactorily. If necessary, anesthetize them, before enucleation, in l/3,000 MS 222. 2. Prepare a completely darkened environment (e.g., photographic dark room) but one in which the temperature does not vary from that of the light environment. This may require the circulation of air with a fan, for any dark Cover will absorb radiant energy more rapidly that a light colored cover. The temperature must be checked at least once dally. Another and most satisfactory method is to coat the outside (sides and bottom) of finger bowls with flat black paint, and provide an overlapping black-painted cover. These darkened finger bowls, along with the controls (unpainted finger bowls), can then be kept together in a conatant-temperature water bath. Daylight (but not sunlight) should be pro- vided. If this is not possible, controlled artificial light (without heat) should be provided. 5. Place in each of 10 blackened finger bowls a single enucleated Amblystoma with 50 cc. of growing medium. Place in each of 10 more blackened finger bowls a single normal (unoperated) Amblystoma. 1|. In a similar manner prepare 10 unpainted finger bowls, placing in each a single enucleated Amblystoma and in each of 10 more unpainted finger bowls, place a single normal (unoperated) Amblystoma. 5. All kO finger bowls should be kept at the same temperature. The water should be changed on alternate days. The young larvae may be fed first on small Daphnla and, as they grow, on small and finally on large Enchytreid worms. The feeding should be identical for all larvae, experlmentals and controls, in dark and in light. 6. The darkened animals may be changed and fed in a photographic darkroom, with a very dim red (photographic) light, in minimum time. The quickest procedure ia to pour the larva and medium through a coarse sieve, to eliminate faecal material. 7. Keep the larvae to the time of metamorphosis (50 to 60 days), using this change as one criterion of growth rate. Meaaurementa of all larvae should be taken at bi-weekly intervals, and notations made on general coloration. Measurements can be made rapidly by pasting millimeter graph paper on the underaide of a flat-bottomed finger bowl (or Petri dish) into which the larva is placed for a quick total length measurement. ■»«»»«**»***»* If facilities and time allow, there are other variables to this experiment: 1. Instead of using a black-painted cover for the darkened finger bowls, cover them with glass (of Wratten celluloid) color filters which allows various mono- chromatic radiations through to the larvae to determine the relative value of parts of the spectrum in pigmentary response. 2. The eyes may be removed and transplanted to heterotopic positions, where they do not acquire nerve connections with the central nervous system, to determine whether they then function in pigmentary response. EYE FIELD OPERATIONS 50I 5. The dark and light-adjusted enucleated (and normal) larvae may be transferred to the opposite environment (dark to light and vice versa) to determine the degree of adaptibility (adjustment) in respect to a definite time interval. (Detwller & Copenhaver in 19'+2 state that dark-adapted eyeless larvae are pale "but darken in moderate lighting, while dark-adapted normal larvae tend to be- come lighter colored In moderate lighting. ) The conclusions from this set of observations should relate both the growth rate and to the pigmentary responses of eyeless larvae. THE EXPERIMENTAL PRODUCTION OF CYCLOPIA Adelmann (I936) states: "The hope of one day attaining an adeq^uate understanding of cyclopia is considerably strengthened by two important considerations, first, the fact that the anomaly may be experimentally produced with considerable ease, and secondly, the fact that experimentally produced cyclopean monsters exhibit essentially the same features as those spontaneously arising." Stockard ( I907 to I9IO) produced cyclopean fish with vari- ous concentrations of NaCl, LlCl, NaOH, and anyl alcohol. Others have used acetone, and butyric alcohol and even physical variables. Amphibian cyclopean monsters have been pro- duced by treating the eggs with lithium chloride, ethyl alcohol, chloralhydrate (LePlat, 1919, Cotronei, 1922, Guareschi, 195*+, and Adelmann, 193*+) phenol and chloretone ( Lehmann, 1935). Cyclopia can also be produced by surgical interference with early cleavage stages up to gastrulation, by constriction (Spemann, 190^), and by excision of parts of the archen- teric roof (Mangold, I93I). The experimental procedure is in general to expose the early blastula of any Amphibian to from 0.2^ to l.O'jt LlCl for periods up to 2k hours, then returning them to normal medium whereupon many of the surviving embryos will develop cyclopia. The effect is essentially one of vegetalization. Since the procedure is described In detail under "The Chemical Separation of Growth and Development" it will not be further discussed here. DISCUSSION: The embryonic eye is made up of two major parts, each of which originates from the ectoderm. The optic vesicle is the first to develop, being an evagination from the dien- cephalon. When this brain ectoderm makes contact with the overlying head ectoderm it "induces" the thickening of this ectoderm to form the lens placode. (See exception under the term "double assurance" in the Glossary.) This placode then invaginates to form a lens vesicle which becomes incorporated into the developing optic cup. The head ectoderm (from which the lens was derived) then closes over the lens to form the ectodermal portion of the (transparent) cornea. In the meantime mesenchyme (mesoderm) invades the whole eye structure, to give rise to the blood vessels, connective tissue, and finally the muscles of the eye. That the mesentodermal substrate has something to do with the development of the eye field has been demonstrated (Adelmann, 1957) • This eye-field is determined prior to the closure of the neural folds, as proven by excision and transplantation experiments. In the heteroplastic and homotopic eye transplantations involving A. punctatum, A. tigrinum, and A. mexicanum (the axolotl) Harrison (I929) has demonstrated that the velocity of growth and, to a certain extent, the ultimate size of the eyes are due to Intrinsic (genetic) factors of the donor tissues. The tigrinum eyes in punctatum hosts often exceeded the donor control eyes, and the punctatum eyes in tigrinum hosts often were smaller than the control eyes, explained by Harrison as due to factors in the circu- lating medium of the host which affected the growth rate of the graft. The form and func- tion of the grafted eyes appeared to be quite normal. Even the intrinsic tendencies of the lens and/or the optic vesicles were maintained when in grafts, when they were from different genetic sources. In all cases where the optic nerve failed to connect, there was marked hypoplasia of the wall of the midbrain on the opposite side. 502 EYE FIELD OPERATIONS Schwind (1957) tas shown that heteroplastic eye grafts between three species of Eana Invariably failed to develop. Eakin and Harris (19'+5) used the optic vesicle as a test object in some xenoplastic transplantations between Urodele donors and Anuran hosts. Grafts from Tri turns or Amblystoma donors to Ifyla hosts never survived for very long, and were eventually destroyed, generally within a week. They state that "Incompatability be- tween host and xenoplastic transplant is regarded as a humoral and cellular antagonism of the host in response to alien substances which diffuse out of the graft into the body of the host." That the eyes have not lost their power of adjustment has been demonstrated recently by Stone and his co-workers (Stone & Ellison, 19^+5) by the exchanging of eyes between adults salamanders of different species. There is apparently a regression of the morphol- ogy and physiology of the eye and a recovery of both the normal structure and fxmction, even in the adult eyes. BEFERMCES: Adelmann, H. B. , I956 - "The problem of cyclopia." Quart. Eev. Biol. Il:l6l & 28i^. Adelmann, H. B. , 1957 - "Experimental studies on the development of the eye. IV. The ef- fect of the partial and complete excision of the prechordal substrate on the develop- ment of eyes of Amblystoma punctatum. " Jour. Exp. Zool. 75:199- Alderman, A. L., I958 - "A factor influencing the bllaterality of the eye rudiment in ^la regllla." Anat. Bee. 72:297. Ballard, W. W., 1959 - "Mutual size regulation between eyeball and lens in Amblystoma, studied by means of heteroplastic transplantation." Jour. Exp. Zool. 8l:26l. Harden, B. B., 19'<-5 - "Changes in the pigmentation of the iris in metamorphosing amphibian larvae." Jour, Exp. Zool. 92:171. Detwiler, S. B., 1929 - "Some observations upon grafted eyes of frog larvae." Arch. f. Ent. mech. Il6:555. Detwiler, S. B. , 19^^ - "Behavior of Amblystoma larvae lacking In forebraln, eyes, and nasal placodes." Proc. Soc. Exp. Biol. & Med. 56:195. Detwiler, S. B. & W. M. CoperJiaver, 19'+2 - "Further experiments dealing with embryonic enucleation In Amblystoma." Proc. Soc. Exp. Biol. & Med. 51:53^' Dinnean, F. L., 19'*-2 - "Lens regeneration from iris and its inhibition by lens reimplanta- tion in Trlturus torosus larvae." Jour. Exp. Zool. 90:'+6l. Dragomirov, W., 1955 - "Determination des Augenkeimes bei Amphlblen." Acad. Sci. d Ukraine, Trav. Inst. zool. et biol. 8:25 (see also 1955 Arch. f. Ent. mech. 129:522). Filatow, D. , 1957 - "Uber die Linsenlnudzlerung nach Entfernung des Chorda-mesoderms bei Eana temporaria." Zool. Jarb. Abt. allg. Zool. u. Physiol. 58:1. Greene, W. F. & H. Laurens, 1925 - "The effect of extirpation of the embryonic ear and eye on equilibration in Amblystoma punctatum." Am. Jour. Physiol. 64:120. Hall, T. S., 191+2 - "The mode of action of lithium salts in amphibian development." Jour. Exp. Zool. 89:1. Handford, S. W. , 19'+5 - "The relation of age and temperature to the relative growth of the eyes of Amblystoma." Jour. Exp. Zool. 98:127. Harrison, B. G., I929 - "Correlations in the development and growth of the eye studied by means of heteroplastic transplantation." Arch. f. Ent. mech. 120:1. Harrison, E, G. , I955 - "Some difficulties of the determination problem." Am. Nat. 68:506. Hewitt, D. C, 195^+ - "Xenoplastic transplantation of amphibian eye rudiments." Jour. Exp. Zool. 69:255. Holtf rater, J., 1955 - "Uber das Verhalten von Anurenektoderm In Urodelenkeimes." Arch. f. Ent. mech. 155:^+27. Kollros, J. J., 19'4-5 - "Experimental studies on the development of the corneal reflex in amphibia. III. The influence of the periphery upon the reflex center." Jour. Exp. Zool. 92:121. Lehmann, F. E., 195^+ - "Die Llnsenblldung von Eana fusca in Ihrer Abhangigkeit von chemlschen Einflussen." Arch. f. Ent. mech. 151:555- Leplat, G., I919 - "Action du milieu sur la developpement des larves d'amphlbiens. Localisation et differentiation des premiers ebauchee oculalres chez les vertebras. Cyclople et Anophthalmle." Arch, de Biol. 50:251. Liedke, K. B. , I9U2 - "Lens competence In Eana plpiens."' Jour. Exp. Zool. 90:551. EYE F lELD OPERAT IONS 305 Lopaschov, G. V., 1956 - "5ye-lnducing suTDstancea . " Biol. Zhurn. 5. Manchot, E., I929 - "Abgrenzung des Aiigenmateriala und anderer Tellbezirhe in der Medullar- platte." Arch. f. Ent. mech. 116:689. Mangold, 0., 1951 - "Das Determinationspor"blem. 3> Teil. Das Wirbeltierauge in der Ent- wicklung und Eegeneration. " Ergetnlsse des Biol. 7:195- Manuilowa, N. A. & M. N. Klslow, 195i)- - "ITber die Einwirkung des Augentechers auf das ventrale und determinlerte Epithel tei AmpMbien bei Horn- und Heterotransplanta- tionen." Zool. Jarhb. Abt. f. Allgem. Zool. 55:521. Mikami, Y., 1958 - "Experiments on the formation of free lenses in Triturus pyrrhogaster with special reference to Harrison's experimental results in Amblystoma. " Proc. Imp. Acad. Tokyo. 14:195. Nicholas, J. S., 1922 - "The reactions of Amblystoma tigrinum to olfactory stimijli." Jour. Exp. Zool. 55:257. Nicholas, J. S., 1950 - "Movements in transplanted limbs innervated by eye muscle nerves." Anat. Eec. h^:2^k, Okada, Y. K. & Y. Mikami, I957 - "Inductive effects of tissues other than retina on the presumptive lens epithelium." Proc. Imp. Acad. Tokyo. 15:285. Patch, E. M., 19'4-1 - "Cataracts in Amblystoma tigrinum larvae fed experimental diets." Proc. Soc. Exp. Biol. & Med. '+6:205. Perrl, E. , 195*+ - "Eicerche sul compartamento dell'abbozzo oculare di Anfibl in conlcloni dl espianto." Arch. f. Ent. mech. 131:115. Piatt, J., 19'+1 - "Grafting of limbs in place of the eye in Amblystoma." Jour. Exp. Zool. 86:77. Popoff, W. M. , 1957 - "Uber den morphogenen Elnfluss des Augenbechers auf verachledene emnryonale Gewebe und auf die Anlage einiger Organe." Zool. Jahrb. 58. Eegnier, J. & A. Quevauviller, I959 - "Quantitative effect of atropln on enucleated eye of the frog." Comp. rendu. Soc. Biol. 150:1215. Beyer, E. W., l'9l+8 - "An experimental study of lens regeneration in Triturus viridescens. " Jour. Exp. Zool. 107. Eichards, O.'W., 19^1 - "Variation and change of sign of the relative growth ratio of larval salamander eyes transplanted on starving hosts." Growth. 5:171. Eotmann, E., 1959 - "Der Anteil von Induktor und reagierendem Gewebe an der Entwlcklung der Amphibienlinse." Arch. f. Ent. mech. 159:1. Sato, T., 191+0 - "Vergleichende Studien uber die Geschwindigkeit der Wilff'schen Llnsen- regeneration bei Triton taenlatus und bei Dlenyctylus pyrrhogaster." Arch. f. Ent. mech. 11+0:570. Scharrer, E., I932 - "Experiments on the function of the lateral-line organs in the larvae of Amblystoma punctatum. " Jour. Exp. Zool. 6l:109. Schotte, 0. E. & K. P. Hummel, 1959 - "Lens induction at the expense of regenerating tis- sues of amphibians." Jour. Exp. Zool. 80:151. Schwind, J. L,, 1957 - "Tissue reactions after homoplastic and heteroplastic transplanta- tion of eyea in the anuran amphibia." Jour. Exp. Zool. 77:87. Sperling, F., 19^+5 - "Extra -epidermal and aupernumerary lensea in aasociation with cyclo- pean eyes in Amblystoma embryos." Anat. Eec. 85:1+15. Spratt, N. T. , Jr., I9I+O - "An in vitro analysis of the organization of the eye-forming area in the early chick blastoderm." Jour. Exp. Zool. 85:171. Stella, E., 1952 - "Eicerche sperlmentali sulla localizzarione del territorio d'origlne dell'occhio in Axolotl e Eana esculenta, mediante trapianti embrlonall." Arch. Zool. Ital. 18:155. Stockard, C. E., I92I - "Developmental rate and structural expreaaion; an experimental study of twins, double monsters, and single deformities, and the interaction among embryonic organs during their origin and development." Am. Jour. Anat. 28:115. Stone, L. S., 1950 - "Heteroplastic transplantation of eyes between the larvae of two species of Amblystoma." Jour. Exp. Zool. 55:195- Stone, L. S., 19l^5 - "Heteroplastic lens grafts related to factors inhibiting lens regen- eration in Triturus." Proc. Soc. Exp. Biol. & Med. 60:10. Stone, L. S. & F. L. Dlnnean, 19l*0 - "Experimental studies on the relation of the optic vesicle and cup to lens formation in Amblystoma punctatum." Jour. Exp. Zool. 85:95. Stone, L. S. & F. S. Ellison, I9I+5 - "Eeturn of vision in eyes exchanged between adult salamanders of different species." Jour, Exp. Zool. 100:217. Stone, L. S., & P. Saplr, I9I+O - "Experimental studies on the regeneration of the lens in the eyes of anurans, urodeles, and fishes." Jour. Exp. Zool. 85:71. 50i^ EYE F lELO OPERAT IONS Stone, L. S. Sc I. S. Zaur, 19'*^0 - "Eelmplantatlon and transplantation of adult eyes in the salamander (Trlturus virideacens) with return of vision." Jour. Exp. Zool. 85:2ii-5. Twitty, V. C, 1952 - "Influence of the eye on the growth of its associated structures, studied by means of heteroplastic transplantation." Jour. Exp. Zool. 6l:535- Twitty, V. C. & J. L. Schwind, 1951 - "The growth of eyes and limhs transplanted between two species of Amhly stoma." Jour. Exp. Zool. 59=61. Twitty, V. C. & H. A. Elliott, I93U - "The relative growth of the an^hlMan eye, studied hy means of transplantation." Jour. Exp. Zool. 68:2^^7- Waddington, C. H., 1956 - "The origin of competence for lens formation in the amphibia." Jour. Exp. Biol. 15. Warren, A. E., 1959 - "Observations on limb development in Rana sylvatica following uni- lateral eye extirpation." Arch. f. Ent. mech. 159:50. Wolff, G. , 1895 - "Entwicklungsphyslologische Studien. I. Die Regeneration der Urodelen- linse." Arch. f. Ent. mech. 1:580. Zalokar, M., 19'4-U - "Contribution a I'etude de la regeneration du cristallin chez le Triton." Eev. Suisse de Zool. '^l-.kk^. UPPER EYELID CIUARY BODY DERMIS CUTIS SUSPENSORY LIGAMENT f CAPSULE LENS I EPITHELIUM- I FIBRES NICTITATING MEMBRANE LOWER EYELID VITREOUS HUMOR ORBITAL CAVITY f-OPTIC NERVE SCHMATIC DIAGRAM THROUGH FROG'S EYE (Redrawn from Mangold I95I) HEART FIELD OPERATION PURPOSE : By excising, transplanting, and 'blocking the fusion of the bilateral prlmordia, to determine the mode of heart formation. MATES IMS : Biological: Anura stages #15-#17: Urodela stages #22 -#25, and #5^-#58. Technical: Standard Ecjuipment METHOD: Precautions: a. Carry out several exploratory dissections of emhryos to determine the color and the extent of heart mesenchyme concerned in the later operations, h. Alcohol sterilization of operating instruments will lessen mortality which is generally high in heart field operations. c. Operations on Anura should he in full strength Standard Solution and on Urodela in Operating Medium. Following recovery from the operation, return the emhryos to appropriate culture media. d. The post-operated emhryos should be kept at constant and low temperatures, Anura from 15°C. to l80C., and Urodela from 10°C. to 15°C. Control: a. For the excision experiments, excision of mesoderm from any other region. b. For production of double hearts by heart block, the same operation should be per- formed but no mesenchyme is removed and no block is introduced. c. For heteroplastic transplantations, similar transplantations of somite mesoderm constitute the control condition. d. For isolation culture, the isolation of somite mesoderm would constitute the con- trol. Procedure: EXCISION OF PART OF THE HEART FIELD At Anuran stage #l6 and Urodele stage #25 the lateral mesoderm is converging from the two sides around the pharynx to form the single ventral heart (see diagrams). If the ma- terial of one of the lateral plates la excised, the formation of a normal, single tubular heart by the bilateral rudiments is prevented. Outline and peel back the ectoderm over the heart field derived from the right side after placing the embryo in a shallow depression in Permoplast, in Operating Medium (Urodele) or in full strength Standard Solution (Anura). Leave a hinge of ectoderm for attachment so that It can be replaced over the wound. With a hair loop and micro-pipette scoop out all visible mesoderm from the one side. The mesoderm is clear white and granu- lar. Replace the ectodermal flap. If the ectoderm is Insufficient, graft some Indiffer- ent ectoderm from the lateral body wall and from a posterior position of another embiyo. Lay a piece of moist lens paper over the wound for 20 to 50 minutes and, after complete healing, return the' embryo to the normal culture medium either in a #2 Stender or a finger bowl. If the excavated space is extensive, it may be partially filled with yolk from an- other embryo of the same species and stage although this should be avoided if possible. THE PRODUCTION OF DOUBLE HEARTS This operation may be performed on the Anura (stage #l6) or on the Urodela (stages #22 -#25) although the latter are preferred because the development of the heart can be followed through the transparent skin of the ventral side without exploratory incisions. * The author acknowledges, with appreciation, the help of Dr. W. M. Copenhaver in organiz- ing this exercise. -505- 506 HEART F lELD OPERATIONS Place the embryo in a Permoplaat depreaalon in such a position that the ventral heart forming areas faces upward. Locate the exact position of the future heart and with sharp glass needles make an incision posterior to the region of the thyroid anlage' and deep enough to reach the grayish endoderm of the pharyngeal floor. Carry this incision pos- teriorly to the position of the liver anlage. With a fine hair loop clean out the loose cells in the mid-ventral line, forming a channel. Leave the lateral mesenchyme intact. NORMAL DEVELOPMENT OF HEART PHARYNGEAL ENDOCERM , ENOOCAROtUM PERICARDIAL CAVITY CONUS ARTERIOSUS VENTRICLE SINUS VENOSUS ■VITELLINE VEIN TUBULAR HEART FRONTAL VIEW OF EARLf HEART BU3CKING TISSUE POINT OF INSERTION OF BLOCK OF INERT TISSUE BLOCKING TISSUE PARTIAL DUPUCATION OF HEART COMPLETE DUPLICATION OF HEART REDRWN FROM EKMAN '25 EXPERIMENTAL DUPUCATION OF HEART From a second embryo of the same species but of a later stage of development, remove a strip of notochord long enough to fill the excavated channel. Insert it into the operated embryo between the lateral heart rudiments, replace the ventral ectoderm, and hold the flap of ectoderm in position for 20 to 50 minutes by means of a Briicke or lens paper bridge. Alternative procedures may include flank ectoderm with underlying somite mesoderm Instead of notochord. Heturn the embryo to the normal culture medium after the wound has healed over. Should the above procedure fail to produce a double-hearted embryo, proceed with a more extensive operation on an embryo one stage younger. Remove the ecto-mesoderm by mak- ing a longitudinal slit from a position between the suckers posteriorly about I/5 of the length of the embryo. Garry the cut doraally on one side of the embryo to the ventral limits of the closed medullary fold, then forward, to complete a trapezoid. Avoid as much of the head ectoderm as possible. After outlining this area, carry the incision deeper until all mesoderm on the side of the operation is circumscribed. Eemove this large mass of ecto-mesoderm. Prom a slightly older embryo outline and remove an area of similar shape, and including ecto- and mesoderm, but from the presiimptive hind-limb region. Trans- plant this mass to the operated (host) embryo wound area, hold it in place for }0 minutes or more, and when healed, return the embryo to normal culture medium. (See Ekman, I925.) TRANSPLANTATION OF HEART FORMING AREAS a. Early stages; In Anuran stage #17 or Urodele stage #25, the heart forming mesenchyme from the bilateral sides has fused ventrally. Bemove a rectangular piece of ventral ectoderm along with all available adherent and underlying mesoderm, and transplant it as one piece to the flank region of a second embryo, previously prepared. The second (host) embryo should be slightly younger than the donor. Such a transplanted heart anlage should give rise to a tubular heart of four typical parts, with its own pulsations but without any circulatory elements. HEART F lELD OPERATIONS 507 b. Late etagea : Use Anura stage #19 or Urodela stage #5i+ to #58 where the heart la well formed. Anesthetize the embiyo in freshly made MS 222 (l/5,000 in operating medium) and graft (transplant) the entire heart mass^ plus liver and foregut, to the flank region of an embryo of the same age or one stage younger. While this operation will not demon- strate self-differentiation (as In "a"), it will show clearly the persistence of function In the absence of innervation. CULTURE OF HEART TISSUE IN ISOLATION After the bilateral heart rudiments have fused ventrally it is possible to remove the mesodermal mass and the overlying ectoderm, and to have It differentiate in isolation In the appropriate medium. In general (see "Isolation Culture" exercise) the culture medium should conalst of the normal culture medium which contains some (coelomlc) body fluids from adults of the same species. In addition, it la now known that 0.5% sodium sulfa- diazine will retard the development of bacteria and hence prolong the life of the Isolate. 25 HEART f^y^t ARE/ AMBLYSTOMA 4TH BR. CLEFT OPTIC VESICLE SENSE PLATE TRAPEZOID AREA HEART AREA GILL ANLAGE PRONEPHRIC AREA RANA PIPIENS « STAGE 15 31 /^ 31 HEART AREA MANDIBULAR ARCH The explanted heart may be cultured in a hanging drop of medium, on the underside of a coverslip sealed over a depression slide; In Standard Solution in a depression slide; or over an agar- embryonic fluid base. If the medium is changed every 5 to 1)- days, the ex- plant may be carried to quite an advanced atage of differentiation. 508 HEART F lELD OPERAT IONS HETEROPLASTIC TRANSPLANTATION OF HEART RUDIMENTS Copenhaver (1950 to 1959) has successfully transplanted Ambly stoma punctatum, Amblystoma tlgrlnum, Amblystoma mexicanum and Triton taenlatus hearts and heart parts' reciprocally. In such transplants it is important to realize that there are intrinsic differences in growth rate and in final pulse rate, in consequence of which the chimeric heart attains functional interest. Specific parts of the heart, such as the ventricle, the sinus venosus, etc. may he interposed between the other parts of the host heart. These are, of course, orthotopic transplants. Variations in this procedure include: a. Eeveraing of the axis of the transplant hut placing it In the otherwise orthotopic position, in order to determine the direction of pulsation in the transplant, and the control of the transplant over the host organ. b. Transplantation heteroplastically to an heterotopic position, such as in place of somites #7 to #10. OBSERVATIONS AMP TABULATION OF DATA: In all instances comparable stage embryos should be carried along simultaneously with the experimentals in order to allow direct comparison of the results of heart field ex- periments. Most of the experiments can be terminated about 8 days after the operation, and the host may be anesthetized in l/5,000 MS 222 emd be dissected (along with the con- trols) to determine the degree of development. In heteroplastic transplants the pulse rates of controls, experimentals, and parts of experimental transplants should be deter- mined. Photographs and drawings will constitute the record of these operations, and his- tological analysis is generally very instructive, providing comparable controls are avail- able. DISCUSSION: lyplcal vertebrates have hearts of bilateral origin. In both the Urodeles and the Anura the prospective heart forming material is derived from the two lateral mesenchymal plates. E^ the time these mesenchymal anlagen have migrated to the ventral position, they have acquired self-differentiating capacities of heart so that if transplanted to an heter- otopic position or explanted into a culture medium they will each give rise to a chambered, primitive heart, often with sinus, auricle, ventricle, and arterial bulb, all of which may exhibit typical rhythmic pulsations. Heart anlagen may be split to give multiple hearts or an extra heart anlage may be superimposed on the host heart material to produce a larger but normal heart, providing the axes of the host and the donor heart anlagen are the same. The heart area of the amphibian is considered as an equi- potential system In that as little as half of the area possesses the requirements for the development of an entire and normal heart. Anterior and posterior portions of the heart area, transplanted to a foreign species (Copenhaver, 1930) will give rise to corresponding specific portions of the ulti- mate heart. The posterior transplant combines with the anterior portion from the host and generally acts as a pacemaker, giving the host the rhythmical control similar to that normally found in the donor species. HEART FIELD OPERATIONS 309 EECOBD OF HETEROPLASTIC HEABT TEMSPLAKTATIONS 510 HEART FIELD OPERATIONS IHOD • - «J O H ^CO 1 S^ Zj in r* 31 >H Tj« CC r^ • CM ca N « ri CO rf ^45 =fc % % :% * ^ ^ a 0) 01 0) o; OJ 0) c r^ aC iO ^ af ■* bC 05 ed « oJ 4 « (d 4J jj 4J tJ w •>-> 5";1 u; « « lA tn (fl (A 2 S g 1 1 1 a 5 7) c^ i°& « o o O £=3 CO SI S ^ 00 o 4-1 00 •rt 1-3 s « CA (A w « w M 4h y} iC :* ^ so qO aO © O o H 00 3 CD Cb :x. LX. E! ^ Cb Lz. 0 ON s ^a HEART F lELD OPERATIONS 311 BEFERENCES : Bacon, B. L., 19'+5 - "Self-differentiation and induction in the heart of Amblyatoma. " Jour. Exp. Zool. 98:87. Copenhaver, W. M., 1959 - "Some ohservationa on the growth and function of heteroplastic heart grafts." Jour. Exp. Zool. 82:259 (see ibid, 80:192). Copenhaver, W. M., 19^+5 - "Heteroplastic transplantation of the sinus venosus between two species of Amblyatoma." Jour. Exp. Zool, 100:205. Ekman, G. , I925 - "Experimentelle Beitrage zur Herzentwicklung der Amphibien." Arch. f. Ent. mech. 106:520. Ekman, G. , I929 - "Experimentelle Untersuchungen uber die fruheste Herzentwicklung bei Bana fusca." Arch. f. Ent. mech. 116:52?. Fales, D. E., 191+6 - "A study of double hearts produced experimentally in embryos of Ambly- atoma punctatum. " Jour. Exp. Zool. 101:281. Goerttler, K. , I928 - "Die Bedeutung der ventrolateralen Mesoderm-bezlrke fur die Herzen- lage der Amphibienkeime. " Anat. Anz. Erg. Heft. 66:152. Goss, CM., 1955 - "Double hearts produced experimentally in rat embiyos." Jour. Exp. Zool. 72:55. Graper. L., I907 - "Untersuchungen uber die Herzblldung der Vogel." Arch. f. Ent. mech. 2i^:375. Hegre, E. S., 19^+5 - "Intracardiac tranaplantatlon in the urodele." Science. 101:1+69. Hilton, W. A., 1915 - "The development of the blood and the transformation of some of the early vitelline vessels in Amphibia." Jour. Morph. 2U:559. Knower, J. McE., I907 - "Effects of early removal of the heart and arrest of the circula- tion on the development of frog embryos." Anat. Bee. I:l6l. Lehmann, F. E., I929 - "Die Entwicklung des Anlagenmusters in Ektoderm der Tritongastrula. " Arch. f. Ent. mech. 117:517. Mangold, 0., I92I - "Situs inveraua bei Triton." Arch. f. Ent. mech. 1*8:505. Schubel, K. & J. P. Stohr, I92I+ - "Ein Beitrage zur Pharmokologle transplanttatierten Amphibienherzen. " Arch. f. Exper. Path. u. Pharm. IOI+. Spemann, H. & H. Ihlkenberg, I919 " "Uber asymmetriache Entwicklung und Situs Inversus viacerum bei Zwlllingen und Doppelbildungen. " Arch. f. Ent. mech. 14-5 :571. Stohr, J. Ph., I92I+ - "Uber Explantation und Tranaplantatlon embryonaler Amphibienherzen." Die Naturwlss. 18. Stohr, J. Ph., 1925 - "Experimentelle Studien an embryonalen Amphibienherzen. III. Uber die Entstehung der Herzf orm. " Arch. f. Ent. mech. 106:l+09. Stohr, J. Ph., 1929 - "Zur Embryonalen Herztransplantation." Arch. f. Ent. mech. 116:500. "Meaning can only be introduced into our knowledge of the ext e rnal univer se by the simultaneous prosecut ion of research at all levels of complexity and organization, for only in this way can we hope to under s tand how one is con- nected with the others. " J. Needham 19i2 Aristotle in his "De Partibus Animalium" maintained that the essence of a living animal is found not in what it is, or how it acts, but why it is as it is and acts as it doe s . REGENERATION* DEFINITION : The restitution of a lost organ In such a form that It Is both structurally and functionally complete. PURPOSE : To determine the ahllity of an emhryo or larva to repair or replace a lost part. MATERIALS : Biological: Rana: stages #10 to #17 and 5 5 to k.^ cm. tadpoles (may he used up to 8 cm. ) . Amblystoma: stages #10 1:0 #29 and larvae with llmhs, before metamorphosis. Triturus: stages #10 to #29 and late larvae. Technical: Standard operating equipment. Sodium sulfadiazine - 0.5^ aqueous (to prevent post-operative infection). METHOD: Precautions: 1. Moderate precautions should be taken against bacterial contamination. Animals may be reared in 0.5^ sodium sulfadiazine as protection. 2. Keep operated animals in sopara^ce containers. The Urodeles particularly tend to snap at any moving object and will bite off each others tails or appendages. Provide adequate volume of medium and space for each specimen. 5. After the wound is healed over all animals must be maximally fed (Enchytrea for the Urodela and spinach for the Anura). Controls : 1. Tall-fin controls consist of unoperated embryos of the same age and stage. 2. Limb regeneration control consists of the limb of the opposite side. 5. Blastema control consists of the transplantation of some other and neutral area (unknown anlage) Into the blastema. Experimental procedure: TAIL-FIN REGENERATION Using either anuran tadpoles (stage #25 or older) or urodele larvae (stage #14-5 or older) with distinct tail fins, select as many specimens as possible of the same age and at the same stage of development. If anuran tadpoles are used they may be placed in groups of 5 in single finger bowls containing a minimum of 25 cc. of medium. If Urodele larvae are used they must be kept in separate containers such as finger bowls, Lily cups, or #2 Stenders. The following directions are for Anuran tadpoxes: 1. Prepare I8 finger bowls, 6 each with the follovrtng solutions (50 cc. each). a. 10^ Standard Solution (hypotonic) b. Standard Solution (isotonic) c. 200^ Standard Solution (hypertonic) 2. Into each finger bowl place an equal number of tadpoles, a minimum of 5. 5. Mark the 6 finger bowls containing the same medium as follows: a. Vertical cut. b. Cut angled toward the dorsal body wall. c. Cut angled toward the belly. d. V-shaped cut, apex toward body. e. V-shaped cut, apex away from body f. Control - no cut. * The author acknowledges, with appreciation, the critical suggestions of Dr. H. S. Emer- son in organizing this exercise, particularly that part relating to inductions within the blastema. -512- REGENERATION 315 FROG TADPOLE (STAGE #25) SHOWING LEVELS OF CUTS FOR TAIL-FIH REGENERATION STUDIES A - V-shaped cut with apex away from the body. B - Transverse cut angled toward dorsal body. C - Transverse cut angled toward belly. D - V-shaped cut with apex toward the body. h. Consult the accompanying diagram of a frog tadpole (stage #25) to determine the angles of the various cuts prescribed. All but "a" and "f" (the con- trol) are Illustrated. 5. Prepare an operating Syracuse dish with Permoplast base. Fill the dish with one of the above (5) solutions, beginning with the 10^ Standard. Transfer all (5) tadpoles successively from each of the finger bowls containing the 10^ Standard to the operating dish and cut the tail fin in the manner Indi- cated on the previously-marked finger bowl. That is, there will be finally 18 finger bowls, containing six (6) different solutions, and representing five (5) different cuts, and a set of controls. The cuts should be made with a sharp scalpel while the tadpoles are immobilized with l/ 10, 000 MS 222 ( made up in the same medium) . If the cuts are made on the group from a single finger bowl^ while in the same Syracuse dish, It will be somewhat easier to insure the cuts being similar. 6. Immediately return the tadpoles to the finger bowl with the appropriate medium, and properly marked. It Is best to make the cut in the medium to be tested. (Do not save the tail tips unless for incidental chromosome counts - see -Tail Tip Technique".) 7. Place all I8 finger bowls under Identical environmental conditions of light and temperature, and minimize evaporation by keeping them covered. The tall fins should be examined under the dissection microscope dally for about ten days, and the record consists of dally sketches beginning with a sketch Immediately after cutting. Note the color of the regenerating (blastema) tissue and the angle of the re- generate. SKETCHES OF KEGENERATIfiG TAIL FINS 5lU REGENERATION THE DEVELOPMENT OF ORGAN ANLAGEN IN REGENERATING BLASTEMAS This experiment Involves regeneration, transplantation, and possibly some Induction. It is based upon the assumption that the regenerating tissue (the blastema) is essentially embryonic in nature and will either supplement an implanted organ anlage or will, under tJie influence of such an anlage, bs induced to form certain organs. 1. Select 20 to 50 frog tadpoles measuring at least 3.5 cm In total length. They may measure as much as 8 cm. The younger ones are generally better, even though the blastema is, in consequence, smaller. 2. Anesthetize the tadpoles with 1/10,000 MS 222 and, with a sharp scalpel, cut off the tail fin at an angle toward the dorsal side of the body about 5 mm. from the body. The older tadpoles may exhibit some hemorrhage but this can be stopped, if necessary, by brief exposure to hypertonic medium. Record the ex- act date. 5. Allow these tadpoles (in any community crystallizing dish) to regenerate their tails. After about 5 days (and until about 8 days) there will appear a whitish growth bulging from the cut surface. This is the blastema. Regions of the early ^astrula to be implanted into the blas- tema, (From Emerson 19^+1: Jour. Exp. Zool. 8T:'+03) Regions of the late gastrula to be implanted into the blastema, M - the anterior limit of the presumptive neural plate, ( From Emerson 19i^2 : Jour. Exp. Zool. 90:553) Prepare aonor material, consisting of various organ anlagen. Consult the Vogt map of organ fields of early gastrula. The eye and the sucker anlagen are generally the most satisfactory, and these may be taken from late tall-bud stages. A suggested list of satisfactory organ anlagen follows: Early and late gastrula ectoderm areas (see accompanying diagrams). Optic vesicle, with and without lens ectoderm. Auditory (otic) vesicle. Olfactory vesicle. Sucker Gill bud Limb bud Forebraln vesicle. Medullary plate or neural fold. Hypophysis Prepare the host as follows: Anesthetize a host (with pronounced blastema) in l/lO,000 MS 222 in Standard Solution or Spring Water. With a sharp-pointed lancet (or Iridectony scissors) gently cut between the blastema and the old tissue (of the tall) along the side of the tadpole. Then make two shallow cute, one along the dorsal and the other along the ventral margins of the blastema. This will form a V-shaped cut with the apex of the "V" at the most posterior limit of the regenerating blastema. The operating area Is small and this is a delicate operation. REGENERATION 315 Quickly excise the anlage to be transplanted and insert it beneath this flap of hlastema tissue, orienting it so that its ectodermal layer is outermost. Ee- place the flap of blastema ectoderm and hold it down for 1 to 2 minutes with a hall tip. The sticky blastema cells will become attached to the transplant and will hold It in place. The transplant may be fixed any time from 18 hours to a month after the transplantation, sectioned and studied to determine the degree of differentiation and induction. SKETCHES OF OEGAN MLAGE TRANSPLANTED TO TAIL BLASTEMA LIMB REGENERATION The Urodelee show regenerative powers throughout life but the Anura exhibit them until metamorphosis and then only to a limited degree thereafter. It is Instructive to study the regeneration of limbs and digits of Urodele larvae. It must be remembered, how- ever, that the post-operative larvae must be treated normally with respect to food, oxygen, space, etc. If x-ray facilities are available, the effect of such irradiations upon re- generating limbs can be studied (see Butler, 195lt and Butler & Puckett, 19^*0, and illus- trations on the following peige). Procedure : 1. Select Amblystoma or Triturus larvae beyond stage #38, when the limbs are developing or are already developed. It is essential that larvae of the same age and stage be used in order to determine the effect of cuts at various levels and angles. 316 REGENERATION Anesthetize the larvae In l/ 10, 000 MS 222 and make the following cuts: a. Cut off the right forelimh at ;he level of the wrist. t. Cut off the right forelimb halfway between the wrist and the pectoral gli^le, above the bend of the elbow. c. Repeat the above two cuts but make them at the greatest possible angles. d. Make the cuts described in "a" and "b" but extend the cut only half way through the limb {or wrist), leaving all other parts attached. ^^- Left forelimb of advanced Amblystoma, indicating possible levels of amputa- tion. (From Schotte' & Butler I9I+I: Jour. Exp. Zool, 87:297) RECOED OF LIMB REGENERATIONS IN AMBLYSTOMA OB TBITUBU5 REGENERATION 517 OBSEBVATIONS AMD BECORDING OF DATA: There are three distinct parts to this exercise on Regeneration and for each specimen studied there must be a complete and separate record. Such a record should consist of drawings or photographs taken at stated Intervals (not more than 2k hours, in most cases) ■beginning Immediately after the operation. The dates for all drawings, the temperature of the medium, the conditions of food, light, space, etc., must all he recorded In the vari- ous places provided. DISCUSSION: Self-repair is a characteristic of all protoplasm, a necessary prerequisite in a com- petitive environment where natural selection plays such an inrportant role. The exact method of this repair is not thoroughly understood. It is not estahlished that such re- pair is the same for all animals, or at all stages within the life span of a single organ- ism. There are still two main concepts relative to the method of restitution. There are some who hell eve that there are reserve, mesenchyme-type cells in all organs, awaiting call for the specific function of regeneration. Ther« is no doubt that an injury calls for the marshalling of active cells in the vicinity of the cut, hut many of these calls are of vascular xsrigin and may have nothing to do with regeneration. The second concept is that the injured cells at the cut surface, and nearby, undergo a period of de-differen- tiation, to be followed by an indifferent (embryonic) period, and then a re-differentia- tion, either into similar or dissimilar tissues. Some rigid adherents to this concept be- lieve that re-differentiation can only be along the original lines of differentiation, implying incomplete de-differentiation. There is, of course, controverting evidence against this. In general the regenerated part does resemble in structure and In function the lost part. Buchanan ( 19^+0) says: "Perhaps the more widely held view, is that organ- ismic control is established and maintained by reason of the diffusion of specific organ- izing substances arising as the result of specific metabolisms of organizing or inducing centers." Regeneration is not limited to the structures or anlagen listed in this exercise, and minor experiments in regeneration are listed in other excercises which deal specifical- ly with certain organ systems such as THE EYE, THE HEART, THE LIMB FIELDS, etc. However, this exercise will illustrate the principles Involved and also the fact that Amphibian larvae do exhibit remarkable powers of regeneration. Emerson {19'+1) has shown that parts of the early and late gastrula ectoderm can be Implanted into a tail blastema and will differentiate into recognizable organs. This is therefore another method (in addition to isolation culturing) of determining the prospec- tive potencies of various fields or areas of the early gastrula or, in fact, any early embryonic stage. Grafts into the larval tall blastema may survive as long as 100 days after transplantation (Emerson, 19'+'+), and the differentiations may include eyes with lenses, brain parts, striated muscle and cartilage. This period of 100 days is long after the blastema has become an integral part of the tall. Differentiation is achieved within a week or two, and many of the grafts will disintegrate at this time. In most cases re- sorption of the graft is accomplished along with the resorption of the tall at the time of metamorphosis. Sometimes, however, a part of the graft may persist and will be found at the tip of the urostyle of the metamorphosed frog. The tadpole tail regenerates rapidly. This may be due in part to the embryonic nature of the cells contiguous to the cut surface. The initial axis of regeneration is generally at right angles to the cut surface but this is rectified subsequently. Twitty and Delanney (1959) found that the tail of Amblystoma is capable of repeated regenerations after successive amputations even during long periods of continuous starvation. There is a gradual decrease in the rate (and degree) of regeneration as amputations are performed later in larval life (Goodwin, 19'+6) and newly hatched larvae respond the better because they have incompletely differentiated tissue which can be more simply de-differentiated to form a blastema, and then re-dif ferentlate as required. 518 REGENERATION The Urodelea retain their powers of regeneration throughout life, hut to a iBsaer degree after metamorphosis. The Anura, on the other hand, lose most of their regenerative powers after metamorphosis. However, there is recent evidence (Butler, Eose, Schotte'', and Singer) that even the Anuran appendage will exhibit some powers of regeneration providing (Eose) the cut surface is continually irritated by hypertonlclty with NaCl, or (Butler, Schotte', Harland and Singer) the connection of the appendage with the central nervous sys- tem is not interrupted. Schotte and Butler (19^1) have demonstrated the regression of the humerus in a denervated limb of Amblystoma. In fact, these investigators make the state- ment that: "No blastema is ever established on a completely nerveless amputated limb." Auo.l6 Regression of skeleton of fore- limb (Amblystoma) following de- nervation. (Total time 32 days) m c^ ^~— i=a3!g:4^ ( From Schotte' & Butler I9U1 : Jour. Exp. Zool, 87:279) Oct. 7 Regeneration of foot and digits of Rana piplens pre-metaraorphlc tadpole hlndllmb. Photo taken 24 days after transverse cut made at shank level. (P. Bernstein) REGENERATION 519 In an early embryo the nervous ayatem la not involved. However, when a formed em- bryonic field ia extirpated, there is generally no regeneration. This means that at this stage regeneration as a property of the embiyo ia restricted to local fields, and is not related to "the organism as a whole". There is still a great deal of room for further study of regeneration, particularly as it may be affected by mitotic inhibitors, colchicine, dlnitrophenols, specific iona, pH, temperature, light (where pigment cells are involved). Radiated ol time of amputation Radiated 12 days after amputation Fig. 1. A larva showing the stage in which the right fore limb was amputated. The double pointed arrow Indicates the level of amputation. Figs. 2 and 3. Larvae which were given a single exposure to x-rays Immediately after limb amputation. Limb regeneration has been completely suppressed. Figs. 4 and 5. Control larvae showing the progress of normal regeneration 12 and 25 days after limb amputation. Stip- pling on the limb stump in Figure 4 Indicates the approxi- mate extent of the normal re- generation blastema on the twelfth day. Figs. 6 and 7. Larvae which were given a single exposure to x-rays on the twelftli day after limb amputation. A blastema was present at the time of radiation, as shown by stippling. Limb regeneration was completely suppressed. (From Butler & Puckett I9U0: Jour. Exp. Zool. 81i:223) days EEFERENCES : Abeloos, M., 1952 - "La re'generatlon et les problemes de la morphogenfese. " Gauthier- Villars, Paris. Barbel', L. W. , 19*+^ - "Correlations between wound healing and regeneration in fore-limbs and tails of lizzards." Jour. Exp. Zool. 89:l4-Ul. Brachet, J., 19^+6 - "Aspecta biochimique de la Eegene'ratlon. " Experientia II/2 . Brunst, V. V. & E. A. Scheremeti Jewa, 1955 - "Unterauchung des Einflussea von Eontgen- strahlen auf die Eegeneration der Extremltaten beim Triton." Arch. f. Ent. mech. 128:181. Buchanan, J. W. , I9I+O - "Eegeneration." Am. Nat. Ih:k8l. 520 REGENERATION Burr, H. S., M. Taffel, & S. C. Harvey, 19^0 - "An electrometrlc study of the healing wound In man." Yale Jour. Biol. & Med. 12:k3^. Butler, E. G., 1955 - "Studies on limb regeneratl'on In x-rayed Amblyetoma larvae." Anat. Bee. 62:295. Butler, E. G. & J. 0. O'Brien, 19i^2 - "Effects of localized x-radlatlon on regeneration of the Urodele limb." Anat. Bee. 8U:l+07. Butler, E. G. & 0. E. Schotte', I9U1 - "Histological alterations in denervated non-regener- ating limbs of urodele larvae." Jour. Exp. Zool. 88:507. Cameron, J. A., I957 - "The mitotic rate in tadpole skin after repeated injury." Biol. Bull. 72:57. Child, C. M., I9I+I - "Patterns and Problems in Development." Univ. Chicago Press. Clement-Noel, H., 191+14- - "Les acides pentosenuclelques et la regeneration." Ann. Soe. Boy. Zool. de Belg. 75:25. David, L. , 1951+ - "La contribution du materiel cartilaglneux et osseujc au blastem de regeneration des membres chez les Amphiblens urod^les." Arch. d'Anat. Microse. 50:217. Detwiler, S. R., I9I+6 - "Midbrain regeneration in Amblystoma." Anat. Bee. 9l+:229, Emerson, H. S., 19414- - "Embryonic grafts In regenerating tissue. III. The development of dorsal and ventral plate ectoderm of Eana plpiens gaitrulae." Jour. Exp. Zool. 97:1- Gldge, N. M. & S. M. Rose, 19'+'+ - "The role of larval skin in promoting limb regeneration in adult Anura." Jour. Exp. Zool. 97:71. Godlewskl, E., I928 - "Untersuehungen iiber Auslosung und Hemung der Regeneration belm Axolotl." Arch. f. Ent. meeh. Ill4-:108. Goodwin, R. A., 19^+6 - "A comparison of regeneration rates and metamorphosis in Triturus and Amblystoma." Growth. 10:75. Guyenot, E. & K. Ponse, 1950 - "Territolrea de regeneration et transplantations." Bull. Biol. 61+: 252. Harrison, R. G. , I955 - "Some difficulties of the determination problem." Am. Nat, 67:506. Helff, 0. M., 1957 - "The relation of the dorsal nerve cord and notochord to tall regener- ation in -Anuran larvae." Anat. Bee. 70:(3uppl.) 101. Hellmich, W., I929 - "Untersuehungen uber Herkunft und Determination des regneration Materials bei Amphiblen." Arch. f. Ent. meeh. 121:155. Herrell, W. E., 195^+ - "Growth and regeneration of tissue in frog tadpoles following the administration of an extract of the anterior pituitary gland." .Anat. Rec. 59:'+7. Hertwig, G. , I927 - "Beitrage zum Determinations-und Regenerationsproblem mlttels der Transplantation haploidkerniger Zellen." Arch. f. Ent. meeh. 111:292. Holtfreter, J., 1958 - "Differenzlerungspotenzen isollerter Telle der Anurengastrula. " Arch. f. Ent. meeh. 158:657. Hooker, D., 1950 - "Studies on regeneration in the spinal cord. TV, Rotation about its longitudinal axis of a portion of the cord in Amblystoma punctatum embryos." Jour. Exp. Zool. 55:25. Horn, E. C, 19^4-2 - "An analysis of neutron and x-ray effects on regeneration of the fore- limb if larval Amblystoma." Jour. Morph. 71:185. Karczmar, A. C, 19^+7 - "The effect of repeated amputation and blastema formation in re- generation." Anat. Rec. 99: suppl. #19. Korschelt, W. , I927 - "Regeneration und Transplantation." Bomtraeger, Berlin. Lecamp, M., I9I4-2 - "Effect of amlno-acids on regeneration." Comp. rendu. Soe. Biol. 211+ : 550. Liosner, L. D., 1958 - "Untersuehungen iiber die Eigensehaften der Regeneratlonsknoape des Amphlbienachwanzes." Bull, de Biol. et. Med. Exper. 6:262. Litwlller, R., 1959 " "Mitotic index and size in regenerating amphibian limbs." Jour. Exp. Zool. 82:275. Mllojevle, B. D., I92I4 - "Beitrage zur Frage uber die Determination der Eegenerate." Arch. f. mlkr. Anat. u. Entwmech. 105:80. Morgan, T. H. , I927 - "Experimental Embryology." Macmillan. Navllle, A., I92U - "Recherches sur I'histogendse et la regeneration chez les Batraelens Anoures." Arch, de Biol. 5l+:255. Needham, J., I9I+2 - "Biochemistry and Morphogenesis." Cambridge Univ. Press (p. '+50ff. ). Polezhayer, L. W. , 19'+6 - "The loss and restoration of regenerative capacity in the limbs of tailless amphibia." Biol. Rev. 21:11+1. Prutscher, L., 1959 - "Histological mechanism of regeneration of striated muscle in Anura." Magyar Orv. Arch. 1+0:1. REGENERATION 52I Puckett, W. 0., 1958 - "The effects of Intra-peritoneal injections of pltviitary substances on the rate of tail regeneration in frog tadpoles." Anat. Bee. 71:537. Eichardson, D., 19't-5 - "Thyroid and pituitary hormones in relation to regeneration. II. Begeneration of the hind leg of the newt, Triturua viridescens, with different com- binations of thyroid and pituitary hormones." Jour. Exp. Zool. 100:i^l7. Eose, S. M., I9U5 - "The effect of NaCl in stimulating regeneration of limbs of frogs." Jo\ir. Morph. 77:119. Sanders, F. K. & J. Z. Young, 19^+6 - "The influence of peripheral connexion on the dia- meter of regenerating nerve fibers." Jour. Exp. Biol. 22:203. Schotte, 0. E., 19'*^0 - "The origin and morphogenetic potencies of regenerates." Growth Supplement . 59 " 76 . Schotte, 0. E., & E. G. Butler, 1914-4 - "Phases in regeneration of the lUTOdele limb and their dependence upon the nervous system," Jour. Exp. Zool. 97:95. Schotte, 0. E., & M. Harland, 19'4-3 - "Amputation level and regeneration in limbs of late Eana clamitans tadpoles." Jour. Morph. 75:529. Schotte, 0. E., & A. G. Karczmar, 19^5 - "Temperature and regression rates in denervated amputated limbs of urodele larve." Jour. Exp. Zool. 99:255. Singer, M., 191+7 - "The nervous system and regeneration of the forelinb of adult Triturus. VII. The relation between number of nerve fibers and surface area of angiutation. " Jour. Exp. Zool. 10l+:251. Speidel, C. C, 191+7 - "Correlated studies of sense organs and nerves of the lateral-line in living frog tadpoles. I. Begeneration of denervated organs." Jour. Com. Neur. 87. Swett, F. N. & M. Wallace, 191+1 - "Growth potencies and polarity relations in the cells which replace the extirpated embryonic limb rudiment." Jour. Exp. Zool. 86:51. Thornton, C. S., I9I+2 - "Studies on the origin of the regeneration blastema in Triturus viridescens." Jour. Exp. Zool. 89:575. Twitty, V. E. & E. Delanney, 1959 - "Size-regulation and regeneration in salamander lanrae under complete starvation." Jour. Exp. Zool. 81:599. Vallette, M., I929 - "Begeneration du museau et territoires de regeneration chez les Urodeles." Bull. Biol, de la France et de la Belg. Weiss, P., I9I+I+ - "The technology of nerve regeneration. Sutureless tubulatlon and re- lated methods of nerve repair." Jour. Neurosurgery. 1:1+00. Williams, W. L. & M. Frantz, 191*8 - "Histological technics in the study of vitally stained normal and damaged cells." Anat. Eec. 100. Woronzowa, M. A., I958 - "Die Eegenerationspotenzen der Schultergurtelmuskulatur beim Axolotl." Bull, de Biol, et Med. Exper. 6:82. Wright, M. B. , I9I+7 - "Begeneration and degeneration experiments on lateral line nerves and sense organs in anurans." Jour. Exp. Zool. 105. Zwllling, E., I9I+I - "The determination of the otic vesicle In Eana plplens." Jour. Exp. Zool. 86:355. "Nothing in the whole world is more wonderful than this ability of organisms to overcome adverse condit ions , to neutralize violent poisons, to restore lost parts. " E. G. Conklin 19Uti "Observe always that ever yt hing is the result of change, and get used to thinking that there is nothing Nature loies so well as to change forms and to make new ones like them. Marcus Aure I lus EFFECT OF THYROID AND OF IODINE ON AMPHIBIAN METAMORPHOSIS* PURPOSE: To demonstrate the relationship of the thyroid hormone, or its major constituent (iodine), to metamorphic changes in the Amphibia. MATERIALS : Biological: Hatched tadpoles of Eana, Bufo, or ^la Neotonous Urodele larvae (Necturus, Axolotl, etc.) Technical: Thyroid tablets (Armour & Company) Thyroxin crystals (Hoffman-La Boche Company) Freshly dissected thyroid glands (frog, rat, sheet, etc.) Iodine crystals dissolved in 95^ alcohol Millimeter ruled graph paper on underside of Petri dish (for measuring) METHOD: Precautions: 1. The number of tadpoles per unit of volume per finger bowl must be the same for all observations. The best ratio is 25 tadpoles per 50 cc. of medium in each finger bowl. 2. The medium for the experimental animals should be changed daily except where thyroxin is used. The thyroid tablets or glands provide an excellent medium for bacterial growth which, in itself, would eventually kill the tadpoles. 5. Avoid overdosing or overfeeding, particularly of iodine or thyroid material. The effect of thyroid is an acceleration of development which may be carried beyond the tolerable limit and the badpoles will be literally "burned up". Controls: Since the treatment of the tadpoles involves feeding, a basic diet of boiled spinach (1 square inch per tadpole per day) should be provided for both the control and the experimental animals. The controls and the experimentals should be from the same batch of eggs, at exactly the same stage and size at the beginning of the ex- periment. Procedure: PREPARATION OF EXPERIMENTAL MEDIA TfflTBOXIN: Dissolve 10 mgm. of crystalline thyroxin in 5 cc, of l NaOH (it is soluble only in alkaline media) and then add distilled water to make 1 liter. This will be a l/lOO,000 concentration of thyroxin and may be kept in the refrigerator almost indefinitely and may be considered the stock solution from which lower con- centrations of experimental media are made. TEfROID TABLETS: Dissolve five 2-grain tablets in about 5 cc. of distilled water, using a mortar and pestle. Add an equivalent amount (in weight) of whole wheat flour, and grind together thoroughly. With a spatula, spread the paste thin- ly onto clean lantern slide covers and allow it to dry. When dry, chip the mixture off of the glass, powder and store it in stoppered bottles in the refrigerator. IODINE SOLUTIONS: Dissolve 0.1 gram of pure crystalline iodine in 5 cc. of 95^ alcohol, and then dilute to 1 liter with distilled water. This will provide a concentration of l/lO,000 as a stock solution, which can be further diluted when required for experimental uses. * The author acknowledges with appreciation, the help of Dr. S. A. D'Angelo in organizing this exercise. -522- THYROID AND IODINE AND METAMORPHOSIS 523 FRESHLY DISSECTED THYBOID GLAMS: Thiee sources of fresh glands are reconnnended. a. From the frog: Since the amphibian thyroid gland is difficult to locate, use the large hullfrog ( Eana catesblana) if available. Eana pipiens glands can be used, however. Eemove the lower Jaw by cutting through the angles of the Jaw and posteriorly to the xiphi sternum. Deflect the ventral akin forward and expose the underlying muscles in the vicinity of the glottis. Clip off the anterior end of the xiphistemum, exposing the hypoglossal muscle which should be cut. With forceps, strip these muscles forward, locate the hyoid cartilage. The thyroids will be seen posterior to the lateral hyoid processes and close to the Jugular veins. The preliminary dissec- tions should be checked by microscopic examination of the removed gland, for there are other glands in the same general vicinity. When the dissection technique has been perfected, add a known number of crushed glands to each of the experimental ( finger) bowls each day. It is difficult to control the amount of thyroid tissue consumed by a tadpole, but if the glands are thoroughly crushed the distribution will be the more homogenous. b. From the rat : Experimental rats are generally available in the laboratory and fresh rat thyroids can be excised, crushed (with clean sand, if neces- sary) and fed to tadpoles directly. Again it is iii5)ortant to reduce the size of the pieces of thyroid tissue to a minimum. c. From the slaughter house: Fresh thyroids of large mammals (sheep, pig, cow) are generally available. Such thyroids may be weighed, macerated in 1^ NaOH, and squeezed (broken up with mortar and pestle) and the mash made up to a known volume (e.g., 100 cc.) with Standard Solution. The maceration liberates the thyroid colloid into the surrounding medium and the fresh and homogenous thyroid mixture may be added in known quantities to the various experimental finger bowls. Such a freshly made thyroid mash will remain usable for several days if frozen quickly. A second procedure is to dehydrate the fresh glands in acetone, freeze them quickly and solidly, later to chop the pieces Into small bits which can be ingested by the tadpoles. PREPARATION OF THE (NORMAL) CONTROL FOOD Anuran tadpoles can be reared to and through metamorphosis on a variety of foods. The most consistently satisfactory diet is washed and par-boiled spinach or lettuce. Spinach must be washed to remove arsenic powder used to destroy insects and must be boiled to soften the tissues. Such spinach cannot be kept more than 2h hours at refrigerator temperatures as it becomes acidified and will kill the larvae. A rough estimate of the amount to provide is 1 square inch of spinach leaf per tadpole, until they are about a month old when they will require more, per day. The larger bullfrog tadpoles naturally require more food. The Urodele larvae (e.g., Necturus or Amblystoma) are fed small Daphnia, and later small white worms, Enchytrea. Other "normal" foods are rolled oats, oatmeal and dried shrimp, liverwurst, etc. Some investigators use a mixture of wheat flour with egg yolk or alfalfa. Since all foods are a source of bacterial infection and growth, the food and medium should be changed dally and the culture should be kept at a uniform and fairly low ( ISOQ. ) temperature. EXPERIMENTAL PROCEDURE 1. Inseminate eggs of Bana pipiens and separate them Into groups of 5 to 10 eggs, and place about 200 in a flat, white enamel pan measuring about U x 12 x 20 inches. Cover the pan with a glass plate and allow the eggs to develop at about 23-25°C. At the 11 mm. stage (about ll+ days) begin to feed the tadpoles a uniform diet, preferably of fresh, washed, and boiled spinach. Change the water and add fresh ^ THYROID AND IODINE AND METAMORPHOSIS food three times per week. If available, separate the tadpoles ao that there are no more than 100 per enamel pan and they will grow faster. 2. When the hind limb buds have attained a length of about 1 mm., select as many as are available at exactly the same stage of development. Place 5 such tadpoles in each finger bowl containing 50 cc. of Standard Solution or Spring Water. (If available, the 12 inch crystallizing dishes may be used with 25 such tadpoles.) In any case the stage of development, volume of medium, and size of container must be identical for all groups of tadpoles. 3. Treat the experimental animals as follows (the controls receiving the spinach diet only while the experimentals receive, in addition, the following). THYROXIN : Place tadpoles in various concentrations for 1 week. Do not suspend normal feeding. a. Concentration of l/l,000,000 b. Concentration of l/l0,000,000 c. Concentration of l/l00,000,000 Doses of 1/500,000,000 have been known to accelerate development. THYEOID TABLETS: Add approximately 50 mgm. of thyroid-wheat mixture per day per tadpole, for a period of one week. Do not suspend normal feed- ing, and change the culture medium daily just before adding thyroid. lODIME: Add concentration and normal food but add no further iodine unless the medium is changed. a. Concentration of l/500,000 for 7 and for Ih days. b. Concentration of l/l,000,000 for 7 and for l4 days. FRESH MAMMALIAN THYBOID: This experiment can have only qualitative significance because it is difficult to control the dose of the thyroid colloid expelled from the living (fresh) glands or the amount ingested by each of the tadpoles. It will be significant, however, if the stu- dent can demonstrate any acceleration of amphibian metamorphosis by the use of mammalian thyroid gland tissue or colloid. It is possible simply to squeeze fresh thyroid glands directly into the experimental dishes, thus liberating some of the colloid to be Ingested. If bullfrog tadpoles are available, their thyroids may be used in the same manner. Such tadpoles can also be used as test animals, providing they are second -year tadpoles and have begun the develop- ment of their hind limbs. h. Neotony is a condition of permanent larval state, during which the forms can re- produce. Examples are Necturus and the Mexican Axolotl. Such forms can be caused to complete their arrested development, and metamorphose, by treating them with the thyroid hormone or iodine. If available, attempt to get rid of the otherwise permanent external gills of such forms by treating them with thyroxin. Evan A. punctatum and A. tirgunum may be hastened through metamorphosis by thyroid treatment. OBSERVATIONS AND TABULATION OF DATA: There is high mortality in this type of an experiment. Mount some millimeter graph paper on the underside of some flat-bottomed Petri dishes to use as a guide in determining the size changes in tadpoles. The important criteria are: a. Hind limb length b. Total length, i.e., from snout to tip of tail c. Body length, i.e., from snout to base of tail THYROID AND IODINE AND METAMORPHOSIS 525 PEOTOGBAPES AND DRAWINGS OF ACCELEBATED METAMOBPHOSIS 526 THYROID AND IODINE AND METAMORPHOSIS Other items to note will be changes in the mouth, the shape and size of the head, the ap- pearance of the forellmbs, etc. 5y means of averages of these size changes, determine the body proportions that show maximum response to the thyroid. While the tadpoles are exposed to the thyroid or iodine treatment for only a short time (1 to 2 weeks) the final data should not be collected for from 1 to 2 weeks (or more) after the cessation of the experimental conditions. Some of the tadpoles should be kept until they achieve metamorphosis and the time of emergence from the water of the experi- mentals and parallel controls should be noted. Normally Eana pipiens larvae will reach stageg of metamorphosis in about 75 days after the eggs are fertilized, if kept at labora- tory temperatures of 23°-25°C., fed well, and not crowded. Arrange your data in tabular form on preceding page, and illustrate with drawings or photographs . DISCUSSION: It is not within the province of this Manual to carry an endocrine study beyond the macroscopic examination. The relation of the thyroid and/or the pituitary gland to meta- morphosis has been the subject of long and thorough studies. There are, however, other embryological approaches to the problem which might bear investigation. These are sug- gested in the form of questions, as follows: 1. Will thyroid or iodine affect stages of development prior to the limb-bud stages, or stages prior to the normal functioning of the host thyroid? Is there any ef- fect on the blastula or gastrula or neurula, for instance? 2.- The pituitary is known to have thyreotropic function, but which of these glands is the ontogenetic precursor? 5. What would be the effect on the embryos (larvae) following extirpation of either or both the thyroid and the pituitary anlagen? h. What is the effect on them of grafting thyroids (or pituitaries) of the same and of advanced ages into the tail blastemas of tadpoles. 5. What is the effect of thyroid and of thyroidectomy upon the regenerative processes of the larva? Some of these questions have been answered, but others should be. (See Beferences.) BEFERENCES : Adolph, E. F. , 1951 - "Body size as a factor in the metamorphosis of tadpoles." Biol. Bull. 61:376. Allen, B. M., 1938 - "The endocrine control of amphibian metamorphosis." Biol. Bev. 15:1. Bower, C. M., 1958 - "Growth rates of the hind limbs of Eana sylvatlca during normal and induced metamorphosis." Anat. Bee. 72: suppl. 99- Clements, D. I., 1952 - "Comparative histological studies of the thyroid and pituitaries in frog tadpoles in normal and accelerated metamorphosis." Jour. Boy. Micr. Soc. 52:158. D'Angelo, S. A., A. S. Ckirdon, H. A. Charipper, 19^1 - "The role of the thyroid and pituitary gland In the anomolous effect of Inanition on amphibian metamorphosis." Jour. Exp. Zool Etkln, W., 1956 - "The phenomenon of amphibian metamorphosis. III. The development of the thyroid gland." Jour. Morph. 59=69. Gudernatsch, J. F., I9I2 - "Feeding experiments on tadpoles." Arch. f. Ent. mech. 55:'+57. Gunthorp, H., I952 - "Results of feeding thyroid glands of various vertebrates to tad- poles." Physiol. Zool. 5:597. Ingram, W. B. , 1929 - "Studies on amphibian neotony. I. The metamorp}josis of the Colorado axolotl by Injection of inorganic iodine." Physiol. Zool. 2:1^9. Schneider, B. A., 1959 - "Effects of feeding thyroid substance." Quart. Bev. Biol. 114^:289. Spaul, E. A., 1925 - "Iodine and amphibian metamorphosis." Proc. Zool. Soc, London. 995- Swingle, W. W., I926 - "The effect on amphibian differentiation of feeding iodofibrln, lododestln, and iodogliadin." Anat. Rec. 5^:150. Uhlenhuth, E. & H. Kama, I928 - "The morphology and physiology of the salamander thyroid gland. III. The relation of the number of follicles to development and growth of the thyroid in Amblystoma maculatum. " Biol. Bull. 5'+:128. THYROIDECTOMY AND EARLY AMPHIBIAN DEVELOPMENT PUBPOSE : By meana of surgical extirpation to determine the functional relationship of the thyroid anlage in the early larra to met amorphic changes in the later tadpole. MATERIALS : Biological: Anura (stage #1?) or Urodele (stage #51) larvae. Technical: Standard Equipment. METHOD: Precautions: a. Avoid Injury or removal of heart anlage which Is Just posterior to the thyroid anlage . b. Avoid post-operative bacterial infection. If necessary, use 0.1^ sodium sulfa- diazine in operating and in culture media. Control: The control consists of embryos of similar age and stage in which similar surgical incisions are made but without the removal of any tissue. Such animals should be given Identical treatment as the experimentals. Procedure: 1. Bemove the embryos from their Jelly capsules and fertilization membranes. If there is any muscular activity, anesthetize them ini/5,000 MS 222 (freshly made up). Ciliary movement cannot be reduced by narcosis. 2. Make a shallow depression in the Permoplast (or paraffin) of the operating dish. Use Urodele Operating Medium for Urodeles and 2X Standard Solution for Anura, adding I'jt sodium sulfadiazine if there is difficulty with infection. Place the embryo on its left side, head away from the operator. 5, Insert the point of a double-edged leincet (or operating glass needle) between the position of the thyroid and the heart anlage, (see diagrams) and make an outward cut through the throat ectoderm. Bemove a wedge of tissue, the apex of which reaches the floor of the pharynx Just at the point of the slightly pigmented thyroid evagination. Carefully excavate the cells with the hair loop, avoiding particularly the heart mesoderm. Part of the pharyngael floor will, of necessity, be removed with the thyroid. (If the student finds this operation difficult, re- fresh his memory of the position of the thyroid anlage by studying both trans- verse and sagittal sections of tall-bud stages. A complete dissection study of the living tall-bud stage prior to thyroid extirpation is definitely recommended, for this is a delicate operation.) h. Transfer the operated embryo to an agar- base in a #2 Stender filled with operat- ing medium for about 50 minutes during which the wound will heal. Then transfer the embryo to Urodele Growing Medium or Standard Solution (depending upon the genus) for further development, preferably at a temperature slightly below that of the laboratory. Begin feeding at appropriate stage of development. OBSERVATION AMD TABULATION OF DATA: Operated and control embryos must be given identical treatment with respect to volume, medium, light, food, etc. 1. Make sketches at 15 minute intervals of the wound healing of the operated animals. 2. At weekly intervals following the operation, make drawings (or photographs) of thyroidectomlzed and control embryos. 5. Select some embryos that show definite effect of thyroidectony at a time when metamorphlc changes are beginning to appear in the controls, and treat them with 1/1,000,000 thyroxin to determine whether It is possible to compensate for the loss of the thyroid gland in bringing the larvee through the critical stage of metamorphosis. (Consult exercise on "Thyroid and Amphibian Metamorphosis".) -327- 528 THYROIDECTOMY AND EARLY DEVELOPMENT RANA STAGE 17 THYROID HEART AMBYSTOMA' STAGE 31 DISCUSSION: HEART THYRCMD ANLAGE Thyroidleaa tadpoles will not generally go through metamorphosis. This stage In Eana piplens controls should he attained at ahout 75 days at lahoratory temperatures, pro- viding the food, space, and other factors are adequate. There is no douht hut that the pituitary gland is closely related to the thyroid in function. The active element of the thyroid gland is iodine, and this may he administered in different forms (e.g., crystal- line iodine, KI, or in thyroxin). Thyroldless emhryos stimulated hy thyroxin may occasion- ally surpass the controls in metamorphic changes. IHFUN0I3ULUH X. ■ » JL ENDOCRINE ANLAGEN AT THE 5 mm. STAGE (FROG) RKJf'KHENCES : Allen, B. M., I918 - "The results of thyroid removal in the larvae of Bana pipiens." Jour. Exp. Zool. 2lt:i^99 (see ihid. 50:201). Allen, B. M., I958 - "The endocrine control of amphibian metamorphosia. " Biol. Kev. 15:1. Bauman, G., 1956 - "Modifications des premiers stades du developpement dea oeufa de ba- traclens anourea sans 1' Influence de la Thyroxine." C. B. Soc. de Biol. 121:1052. Hoskine, E. E. & M. M. Hoskina, I919 - "Growth and development of Amphibia as affected by thyroidectomy." Jour. Exp. Zool. 29:1. Larson, M. E., 192? - "The extirpation of the thyroid gland and Ita effecta upon the hypo- physia in Bufo amerlcanus and Bana pipiens." Sci. B^lll. Univ. Kansaa. 17:519' Stokes, M., I9U0 - "Esrly localization of the thyroid anlage in %^la regllla." Proc. Soc. Exp. Biol. & Med. 1+5:681. Taylor, A., 195'+ - "Athyroidism in the salamander, Trlturus toroaus." Jour. Exp. Zool. 275:155. HYPOPHYSECTOMY AND EARLY AMPHIBIAN DEVELOPMENT PURPOSE : By means of surgical extirpation to determine the relation of the epithelial hypophysis to early amphlhian development. MATERIAI^ : Biological: Early tail-bud stages of any amphiblEin (Anura stage #l8, Urodela stage #29). Technical: Standard equipment. METHOD: Precautions: a. Use only embryos that are not injured during removal from their Jelly capsules. b. Avoid bacterial contamination following the operation. If this becomes a factor, operate in 0.1^ sodium sulfadiazine in Standard Solution (or Operating Medium for Urodeles) . c. Endeavor to remove the hypophysis completely, but no other tissue. (The success of this operation can be tested only by subsequent histological examination. ) Control: The control for this experiment consists of surgical cutting in the vicinity of the hypophysis, but without removal of any cells. Procedure: a. Bemove all coverings, including the vitelline (fertilization) membrane, from a group of embryos at the appropriate stage (see above). Place them in 2X Standard Solution or in Spring Water (Anura) or in Urodele Operating Medium (Urodeles) over a base of agar. The agar will prevent adhesion of the epidermis to the glass bottom of the dish. b. Prepare an operating dish with base of soft paraffin or of Permoplast, and, with a ball tip, mould a depression so that the embryo can be held securely with its face looking upward toward the operator. c. Locate the hypophyseal groove (from stomodeum to hypophysis) and its dorsal hypo- physeal pit. This is the region of ingrowing of the pigmented, ectodermal cells which constitute the hypophysis. Use a double-edged lancet, or micro- (glass) needles to remove the wedge of pigmented hypophysis. This anlage grows inward between the roof of the pharynx and the floor of the brain ( Infundibulum) but neither of these other tissue areas should be disturbed, if possible. With small hair loop excavate all pigmented cells from the hypophyseal pit.* d. Leave the embryo in operating medium until the wound heala, about 50 minutes, then return it to the normal culture medium (i.e., Standard for Anura and Growing Medioun for Urodeles). Keep operated embryos separately in #2 Stenders, with agar bases, preferably at temperatures slightly below that of the laboratory. OBSERVATIONS AND TABULATION OF DATA: a. Make sketches immediately after the operation and at 15 minute intervals during the healing process of the hypophyseal area. Healing should be complete within 1 hour . b. At weekly intervals after the operation make drawings (or take photographs) of the operated and control embryos, side-by-side, to show any 1. Changes in pigmentation 2. Differences in rate of development (i.e., size differences) Eemember that these embryos must be fed after the stage #1+2. c. Embryos which show prono\inced effects of hypophysectoipy (silvery appearance), stunting, etc.) should be sectioned (transversely and sagl tally) to determine the extent or success of the hypophyseal extirpation. Control embryos of the same age should be sectioned for direct comparison. Note also any variations in the development of the thyroid glands. * See section on'^Thyroidectoiny and Early Development" for photogi-aph of sagittal section of Anuran embryo showing hypophysis. -529- 550 HYPOPHYSECTOMY AND EARLY DEVELOPMENT RANA PIPIENS RANA PIPENS HYPOPHYSEAL INVAGINATION HYPOPHYSEAL INVAGINATION STOMODEAL CLEFT STAGE 19 STAGE 17 HYPOPHYSEAL ANLAGE HYPOPHYSECTOMIZED AT STAGE 18 CONTROL HYPOPHYSECTOIY (Causing stunting and failure to develop pigment) HYPOPHYSECTOMY AND EARLY DEVELOPMENT 53I DISCUSSION: The hypophyela is the anlage of the para anterior, the pars intermedia, and the pars tuheralis of the adult pituitary gland. The gland is derived entirely from ectoderm, but these portions are derived from head rather than brain ectoderm. Eemoval of this ectoderm (epithelial hypophysis) after its ingrowth has begun seems to prevent regeneration of the same type of tissue so that the relationship of the pars intermedia to the pigmentary sys- tem is clearly indicated. Such embryos should survive for many weeks. The method of transplantation might be super-imposed upon this procedure of fextirpa- tion. The host hypophysis might be transplanted in toto (if so removed) to the flank or tall-bud region of the same embryo, to determine whether It could support the pigmentary system although separated from the Infundibular portion of the pituitary. This double- treatment of a single embryo is rather drastic, and it is not always possible to remove the hypophysis Intact. The initial attempts might therefore be a direct transplantation of an hypophysis excised from another Individual, after removal of the original host anlage. Atwell and Holley (1956) have shown that if the epithelial hypophysis of Eana sylvatlca is removed at the tall bud stage, some of those which became silvery would, nevertheless, metamorphose. The thyroids, gonads, and adrenals of such forms showed no ef- fect of hypophysectoiny. The silvery tadpoles which achieved metamorphosis lacked the pars intermedia but possessed sufficient of the anterior lobe to stimulate the normal thyroid to carry the animals through the critical period of metamorphosis. BEFERENCES: Allen, B. M., 1929 - "The Influence of the thyroid gland and hypophysis upon growth and development of Amphibian larvae." Quart. Bev. Biol. l+:325. Atwell, W. J., 193**^ - "On the metamorphosis of silvery tadpoles of Bana sylvatlca." Anat. Bee. 58: suppl. h8, Atwell, W. J. & E. Holley, I956 - "Extirpation of the pars intermedia of the hypophysis In the young amphibian with subsequent silvery condition and metamorphosis." Jour. Exp. Zool. 75:25. Blount, E. F., 1955 - "Total growth and body proportions as influenced by pituitary rudi- ment implantation and extirpation in Urodele embryos." Anat. Bee. 55:^6. Greenwood, A. W., 192^*- - "The growth rate in hypophysectomized salamander larvae." Brit. Jour. Exp. Biol. 2:75. Hogben, L. T., 1924 - "The Pigmentary Effector System." Oliver & Boyd, Edinburgh. Klatt, B., 1955 - "Weitere Versuche ( gypophysesextlrpatlon und Implantatlonen) an Triton- larven." Arch. f. Ent. mech. 150:79. Schotte, 0. E., 1926 - "Bypophysectomle et Metamorphose des Batraciena Urodeles." C. B. Soc. Phys. et Nat. Hist., Geneve. ^5:95. Smith, P. E., 1920 - "The Pigmentary, Growth, and Endocrine Disturbances Induced in the Anuran Tadpole by the Early Ablation of the Para Buccalis of the Hypophysis." Am. Anat. Memoires, #11:1. Swingle, W. W., I92I - "The relation of the pars intermedia of the hypophysis to pigmenta- tion changes in /nuran larvae." Jour. Exp. Zool. 5'+:119. CYTOCHEMICAL TESTS FOR GAMETES AND EMBRYOS OF AMPHIBIA The following tests have all teen tried and proven successful, and should he used on small and large ovarian eggs, Isolated germinal vesicles, testes, the gastrula and neurula stages. The tests are largely for proteins, with emphasis on the amino acids and nucleo- proteins. Some testa for carbohydrates, lipids, and enzymes are Included. Specific suggestions are as follows: 1. Stain immature or post-ovulatlon ovary of Rana pipiens with a. Safranin and light green (note yolk nuclei). h. Iron haematoxylin (note chromosome and nucleolar structure) c. Toluldine hlue - stains both nucleic acids. d. Unna's methyl green-pyronine (combine with enzymes). Thymonucleic acid green, ribonucleic acid red. e. Feulgen - specific for thymonucleic acid. Plasmal reaction. 2. Glycogen and lipid distribution in oogonia and early oocytes. 3. Protein tests : a. Nucleoprotein extraction - Mlrsky & Pollister (Frog testes). b. Biuret - Xanthoproteic - Ninhydrin tests (Frog testes). c. Arginine - lyrosine - Tryptophane tests (Frog testes smears; frog ovary). d. Test for - SH proteins, nitroprusside -test (Frog testes smears, embryos). h. Tests for enzymes: peroxidases - Indolphenoloxidases (Frog testes smears). 5. Miscellaneous: Test for Phosphorus and Oxygen. PROTEIN TESTS NUCLEOPBOTEINS : (Mlrsky & Pollister, I9I+2 ) Grind up a large number of whole frog testes in neutral sodium chloride solutions of three concentrations, in two of which the nucleoproteins are soluble and in the third they are Insoluble. 1. Extract with 1 M NaCl-volume about 10 X that of testes mash. Becomes viscous. 2. Centrifuge at 10,000 r.p.m. , providing a viscous, slightly opalescent super- natant fluid. Viscosity due to nucleoprotein dissolved therein. 5. Add 6 volumes of distilled water - nucleoprotein percipitatea in a fibrous mass, settling rapidly so that the supernatant fluid can be siphoned off. k. Wash percipitate in 0.1^*- M NaCl (in which it la insoluble). 5. Redisaolve in 1 M NaCl. 6. Centrifuge again at high apeed to remove the suspended material. 7. Be-percipltate by adding 6 volumes of distilled water. 8. Stir the mixture with a glass rod with a crook at its end, collecting a fibrous nucleoprotein which will wind around the glass rod. (Further purification can be achieved by repeating the above process) ************* NUCLEIC ACIDS:* TOLUIDINE BLUE: 1. Stain sections (ovary) for 20 minutes in saturated aqueous solution of Toluldine Blue. 2. Differentiate twice for 10 minutes in 95']^ alcohol. 5 . Mount as usual . Toluldine blue is taken by both nucleic acids, an orthochromatic (blue) color la given only by nucleic acids, found in both the cytoplasm and nucleus. ♦The cytoplasm of cells contains principally ribonucleic acid (also known aa yeast or phytonuclelc acid) while the nucleus contains largely desoxyribonuclelc acid (alao known as thymonucleic acid). The ribonucleic acid la transformed Into thymonucleic acid during development. -552- CYTOCHEMICAL TESTS ON EMBRYOS 555 B. THE PLASMAL BEACTION: The following procedure haa been used successfully with the amphlhian ovary, particularly the iimnature and post-ovulation ovary. It is haeed on Voss (1922 and 1951) and Lison (I956). The reaction is given by a special type of phospholipid in which an aldehyde group appears upon treatment with the sublimate. The pro- cedure follows : 1. Fix the ovary in saturated corrosive sublimate - 5 minutes. 2. Wash in distilled water. 5. Dehydrate, clear, embed, section (10 ^), and then hydrate. k. Place directly In Feulgen reagent for I5 minutes. 5. Wash with 5 changes of distilled water saturated with SO^. 6. Einae in distilled water. 7. Mount in pure glycerine and observe under the microscope. Plasmalogen is a component of the cytoplasm which gives a positive Feulgen test. Being a phospholipid, the control for this test consists of fixing the ovary with Carnoy's fluid and washing twice with alcohol for 15 minutes each. This extracts most of the lipids. C. THE NUCLEAL BEACTION: This reaction depends upon the combination of the N-sulphinlc acid in fuchsin- sulphurous acid with the aldehyde component released from a molecule by mild and partial hydrolysis. However, it nnist be remembered that the failure of any tested tissue to give this reaction may be due to any of four following causes (Gardiner, 1955). 1. The substance may not contain the aldehyde component. 2. The hydrolysis may be insufficient to release the aldehyde group from the molecule . 5. The hydrolysis may result not only in splitting off of the aldehyde group but also in its disintegration, so that it cannot react with the sulphinlc acid to form the new compound. k. The aldehyde-containing substance, and consequently the aldehyde set free on hydrolysis, may be so small in quantity that, although the reaction actually occiu^s, the compound la too minute in amount to be visible even with high magnification. Gardiner (1955) saya further: "lypically chromatin gives the reaction, but it does not invariably do so, nor la it the only cellular substance capable of it." Stowell (19^6) says; "The preponderance of evidence indicates that with the proper precautions the Feulgen technlc for thymonucleic acid la one of the most specific histochemlcal reactions." THE PROCEDURE The procedure given here is baaed on the paper by Eafalko (19^+6): hydro- chloric acid is neceaaary only to release the sulphur dioxide from the sulphites used in the formation of the leuco-baalc fuchsin and in the sulphurous acid bath following atainlng. Both the acid and the sulphite were eliminated and direct charging of both the basic fuchsin and the bath water with sulphur dioxide gas was substituted. Bubble sulphur dioxide gas from a small aperture In glass tubing into 100 cc. of 0.5^ basic fuchsin, beneath a hood. Decolorlzatlon takes place in 1 hour and the reagent is ready for use. Distilled water la aimilarly aaturated, and may be stored In tightly corked bottlea for weeka. To get SOg, use simple flask-and- funnel generator and sodium bisulphite and dilute aulphurlc acid. 1. Fix tiaaues in Zenkera (or Bouln) for 2-20 mlnutea. 2. Wash not more than 20 mlnutea, embed and aection in the usual manner. 5. Place in distilled water - 2 minutes. k. Normal HCl at room temperature - 2 minutes. 5. Normal HCl at 60OC, for 8 to 10 mlnutea. 55l^ CYTOCHEMICAL TESTS ON EMBRYOS 6. Normal HCl at room temperature, rinse only. 7. Distilled water - rinse. 8. Sulphurous acid - 2 minutes. 9. Leuco basic fuchsin - Ij to 2 hours. 10. Sulphurous acid bath for sufficient time to remove the free, unreacted leuco basic fuchsin; three 1 minute changes should be sufficient. 11. Tap water for 10 to 15 minutes. It is possible, and even advisable, to counterstaln with fast green in aque- ous or alcoholic solutions. Dehydration ia accomplished either through the alcohols or from water through triethyl phosphate (Nelsen, I'^k^: Stain Techn. 20:131) directly unto xylene. The latter is a shorter method and does not ap- preciably remove the aqueous counterstaln. The Feulgen reaction is essentially Schiff's aldehyde teat applied to a tis- sue cell. The aldehyde is the carbohydrate released from the nucleic acid com- ponent of chromatin after hydrolysis with normal HCl; this carbohydrate, a d-ribodesose (Levene, 1951), combines with the active principle of fuchsin- sulphurous acid, an N-sulphinic acid with the formula H .H<^ (Welland and Scheurlng, I92I) SO2H to form, a blue- red color, ofteh almost purple. The validity of the test rests upon the absence from the tissues of any aldehyde other than that tested, which might combine with sulphlnlc acid. If the fuchsin-sulphinic acid ia oxidized, the color may be restored to act as a stain rather than as a reagent. Gardiner (1955) says: "It la impossible in the Bouin material to diatinguiah the chromatin from other cell constituents taking haematoxylin, but the Feulgen preparations show clearly that this perinuclear substance ia not chromidlal." ************* UrmA'S (1921) METHYL-GREEN FYBONINE STAIN FOB NUCLEIC ACIDS: Methyl green stains thymonuclelc acid green while pyronlne stains the ribo- nucleic acid red. It is important that Gruebler's Pyronlne be used and this pro- cedure works best on late embryonic or adult tisauea rather than the oocytes and early cleavage stages. The stain is aa follows: Gruebler's methyl green O.I5 gm. Pyronlne B 0.25 gm- Alcohol (95^) 2 .50 cc. Glycerine 20.00 cc. Carbolic acid (0.5^ aqueous) 77-50 cc. Fix the tissues in 95^ alcohol, embed eind section in the usual manner, and stain in the above (Unna's) stain for 20 minutes. The albumins and globulins are stained red by the pyronlne, while the nucleo- proteins are stained blue-green with the methyl green. To separate the albumins and globulins one can take advantage of the differential solubility. The albumins and pseudoglobulins are soluble in water, the globulins are not soluble in water. Both are soluble in salt solutions. Failure to stain with methyl green would indicate the absence of nucleoproteins. CYTOCHEMICAL TESTS ON EMBRYOS 555 0 H OH ,0(P05%) Guanln - d - ribose nucleotid (Ribosnucleic acid) The distribution of ribonucleic acid at three stages in the oogenesis of the amphibian egg. (Redrawn from Brachet, 1944.) OlPOj^) Guanln - 2-desoxy - d - ribose - nucleotlc (Thymonucleic acid) IDENTIFICATION OF NUCLEIC ACID BY TEE USE OF ENZYMES:* 1. Rlbonuclease (crystalline) can be used to digest away the ribonucleic acid, following which Toluldine Blue will give blue nuclei and cytoplasm colorless or following which Unna's stain would give green nuclei and colorless cyto- plasm. See Kunltz (19'+0) for simple method of preparing crystalline rlbon- nuclease. A concentration of 0.1 mgm./ cc. of water, properly buffered (Mcllvalnes buffer to pH 7.0), of the crystalline rlbonuclease should be used with the tissue. Incubated at 50OC. for 5 hours. Wash with distilled water; stain with Toluldine Blue or Unna. The best fixatives for this test are Camoy, or the a Icohol-formol- acetic mixtures, Zenker's without formol but with acetic acid. Fixation no more than 1 hour for Amphibian eggs. A positive stain following this procedure identifies thymonucleic acid, and will be essentially nuclear. ************* 2. Thymonuclease (McCarty, I9I+6: Jour. Gen, Physiol. 29:125) acts In a few minutes at room temperature. If the tissues are rinsed and followed with Toluldine Blue or Unna, the positively staining elements will represent the ribonucleic acid which is essentially nucleolar and cytoplasmic. (It must be remembered, however, that the specificity of these enzymes has not been conclusively demonstrated, and certain variables are Involved in their use.) Brachet {Private Com.) reports that Miss MacDougall at Cold Spring Harbor stated that she obtained rlbonuclease completely free of proteolytic action and that it worked Just the same on sections. Brachet was unable to find a decrease In the arglnine and pyronlne reaction after digesting tissues with rlbonuclease. ************* ♦These enzymes are still very expensive. 556 CYTOCHEMICAL TESTS ON EMBRYOS NOTE: The student ia advised to run controls for all of the preceding page proce- dures, and to gain a prior acquaintance with the standard procedures with safranln-llght -green and the iron haematoxylln. It must be remembered that the chromosomes consist of more than thymonucleic acid; that both types of nucleic acids may be found in both the cytoplasm and the nucleus; that stain- ing reactions which are essentially chemical reactions involving the use of enzymes rather than adherent staining, depend on fixation, pH, and tempera- ture, and concentration of the enzymes (Stowell and Zorzoll, 19'+7); and that the technique of combining the use of enzymes with specific staining proce- dures has not yet been fully checked for reliability. Another approach which, if combined with the above, would give more re- liable results, la the U.V. absorption photography ( Caspersson, Lavin and others). Both nucleic acids absorb in the ultraviolet range, but the bands are sufficiently far apart so that if the tissues are pre-treated with enzymes, then the absorption Is read, an analysis can be made. F. GENERAL PBOTEIN TESTS ( STAHDARD) : 1. Biuret Test for Peptides - this ia a crude and relatively Insensitive teat for peptides or proteins in general, but has the advantage of being rapid and rather simple. The procedure is aa follows: a. Harden tissues In 10^ formaldehyde for 2k hours if formalin is not Included in the fixative. Wash thoroughly- b. If the material la to be sectioned, it should be cut thickly. The reactions must be carried on in an alkaline environment which tends to macerate the tlaauea. It ia a more satisfactory test for the pre- sence f^f these substances in relatively large pieces of tissue. c. Place the tissue In 1^ NaOH or 1^ KOH in a watchglaas; add a few drops of 1^ aqueous CuSOi^, and stir. A red color develops in the presence of simple peptides: a blue-violet color with the higher peptide and proteins. 2. Xanthoproteic Beaction - this is also a crude but simple test for proteins and is positive in the presence of tyrosine, phenylalanine, tryptophane, all phenolic compounds, and all the peptides except the protamines. a. Harden the tissuas in 10^ formaldehyde for 24 hours, if formalin is not included In the fixative. Strong fixation is necessary. b. Immerae the tissue in concentrated HNO^ for some minutes, until it be- comes intensely yellow. c. Wash in distilled water. d. Immerse in diluted ammonia, or expose the tissue to ammonia vapors. An orange color indicates a positive teat. e. Mount directly and examine in pure glycerine. 5 . Ninhydrin Beaction for Amino Acids and Lower Peptides - this test glvea a blue or violet color in the presence of amino acids, free or bound peptides and proteins. The reaction ia not highly specific, however, for it is negative to amino acids proline and hydroxyproline, and it Is positive to certain amines, aldehydes, augars with free aldehyde or keto groups and ammonia compounds. With these non-protein and non-amlno acid compounds, the color reaction is much less intense and tends to be reddish instead of blue. Serra (19'+6) points out the importance of hard fixation to prevent the color moving about within the tissues and becoming adherent to unnatural cell structures. The reaction is aa follows (Serra and Lopea, 19'+5)*. a. Sectioned material Is immersed in equal volumes of O.U^ solution triketohydrinden-hydrate (ninhydrin) in distilled water and any phos- phate buffer held to pH 6.98. The phosphate buffer may be made by adding 6 cc. of a M/15 secondary sodium phosphate to ^4. cc. of M/ 15 primary potassium phosphate. CYTOCHEMICAL TESTS ON EMBRYOS 337 b. Place the material in a watchglasa and over a 'boiling water tath, among the vapors, for 1 to 2 minutes after the water 1)0118. c. Mount in pure glycerine. If the- tissues are thick, compress teneath a coverslip to separate the cells from each other. The color will fade within a few hours. G. TESTS FOB VABIOUS COMMON AMINO ACIDS: 1. Arginine: The development of histo-chemlcal teats of great specificity has immeasurahle significance in relation to an understanding of cell morphology and physiology, particularly in respect to the nuclear inclusions. Thomas (191+6) and Serra (19^+6) following Sakaguchi (I925) have perfected the test for arglnlne. The enjjirical formula for arginine is H3N NH \/ / OGH N]^ According to Thomas {19'+6) "when the Sakaguchi reaction is applied to a protein, a color is Imparted to the protein molecule." Ejy the methods of Thomas and of Serra, the red color of the following reactions may he re- garded as proof-positive of the presence of arginine. It is suggested that sections of the testes and of the ovary be tested for arginine. The red color generally develops in both the cytoplasm and the nucleus of most cells, but there is considerably more color in the chromosomes, nucleoli, and intermltotic chromatin than in the remainder of the cell. The spermatozoan heads, representing concentrated nuclear material, give a most intense reaction (see Thomas, 19'+6). Thomas (I9U6) and Serra (191*6) have independently modified the original Sakaguchi (1925) reaction as a specific test for arginine in biological materials. The Thomas procedure is negative for guanidine, urea, creatine, creatinine, and other amino acids that might be encountered in biological tissues. The presence of arginine is demonstrated by a strong red or red- orange color which is transient but can be prolonged for several hours by proper dehydration. The ufjual ethyl alcohol tends to extract some of the color, as do most of the other dehydrants, but Thomas il9k6) has found that tertiary butyl alcohol will dehydrate the tissues without the removal of color and aniline oil is used to clear. The system must be kept alkaline because the color fades in either neutral or acid media. Serra (19'+6) now advises the use of glycerine in which the color seema to be stabilized for many months. a. THE METHOD OF SERRA (!946) a. Harden fixed tissues in 10^ formaldehyde for 2U hours unless formalin was included in the original fixative. b. Prepare an alkaline a-naphthol-urea mixrixre and bring it to 0° to 5°C., in a watchglass. The mixture is as follows: 1. 0.5 cc. diluted a-naphthol. Use stock solution 1^ crystallized a-naphthol in 96^ alcohol and. Just before using, dilute to 1/10 with 1+0^ alcohol. 2. O.'? cc. normal NaOH. 5. 0.2 cc. of hCfjii aqueous urea solution. 558 CYTOCHEMICAL TESTS ON EMBRYOS c. After 12 to 15 minutes in the above mixture, add 0.2 cc. of 2'jt NaOBr, and stir well for 5 minutes. This solution should be freshly made up by pouring 0.7 cc. of liquid bromine into 100 cc. of 5^ NaOH, agitat- ing, and cooling. d. Add 0.2 cc. of kOf, urea, stir. e. Add 0.2 cc. of 2^ NaOBr and again stir well. The color will now develop if arginine ia present and should attain its maximum intensity in about 5 to 5 minutes. The color can be somewhat stabollzed by passing the tissue through h changes of pure glycerine. Serra claims that if the tissue is placed In pure NaOBr solution for 5 minutes after step "e" (above) the color becomes more intense and is stabilized in glycerine. He uses this test for basic proteins, for proteins in general which contain arginine, and for guanldine derivatives in which only one H-atom of one amino group is substituted by a radical of the alkyl or fatty acid type. b. THE METHOD OF THOMAS {I9t6) With this method the red color develops when a solution of arginine or a protein solution containing arginine is treated with a-naphthol, alkali and hypochlorite. Guanidine itself is negative but when one of the guanidine H-atoma is substituted by an alkyl, fatty acid, or cyano radical, then there is a positive color reaction. The color develops and fades rapidly but the addition of urea helps to hold the color for several minutes. Bapid dehy- dration is critically important, and is accomplished by tertiary butyl alcohol followed by aniline oil. a. Fix tissues in Bouin'a fluid. Carnoy's or 10^ formalin are equally good. b. Sectioned material must be firmly affixed to slides, with the usual paraffin albumin method. The slides may be left in 70^ alcohol until ready for the tests. c. Hydrate the tissues down to distilled water, then place the slide in a-naphthol in 10^ alcohol, by volume. d. Transfer to sodium hypochlorite solution for 20 seconds. This is a 0.15 normal solution of sodium hypochlorite in 0.05 normal sodium hydroxide. This solution can be made from commercial "Clorox" (see Albanese & Frankston, I9U5) or by the method of Van Slyke & Hi Her (1955). e. Transfer to urea for 5 seconds. This is an alkaline urea made up by adding 20^ urea to 0.05 normal sodium hydroxide. f. Dehydration: 80^ tertiary butyl alcohol for 50 seconds. To the BO cc. of tertiary butyl alcohol add 1 cc. of 5 normal sodium hydroxide and 19 cc. of distilled water. Transfer to 100^ tertiary butyl alcohol for 2 minutes. Amblystoma testes, Bouin fixation showing positive Arginine reaction. (Courtesy L. JE. Thomas 191+6: Jour. Cell. & Comp. Phyelol. 28:11+5) CYTOCHEMICAL TESTS ON EMBRYOS 339 g. Clearing: 100^ aniline oil for 2 minutes. 100^ toluene for 5 seconds, h. Mounting: clarite. ************ 2. lyroaine: According to Serra (19^6) "lyrosine seems to be present in almost all natural proteins in amounts which are not very different for the various classes, excepting principally silk fihroin, pepsin and insulin. " The formula for tyrosine is OOH HO. N^ ^ ^ k- K.' i^.p\ J OOCYTES OF RANA RIOIBUNOA Fig. 4. Tyrosine reaction, photographed wlt}iout filter. Fig. 5. Tyrosine reaction, photographed at higher magnification than Fig. 4 and without filter. *Flg. 6. Arglnlne reaction, photographed with green filter. Slight differential between cytoplasm, smd nucleus. *Flg. 7. Same as Fig. 6 except that tlie nuclei have been separated from the cyto- plasm by compression after tlie arglnlne reaction. Photographs hy courtesy J. A. Serra ♦Figures unpublished. 3W CYTOCHEMICAL TESTS ON EMBRYOS A modified Mlllon's reaction, devised by Serra and Lopes (19^5) lias been used to detect the presence of tyrosine, in tiie protein molecule, but the color Is also produced by other phenolic compounds. A transient color is produced by tryptophane but the tyrosine color attains Its maximum In- tensity in about 5 minutes and lasts for some months, fading gradually with time. Their procedure follows: a. Immerse tissues in mercuric solution for 30 minutes. This la made up of 7.5 gms. BgSOjj.; 5.5 gms. HgClg; and 7.O gms. NagSOl^ dissolved In 85 cc. of distilled water to which 12.5 gnis. of cone. :^SOi+ had been added, the whole being made up to 100 cc. volume with distilled water. The reaction should take place in a glass stoppered bottle in water bath at 60OC. b. Cool the bottle in running water for 10 minutes. c. Add to the mercuric solution in the bottle an equal volume of distilled water. d. Develop the color by adding a few drops of -1 M NaNC^ (6.9^ aqueous), freshly prepared. The color may last for months if the tissues are mounted in glycerine. Compress cells apart beneath coverslip. A second procedure may be used and is known as the diazo reaction for hlstidine and tyrosine. This test gives an orange or yellow color in the presence of hlstidine and tyrosine of the proteins. It is the hlstidine which imparts the reddish tinge. Lison (I956) follows a different procedure to demonstrate the presence of phenolic compounds and axoproteids. Serra' s (19't-6) procedure follows: a. Treat tissues for 2 to 3 minutes with saturated aqueous solution of sodium carbonate. b. Add a few drops of diazo reagent, stir well, and observe in pure glycerine. The diazo-reagent (prepare Just before using, keep cool) Place 1.5 cc. of sulphanlllc acid In a 50 cc. flask in an Ice bath. This Is made by dissolving O.9 gms. pure sulphanlllc acid in 9 °c« of concentrated HCl, and adding water to make 100 cc. Add 1.5 cc. of a 51^ aqueous solution of NaNO^, shaking well. Add 6 cc. of NaNOg after 5 minutes, constantly shaking mixture. Add cooled distilled water to make 50 cc. total volume. ********^*** Nuclei of spermatozoa vH>:^ Tails of spermatozoa L J Modification of the Sakaguchi test for Guanldine derivatives. Trlturus vulgaris teatls. Courtesy J. R. Baker, I9U7: Quart. Jour. Microsc. Science. 88:115. CYTOCHEMICAL TESTS ON EMBRYOS 5i^l 3. Tryptophane : Tryptophane compriaes from 1 to 5^ of the great majority of proteins. Its formula follows: H • - C - C^ — CH(N^) - COOH H H Fig. 8. Testis of Rana ridibunda perezi. Arginine reaction (green filter). Fig. 9. Spermatozoa of Helix aspersa, heads to upper left. Arginine reaction. Fig. 10. Salivary gland chromosomes of Chlronoraus larvae. Tyrosine reaction. Fig, il. Salivary gland chromosomes of Chironomus larvae. Tryptophane reaction. Photographs by courtesy of J. A. Serra Since this amino acid results from the hydrolysis of many proteins and the test is sensitive, a satisfactory procedure is available for animal tis- sues. a. Harden the tissues in 10^ formaldehyde, if they were not previously fixed In a formalin fixative, for 5 hours; wash well. b. Immerse for 5 to 5 seconds in an aqueous solution of sodium silicate. c. Immediately Immerse the pieces In Volsenet reagent for 10 to 15 minutes, in a small glass-stoppered bottle. This reagent is made by adding 1 drop of 2% aqueous formol and 1 drop of 0.15^ aqueous NaNC^, with stirring, to 10 cc. of concentrated HCl. Solution is freshly made before using. d. Mount in glycerine and observe directly. Tissues may be compressed apart between coversllp and slide. The color fades hence the tissue must be examined within a few hours. »*»**»*»**■)(■* 3h2 CYTOCHEMICAL TESTS ON EMBRYOS H. TEST FOB THE -SH GROUPS: The following test gives a stable red coloration in the presence of the tri- peptlde glutathione. According to Serra (19i;6): "it is possible not only to demonstrate the existing -SH groups but also to reduce SB groups to SH groups by means of a pre-treatment of the naterials with a solution of 10^ KCN for 10 minutes." Of course, the arginine test is also positive for the -SH group pro- teins. An intense reaction for protein -SH presumably demonstrates the existence of active metabolic and synthetic changes in the proteins (Brachet, 19i4-0). Pro- tein denaturation involves an unfolding of polypeptide chains and an increase in -SH reacting groups. Hence, a positive -SH reaction might indicate either an active synthesis or a breakdown of proteins. The procedure follows: a. Fix tissues for no more than h hours at room temperature in 10^ formaldehyde. Rlnae in distilled water. b. Immerse tissues or sections in 51^ aqueous zinc acetate - 50 seconds. Rinse in distilled water. c. Treat with 10^ aqueous solution sodium nitroprusside containing 2^ concen- trated ammonia. Brilliant red color develops within 5 minutes. Wash in distilled water and mount in glycerine. The glutathione is partially soluble, hence the reaction will vary with fixa- tion and other preliminary treatments. Fresh material, without fixation, gives even more reliable results. EARLY BLASTULA GASTRULA Distribution of -SH proteins (nitroprusside test) at the beginning (a) and end (b) of oogenesis. (Redrawn from Brachet, 1944) . The distribution of basophilic granules (dots) and the sulfhydril proteins (dashes) (after Brachet, 1938) . Location of the -SH proteins by the nitroprusside test during various stages In the early development of the amphibian embryo (Redrawn from Brachet, 1944) . CYTOCHEMICAL TESTS ON EMBRYOS 5I15 THE GLYCOGEN TESTS The plaamal reaction is generally used for plasmalogen (a phoaphollpln) in the cyto- plasm and consists of the Feulgen reaction. But this can also he used for glycogen if the proper preliminary treatment is given to the tissues, as follows: 1. Fix pieces (small) of ovary (amphibian) for 1 hour in k-'f> chromic acid. 2. Wash 5 minutes in running water. 5. Immerse for 15 minutes in Feulgen reagent. h. Wash 3 times in water saturated with SC^. 5. Einse in water and mount in glycerine. Glycogen makes its first appearance soon after the first fat glohules of the vitellus appear, with a concentration in a ring about the nucleus. There is none in the germinal vesicle. Bevelander and Johnson (19^+6) give a simple method of hletochemlcal localization of glycogen, as follows : 1. Fix tissue in Carnoys for 2k hours. 2. Mount sections on albumen smeared slides, flooded with Lugol's solution at ^QOC. 5. Eemove the paraffin with xylol. h. Flood the sections with a saturated solution of Iodine In 100^ alcohol. 5. Mount in clarite to study. Bensley (1959) describes a stain for glycogen as follows: 1. Boll the following gently until the color darkens, then cool. Carmine 2 gma. Potassium carbonate 1 gm. Potassium chloride 5 gms. Distilled water 60.O cc. 2. Add 20 cc. of concentrated ammonia. 3. Allow to ripen for 2h hours. This becomes the stock solution. THE LIPID TESTS Identification of the various lipid subatances in the cell ia very difficult (Liaon, 1936). Solubility tests are unreliable, and formalin fixation alters normal solubility of some fatty subatances. Cytochemlcal and macrochemical teats may vary, even with the iden- tical lipids. Glycerides and fatty acids are never birefringent in the dissolved condi- tion when examined in vivo, but after treating with formalin or freezing they may become crystalline and birefringent . Tests with Osmlc Acid, Sudan III, when coupled with other (physical) tests will give substeintially reliable analytical results. Serra and Lopes (19'*-5) in studying the cytophyslology of the nucleolus give the following procedure: 1. Fix for 16 hours in 10^ formol. 2. Wash tissues well in running water. 5. Stain with Sudan III in alcohol: At 70° stain for 25 to 75 minutes. At kO° stain for 22 hours. The tests for lipids is not quantitatively reliable. ENZYMES* 1. Hlatochemical Teat for Peroxidase: This test should be applied to immature or post-ovulatlon amphibian ovaries containing oocytes of various alzea. a. Fix the ovary in 10^ formol for 10 minutes. This destroys the catalase. b. Wash thoroughly in distilled water. c. Immerae the tiaaue in saturated aqueous solution of benzidine containing a few drops of acetic acid per 10 cc. d. Immerse in 1^ hydrogen peroxide (perhydrol diluted to 1^). Note the oxygen bubbles and the blue followed by brown coloration. The reaction will appear most intense in the ovarian capillaries. *See Sumner and Somers 19^7. 5^1^ CYTOCHEMICAL TESTS ON EMBRYOS 2. Hlatochemlcal Test for Indophenoloxldase : This teat should be applied to the im- mature or post-ovulatlon ovary of any amphibian. The method is essentially that of Voss (1921+). a. l^ke up Solution A by dissolving 0.5 gm- of alpha-naphthol in 100 cc. of boiling distilled water; boll for 5 minutes; cool and filter. b. Make up Solution B by dissolving 0.5 gm. dimethylparaphenylenedi amine* in 100 cc. of cold distilled water and allow it to stand for 2U hours. Filter. c. When ready to make the test, mix equal parts of "A" and "B"; add an equal volume of 0.64^ NaCl and mix thoroughly. d. Pour some of the mixture in a Stender and add the tissue to be tested. The mixture can be further diluted with physiological saline solution to prevent too intense a reaction. This procedure has been modified by Child by diluting the reagent as follows: a. To 10 cc. of distilled water add 1 drop of dlmethylparaphenylenediamlne.* b. To 10 cc. of distilled water add 1 mgm. of alpha-naphthol. c. Place the fresh tissue to be tested in physiological saline and to each 1 cc. of this solution add 1 drop of each of the solutions "a" and "b". Note which of these methods stains the yolk nucleus of the small oocytes. THE TEST FOR PHOSPHORUS Phosphorus is found in the nucleolus, the chromosomes, and the protoplasm generally. It is in the thymo- and ribonucleic acids, in conjugated pliosphoproteins and in the nucleo- proteins. Serra and Lopes ( 19^+5 ) say: "It seems that we can safely conclude that the phosphorus reaction, the coloration witn basic and acidic stains and the nuclease reac- tion, show the existence of nucleotides of the riboae type in the nucleolus." The nucleo- lar Inclusions probably have a greater concentration of these nucleotides than does the remaining part of the nucleolus. The nucleoli are richer in nucleotides, as determined by this phosphorus teat, when they are young. The method of Angell, A., (1955 Elv. di Biol. 10:702) a. Sections treated for 20 minutes with solution made up of 5 gms. ammonium molybdate 20 cc. distilled water 20 cc. of 50^ aqueous hydrochloric acid • b. Eeduced in N/50 stannous chloride. c. Blnsed quickly in distilled water. d. Washed in 2.5^ aqueous ammonia. If phosphorus is present in any form there will develop a blue-green color. The method of Serra and Lopes ( 19'*-5 ) a. Fix small pieces of tissue in a mixture of 2 volumes of 95^ alcohol, 1 volume of formol, and a few drops of glacial acetic acid per 10 cc. of total volume. Carnoy's fixative is also satisfactory. b. Wash In running water, then in distilled water. c. Hydrolyze for 5 days or more at 10° to 12°C. and in darkness, if possible. Use 5 cc. of the following reagent: to 20 cc. of diatilled water add 0-5 gms. ammonium molybdate and 10 cc. of 50^ HCl, dilute the mixtxire to a total of 50 cc. with distilled water. d. Transfer to temperature of 20° to 25° for several days. e. Add 1 drop of acetic benzedine. (Dissolve 25 mgms. of benzedine in 5 cc. of pur^ glacial acetic acid. Dilute to 50 cc. with diatilled water. Stir or agitate for 5 minutea.) f. Add 2 drops of pure saturated sodium acetate. The tissue, if phosphorus is present, will rapidly develop an intense blue color. The color is durable. g. Mount In glycerine to which has been addec" a few crystals of sodium acetate. * If the reagent ie in solid form. It should be heated on a water bath until it melts. CYTOCHEMICAL TESTS ON EMBRYOS 3j^ THE WINKLER METHOD OF MEASURING OXYGEN CONSUMPTION* Thia Is a tltrimetric method of measuring dissolved oxygen, first used in I908 ty Warburg in a study of the change in rate of ojtygen consumption of the Arbacia egg follow- ing fertilization. It has been used recently by Earth ( 19^+2) in a study of the oxygen con- sumption of fragments of the amphibian gastrula. STOCK SOLUTIONS: (For Class Use) 1. Manganese chloride {kCf^, iron-free) 500.0 cc. 2. Potassium iodide (15^) in NaOH (56^) kept in dark 500.0 cc. Dissolve 180 grams of NaOH in distilled water, cool, add 75 grams of KI, make up to 500. cc and keep in cool dark place. 5. Hydrochloric acid ( C. P. cone, no free Clg) 500.0 cc. h. Sodium thiosulphate (n/iOO). 5000. Occ. For each liter dissolve 2.1*82 grama of C, P. grade Na2S20i'5^0 in distilled water. If solution is to be kept several days, add h cc. of In-NaOH per liter. 5. Starch solution (0.5^) 500.0 cc. Emulsify 1 gram of potato starch with 25 cc. of water and pour slowly into 175 cc. of boiling water, boil a few minutes, allow to settle, de- cant off the clear supernatant fluid. If it is to be kept for several days add a few drops of chloroform. OXYGEN CONSUMPTION DURING THE FIRST CLEAVAGE OF THE FROG'S EGG 1. Prepare an ovulating female ( Bana pipiens) and secure several mature males. 2. Prepare 5 respiration bottles consisting of 125 cc. glass -stoppered bottles. Mark them A, B, and C. 5. Prepare 5 Erlenmeyer flasks, 250 cc. capacity, and into each introduce an equiva- lent number of glass beads or small marbles. Mark them A, B, and C, and cork them. k. Prepare 3 finger bowls, mark them A, B, and C, and into each introduce exactly 10 cc. of Spring Water (Standard Solution) or any medium in which frog's eggs are normally inseminated. Into finger bowl "C" only, introduce and macerate one pair of adult frog testes. Allow these bowls to stand for 10 minutes. 5. By stripping, remove a few eggs from an ovulating female and discard them. Then strip about 200 eggs into finger bowl "B" (no sperm) and "C" (sperm suspension), and see that the eggs are completely covered with the medium. Avoid transfer of any sperm from bowl "C" to bowl "B". Allow them to stand for 2 minutes, then add to each of the three finger bowls exactly 250 cc. of the same medium (i.e.. Spring Water). This will provide a total volume in each bowl of 260 cc, plus eggs in two of the bowls. 6. Using a clean section lifter, gently separate the eggs from the bottom of bowl "B" and then bowl "C" after 5 minutes. (Avoid possible insemination of eggs in bowl "B" by washing off and drying the section lifter each time it is used.) Allow the Jelly on the eggs to expand another 5 minutes. 7. Fill Erlenmeyer flask "A" with the medium from finger bowl "A" to overflowing, and add the cork stopper without introducing any air. Fill flask "B" with the super- natant fluid from finger bowl "B", then carefully count out 100 eggs from bowl "B" and add them to the flask, then cork without introducing any air. Similarly fill Erlenmeyer flask "C" with the supernatant fluid from finger bowl "C", count out 100 eggs from finger bowl "C" and add them to this flask, insert the cork *Thls is a modification of the method used by Dr. A. lyier in the course in Marine Embryology given at the Marine Biological Laboratory, Woods Hole, Mass. 5U6 CYTOCHEMICAL TESTS ON EMBRYOS without introducing any air. Betain finger bowls "B" and "C" for the duration of the experiment to determine the developmental changes in the eggs. During the suhsequent 5 hour period there should he variable oxygen consumption in "B" and "C" as compared with "A" in which there should be no oxygen change. 8. Make the stoppers in the Erlenmeyer flasks secure by placing a heavy rubber band lengthwise around the entire flask and stopper. Note the time and temperature and place the three flasks on a standard shaker, agitating about 5 to 25 round trips per minute at 2 to 10 inches amplitude. This will facilitate oxygen con- sumption. Agitate for 3 hours, or until after the control eggs (in finger bowl "C" have completed the first cleavage). 9- Determination of the oxygen consumption: a. Transfer with minimum agitation and exposure to air, supernatant medium from each of the flasks to 50 cc. calibrated bottles, marked A. B. and C. b. Using 1 cc. measured pipettes, transfer 0.2 cc. of the Manganese chloride solution (listed above) and 0.2 cc. of the KI-NaOH solution into each of the calibration bottles, inserting the tip of the pipette about halfway down the bottle. Avoid air bubbles in replacing the glass stopper. c. Agitate the bottles for several minutes, then allow the precipitate to settle 30 that there Is some clear fluid at the top. d. Carefully remove the stopper and introduce O.h cc. of HCl Just below the sur- face of fluid in each of the calibrated bottles. Stopper (without air) £ind shake until the precipitate is dissolved. e. Transfer the fluid from each of the bottles to similarly marked (clean) 125 cc. capacity Erlenmeyer flasks for titration. Solution "C" at least con- tains some free iodine which must be titrated soon In order to avoid loss due to volatility. f. Titration procedure for each sample (A, B, and C) : 1. Add sufficient sodium thiosulphate to cause most of the yellow (iodine) color to disappear. 2. Add U or 5 drops of the starch solution to give a distinct blue color. Continue the titration until the blue color Just disappears. Each cc. of N/100 sodium thiosulphate corresponds to 0.0025 mllllmoles of Og. The relative values of solutions A, B, and C should be determined, and since the number of eggs In B and C is known, the oxygen consump- tion per fertilized and unfertilized egg can be determined. MnClg * aNaOH = Mn(0H)2 + 2NaCl l+Mn(0H)2 + Og = 2Mne05 * k^O MngOj * 6HC1 = = 2 MnClg * 5%0 * Cl^ + 2K1 = gKCi + l2 This procedure seems at first a bit crude and yet very accurate results can be ob- tained. The reactions in the procedure are as follows: CI2 Therefore, for each molecule of Og present, two molecules of Ig are liberated. In titrating, the free iodine reacts with the sodium thiosulphate to form tetrathionate and sodium Iodide as follows: 2Na2S20j ■>- Ig = NagSi^Og + 2 Nal, which are both colorless, allowing the end point to be determined by the disappearance of the blue color that forms when iodine reacts with the starch indicator. Once this procedure is stabilized, and the student has achieved reproducible results, it is suggested that the following additional tests be made: a. Oxygen consumption at various stages of development, particularly during a series of early cleavages and at gastmlatlon. b. Effect of cyanide, dlnitrophenol, iodoacetate, colchicine. c. Effect of low and of high pH. d. Effect of temperature over definite time Interval. e. Oxygen consumption of artificially activated eggs (parthenogenetic) and of androgenetlc eggs. CYTOCHEMICAL TESTS ON EMBRYOS 3^7 BEFEEENCES : Astbury, W. T., I9I+I - "Proteins." Chemical Industry, 60:491. Barth, L. G. & L. Jaeger, 19^7 - "Phosphorylation in the frog's egg." Physiol. Zool. 20:135. Behrens, M., 1958 - "Ifber die Verteilung der Lipase und Arglnase zwischen zellkem und protoplasma der Leter." Zeit. f. Physiol. Chemie. 258:27- Bensley, B. B., 19^+5 - "Chemical nature of cytoplasm." Biol. Symposium. 10:523. Bevelander, G. & P. L. Johnson, 19^+6 - "The histochemical localization of glycogen in the developing tooth." Jour. Cell. & Comp. Physiol. 28:129. Boyd, E. M., 1958 - "Lipoid suhstances of the ovary during ova production in Bana pipiens." Jour. Physiol. 91:59l4-. Brachet, J., 19^'<^ - "Embryologie Chimique." Maseon et Cie, Paris. Brachet, J., 19^6 - "Evolution de I'acide rihonucleique au cours de la cytolyse chez lea oeufs d'Amphihiena." Compt. rendu. Soc. Biol. lU0:1125. Brachet, J. & B. Jeener, 19^+6 - "Phosphatase alcaline des noyaux et vitesse de remplace- ment du phoephore de I'acide thymonucleique." Comp. rendu. Soc. Biol. 11+0:1121. Bragg, A. N., 1939 - "Observations upon Amphibian deutoplasm and its relation to embryonic and early larval development." Biol. Bull. 77:268. Caspersson, T., 19^+0 - "Methods for the determination of the absorption spectra of cell structures." Jour. Boy. Micr. Soc. U0:8. Caspersson, T. & J. Schultz, 191+0 - "Bibonucleuc acids in both nucleus and cytoplasm and the function of the nucleolus." Proc. Nat. Acad. Sci . 26:507. Chantrenne, H., 191+3 - "Becherches sur dea particules cytoplasmlques de dimensions macro- moleculalres riches en acide pentosenucleique. " Enzymologle. ll:ll+. Davidson, J. N. & C. Waymouth, I9I+6 - "Establishment of cytochemical techniques." Nature. 157:755- Dusplva, F. , I9U2 - "Die Verteilung der Peptidase auf kern und plasma bel Froschoozyten im Verlauf der Zweiten Wachstumsterolde." Biol. Zentrlbl. 62:1+03. Femandes, A. & J. A. Serra, 191+1+ - "Euchromatine et heterochromatine dans leurs rapports avec le noyau et le nucleole." Bol. Soc. Brot. 19:67. Feulgen, B., I926 - "Die Nuclealfarbung. " Abderhalden Handb. der blol. Arbeit. 5:1055. Fruton, B., I9I+6 - "Currents In Biochemical Besearch." Interscience Pub. House, N. Y. Gardiner, M. S., 1935 - "The origin and nature of the nucleolus." Quart. Bev. Mikr. Sci. 77:525. Gregg, J. B. & B. Ballantlne, 191+6 - "Nitrogen metabolism of Bana pipiens during embryonic development." Jour. Exp. Zool. 103 : 1^+3 • Gregg, J. B. & C. M. Pomerat - "The glycogen content of the embryo of Bana pipiens during development." Growth. 6:231. Gudematach, F. & 0. Hoffman, I936 - "A study of the physiological value of a-amlno acids during the early periods of growth and differentiation." Arch. f. Ent. mech. 135:136. Harris, D. L., 19!+!+ - "Phosphoprotein phosphatase, a new enzyme from the frog egg." Blol. Bull. 87:l6l+. Heatly, N, G., C. H. Waddington, & J. Needham, 1957 - "Studies on the nature of the amphibian organization center, VI." Proc. Boy. Soc, London B. 122:1+03. Hotchkisa, B. D., I9I+8 - "A Mlcrochemlcal Eeaction Besultlng in the Staining of Polysac- charide Structures in Fixed Tissue Preparations." Arch, of Biochemistry, l6:131. Jeener, B. , I9I+6 - "Sur quelques proprieties physique dea proteines du noyau' cellulalre." Comp. rendu. Soc. de Blol. 11+0:1101 (See ibid. 11+0:1103). Kunitz, M., 191+0 - "Crystalline ribonuclease. " Jour. Gen. Physiol. 2l+:15. Lison, L., 1956 - "Hlstochlmie animale Methods et problemes." Gauthier-Vi liars, Paris McCarty, I., I9I+6 - "Purification and properties of desoxy-ribonucleic acid isolated from beef pancreas." Jour. Gen. Physiol. 29:123. Mirsky, A. E. & A. W. Pollister, 191+5 - "Studies on the chemistry of chromatin." Trans. N. Y. Acad. Sci. 5:190 (See also 19^+5: Blol. Symposia, 10:21+9) Needham, J., I9I+2 - "Biochemistry and Morphogenesis." Macmillan. Nowinski, W. W., 1939 - "Intermediary carbohydrate metabolism in an^ihlbla. I. Carbohydrate breakdown before metamorphosis." Bioch. Jour. 35:978. Painter, T. S. & A. N. Taylor, I9I+2 - "Nucleic acid storage in the toad's egg." Proc. Nat. Acad. Sci. 28:311- Polliater, A. W. & A. E. Miraky, I9I+I+ - "Distribution of nucleic aclda." Nature, June 10, I9I+I+. 5U8 CYTOCHEMICAL TESTS ON EMBRYOS Polllster, A. W. & H. Ells, 19^+8 - "Nucleoproteln determination In cytological prepara- tions." Cold Spring Harbor gymp. Biol. 12:114-7. Eafalko, J. S., 19^6 - "A modified Feulgen technique for email and diffuse chromatin elements. Stain Techn. 21:91. Sakaguchl, S., 1925 - "ITber eine neue Farhenreaktion von protein und arginine." Jour. Bloohem. (Japan). 5=25. Sawyer, C. H., 19lt-2 '- "Cholinesterase in developing Amblyatoma." Proc. Soc. Exp. Biol. & Med. 1+9:37. Schmitt, F. 0., 1959 - "The ultrastructure if protoplasmic constituents." Physiol. Eev. 19:270, Schultz, J., I9UI - "The function of heterochromatin. " Proc. 7th Int. Gen. Cong. 257-262. Serra, J. A., 19k6 - "SLstochemical teats for proteins and amino acids; the characterisa- tion of haslc proteins." Stain Techn. 21:5- Serra, J. A.. & A. Queroz Lopes, 19'<-5 - "Chemical constitution of the nuclear inclusions In grovring oocyte cells." Nature. 155 •"792. Splegelman, D. & M. D. Kamin I9I+6 - "Genes and nucleoproteins in the synthesis of enzymes." Science. 10i|-:58l. Stacey, M. , E. E. Deriaz, E. G. Teece, & L. T. Wiggins, I9I+6 - "Chemistry of the Feulgen and Dische nucleal reactions." Nature. 157:7^*^0. Stowell, E. E., 1914-6 - "The specificity of the Feulgen reaction for thymonucleic acid." Stain Techn. 21:157. Stowell, E. E. & A. Zorzoli, 19^4-7 - "The action of rihonuclease on fixed tissues." Stain Techn. 22:51. Sumner, J. B. & G. F. Somera, I9I+7 - "Chemistry and Methods of Enzymes." Acad. Press Inc., N. Y. Thomas, L. E., 1914-6 - "A hiatochemical test for arginlne-rich proteins." Jour. Cell. & Comp. Physiol. 28:11+5. Unna. P. G., 1921 - "Chromolyse. " Abderhalden Handt. der Biol. .Arbeit. 5:1. Voss, H. , 1951 - "Unterauchiuigen mlt der Plasmalfarbung am Axolotei." Zeitschr. Anat. Entw. Gesch. 9l+:712. Wrinch, D. , 1914-2 - "Native proteins, flexible frameworks and cytoplasm organizations." Nature. 150:270. Zielinski, M. A., 1957 -"Phosphagen and creatine in frog's eggs." Jour. Exp. Biol. • Il4:l48. HETEROPLOIDY INDUCED BY VARIATIONS IN TEMPERATURE PURPOSE: To intercede in the kinetic movements of maturation and amphimixis "bj utilizing extremes (high and low) of temperatures on amphibian eggs immediately after insemina- tion, thereby producing variations in the numbers of chromosome sets within the somatic nuclei (i.e., heteroploidy) . MATERIALS: Biological: Recently layed eggs of Urodeles; ovulating Anura and mature males of the same and different species. Technical: Refrigeration controlled at 0.5°C. to 5.0°C., and 3k°C. to 37°C. Histo- logical technique equipment listed under "Tail Tip Chromosomes". METHOD: Precautions: 1. The transfer of eggs to and from the extremes of temperature must be abrupt. 2. The eggs must be cold (or heat) treated prior to the normal con^letion of matura- tion. This means immediately upon egg-laying (for the Urodeles) or within 20 minutes of insemination (for the Anura). 5. Heteroploids (particularly haploids) are less viable than diploids, and must be given special care. Controls : These consist of eggs from the same source, fertilized in the same manner, but kept within the temperature range for normal development. For Urodeles this is generally between 15° and 20°C. and for the Anura between 18°C. and 25°C. Procedure: The procedure is very simple, but it varies slightly with the different species and the temperature used. In general, a short exposure at the higher temperatures is equivalent (and often better) than a long exposure at the lower temperatures. Triturus viridescens is better than T. pyrrhogaster, which, in turn, is better than the white Axolotl. The Anura have not been used in this type of experiment until recently (Briggs, 19^7), partly because their tall-tips do not yield such satisfac- tory chromosome figures as do the tail- tips of most Urodeles (the Axolotl is the poorest: Fankhauser & Humphrey, 19*^2). The second polar body is given off from the Anuran egg about 25 to 50 minutes after Insemination while it seems to take about 1 hour to emerge from the Urodele egg (Griffith, 19'*-0). The extreme of temperature used supposedly suppresses this second polar body formation. FOR URODELA Urodele eggs are layed singly, and are fertilized as layed by spermatozoa with- in the female genital tract. They should be picked off of the greens (Elodea etc.) and transferred individually to the low (or high) temperature in a marked #2 Stender. After the prescribed exposure, transfer the eggs directly to another container at a temperature of 15° to 20°C. The culture medium is generally Urodele Growing Medium, or Spring Water. Do not crowd the eggs, allowing about 5 cc. of medium per egg. FOR ANURA Anuran eggs should be secured from an ovulating female, pituitary-induced. The eggs should be Inseminated in the normal manner, flooded within 5 minutes, and trans- ferred abruptly to the low (or high) temperature in Standard Solution or Spring Water. The eggs will stick to the bottom of the container (e.g., finger bowl or Petri dish) and the water may be poured off, and the water of a different temperature added directly. The Anuran eggs need not be separated until after the drastic ten^erature treatment, but at that tine they must be separated into finger bowls containing no more than 25 eggs per 50 cc. of medium, kept at temperatures of 18°C. to 25°C. 550 TEMPERATURE INDUCED HETEROPLOIDY TEMPERATURES AND EXPOSURE PERIODS Temperature Species Ean^e 16 Time - 26 hrs. Eeference T. vlrideacens 0.5° - 5.0°C, Griffiths I9I+O II tt M 5 hre. 19i+l It 0.0° - 6.58°C. 5 - 2k hrs. Fankhauser & M 0.0° - i+.o°c. 5 hrs. Watson I9I+2 Axolotl 1.0° - 5.0°C. 9 - 2h hrs. Fankhauser & Humphrey 1914-2 T. vlrideacens 5i*.20 - 57.2° 5 -'50 mln. Fankhauser & Watson I9I+2 It 35.0OC. 10 - 19 mln. ri It 57.0OC. 5 mln. H B. pi pi ens 37.0°C. k mln. Brlggs I9U7 Chromosome (2N) number 22 28 26 ************* The time/temperature ranges for optimum results (highest heteroploidy with lowest mortality) have not been determined. However, for the Urodele eggs an exposure of about 5 hours at about 3'0°C. or an exposure of about 15 minutes at 35.0°n. may be considered as optimum until exact data are available. For the Anura, the only reference is Brlggs (191+7) who suggests that h minutes at 37.0°C. seems to be highly productive of triploidy. Rostand (1953, IS'ik^ 1956) found that hybrids between Qyla, Bufo, and Rana which normally go to pieces during cleavage or early blaatula stages, will respond to the cold treatment by producing some larvae. This type of experiment indicates that the Anura (as well as the Urodela) will react to low temperatures by altering the chromosomal conditions rcRTIUZEO ECC RCfRIGUATCD secoNo MAivftArioH DfVISIOH AUPPRC&iCO Diagram of hypothetical effect of low temperature treatment on freshly fertilized salamander eggs. At the time of fertilization, tlie ampliiblan egg, has given off the first polar body; the second matura- tion division has reached metaphase and remains in this stage until fertilization occurs. Refrigera- tion presumably suppresses the second maturation division and produces a diploid egg nucleus. From Fankhauser I9I+2 : Biol. Symposia 6:21 TEMPERATURE INDUCED HETEROPLOIOY 351 METHODS FOR OBTAINING HAPLOID EM3RY0S EGG ACTIVATED BY ARTIFICIAL MEANS EGG NUCLEUS DIVIDES PARTHENO- GENESIS EGG NUCLEUS ALONE DIVIDES GYNOGENESIS RADIATED EGG NORMAL SPERM SPERM NUCLEUS ALONE DIVIDES ANOROGENESIS EGG NUCLEUS ELIMINATED BY DIVIDING EGG INTO TWO PARTS UPPER HALF DEVELOPS WITH SPERM NUCLEUS ALONE MEROGONY FOUR WAYS OF PRODUCING HAPLOID EMBRYOS Above are shown rilagrammatlcallj the ways of producing embryos having only one set of cliromosomes Instead of the two sets found ordinarily in the higher plants and animals. From Fankliauser 1957: Jour. Heredity 28:1 and thereby obviate the deleterious effects of foreign chroniosoines. If various genera (or species) of Anura are available, such attempts should be made to carry normally non- viable hybrids past the critical stage of gastrulatlon. OBSERVATIONS AND TABULATION OF DATA: A complete record must be kept of the number of eggs treated, the exact time ( In re- lation to maturation) and duration of exposure, the number of eggs that cleave, and the number of eggs that reach the various stages of early development. The final test of heteroploldy rests with the cytologlcal analysis of the tall fin (tip). For the Urodela the tall tip is in its best state for study at from 2|r to 5 weeks (just before the ex- 552 TEMPERATURE INDUCED HETEROPLOIDY hauatlon of the reserve yolk at the third finger- bud stage), eind for the Anura some time after the external gills have been resorbed. LATEBAL LINE ORGANS PRIUOROIA OF LATERAL LINE SYSTEM RED BLOOO CELLS BLOOO ISLAND Dia;i,raramatlcal drawing, of a salaman- der larva to show the origin of the rudiments of the lateral line organs and of the red Dlood cells which are present in the cailtip. The ampu- tated tailtip includes cells from widely separated regions of the embryos . From Fankhauser I958: Proc. Am. Philosoph. Soc, 79:715 Fix the tall tip In Bouln's fluid (it is not necessary to kill the larva) after clip- ping It off about 1/5 the distance from the tip to the base of the body. After about h hours, wash In the usual manner (decolorize in 0.51^ NHj^OH), wash in running tap water to neutralize the tissue, and then stain for 7 to 10 minutes in about 531^ Harris' acid haemalum (freshly acidified). Blue In tap water (alkaline), then dehydrate in the usual manner, mount, and study. The percentage of cells showing chromosome figures will be small, best in Triturus vlrldescens and poorest in the Axolotl and Eana. Drawings and photographs of chromosome groups constitute the record, in addition to which drawings and photographs of haplold, diploid, and trlplold larvae, their chromatophores and relative cell sizes would be convincing (See figures on following page). PHOTOGRAPHS OR DRAWINGS OF TAIL TIP CHRCMOSOMES TEMPERATURE INDUCED HETEROPLOIDY 555 DISCUSSION: Heteroploldy in plants has long been recognized but not until recently has its normal incidence among animals been determined (See Fankhauaer's excellent review In 19'*^5 Qwart. Bev. Biol. 20:20). It is now believed that triploidy in Triturus viridescens occurs naturally to an extent of about O.T^. In some batches of eggs (or, more accurately, eggs from certain females) will respond to cold treatment by producing almost 100^ triploids, among those which survive the treatment. It must be emphasized that the mortality of all eggs, treated by these extremes of temperature, is very high, often as much as 50^. ABNORMALITIES FOUND IN HAPLOID EMBRYOS AND LARVAE OF AMPHIBIANS Species Author Broader head and body Triton taenlatus Triton palmatus P. Hertwlg, 1916, Baltzer, 1922, fankhauser, 1937a (Fig. 6) Fankhauser, 1937a (Fig. 7) Dorsal flexure of body, widening of tail Bana pipiens Rugh, 1959 Shorter gills Bufo, Rana Triton taenlatus Triturus viridescens G. Hertwlg, 1913 0. Hertwlg, I915, P. Hertwlg, 1923 unpublished Deformed lower Jaw Triturus viridescens Triturus pyrrhogaster unpublished Fankhauser, 1957a, Kaylor, 19i4-Oa Circulation seldom functional Bana pipiens Porter, 1939 Neural plate one -third shorter Bana pipiens Porter, 1939 Microcephaly Abnormal development of brain Bana fusca Bana pipiens Bana pipiens Triton taenlatus Dalcq, 1932 Rugh, 1939 Porter, 1959 P. Hertwlg, 1923 Small or abnormal eyes Bana pipiens Bana fusca Triton taenlatus Triton palmatus Porter, 1959 Dalcq, 1932 P. Hertwlg, 1923, Book, 19iH Fankhauser, 1937a Table from Fankhauser I'^k'i: Quart. Rev. Biol. 20:20 It is now believed that the temperature shift at this particular time in maturation affects the kinetic movements of maturation so that the formation of the second polar body is suppressed. It is difficult to explain the infrequent haploids achieved by this treat- ment, but the vast majority of abberatlons are in the direction of triploidy, tetraploldy, etc. The survival of Bostand's (I936) hybrids beyond the normal stage of termination of development, following exposure to cold, fits in perfectly with the concept of polar body retention and the further possibility that amphimixis (in this case, with the foreign sperm) is prevented. If the drastic temperature treatment is delayed for more than 30 minutes (Triturus viridescens) the larvae which result are all diploid. DIRECT EVIDENCE OF HAPLOIDY Diagrammatic drawing of the metaphase chromosomes of a dividing, epidermal cell from the amputated tail tip of 19-day haploid larva. The haploid chromosome number in this species Is twelve; normal diploid tissue having twelve pairs of chromosomes. (Triturus pyrrhogaster) j)rom Fankhauser 1957: Jour. Heredity 28:1 551+ TEMPERATURE INDUCED HETEROPLOIOY Metaphase chromosomes from epidermal cells of a haploid, a diploid and a trlploid tail-tip (11, 22 and 33 chromosomes). Tracinji,s of enlarged photomicrographs. Nuclei of epidermis cells from a haploid, a diploid and a triploid tail-tip. The size of the nuclei is rouglily proportional to the number of chromosomes they contain. Camera lucida drawings. Pigment pattern on the head of a haploid, a diploid and a triploid larva, 4 weeks old. Tracings of enlarged photomicro- graphs. The same larvae as shown in Figure 3. Tlie haploid larva (left) is dwarfed and edema- tous, the triploid is slightly larger than the diploid. Tracings of enlarged photo- micrographs. TRITURUS VIRIDESCENS From Fanichauser 1939: Proc. Nat. Acad. Scl. 25:255 Fanlchauaer and his students have analyzed the nucleoli, nuclei, cells, and organs of the heteroplolda. They find that when there are extra sets of nucleoli and of chromosomes, that the Increase in nuclear and cell size is compensated for hy a corresponding decrease In cell number. This decrease must occur early, for in the later stages of development the mitotic rate of diploids and triploids seems to he much the same. There are corre- sponding differences in organ size except for the gonads and the notochord. Further "The diameter of the wall of the pronephric tubules and pronephric ducts, and the thickness of the epithelium of the lens of the eye thus remain about the same from the haploid to the pentaploid levels." and "These observations show that in the amphibian embryo both cell number and cell shape may be modified to allow the formation of organs of normal size and structure. This indicates that both are subject to some control by the developing organ- Ism." ( Fankhauser, I9U5). There is evidence that triploldy affects the females only, the triploid larvae being undifferentiated or males. This field of investigation is extremely important from the physiological, morphologi- cal, and genetical points of view. TEMPERATURE INDUCED HETEROPLOIDY 355 BEFEREWCES : Artom^ C, I928 - "Le polyploldie dana ees correlations morphologlques et 'biologlquea. " Compt. rendu. Soc. Biol. 99:29- Barter, H. N. & H. G. Collan, I9I+3 - "The effects of cold and colchicine on mitosis in the newt." Proc. Boy. Soc. London B. 151:258. Beal, J. M.J 19^2 - "Induced chromosomal changes and their significance in growth and development." Am. Nat. 76:259. Book, J. A., 19^0 - "Induction of haploldy in a cold treatment experiment with egg cells of the salamander Triton taenlatus." Kungl. lysiogr. Sallsk. Lund. Forh. 11, #12:1 (See also I9I4-O Hereditas 26:107). Brachet, J., I'^hk - "Acidee nucleiques et morphogenese au coura de la parthenogenese la polyspermie et 1' hybridation chez les Anoures." Ann. de la Soc. Boy. Zool. de Belg. 75:^^9. Briggs, B. W., 19'*-7 - "The experimental production and development of trlploid frog embryos." Jour. Exp. Zool. I06. Costello, D. P., I9I+2 - "Induced haploldy and trlploidy In the California Tri turns." Anat. Bee. 81+: Buppl. 60. Dalcq, A., 1952 - "Contribution a I'analyse des fonctlons nucleaires dans I'ontogenese de la grenouille. TV. Modifications de la formule chromosomiale. " Arch. Biol. '+5:5^5- Dal ton, H. C, 1914-6 - "The role of nucleus and cytoplasm in development of pigment patterns in Triturua." Jour. Exp. Zool. 105:169. Dermeri, H., I958 - "A cytological analysis of polyploidy." Jour. Heredity. 29:211. Fankhauser, G-., 1957 - "The production and development of haplold salamander larvae." Jour. Heredity. 28:5. Fankhauser, G., 191+1 - "Cell size, organ sind body size in trlploid newts (Trlturus virldescens) ." Jour. Morph. 68:l6l. Fankhauser, G., 19'+5 - "Maintenance of normal structure in heteroploid aalamander larvae through compensation of changes in cell size by adjustment of cell number and cell shape." Jour. Exp. Zool. 100:1+45. Fankhauser G., I9I+5 - "The effects of changes in chromosome number on amphibian develop- ment. Quart. Bev. Biol. 20:20. Fankhauser, G. & B. B. Humphrey, I9I+5- "The relation between the number of nucleoli and number of chromosome sets In animal cells." Proc. Nat. Acad. Sci. 29:54'*'' Fankhauser, G. & B. Watson, I9I+2 - "Heat-Induced trlploidy in the newt, Trlturus virldescens." Proc. Nat. Acad. Scl. 28:1+56. Griffiths, E. B., I9I+I - "Trlploidy (and haploldy) in the newt, Trlturus virldescens, in- duced by refrigeration of fertilized eggs." Genetics. 26:69. Heilborn, 0., I95I+ - "On the origin and preservation of polyploidy." Hereditas. 19:255. Hertwlg, G. & P. Hertwlg, 1920 - "Trlploide Froschlarven. " Arch. mlkr. Anat. 9h:'^k. Humphrey, B. B. & G. Fankhauser, I9I+6 - "Tetraploid offspring of trlploid axolotl females from matings with diploid males." Anat. Bee. 9I+: suppl. 95. Huskins, C. L., I9I+I - "Polyploidy and mutations." Am. Nat. 75:529- Kawamura, T., I9I+O - "Artificial parthenogenesis in the frog. III. The development of the gonads In trlploid frogs and tadpoles." Jour. Scl. Hiroshima Univ. Ser. B. 8:117- Kaylor, C. T., I9I+O - "Studies on experimental haploldy in salamander larvae. I. Experi- ments with eggs of the newt. Trlturus pyrrhogaster. " Biol. Bull. 79:597- Llndstrom, E. W., I956 - "Genetics of polyploidy." Bot. Bev. 2:197- Miintzing, A., I956 - "The evolutionary significance of autopolyploldy. " Hereditas 21:265. Parmenter, C. L., I955 - "Haplold, diploid, and tetraploid chromosome numbers and their origin in parthenogenetlcally developed larvae and frogs of Bana plpiens and Sana palustrls." Jour. Exp. Zool. 66:1+09. Eostand, J., I956 - "Gynogen^se par refroidiasement des oeufa chaz ^la arborea." Comp. rendu. Soc. Biol. 122:1012. Sansome, F. W. & S. S. Zilva, 1955 - "Polyploidy and vitamin C." Blochem. Jour. 27:1955. Vandel, A., 1957 - "Chromosome number, polyploidy and sex in the animal kingdom." Proc. Zool. Soc. London. 107A:519. HYBRIDIZATION AND EARLY DEVELOPMENT PURPCSE: To demonstrate the effects upon early development of the egg when activated 'by- foreign spermatozoa, and to determine the relative influence of the maternal and pa- ternal factors in hybrids which pass the critical period of gastrulatlon. MATEBIALS: Biological: Ovulating females of any Amphibian species, and sexually mature males of a variety of species. ( Pre- breeding, sexually mature females of any species of Amphibia can be induced to ovulate by pituitary injection.) Technical: Standard equipment- METHOD: Precautions : 1. Avoid contamination with sperm suspensions other than that intended for use. All instruments and glassware should be sperm-sterile. 2. Examine sperm suspensions under the microscope for motile spermatozoa. 5. Cleaving eggs should be separated from others, and placed in finger bowls of Standard Solution (or Urodele Growing Medium) at cool temperatures, not more than 25 eggs per 50 cc. of medium per finger bowl. Controls: 'Egga inseminated with spermatozoa of the same species. This should be done after the hybridization experiments, to avoid contamination. Procedure : A summary (Moore, 19'*1) of the hybrid crosses between American species of Bana follows : NO CLEAVAGE OCCUBS FEMALE Bana clamitans Bana clamitgins Bana clamitans Bana clamitans Bana clamitans Bana clamitans Bana septentrionalis Bana septentrionalis MALE Bana sylvatica DEVELOP TO Bana sylvatica BEGINNING OF Bana sylvatica GASTBULATION Bana sylvatica ONLY * Bana pi pi ens ( Cleavage Bana pi pi ens rate Bana pi pi ens bumsi maternal) Bana palustris s^Bana cateabiana ''Bana pi pi ens WILL DEVELOP Bana pi pi ens PAST THE Bana piplens STAGE OF GASTBULATION: Bana pi pi ens bums! Bana pi pi ens burnsl MAY EVEN Bana sphenocephala METAMOBPHOSE Bana sphenocephala .^Bana palustris X X X X X X X X X X X X X X X X X X X X X X X X X Bana sylvatica Bana pipiens Bana palustris Bana catesbiana Bana septentrionalis Bana hechscheri Bana clamitans Bana pipiens Bana pipiens Bana palustris Bana catesbiana Bana sphenocephala Bana sylvatica Bana catesbiana Bana sylvatica Bana sylvatica Bana pipiens Bana palustris Bana sphenocephala Bana pipiens burns i Bana pipiens Bana palustris Bana pipiens Bana palustris Bana pipiens If 5yla or Bufo eggs or sperm are available, hybridization with Bana should be at- tempted. In most, if not all, cases these hybrids would fall into the second group above. -556- HYBRIDIZATION AND EARLY DEVELOPMENT 357 RECORD OF THE EXPERIMENTAL DATA FROM HYBRID CROSSES Kgg source Sperm source Condition* Cleavage $ Gastrulation $ Neurula ^o * Condition refers to any variables concerning either of the gametes such as prior irradi- ation with x-rays, or ageing of the eggs, etc. NCUBU. JIOTOCMORO OP' jaiic •*ru>» Embryo formation in normal development and in the piplens 9 X sylvatica o a O r o r o .r^^ X ' •/i'v <^ "S °^ '# ° ^ S3 '■ y; ,-^ i\ ^ ^ rw = ■•m o ^ V ^T" ^i I < < Q LjJ llJ Ijj z 2 < a o Sj (0 UJ Q. 3 O I J1 O r o EXPERIMENTAL FISH EMBRYOLOGY 377 JUST FERTILIZED CONCENTRATION OF PROTOPLASM AT POLAR CAP 2 CELL - I HOUR BLASTULA - 3 HOURS 1^ -ri*^'- I s I 3 HOURS -HP 10 HOURS 12 HOURS TWO D/ /r-r-1-n > L. il^ ^ 1 n ^y DAYS TO HATCHING YOLK SAC- HATCHED EARLY DEVELOPMENT ^' OF '* ORYZIAS LATIPES -^ THE JAPANESE MEDAKA L^V^ OPTIC VESICLES Photographs by L. Roth TAIL SOMITES 578 EXPERIMENTAL FISH EMBRYOLOGY EARLY DEVELOPMEMT OF OBYZIAS LATIPES: In an aquarium containing several dozen fish you will find eggs (I-80 per female) every morning. Since the eggs are laid shortly after daylight these ohservations must he made very early in the day. Using a dip-net, catch a female which has a cluster of eggs attached and transfer it to a finger howl of aquarium water. Pick off the eggs either with a wide-mouthed pipette or a pair of forceps. 'Egga which appear to be opaque within 10 minutes after removal are very likely to be dead. Separate the eggs so that there are about 10-20 per finger bowl. Keep no vegetation with the eggs. Observations should be made under dissecting microscope (mag. x 1? or more) with both direct and transmitted lighting. Since the development is rapid (see diagrams and photo- graphs by Eoth) and hatching generally occurs within 6 days, it will be necessary to make rather constant observations or to supplement any series with observations on other eggs. The schedule of development follows: TIME SCHEDULE OF DEVELOPMENT: 1. Egg becomes translucent , perivitelllng space widens, germinal disc becomes lens- shaped: 10 minutes after fertilization. 2. First cleavage: 1 hour after oviposition; cleavage meridional, nuclei visible. 5. Second cleavage: I5 hours, meridional, right angles to first; blastomeres equal. 4. Third cleavage: 2 hours, plane parallel to first cleavage; blastodisc appears rectangular in outline with no space beneath. 5. Fourth cleavage : 3 hours, parallel to the second; central blastomeres smaller than peripheral ones. Note margins of boundary cells. Is there any incorpora- tion of yolk? 6. Fifth cleavage: Js hours, unlike most teleost eggs, this cleavage does not give rise to layers of cells. 7. Sixth cleavage: h hours, after which the cleavages are no longer synchronous but give rise to a many-layered blastodisc. 8. Blastula: Ely 6-8 hours the sub-germinal cavity first appears. The blastodisc is elevated and a thickened marginal periblast of syncital nature may be seen. Ob- serve in optical section if possible. Coinpare the periblast nuclei with those in the center of the blastodisc in regard to shape and color. Ely 2k hours there is nucleation of these periblast cells and the marginal thickening is accentuated to become the germ ring. This germ ring will grow down over the yolk in much the same manner as a bathing cap is pulled down over the head. 9. Gastrula: It ia difficult to designate a specific time when gastrulation begins but it is accomplished by invagination, accompanied by delaminatlon and over- growth at the time the germ ring has grown around about a third of the yolk mass. The blastopore is represented as the uncovered portion of the yolk mass and embryonic structures first appear with the closure of this blastopore. 10. Embryonic development: First day: The embryonic shield appears as a result of the thickening of the cells along a region which represents the axis of the future embryo. This appears dur- ing gastrulation as a result of migration of cells and will extend toward the center of the blastodisc from a point along its margin. This embryonic shield should be observed from all aspects. If possible, determine the regions of most active growth and the source of cells that go into the various parts of the em- bryonic shield. During the first 2U hours the embryo will encircle 5/8 of the yolk mass, the optic vesicles will appear and there will be fewer than 10 somites. Second day: The embryo extends 5/8 around the yolk; the heart beats about 5O-60 times per minute; the blood is colorless; Kupfer's vesicle is seen at the caudal end; and there may be 18 somites. Third day: The embryo extends ^/h around the yolk; the heart is beating as in the adult, about 150-170 per minute; the blood Is slightly pink In color; pigment ap- pears in the optic cup; and there are 20-28 somites. EXPERIMENTAL FISH EMBRYOLOGY 379 Fourth day: Embryonic movementa begin; the blood is red; somites number 50-58. Sixth day: Numerous chromatophores appear, hatching may occur on the 6th day, depending upon temperature. In the hatching process the tail moves violently to break the membrane, and the tail region emerges first. The size schedule is as follows: Just hatched: h.3 - 5.0 mm. in length. One month: 11.5 " 15.0 mm. in length. Oryzias latipes reaches maturity in 1-1^ months, and the life span is 1-2 years. Generally a fish hatched in one summer dies after it breeds during the following summer. The life span of laboratory fed fish has not been determined. Care of Material: Avoid crowding, and observe eggs without undue handling. They may be left in Stenders or finger bowls throughout development. When the fry hatch they should be fed #0 (finest grain) baby fish food and protozoa. While the range of temperature tolerance is 7°-59°C. the optimum for all stages is between 20°-25°C. In studying the normal series, use a constant and recorded temperature and compare with the accompanying drawings and photographs. fixation of any fish embryonic material may be in Bouin-Dioxan .or in Stockard's solution (5 pints formalin, h pints glacial acetic, 6 pints glycerine, and 85 pints water) and after 5 days may be transferred permanently to 10^ formalin. Sectioning la difficult but can be accomplished if the chorion is removed after fixation. Note: A synthetic medium of balanced chemicals may be used for Oryzias if conditioned tap water proves to be deleterious. This solution will also inhibit the growth of molds and bacteria. NaCl 1.0 gram KCl 0.05 grams CaClg 0.05 grams MgSOij. 0.08 grams Distilled Water to 1 liter OBSERVATION AND TABULATION OF DATA: It is Important that the student become thoroughly acquainted with the reproductive physiology of the fish particularly since there are very few fish with which this can be done (see Robinson and Rugh, 19'*-5). Compare any observations with those on the frog. The observations on early embryonic development are to be made under low magnifica- tion almost continuously during the first 2-5 hours, then several times daily thereafter until the embryo hatches (6th day). Note particularly the following: 1. Somites : number as criterion of age; manner of formation; positional relation to future parts of the central nervous system. 2. Development of the central nervous system: neuromeres; fore, mid, and hind-brain; cerebrum, cerebellum, medulla and cord; optic vesicle, lobe and lens; olfactory pit and otocyst. 5. Circulatory system: appearance and shape of the heart; the initial heart beat; development of blood vessels including sketches of the circulation on the Uth and 6th days; changes in blood color from day to day. k. Numerous wandering mesenchyme cells found toward the posterior end in 2-5 day old embryos; observe movement and mitosis. 5. Chromatophores : appearance, behavior, function; color and shape; contractility; response to light. 6. Movement : blastodisc; body; and finally the fins. 7. Fry: behavior in response to various types of stimuli. DISCUSSION: Oryzias ( Aplochellxis) latipes, the Japanese Medaka (also known as the Geisha Girl fish) has been adapted so satisfactorily to laboratory conditions that it promises to con- tribute much to the field of experimental embiyology. For this reason a rather thorough description of the adult fish, the reproductive physiology, and the egg will be given here. 580 EXPERIMENTAL FISH EMBRYOLOGY o >- >- 1— •< _l a. 'ei^i 5 » S 3 « « s ID +i h » ,o C) » ^ oH CM K^ (11 tji; irt rH o () •H (> W) 01 () H .-1 o 01 ^ •H rH 1^ .H -=)■ f-i fTN •js .H EXPERIMENTAL FISH EMBRYOLOGY 381 O > a m o o Pblyboecitui macuLtlus "■V V ill it 10 THEORETICAL AGE IN DAYS Fig. 21. Chart showing age variations in 21 embryonic broods. Each vertical bar represents an entire brood removed from a female. Dotted line connects the mean morpliolo^ical a^es for eacli theoretical a^e group. (See detailed desnription in text.) RROI(BEK;KI>HALDIt USEHCtPHALON tw)aiw;tpnALOii PERICARDIAL SOtUTOPLEUni TOLK SAC - miCARDIAL SPLANCmOPLCUTU SmS VSMQSDS KXTM-IOIITOnC SPLAKtaOPLBU 23. Fig. 22 Fig. 23 Fig. 24 Fig. 25 Diagrammatic sagittal section of Funriulus embryo before development of pericardial sac. Diagrammatic sagittal section of Fundulus emoryo at maximum development of pericardial sac. Diagrammatic sagittal section of PlatjTioecilus embryo at beginning of formation of extra-embryonic pericardium. Diagrammatic sagittal section of Platypoecilus embryo at maximum development of the pericardial amnion and peri- cardial serosa. 582 EXPERIMENTAL FISH EMBRYOLOGY DEVELOPMENT OF THE PLATYFISH, PLATYPOEC I LUS MACULATUS DESCRIPTION OF NORMAL DEVELOPMENT STAGES Stage I. Mature Ovum (Fig. I) The mature Infertile ova, after the yolk has teen deposited, average 1.5 nun. In di- ameter. They are of a clear yellow color with peripherally arranged fat globulea of var- ious sizes. These globules vary somewhat in size and numher depending on the individual ovary. However, the eggs in any one ovary are all very similar in this character. When the egg is damaged, the globules are found to be adherent to the peripheral membrane; they are composed of a colorless fluid somewhat less viscous than the yellow colored matrix. The germinal vesicle cannot be seen in the living egg, but it has been demonstrated by Hopper (19^+5) to be peripheral in position in sectioned material. The vitelline membrane is probably present since a fertilization membrane is subse- quently demonstrated. No tertiary membrane, such as is found in oviparous species, is present around the egg. Immature eggs appear to be more opaque than mature fertilized ones. It may be that this change takes place at fertilization as it does in Fundulus ( Oppenheimer, 1957), but in this viviparous species it is difficult to substantiate. Staqe 2. Cleavage (Fig. 2) Cleavages may be seen only occasionally, and only in eggs preserved in formalin. The cleavage cells are very thin, broad and flat, and since they are not raised above the yolk surface to any visible extent, this stage is poorly distinguished from the previous one. Fig. 2 shows the cleavage stage more distinctly than it actually appears. Using a glass needle and a pair of sharpened watchmaker's forceps, the fertilization membrane can be re- moved from such eggs while in the saline solution, and the contents left in place. Such a membrane cannot be demonstrated around infertile ova. This fertilization membrane per- sists throughout the gestation period and is ruptured together with the follicle Just prior to parturition. Stage 3. Compact Blastula (Fig. 3) This is the earliest stage which can be identified readily by gross study. The cells are small and tightly packed into a small grayish protoplasmic disc, which is slightly raised above the yolk surface. A segmentation cavity has been described beneath the disc (Hopper, 191^5 )• Stage 4. Diffuse Blastula (Fig. n) Gastrulation begins at this stage with the blastodisc flattening out into a thin mem- brane of cells. The periphery of the blastodisc Is uniformly thickened, indicating the region of proliferation and probable involution. Stage 5. Early Germ Ring Gastrula (Fig. 5) Gastrulation continues during stage 5 with a peripheral spreading of the blastodisc in all directions. The embryonic shield is visible as a widening and thickening of a sector of the rim of the blastodisc. Stage 6. Late Gastrula - Early Neurula (Fig. 6) The embryonic shield takes on an elongate form and becomes raised from the yolk sur- face, indicating the antero-posterior axis of the developing embryo. The notochord is present, and the anterior end of the neural keel can be seen. The nerve cord is formed EXPERIMENTAL FISH EMBRYOLOGY 383 from a solid core of invaginating tissue; the neurocoele appearing after Invagination is completed, as seen in sectioned material. This type of neurulation is typical of teleosts. Stage 7. Late Neurula (Fig. 7) The germ ring at this stage is somewhat helow the equator and the embryo has hecome further elongated. Since elongation takes place principally in the posterior portion, a region roi;ighly corresponding to the dorsal hlastopore lip of amphibian gastrulae, the anterior end of the embryo lies in much the same position as did the original emijryonic shild of stage 5. . The neural keel has invaginated throughout the greater length of the embryo, and a neurocoele is present in the anterior one-fourth. Stage 8. Head Fold (Fig. 8) A prominent head fold is present by stage 8. The neurocoele is open for about the anterior half of the length of the embryo. The optic buds are present and attached to the short, thin stalks, and they are, at this stage, without a cavity. Two pairs of rather diffuse somites are evident, but there is considerable variation in the time of their first appearance. Somites sometimes appear as early as stage 7- Stage 9. Optic and Otic Vesicles; I.I mm. (Fig. 9) The head fold has now begun to elongate anteriorly. The blastopore is still a wide open structure and the caudal end has not progressed back much farther than its position in stage 8. The optic prlmordla now possess cavities, and are usually still attached to the prosencephalon by thin optic steilks. The brain is divided into three general regions: a narrow prosencephalon, a slightly wider mesencephalon, and a short rhombencephalon. Otic vesicles have Invaglnated at the level of the rhombencephalon, but are still connected to the exterior by the endolymphatic ducts. Usually, 7 pairs of somites are visible at this stage. The pericardial sac, which develops very early, closely enfolds most of the head fold at this stage. Stage 10. Tail Gud; 1.5 mm. (Fig. 10) The optic vesicles are detached from the brain and are slightly flattened around the invaginating lens prlmordla. The mesenchephalon and rhombencephalon have become widened and more thin walled. The otic vesicles are slightly ellipsoid and are completely cut off from the superficial ectoderm. There are ten pairs of compact somites visible. The tail bud has begun to form and extends slightly over the region of the dorsal lip of the open blastopore. The region of the pericardial sac that is extra-embryonic is easily distinguishable, and, upon dissection, the heart can be found as a straight tube on the floor of the peri- cardial sac. The vascular system is apparently complete at this time, but the blood is- lands are never visible under gross examination. The heart exhibits no regular beat, only an occasional twitch. Stage II. Pectoral Fin Buds (Fig. II) The optic vesicles partially envelop the lens prlmordla. The prosencephalon shows little differentiation, but the mesencephalon has widened out considerably. Indications of neuromeres can be seen In the rhombencephalon. The entire brain possesses a thin roof, and this is especially true at the hind-brain level. In later stages, the roof of the mesencephalon becomes thickened, but that of the i^yelencephalon remains thin as the pos- terior tela chorloldea. The otic vesicles show little or no change, aside from a general growth, in this and several of the following stages. Fig. 11 shows the presence of the anterior fin buds. 53U EXPERIMENTAL FISH EMBRYOLOGY Posteriorly, ib to 20 small, compact somite pairs blend into a poorly differentiated region in the now prominent tail bud. It is noteworthy that, although a sizeable tail bud is present at this stage, the blastopore is open in majority of the embryos. This is in contrast to the case in most teleosts, and even in the closely related Fundulus. In the heart, the ventricular and atrial portions are distinct, and at the anterior end, the sinus venosus projects In front of the head. The heart exhibits a fairly rhyth- mical beat at this time. The color of the blood is light pink, but barely perceptible. Stage 12. Regular Heart Beat; 1.8mm. (Figs. 12, 13) The optic cupa envelop the lenses closely. Olfactory placodes are visible. The brain has undergone further development; the telencephalic region is slightly expanded; the mes- encephalon has a thicker roof; the rhombencephalon is greatly widened. The somites are more closely packed and less distinct. The vascularization of the pericardial membrane is in the form of small capillary-size vessels. The extra -embryonic circulation can be followed at this stage. The blood leaves the embryo through the ducts of Cuvier at the posterior ventral margin of the pericardial membrane, drains into the yolk portal system and the vascularized pericardial membrane, and collects at the elongated sinus venosus. The mid-gut is broad and extends under about one-third of the embryo. The hind-gut is short, and the fore-gut, upon dissection, is shown to possess a distinct first pharyn- geal pouch and a corresponding visceral furrow. Stage 13. Early Retinal Pigment; 2.1 mm. (Figs. 14, 15) Olfactory pits are distinct. Pigment can be seen in the retina as a thin gray band. The brain and the head are further enlarged. The pericardial sac has increased to its maximal size. In the future stages the head enlarges to fill the serosa-llke cavity and sinks down into the yolk mass. In side view, the stomodaeum, five gill clefts and the sixth furrow can be seen. Stage 14. Early Motilitv; 2.8 mm. (Figs. 16, 17) The head is expanded to almost 0.5 nun- across the mesencephalon. The eyes exhibit more pigment and are pushed forward by the expanding mesencephalon. The latter possesses a thickened roof where the optic lobes are developing. The telencephalon has a somewhat rhomboidal- shaped cavity and the diencephalon is small and hardly distinct; this is typi- cal of the teleosts. Both the metencephalon, which is poorly defined, and the nyelen- cephalon have thin roofs. The neuromeres are still visible in the latter. The heart possesses a long sinus venosus and a narrow atrium that has been twisted to the left of the thick-walled ventricle. The blood vessels of the pericardial membrane are enlarged to a size equal to almost one-half the diameter of the ducts of Cuvier. The anterior fin-buds are club-shaped and rounded. The somites have taken the form of nyotomes, and, when the living embryo is removed from its membranes, the posterior por- tion exhibits a slow twitching motion. The tail is conical and acuminate. All six gill slits are distinct and open at this stage. The mid-gut is narrowed to- ward the posterior portion of the embryo, and fore-gut is an undifferentiated tube. Stage 15. Otoliths in Ear Vesicles; First Extra-ocular Melanophores; 3.2 mm. (Figs. 18, 19) In this stage the telencephalic vesicles are beginning to show as lateral bulges. The diencephalon Is shorter than the telencephalon and less distinct. The optic lobes possess a solid roof. The metencephalon is more distinct and thickened, and the nyelen- cephalon is somewhat narrowed. EXPERIMENTAL FISH EMBRYOLOGY 385 The eye pigment has become considerably darker and some iridiophores are present. The pupil is ellipsoidal. The olfactory bulbs have completely invaginated. The otic vesicles are enlarged and three crystal-like otoliths are present in each. The fin buds are laterally flattened. The caudal tip of the notochord is slightly upturned and the tail tip is laterally compressed, exhibiting a rudimentary sign of a heterocercal type of tail structure. A few stellate melanophorea are usually found in the connective tissue above the mid- dorsal, posterior region of the mesencephalon. This is the first indication of extra- ocular melanophores . The gut is completely separate from the yolk and the anterior intestinal portion is twisted into two coils. The posterior portion is straight and ends in a somewhat long post-anal region. The gill slits, except the first, are beginning to sink into a common cervical sinus, the forerunner of the opercular cavity. Stage 16. Fin Rays; 3.2 mm. First indications of fin rays in the caudal and pectoral fins are present. Melano- phores are spreading to the myelencephalon region. Stage 17. Anal and Ventral Fins; 3.H mm. Anal fin and the skeletal elements of the ventral fins are beginning to appear. Smaller, dot-like, melanophores appear on the lateral body folds. Head la further en- larged and fills the entire pericardial membrane tightly. The operculum is formed at this time. Stage 18. Dorsal Fin; 3.7 mm. Primordium of dorsal fin becomes visible, but there are no skeletal elements within It. Melanophores have spread over the entire mid- and hind-brain regions. Embryos at this stage are capable of swimming about, although the yolk sac prevents them from rising from the substrate. Stage 19. Eyes and Mouth Mobile; 3.9 mm. Through the enveloping pericardial membranes, the eyes may be seen to move and the mouth to open. The operculum is functional here. Fascial and peritoneal melanophores ap- pear as small black dots. Stage 20. Pericardial Sac Splitting; 1.2 mm. The pericardial extra- embryonic membrane begins to split down the dorsal midline, starting at the anterior margin Just above the sinus venosue. (This Is the first step In the formation of the "neck strap," described by Turner (19U0a) in many viviparous cyprinodonts. ) Stage 21. Mouth Protrudinn; 1.6 mm. The pericardial sac has split open as far as the anterior margin of the eye, allow- ing the mouth to protrude. The peritoneal melanophores are more numerous and small fascial melanophores are concentrated around the notochord. Stellate cutaneous melanophores are very sparsely scattered over the entire embryo, and many are concentrated in the mid- and hind-brain regions. 586 EXPERIMENTAL FISH EMBRYOLOGY Stage 22. Broad "Neck Strap"; 5.1 mm. (Fig. 20) The pericardial membrane has split as far hack as the posterior third of the eye, exhibiting a broad, vascularized "neck strap". The appearance of sclerotomes is here ac- centuated by concentrations of small melanophores in the fascial tissue around them. Cu- taneous melanophores are more numerous, sometimes present in the caudal fin rays. Stage 23. Fin Rays in Dorsal Fin; 6.1 mm. The "neck strap" (pericardial membrane) has been reduced to about one-half the width of the eye, and Is situated back of the posterior margin of the eye. Fin rays begin to appear in the dorsal fin. Large cutaneous melanophores are thickly scattered over the en- tire embryo. The yolk sac begins to show a rapid reduction in size, measuring 1 mm. In diameter. It is noteworthy that the yolk sac begins to Involute at about the same time that the pericardial membranes are in the process of accelerated regression. Embryos at this stage, if removed from their mothers, will feed readily on small Daphnia. Stage 2^. "Neck Strap" Breaking Down; 6.5 mm. The "neck strap" may be completely broken down at this stage, but it Is sometimes present as a narrow band of tissue. The general shape of the embryo is determined by the condition of the "neck strap," the cephalic flexure straightening as the head lifts up in- to the main body axis. The melanophores In the dorsal head region are stellate and more closely packed. Stage 25. Pre-Partur it ion; 6.9 mm. The extra-embryonic membranes and the yolk flanges are absent. The yolk has been re- duced to a mean diameter of .8 mm. No trace of the adult color pattern is yet visible, there being only a general Increase in the number of melanophores on the peripheral areas. This is true even in embryos of Culture Nos. l87 and 195, where the adult pattern (Induced by the gene Sp for spotting and St for stippling) is composed of large masses of macro- melanophores and mi cromelanopores . Nor can these two types of melanophores be distin- guished. Stage 26. One Hour after Birth; 7.9 mm. Birth activity begins with a rupture of the fertilization and follicle membranes by the violent movements of the embryos. The embryos break into the ovarian sac and then one by one they are extruded through the oviduct into the water. In earlier stages, the heart extends forward from the conus, and the sinus venosus lies directly beneath the tip of the head. As the yolk mass becomes reduced, the heart pivots on the conus and the yolk sac portal system shrinks until the ducts of Cuvler drain directly into the sinus venosus, which eventually moves into place posterior to the conus. Growth proceeds rapidly and within 2l+ hours after birth the young fry reach an aver- age length, of 8.7 dmi- Rate of Development In order to obtain some estimation of the developmental rate in Platypoecilus macu- latua, records were kept on the number of embryos and their stages found in each timed gravid female. The morphological age of the embryos was determined by comparing each with the twenty- five established graded stages. The following terms are used in this section: Theoretical age is the value determined for the entire embryonic brood from the date of the previous brood, less the seven day in- terval (as determined by Hopper, 19'<-5). Morphological age for each embryo is established EXPERIMENTAL FISH EMBRYOLOGY 58? by comparlaon with the graded series of stages. Chronological age represents the actual developmental rate for each stage. The theoretical age of all the memhers of a hrood was determined hy recording the date of birth of a previous brood. This is based upon the fact that fertilization of a successive complement of eggs within a gravid female takes place on about the seventh day after the birth of its previous brood ( Happer, 19^+3 )• Theoretically then, the embryos carried by a gravid female, which had dropped a brood eight days previously, are 2h hours old. This theoretical age value, it must be noted, la only an approximation, since matura- tion and. fertilization of a complement of eggs is spread apparently over a period of two or three days. The seven day interval, as determined by Hopper (19'*'3), has been found to be only an average time lapse. The estimation of the true chronological age may be de- termined by comparing the theoretical and the morphological age values. A reliable estimation of the theoretical age was obtained by study of those broods from fully matured females which contained 25 or more embryos, and which had given birth to at least, two previous broods at an interval of approximately 28 days. Only 21 out of 55 females examined had these qualifications. Data on many young females were found to be unreliable since many of them had run highly irregular reproductive cycles, varying from 35 to 90 days between broods; and a large percentage of their embryos were dead or abnormal. For these purposes, too, data on exceptionally small embryonic broods (those containing less than 10 embryos) were not considered. The chart (Fig. 21) siimmarlzea the data on 21 embryonic broods plotted in the follow- ing manner: Each vertical bar represents all the members of one entire brood carried by a single gravid female platyfish. The length of each vertical bar, projected on the ordi- nate, shows the range of morphological ages found in each embryonic brood. In some cases, especially during the later portion of the gestation period, the embryos are all of a single morphological age; these are represented by plus (+) signs. The embryos are divided into theoretical age groups according to the number of days that have elapsed since the birth of the previous brood (leas the seven day interval) and are arranged along the abscissa. Usually there is more than one brood in each age group. The mean morphological ages for all the embryos of each theoretical age group are also plotted on the chart, and these values are connected by the dotted line. From the chart, it may be seen that there are two kinds of variations. First, there is the wide range of morphological stages among the embryos found within any one gravid female; and second, the variationa of the average morphological age of a brood with re- spect to its theoretical age. The greater apparent spread of morphological stages in the earlier broods may be at- tributed to the unequal time lapse between stages distinguished on the basis of morphology alone. Using the Information described previoualy on the reproductive cycle of the platy- fish, it was thought that not only a graded series of morphological stages but also a chronological series could be obtained. On the basis of these data, some estimations of the time of development of each stage could have been made. However, the variation, aa demonstrated by the chart, proved to be so great that an estimation of the true chrono- logical age was in^osaible. 588 EXPERIMENTAL FISH EMBRYOLOGY THE NORMAL DEVELOPMENT OF THE TELEOST FISH Within the last decade a considerable volume of research has been published on the brook-trout (Salmo fario) and the rainbow-trout (Salmo Irldaeus) in Europe; on Oryzias latipes in Japan and the United States; and finally, on Fundulus in this country. Before proceeding to experimental techniques It is in order that we describe, in summary form, some of the characteristic features in the development of the Teleost egg. The egg consists of a large mass of fluid yolk surmounted by a disc of protoplasm, both contained within a plasma membrane and protected by a heavy chorion. The embryo is derived from the blastodisc which alone divides, ultimately to form a sheet of cells whiCh will encompass the yolk (eplboly). The aemi-gel mediiom which contains the blastoderm cells is bound to the yolk by an encircling gel layer (Lewis, 19'+3)' With expansion of the blastoderm there is a thinning of its center and a thickening of the "Eandring" or genn ring around its periphery. From a specific point on this germ ring the thickened embryonic-shield extends toward the center of the blastodermic area. With continued eplboly of the circumferential germ ring, there is an interruption at a point near the origin of the embryonic- shield, and an infolding (involution) of cells to form the endodermal roof of the archenteron. Differentiation of the embryonic -shield into brain, optic and otic vesicles, and somites then occurs. Oppenheimer (I956) has eonfinned the earlier statement by Morgan (I895) that the (Fundulus) embryo could survive without its yolk, by explanting the blastoderm alone into concentrated Holtfreter's solution to find that if the excision Is made prior to the 52- cell stage hyperblastulae developed but after that stage, embryonic structures and fre- quently embryos were differentiated. Devillers (19'+T) cultured the small pike egg in 5 times Holtfreter's solution beginning at the blastula stage to secure definitive em- bryonic structures. The trout egg (Salmo fario) did not give comparable results, indi- cating ontogenetic stage and differentiation may not be the same in different species. Oppenheimer (19*+?) believes that the yolk has a physical relationship to the early morpho- genetic movements which must not be minimized. amZ. /b'^ 1 : 1 2 e; ^ c^^V L L D" c^' c?J'\c3l^ A'" t."' .,.M q2 2 ^Jb^zT IB'l 1 " ;!l \ ll. \ / j / v=\ X ! 1 1 All?l ■ '^:::^r^ A» JJ V^\ ^ AJJ22 T / — /wi *'"\ -.\ B»"i Qil22 /y B' V \ \J A^ B' 1 I ^ Representation in the cleaving blastoderm of the embryonic areas mapped out in t)ie gastru- la. The long axis is here repre- sented by the second plane of cleavage. The nervous system is indicated by vertical liatchlng, notochord by heavy stipple, endoderm by light stipple, and mesoderm by horizontal hatching. 16 K ji.1.1 Bl-l-l ^ ^^.1.1.2.5 Notochord and endoderv Hsrvous ayoton posterior to stldbrsln Hesodera, aildbraln to ?d eomlto Endoderm, neeodera Mesodern posterior to 2d soKlto Nervous e/stea and Dssoder«, Torsbraln Posterior nesodem Mesodern and «xtra-est)i7ordo aeabruw Notocbord and erdodera Norvoufl Bjsten posterior to aldbraln Hesoders, midbrain to 2d somite Endoderm, mesoderm Mesoderm posterior to 2d somite RorrouB systsm and assoderm, forabrmlo Posterior mesoderm Mesoderm and extrs-embrronlo msmbrmne Oerm-rlng •- tall-bud blastema Ejctra-embiyonlc membrane Germ- ring Sxtra-embrjoolc membrane Sxtra-eBbryonlc eofflbrans Germ- ring Extra -embryonic meabranv Germ-ring •• tall-bud blastema £ztra-eBbT7onlc membrane Germ- ring £j(tra-embi7otilc membrane Extm-eabryoMlc membrane Germ- rl Tig Extnt-embrjonlc membrane CELL LINEAGE OF FUNDULUS. SCHEME FOR THE LOCATION OF MATERIALS IN THE BLASTODERM WHEN THE FIRST CLEAVAGE IS TRANSVERSE (From Oppenheimer 1956: Jour, Exp. Zool. 75:'+05) EXPERIMENTAL FISH EMBRYOLOGY 589 FUNDULUS HETEROCLITUS I A B C D ] (d" P" C ' ''1 P" D" c^' C" A- A" B'i B^' Al^ Al 1 &" ^' D"J» A"» r° 12) Ql I 1 C'l' CuV \ /■ 0112 c"» N, c." D" C2' • a" B" • \ Al" all' if J &'" ^ Hi Alii Bill Si. y^ A ^ a X li • - 7 -+- -+- - -♦- ^ V 1 ^ V f ^ y ^1122 A"2lB"*'B"" The typical cleavage pattern of the egg of Fundulus heteroci itus . Schematic. A represents the uncleaved blastoderm, B the two-cell stage, C the four-cell stage, D tlie eight-cell stage and E tlie sixteen- stage. V and G show the direction of the fifth and sixth planes of cleavage in the marginal cells of the sixteen-cell stage; these planes are not represented in tlie central cells of the sixteen-cell stage because they are here horizontal and dif- ficult to follow. A, B, C, D and E are drawn at approxi- mately the same scale; the magnification is greater for F and G. The position of the first four planes of cleavage is in- dicated by niomerals; see text for explana- tion of the designation of cells by letter and number. ^ L' D" A" 112 f~ -~N V. 0" A" ) C ' ■-N, V. u ^ X , c;" > f > \ V. -_A_i_ y Diagrams illustrating the results of ex- periments in which stained blastomeres were followed through to gastrulation. The figures marked "A" in each case represen the blastoderms immediately after staining those marked "B" and "C" are the later stages of the same embryos. The relation of the first or second cleavage plane to the embryonic axis may be determined. Occasionally the embryonic axis is oblique to the early cleavage planes. Usually two of the 8-cell stage blas- tomeres or four of the marginal cells of the 160-cell stage blastoderm give rise to the early embryonic shield, and (in one in- stance) two of the 16-cell stage blastomeres formed the entire shield. The germ ring is formed by peripheral cells of the 32-cell stage and the material for the fore-brain comes from the central cells of the 16-cell stage. From Oppenheimer 1956: Jour. Exp. Zool. 73:'*05) 590 EXPERIMENTAL FISH EMBRYOLOGY NORMAL DEVELOPMENT OF FUNDULUS HETEROCLITUS AT 25"C. Developmental atase Unfertilized egg. 1 cell. 2 cell. k cell. 8 cell. l6 cell. 52 cell. 61+ cell. 128 cell. 256 cell. Early high bias tula Late high blaatula Flat blaatula Expanding blaatula. . Blaatula enlarges. Gaatrulation begins. Early gaatrula. Blastoderm about one- third over surface of yolk. Blastoderm about one-half over surface of yolk. Middle gaatrula. Blastoderm about two-thirds over surface of yolk. Blastoderm about three-fourths over surface of yolk. Embryonic shield condenaes to form keel. Optic vesicle first visible as an expansion of the forebrain. Large yolk plug. Small yolk plug. Blaatopore cloaes. First somites formed. Four somites. Optocoele develops. Auditory placode forma. Optocoele connects across brain. Optic cup forms, and lens develops. Neurocoele develops. About 10 somites. Expansion of the mid-brain to form the optic lobes. Melanophorea first appear on yolk. Melanophorea appear on embryo. Heart pulsates. No circulation. Hind- brain enlarges. Circulation begins, through dorsal aorta and vitelline veaaels. Circulation through ducts of Cuvier. Otolitha develop. 35 somitea developed. Pectoral fin bud appears. Retinal pigmentation begina. Urinary vesicle formed. Caudal fin begins to develop. Liver develops. Cartilage begins to differentiate. Pectoral fin round. Lens of eye Just obscured by retinal pigmentation. Pigmentation of peritoneal wall. Circulation in pectoral fin. Fin rays in caudal fin visible. Air bladder develops. Neural and hemal arches in vertebrae in tail are developed. Head flexure begins to straighten out. Head flexure nearly straightened out. Mouth opens. Hatching. Pigmentation of air bladder. Age in Oppenheimer hours stage 0 1 1 2 4 5 2 k 2i 5 5, 6 5i 7 1^ 8 H 5, 5i-6 7-9 9 10-12 10 15-15 11 16 12 Ifl 191 15 21 22 25 Zk Ih 25 15 26 16 27 28 51 17 35 18 3^ 19 58 20 ko k2 Uh 21 h6 22 k8 60 25 72 78 2k 8k 25 90 26 102 27 108 n't 28 120 29 126 50 Ikk 51 168 192 216 21*0 26U 52 55 (FromSolberg I958: Jour. Exp. Zool. iQ-.kk'y) EXPERIMENTAL FISH EMBRYOLOGY 591 ^t ^)l 'f : Pi s Id •-3 z o CO C3 592 EXPERIMENTAL FISH EMBRYOLOGY The axis of the second cleavage plane tends to coincide with the longitudinal axis of the embryo. Cleavage, In general, tends to be geometric and so predictable that early cell lineage studies are possible. Teleost cleavage Is essentially the non-determinate type. There are variations in the qualitative distribution of parts of the blastodlsc so that Oppenhelmer (I956) says: "This lack of constancy of relationship between particular cells of the cleaving blastoderm and specific parts of the embryo is, of course, strictly compatible with the fact that the development of the teleost is of the Inductive type." Pasteels (I936) and Oppenhelmer (I936) have shown, by means of vital staining, that the fate map of Salmo and Fundulus are not unlike those for the Amphibia; that the periph- ery of the blastoderm furnishes the mesoderm for the embryo; that the prospective endoderm is found in the cellular blastoderm rather than in the yolky portion of the egg; that the prospective chorda lies in a crescentlc area (Fundulus) Just anterior to the prospective endoderm; and that the prospective nervous system lies Just anterior to the chorda-meso- derm. Gastrulation does not Involve differential mitosis but rather (as In the Amphibia) a re-arrangement of the cells, the types of movement being described as involution, ex- tension, and convergence. The initial expansion of the blastoderm is considered as epl- bolic movement. It seems apparent from the work of Morgan (I895), Lewis (I912), Hoadley (I928), Nicholas and Oppenhelmer {l'^k-2) and Tung (19^*^5) that the early blaatodlac can adjust to the removal of single or groups of blastomerea, and still give rise to normal embryos. This suggests that there is no qualitative division in the early stages of Fundulus, and that all regions are totipotent. It now appears that all parts of the trout blaatula are equlpotent (Luther, 1957) and, when the blastoderm of the blastula stage is quartered, each gives rise to tissues and organs (in tissue culture or transplantations) representing all parts of the embryo. Gastrulation is the time when there is qualitative segregation of areas, and Oppen- helmer (1956) and Luther (1955) have shown that the dorsal lip of the blastopore functions In teleosts much as it does In the amphibia. Secondary embryos can be Induced by hetero- topic transplantations of pieces of the dorsal lip. Oppenhelmer (l9'+7) believes that the Fundulus dorsal lip shows regional determination, supported by the studies of Eakln (1959) in Salmo. Transplantations to the extra- embryonic areas stand a better chance of surviv- ing and differentiating than when placed In the embryonic shield. EPIBOLY OF FISH BLASTODERM EXMNSON OF BLASTODERM AMMAL POLE VE0ET4L POLE flNIMAL POL£ VIEW GASTRULATION 1 APPEARANCE Of GERM RING EXTRA EMBRYONIC neMBRANE SIDE VEW rOLK PLUG OeVELOPMENT OF EMBRYONIC SHIELD AND EMBRYO (REDRAWN FROM OPFEfCCIMCR I9KI EXPERIMENTAL FISH EMBRYOLOGY 395 Fig. A and B. Maps showing position of areas for prospective tissues in the early gastrulae of Fundulus (A) and Salmo (B) . Endoderra, light stipple; notochord, heavy stipple; prechordal plate, cross-hatching; nervous system, vertical sliading; mesoderm, horizontal shading. Fig. A after Oppenheimer, 1936; Fig. B simplified after Pasteels, 1936. Drawn by Rosemary Gilmartin. (From Oppenheimer 19'+7-' Quart. Rev. Biol. 22:105) MORPHOGENETIC MOVEMENTS IN THE BLASTULA AND EARLY 6ASTRULA OF THE TROUT AS DETERMINED BY VITAL DYES rtepartition des territoires de la blastula vue de haut et de profil. Pf^intille dense, chorde; pointiile espace, plaque prechordals et entoderrae; croix, lames laterales. Systeme nerveux et ectoderme en blanc. line ligne Interrompue separe le materiel neural cephalique du troncal. Mouveraents morphogenetlques a divers stades de la forma- tion de I'embryon. A gauche, feuillet profond (fleches brisees) . A droite, feuillet superficiel (fleches pleines) . Le fin trait continu represente les formes exterieures de I'embryon; le trait interrompu, la liraite d' invagination. (From Pasteels 195^: Comp. rendu. I'Assoc. des Anat., Bruxelles) 59"+ EXPERIMENTAL FISH EMBRYOLOGY Semi-diagrammatic sketches of preserved Fundulus embryos to show the com- parative stages of organ formation. Made with the assistance of a camera- luclda. 1. Twenty-eight hour old embryo in which the embryonic shield is differen- tiating and invagination has begun. 2. Thlrty-six-hour-old embryo in which the embryonic axis has beguii to be recognizable . 3. Forty-eight-hour-old embryo, Just before the formation of the somites. 4. Ten-somite embryo. Note the beginning of the optic cups and auditory structures . 5. Fifteen-somite embryo. Considerable change has occurred in the organiza- tion of the brain and sense organs. 6. Twenty-five somite embryo, eighty hours of age. Note the increase in size as well as the degree of differentiation. Ctiraera-lucida drawings of sections through the optic vesicles of Fundulus embryos . 7. Kmbryo in one-somite stage. 8. Embryo in seven-somite stage. 9. Embryo In ten-somite stage. Note the flattened optic vesicles and lens anlagen. 10. Embryo in fifteen-somite stage. The optic cups are nearly complete and the lenses are developing. 11. Embryo in twenty-f Ive-somlte stage. The lenses are completely separated from the ectoderm. ( From Jones 1959-" Trans. Am. Microscopical Soc. 58:1) EXPERIMENTAL FISH EMBRYOLOGY 395 EXPERIMENTAL PROCEDURES WITH FISH EGGS AND EMBRYOS Fish material has been generally conaldered too difficult for experimental (oper-Mve) procedures by graduate students of embryology. However, Nicholas, Oppenhelmer, Luther, Eakin and others have demonstrated that the fish egg and embryo can be studied much in the manner of the classical experiments with amphibian eggs and embryos. Through the very generous help of Dr. Oppenhelmer, the following procedures are outlined: (1) Vital stain- ing of presumptive areas, (2) Excision and reconstitutlon, (5) Explantatlon and culturlng in vitro, and (h) Transplantation. In all of this work there are three very important considerations. 1. Preparation of the egg or embryo: The fish egg is provided with a tough chorion, or outer shell. This must be removed for most experiments, or a window nmat be provided through which the vital dye or a graft may be Inserted. The method is described by Nicholas (192?) as follows: a. Mortality is greatest during the cleavage stages. Shell removal after the embryo has formed a distinct cap on the upper surface of the yolk will be the more successful, and should be attempted first. b. It will be necessary to hold the egg In a Permoplast depression. Use very sharp-pointed scissors (iridectomy) and insert one blade between the egg and its shell so that its point is at a tangent to the egg. Enter the shell to the right of the embryo above the omphalomesenteric vein, but forward. If one point of the scissors is longer than the other, this may be used for the puncture and invasion of the shell. If the Invasion is properly made, the embryo should not be Injured. A small amount of fluid will escape from around the embryo. c. Avoid any pressure against the egg or yolk sac, either of which will rupture with the slightest pressure. The chorion is relatively so tough that It will hold the egg to the scissors, so that the latter may now be rotated into such a position that a cut may be made. Assist in the rotation of the egg with a hair loop or spear-point needle. When properly oriented on the scissors, cut through the chorion and continue the cut around the egg {or embryo) dividing the shell, as nearly as possible, into egual halves. d. Discard those eggs or embryos which have been ruptured or are in any way damaged. (If prepared, some parts of such damaged embryos may be used for in vitro experiments described below. ) e. The removed eggs and embryos of Fundulus will develop in distilled, fresh, or sea water providing the yolk membrane la Intact. It is probably best to use filtered water of the normal environment for eggs of the various species used. 2. Operating medium: It has Just been stated that the Fundulus embryo can survive almost any osmotic condition. This may not be equally true of freshwater forms. In general, for marine forms, Holtfreter's (Standard) Solution la used but in con- centrations twice or three times the nonnal and In normal or double concentration for freshwater forms. In general, therefore, the slightly hypertonic media are advised. 5. Asepsis: Prior to decapsulatlng the egg (or embryo) wash it in 8 to 10 changes of large volvunes of sterile water, in a sterile finger bowl. After decapsulatlng, transfer the embryo (gently) with wide-mouthed and sterile pipette through several changes of sterile medium. All glassware and solutions should be autoclaved, in- struments and pipette should be flamed before use. Steel instruments are used exclusively. (Oppenhelmer has reduced mortality from the usual 50^ to almost 0^ by using aseptic precautions - private commurdcatlon. ) 596 EXPERIMEHTAL FISH EMBRYOLOGY VITAL STAINING OF FISH EMBRYOS Procedure: 1. Prepare vital-dye stained cellophane. Grubler'a Nile Blue Sulphate and Neutral Red are to be used separately, and together. The cellophane (thinnest available) should be soaked In 1^ aqueous solutions for a day or more, then dried on clean white paper. The cellophane Is preferable to the agar because it remains in one piece and can be removed. Store the red, blue, and the combination-stained cel- lophane in clean marked envelopes until used. These vital dyes are not considered to be toxic. 2. Pass the eggs (in their sheila) through sterile media. Marine forms are to be treated in double strength Holtfreter's and freshwater forms in normal Holt- freter's (Standard) Solution. When tap or fresh water are used the stain pene- trates too rapidly and may even damage some of the cells. 3. Following the (Nicholas, 192?) technique described above, cut a minute window through the chorion of the egg to be studied. h. With watchmaker's forceps insert a small piece of stained cellophane through the window and maneuver it into the desired position with a fine hair loop. The chorion will generally hold the cellophane in position, where it should be left for from 15 to 1+5 minutes. The penetration and diffusion of the dye can be ob- served directly. The dye penetration is best in hypertonic media. 5. After. adequate staining has occurred, carefully remove the stained cellophane with watchmaker's forceps, wash the egg with chorion in one change of sterile medium and place it in a covered #2 Stender, containing the normal medium, and at appropriate temperatures for that embryo. 6. Observe during the next 56 to 1+8 hours, keeping the embryo at the lower limit of viable temperatures if it is desired to prolong the early stages. The record consists of a series of drawings of the changing position of the stained areas, beginning Immediately after, applying the dye. SKETCHES OF VITALLY STAINED FISH EMBRYOS SHOWING MOBPHOGENETIC MOVEMENTS EXPERIMENTAL FISH EMBRYOLOGY 597 ^ ^ \ fo^ /^ rx ^ Tir^ 0\ (S^ 1 / \ KJ V J II L \. y V y <>- — ^ a A^ ^ a VITAL-STAINING OF THE AREAS OF THE PRESUMPTIVE NERVOUS SYSTEM OF FUNOULUS A, B, C, represent stages in the early development of a single embryo. First embryo - entire nervous area. Second embryo - spinal cord area. Third embryo - mid- and hind-brain areas. Fourth embryo - nervous contributions from either side of the embry- onic axis. Fifth embryo - fore-brain and eyes from material anterior to the shield. VITAL-STAINIHG OF THE AREAS OF THE PRESUMPTIVE MESODERM OF FUNOULUS Drawings A to D represent stages in the development of a single embryo, reading from left to right. Note that the mesoderm is derived from the lateral wings of the embryonic shield, from the germ ring, from the germ ring 180° distant from the embry- onic axis, and from the extra-embryonic membrane. Successive stages In the development of embryos in which the presumptive endoderm has been vitally stained. When stain has been applied to the pos- terior lip of the early embryonic shield (A), the invagination of stained cells may be watched as gastrulation proceeds (B) . Occasionally the stain still remains localized in the region of Kupffer's vesicle after the blastopore is closed (C) . (From Oppenhelmer I956: Jour. Exp. Zool. Ti'-hO^) 598 EXPERIMENTAL FISH EMBRYOLOGY INJURY, ABLATION, AND RECOVERY OF FISH EMBRYOS Nicholas and Oppenheimer (19'+2) have found that up to the l6-cell stage the Pimdulus hlastomerea are totipotent, and that removal of as much as 50^ of the embryonic protoplasm can be survived by the embryo. On the basis of their work, the following procedures are given- The early stages may be operated on through the chorion, (either by direct pricking with a steel needle or through a window), or after the removal of the chorion. The first procedure is not so easily controlled and pressure factors may be involved, and yet it has the advantage of protection of the embryo against bacteria infection. The second method (i.e., decapsulating the embryo) involves the possible hazard of infection but removes the tension factor eind allows more accurate operational technique. (See previous section for method of decapsulation.) Both should be attempted. Use any fish egg available, such as Oryzias, Betta, Paradise or Hemichromus. Scliematic representation of suc- cessive stages in the transformation of the blastoderm to the embrjo. Tlie blastoderm (B) slowly expands over tlie yolk (Y) , as is shown In Figs. A, B and C. As gastrulation commences (Figs. D and E) the cells are piled up at the periphery of the blastoderm to form the germ ring (G.R.) and the embryonic sliield (E. S.); the central portion of the blastoderm becomes the extra-embry- onic membrane (E.M.) . During the course of gastrulation the blasto- derm gradually covers the yolk (dia- grams F, G, H and 1); late in gas- trulation a refractile streaK (N) visible in tlie sliield represents the keel of the central nervous system. Fig. J shows the extent of embryonic differentiation a few hours after the yolk is completely covered; O.V., optic vesicle; F.B., forebraln; M.B. , mid-brain; H.B., liind-braln; N.C., nerve cord; S. somite; M. unsegmented mesoderm. The egg Is drawn In profile in all figures except figure E, which represents the stage shown in Fig. D seen from the animal pole. (From Oppenheimer 1956: Jour. Exp. Zool. 75 ••'+05) A. REMOVAL OB DESTRUCTION OF A BLASTOMERE: Two to U-cell stage embryo. 1. With sharp-pointed steel needle invade the chorion directly over one of the blastomeres of a 2 or U-cell stage, and rupture it. Avoid damage to the yolk and contiguous blastomeres. 2. Make a small window in the chorion, directly over the blastomeres. Prepare a micro-pipette with terminal bore slightly smaller than the diameter of a single blastomere. If the edges of the pipette opening are rough, so much the better. Pass this pipette through the small aperture, and gently suck up a single blastomere. This may require first the loosening of the blas- tomere with a slight circular movement of the pipette. If the pipette is attached by a small-bore rubber tube held in the mouth the suction can be the better controlled. By this oral suction method 1 and then 2 blastomeres of the U-cell stages should be removed. EXPERIMENTAL FISH EMBRYOLOGY 399 DRAWINGS OF BLASTOMERE DESTRUCTION AND EXCISION EXPERIMENTS B. REMOVAL OF PARTS OF THE GERM RING: Stagea #10 to #15- The germ ring I3 so adherent to the underlying yolk that the suction proce- dure nniat be used cautiously or the yolk will exude whereupon death follows. 1. Use the window or decapsulation method, and suck out the ectoderm (only) of tne germ ring up to not more than 20$ of the ring. Concentrate on those parts of the germ ring distant from the early shield. 2. Suck out parts of the early neural keel (stage #15), with and without the adjacent germ ring. (Nicholas and Oppenhelmer found that if 25^ of the keel is removed, development ceases.) 1+00 EXPERIMENTAL FISH EMBRYOLOGY DRAWING OF GERM RING AND KEEL EXCISION EXPERIMENTS C, ABLATION EXPERIMENTS ON LATER STAGES: (stages #20 to #25 all decapsulated. ) 1. Remove the pectoral fin as it appears as an anlage' posterior and ventral to the ear vesicle (stage #20). 2. Remove the eye from one side only. This may be accomplished by drawing the eye Into a small-bore pipette and then cutting it off at the base with a fine needle knife. 5. Remove the otic vesicle (stage #20). With controlled oral suction the ear vesicle can be removed without injury to the adjacent medulla, but there is generally no regeneration and the embryo later shows circus movements In consequence. k. Remove small parts of the nerve cord (stages #17 to #20). This may also be done by auction, but it is difficult to limit the amount removed. Not more than the equivalent of 2 somite lengths should be removed, and a minimum of the adjacent notochord and lateral mesoderm. 5. -Remove parts of the brain, also by the suction method. This operation seema to be less drastic for the embryo than some of the others, but it is some- what more difficult to localize the ablation. No two embryos will be iden- tically treated, hence Individual records are required. EXPERIMENTAL FISH EMBRYOLOGY 1|£1 There is a quantitative decrease in regenerative or regulative capacity during early development as revealed by the preceding operations. Nicholas and Oppenheimer, using Fundalus, found no permanent damage up to the l6-cell stage of many of the operated forms. "Up to this point the blastomeres may he regarded as totipotent so long as one-half of the original content of the egg is left untouched." (Nicholas & Oppenheimer, 19^2.) Follow- ing this stage there is rapid reduction in regulation, with progressive differentiation. RECORD OF ABLATION EXPERIMENTS ON LATER STAGES D. TRANSECTIONS OF THE SPINAL CORD OF MOTILE STAGES: (stage #25) The nerve cord is transected (without removal of any tissue) with a sharp steel needle or iridectoiny scissors, avoiding injury to the notochord if possible. The transections should be at different levels gind records should include descrip- tion of the type of movement upon development to stage #26. Histological analysis will determine the degree of damage and reconstitutlon. U02 EXPERIMENTAL FISH EMBRYOLOGY RECORD OF NERVE COED TRANSECTION EXPERIMENTS DISCUSSION: With the Increaalng physiological complexity of the embryo there Is a decrease In the regulative capacity, noted first at the l6-cell stage. When as much as 50^ of the proto- plasmic material of the l6-cell stage Is removed In Pundulus (Nicholas & Oppenhelmer, 19^+2 ) defects appear and few of the fish gastrulate, hut the same proportion of protoplas- mic material can be removed at the previous (8-cell) stage and this will be followed by complete regulation. Following the l6-cell stage there Is a rapid drop In regulation, so that at the blastula stage the embryo can tolerate a loss of not more than about 20^ of the protoplasmic mass. % 1 1 / 30- \ PLO SCHEMA OF RECONSTITUTION / TTEO AGAINSTSTAGE OF OEVEUOPMENT ^ / o \ ,IN FUNOULUS y^ > \ A^X o ^/ 5 40- \ &/ «« \ ^* / 3 . * y ;? \ // - or / SS " \ if/ 3 \ if V .^ CELLUL4R X V < » MULTIPLICATION \ / 03 WITHOUT FIXED ^S^ / SPECIFIC - O LOCALIZATION STRETCHING ^S. / ORGAN TRUE Is '° Of POTENCY AND ^^^ / FORMATION REGENERATION a. o O PROLIFERA- ^^ FIXITY REPAIR 22 5 TION oflCAHl2^1^r^^s,,^ OF PARTS LIKE FROM LIKE 5 • 0 LOpWrfZATION POLARIZATION >%»„^^_^ ZCCIXED ecCLl£0 BLASTUL* 0A3TRULA SHIELD PREMOTILE m\s\ HATCH- 4CELJLE0 16 CELLED RING CMBRYO ING SPCCNHCII* R5-4-3- 6 ro-ll -12 13- 14 - IS 17 - 84 STAGES ( From Nicholas & Oppenhelmer I9U2 : Jour. Exp. Zool. 90:127) EXPERIMENTAL FISH EMBRYOLOGY k03 Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. 1. Dorsal and lateral views of a Kundulus after hiatchlng revealing the shortening of the head with orbital formation following ablation of the eye at stages 21-23. 2. Stage 27 embryo showing the recovery and healing of an embryo after the removal of the eye in Stage 22. Indications of the future mouth and Iiead asymmetry are already evident. 3. The healing after operation at Stage 22 shows a puckering of the opercular region following the removal of fin tissue. The embryo Is slightly bent toward the side of operation. 4. The removal of the fin in Stages 20-22 gives rise always to the t>Tpe of animal here shown at the time of hatching. 5. The ear was removed in its early vesicle stage, Stage 20. At the time of opera- tion slight damage was done to the right half of the brain which is reduced in size. The C curvature is .characteristic of the posture of the earless forms. The operation performed at Stage 17 Involved a slight injury to the mld-braln and the constrictions made by the pipette are still evident at Stage 25 anterior and posterior to the original site of Injury. The result obtained by an injury to the anterior part of the medulla; the circu- lar region shows a deficit extending to the notocord which was slightly en- larged. There is no reconst itution. Operation performed at Stage 18. Development without repair after injury to the left side of the brain. Opera- tion performed at Stage 18. 9. The free portion of the tall bud of a stage 20-embryo was removed. The wound healed and was covered with epidermis. The rounded eminence is a notochordal projection covered with epithelium. Embryo Stage 25. 6 8 ^From Nicholaa & Oppenheimer 19i;2 : Jour. Exp. Zool. 90:127) 1+Oi^ EXPERIMENTAL FISH EMBRYOLOGY Injury or ablations of the embryonic shield and neural keel generally show localized effects, particularly when they occur in the midline of the embryonic axis. After organ differentiation, the damage tolerated must be only a small fraction (less than 10^) of the available tissue. This applies particularly to the formed brain, sense organs, fin, tail, etc. In all of this type of work it must be remembered that some of the results may be due to incidental handling of the embryo, as in decapsulating. Also, it is somewhat more dif- ficult to excise a specific region, and only that region, in fish than in the amphibian (or even chick) embryos. Collateral injuries may be the primary cause of failure in re- constitution. Operational skill with fish material Is achieved only with great patience, persistence, and repetition. THE CULTURING OF FISH EXPLANTS IN VITRO This type of investigation, so readily achieved with amphibian and even with chick material, proves satisfactory but to a lesser degree with fish embryonic parts. Oppen- heimer (1958) cultured parts of Epiplatys fasciolatus in a modified Singer's for from 4 to 7 days and secured tail-like differentiation without histogenesis. Figs. 1 to 7. The types of differentiation attained in isolated blastoderms which have not gastrulated. These are hyperblastulae , masses of cells generally differentiating only a hollow vesicle whose wall represents the ectodermal portion of the yolk-sac epithelium. The stage at which the blastoderms were removed from the yolk, and the number of days they survived before preservation, are indicated in parentheses. Cell walls are represented only in Fig. 1. Fig. 1. Hyperblastula in which the non-differentiated embryonic cells form a compact mass In the center of the vesicle. (16-celled; 4 days.) Fig. 2. Hyperblastula in which the non-differentiated mass of cells is continuous with the vesicle epithelium at one region only and surrounded elsewhere by a columnar epi- thelium. (32-celled; 3 days.) Fig. 3. Explant whose differentiated cells are joined to the vesicle epithelium by a mesenchymatous network. (8-celled; 4 days.) Fig. 4. Explant In which the cellular arrangement suggests tlie occurrence of irregular cell movements. Except for the formation of columnar epithelium no hlstogenlc changes have taken place. TJie cell movements were not those of gastrulation. (16-celled; 38 days.) Fig. 5. Hyperblastula containing two different types of cells, one non-differentiated, the other with denser nuclei and more heavily staining cytoplasm (NV) . These probably present cells which have commenced self-dlfferentlat Ion of nervous tissue without the Inductive stimuli of gastrulation. (64- to 128-celled; 39 days.) Fig. 6. Hyperblastula in wliicli one group of cells has the dense nuclei characteristic of nervous tissue (NV) , while another group (N) is surrounded by a heavy sheath simi- lar to that normally surrounding the notocliord. These cell groups are large splierl- cal masses, surrounded by homogeneously arranged non-differentiated cells; their differentiation has been Independent. (2-celled; 3 days) Fig. 7, Hyperblastula in which a small group of cells, probably epidermal in origin, have begun to self-differentiate nervous tissue (NV) . (8-celled; 6 days.) (From Oppenhelmer 1959: Jour. Exp. Zool. 72:21+5) EXPERIMENTAL FISH EMBRYOLOGY U05 Procedure: Insure complete aaepals, autoclaving the media and sterilizing all instruments and glassware. Culturing can be in deep depression slides, or in covered and sealed #1 Stenders. Culture medium: For marine forma the filtered and sterilized sea water can be used. For fresh-water forma, Standard ( Holtfreter' s) Solution should be used, in normal and in double strength. To vary the medium, it is suggested that O.l'jt glucose or 15^ glucose plus O.l'jt peptone be added prior to autoclaving. (See vari- ous media used with chick explants.) Pass the egg to be studied through 7 to 10 changes of aterlle medium isotonic for that egg, to free it of most of the bacteria. Decapsulate the egg in the manner described above (Nicholas, 1927) at stages #2 to #7. Using sharp, sterile, steel knives dissect away the blastodermic disc of any stage from 1 to 32 -cells. Clean away all adherent yolk graniiles and culture in hanging drop (see chick exercise), in sterile medium. (These will generally form hyperblaatulae. ) From later stages (stages #7 to #12) explant the entire embryonic mass (exclusive of the yolk) Into culture dishes with hypertonic and sterile media and observe for h8 hours or more for differentiations. Histological analyses will be necessary to determine the degree of differentiation. Bisect the entire egg of stage #15 in the manner indicated in the diagram below, using a sharp and sterile steel needle. Aa the needle is pressed through the egg and into the underlying Permoplaat base of the operating dish, the cut surfaces of the two halves are pinched apart in such a manner that the cut surfaces are usiially closed together and the yolk is contained within the half-aized vesicles. When this occurs, the entire halves may be cultured. When there Is rupture, parts of the germ ring and embryonic shield should be further dissected away and cul- tured in isolation. The localization of presumptive nervous system, mesoderm and endoderm in the early gastrula (A) and the middle gastrula (B) , and the position of these tissues in the seven-somite embryo (C) . The position of the nervous tissue is Indicated by heavy stipple (forebraln and optic vesicles), diagonal hatching (mid- brain and anterior hlnd-braln) and vertical hatching (posterior hlnd-braln and spinal cord). The cells whose position Is Indi- cated by horizontal hatching aid In the formation of mid-brain, hlnd-braln and spinal cord. The mesoderm is indicated by the light- ly stippled areas. The areas marked by the numbers 1, 2, 3 and 4 ultimately lie in the regions of the embryo indicated by the ar- rows accompanying diagram C. The endoderra Is represented by open circles. The endodermal cells that have invaginated are not shown. (From Oppenheimer I956: Jour. Exp. Zool. 7^:kO^) Scheme of ope Isolate tlie germ originally locate dorsal lip of tlie are divided into along the plane X Fig. IB shows a d blastoderm. The represent tlie mat grafts of germ rl I8O" from the emb stippling Indicat embryonic shield Ing extra-embryon rations. In order to ring of late gastrulae d 18(i" away from the blastopore, the eggs two parts by cutting -X shown in Fig. lA. orsal aspect of tlie cross-hatched regions erial involved in the ng originally UO" or ryonlc axis. The heavy es germ ring (OR) and (ES) , the light stlppl- Ic membrane. (From Oppenheimer 195S: Jour. Exp. Zool. 79:185) Uo6 EXPERIMENTAL FISH EM3RY0L0GY Parts to be cultured at stages #12 or #15= a. Portion of the germ ring 180° from the embryonic shield. b. Embryonic shield and related germ ring In half embryo. c. Dorsal lip material only, i.e., margin of the embryonic shield. Parts to be cultured at later stages, #15 to #2l4-. a. Embryonic shield alone at stage #15. b. Optic vesicle of stage #17. c. Melanophores of extra- embryonic membrane, stage #20. d. Heart anlage of stage #20. e. Somites and related neiire and notochord of stage #20. f . Pectoral fin of stage f2k. If the period of isolation can be extended to more than h days it will be neceseaiy to renew the culture medium at that time. Generally the limit of differentiation will be achieved before that time, usually within kQ hours. If the cvilture is made in a hanging drop (see chick exercises) the complete process of development can be observed under the dissecting microscope without disturbing the explant, and if It is bacteria-free, it should survive. Figs. 9 to 15. Sections through isolated blastoderms wlilch have undergone gastrulatlon and have formed embryonic structures. Fig. 9. The ear region of the embryo shown In Fig. 8. The organs are In their topo- graphically normal relationships. B, hlnd-braln; E, ear; P, periblast. Figs. 10 and 11. Two sections through em embryo whose structures are in topographically normal relationships although periblast (P) is found only In the eye region. The embryonic axis was curved, therefore the eye region, shown In Fig. 10, was cut frontally, and the ear region, shown in Fig. 11, transversely. In Fig. 10, optic vesicles (0), forebraln and optic lobes (b) , and periblast are seen, and In Fig. 11 medulla (NV) , notochord (N) , gut (G) and ears (E) . Posteriorly in the liead the embryonic axis Is double, and the secondary nervous system Is imperfect. (64- to 128-celled; 4 days.) Fig. 12. Sagittal section through an embryo in which gut (G) , notochord (N) , medulla (NV) and heart (H) show In general typical relationships to each other. One ear is ven- tral (E) rather than lateral, however, due to twisting of the embryonic axis; t)ie notochord Is somewhat contorted, and posteriorly grades off Into a dense group of cells continuous with the nervous system. (2.")f)-celled; 4 days.) Fig. 13. Sagittal section t)irough an embryo In which grain (B) , gut ((!) and ear (R) are in their normal positions, but the notochord (N) is greatly convoluted and forms a mass without association with the nervous system. (64-celled; 4 days.) Figs. 14 and 15. Two sections through a blastoderm in which irregularities of the nervous system have resulted from the distortion of the substrate after gastrulatlon. A floor plate has been formed only in regions where the notocliord (N) and nervous tissue (NV) are In contact. (32-celled; 4 days.) (From Oppenheimer I956: Jour. Exp. Zool. 72:2^7) EXPERIMENTAL FISH EMBRYOLOGY ^01 DRAWINGS AND RECORD OF FISH EXPLAMTS IN VITRO Yamamoto (1959 Fac. Scl. Tokyo Univ. 15:269) gives a formula for a synthetic medium which is isotonic to the Oryzias egg. M/7.5 NaCl 100 oc. M/7.5 KCl 2.0 cc. m/II CaCl^ 2.1 cc. pH to 7.5 To such a medium 0.1^ glucose may he added for nutrition for the explants of Oryzias. Hayes, Darcy and Sullivan (l9'+6: Jour. Biol. Chem. l65-"621) have analyzed the ovarian or coelomic fluid of the salmon, which fluid appears suddenly Just at the time of hatching. They find it to he a clear, limpid, and slightly translucent medium with the following constituents: Ions per 1,000 liter of water: Sodium 151 mllli equivalents Potassium 5-2 Calcium 7 • 1 Magnesimn 2.6 Chlorine II6 .0 HPOi^ 1+ . 0 HCO5 : 13 .'+ Since the ripe eggs of the Salmon lie freely within the body cavity, and hence within this fluid medium, this medium may he considered as isotonic to the eggs at this stage. This fluid is, however, hypertonic to hlood. Sodium chloride is the dominant salt, with other Ions in the approximate ratio that they are found in sea water. In the eggs the potassium dominates, and the calcium and magnesium are not osmotically active. At fertilization, in water, the egg loses osmotic pressure hy about 5^' During development the egg and embryo take up calcium and sodium from the environment so that the final amounts are h and 3 times the initial amounts respectively. Phosphorous intake is markedly increased, probably in connection with sketeon formation. U08 EXPERIMENTAL FISH EMBRYOLOGY INDUCTION OF SECONDARY EMBRYO BY GRAFTING OF DORSAL LIP The fish egg can be uaed as a host for successful transplantations without the neces- sity of a Briicke or bridge to hold the graft In place. Healing and development are so rapid that the whole experiment can he concluded within several days. Procedure: 1. Pass the egg and its chorion through 7 to 10 changes of sterile medium. This should be hypertonic, such as double or triple Standard (Holtfreter' s) Solution. 2. Decapsulate the egg, using the technique of Nicholas (1927) described above. The steel needles or watchmaker's forceps imist be sterile. Use stage #13 when the germ ring has passed over about 5/*+ of the yolk. Similarly prepare the donor, of the same age and stage. 5. Dissect out of the donor the region comparable to the amphibian dorsal lips, i.e., the margin of the germ ring from which arises the embryonic shield. Clear it of all adherent yolk. Use sharp and sterile steel needles. It will help to follow the graft If the entire donor is previously stained in l/lO,000 Nile Blue Sulphate . h. Prepare the host for the graft, using two different sites in as many hosts. Operate in double Standard ( BDltfreter's) Solution, or stronger. a. Loosen the embryonic shield on one side, using sharp, steel needles. The cells will tend to grow together rapidly, and will hold any graft In place. Insert the excised dorsal lip material, pushing it beneath the margin of the embryonic shield. b. Loosen some superficial cells of the extra-embryonic blastoderm at some distance from the embryonic shield, and quickly insert the excised dorsal lip material. 5. After the wound has healed and the graft seems to be held intact, transfer the embryo (by wide-mouthed pipette) to a covered #2 Stender containing sterile, normal (isotonic) medium for that egg. Culture it for a few hours to as many as 7 days, depending upon the success of the take and health of the embryo. Localization in the nerve keel of the late gastrula. Cells removed from the region A dif- ferentiate when grafted on ex- tra-embryonic membrane to form optic lobe, cells from the re- gion C to form spinal cord. A defect in the region U resulted In a deficiency in the region of Mauthner's cell. An embryo In which 180 germ ring from an early gastrula has formed caudal fin dorsal to the brain of the host. Fixed 8 days after operation. (From Oppenheimer 1958: Jour. Exp. Zool. 79:185) (From Oppenheimer 1956: Jour. Exp. Zool. 75:'*05) EXPERIMENTAL FISH EMBRYOLOGY ii09 BECORD OF EMBRYONIC INIXTCTIONS IN FISH THE GENETICS OF FISH* Through an Intenaive series of researches Gordon (1927-19'*8) has demonstrated that the mechanism of inheritance la essentially Mendellan for fishes, as It has heen proven to he for all other groups studied. There are wild types (gray) of the Platy (Platy- poecilus maculatus) and domesticated albino and golden types which can he interbred readily. In all, there are some 150 varieties of patterns in this one species, relative- ly few of which have, as yet, been thoroughly analysed from a genetic point of view. Ee- cently (l^^k-V^kQ) Dr. Gordon has concentrated on the inheritance of melanomas, which study is closely akin to cancer studies on higher forma. The Platy Is readily available at any Aquarium Supply Houae, and the three major varieties (wild, albino, and golden) may be procured for simple genetic crosaes. The golden mutant arose from a wild atock in I92I and the albino appeared first, also from a wild stock, in 195*+ • When these mutanta are Interbred, the wild variety re-appears- Following are a aeries of genetic croasea that have been made by Dr. Gkirdon, indicat- ing clear-cut dominance and recesalveneas of various pigment patterns. These are offered here as suggestive of the type of study in the field of developmental heredity that is now possible. (See also papers by Goodrich et al.) * These figures are kindly loaned by Dr. l^ron Gordon of the New York Zoological Society. It is hoped that Dr. Gordon will aoon make available, in concentrated form, his knowl- edge of the Genetics of Fishes. ^10 EXPERIMENTAL FISH EMBRYOLOGY Fig. 1. Ail early stage In Induction in Fundulus, drawn fii hours after operation. The first visible effect of dorsal lip implantation in Fundulus is concentration of cells in the vicinity of the graft; here host cells (H) stained with Nile blue sul- phate aggregate in the region of the graft (O) stained with neutral red. P, primary embryo. Figs. 2 and 3. Two percli eraoryos (I) induced by dorsal lip grafts implanted into the edge of the blastoderm 180° away from the primary dorsal lip; drawn 2 and 5 days after operation, respectively. P, primary embryo. Fig. 3 drawn by Miss L. Krause. Fig. 4. Axial structures (I) induced from the extra-embryonic epithelium of a Fundulus egg by implantation of young dorsal lip into the edge of the blastoderm; drawn 21i hours after operation. Fig. 5. Embryonic structures (I) induced in Fundulus by implantation of a dorsal lip material into extra-embryonic epithelium; drawn .23 days after operation. E, ear. Fig. 6. Perch embryo (I) induced by dorsal lip implanted into extra-embryonic epithelium; drawn li days after operation. This embryo was probably a lateral hemi-embryo; somites were formed on one side only. Figs. 7 and 8. Two stages in the development of a Fundulus embryo induced by the implanta- tion of very young dorsal lip into a very young gastrula; drawn at 18 hours and 3i days after operation. The right part of the graft, which liad been stained with Nile blue sulpliate, failed to invaginate and formed a knob (0) at the right side of the developing Induced embryo; as a result the induced embryo (I), although two ears were formed, was a lateral liemi-embryo with somites present only on the left side. Fig. 9. Fundulus embryo (I) induced by a dorsal lip graft implanted into the edge of the blastoderm; drawn 3 days after operation. The ears and right fin of the Induced embryo are formed posterior to the corresponding structures in the primary embryo (P). Fig. lu. Head structures of Fundulus (I) induced by a dorsal lip graft which was original- ly implanted into extra-embryonic epit)ielium but which shortly after implantation became incorporated into tiie primary embryonic shield; drawn 3 dasy after operation. Fig. 11. Fundulus embryo (I) induced by the implantation of very young dorsal lip; the graft was incorporated into the primary embryonic shield. Drawn 22 hours after operation. Fig. 12. Somites (I) induced by a dorsal lip graft implanted into the primary embryonic shield; 1 day after operation. The accessory somites persisted for 2 days and sub- sequently were absorbed into the host; compare embryo shown in Fig. 6, where somites induced in an extra-embryonic region exhibited a different segmentation from the host's. Fig. 13. A dorsal lip graft implanted anteriorly in the embryonic shield was partially absorbed into the forebrain, wltli the result that this structure has wider walls than normal . Fig. 14. A graft of cells removed from a lateral part of the shield formed brain (G) in the mesencephalic region of the Iiost. HOL, optic lobes of host. F'lg. 15. A perch embryo In which dorsal lip implanted into the embryonic region was trans- formed to brain; drawn 6 days after operation. (From Oppenhelmer I956: Jour. Exp. Zool. 72:1+09) EXPERIMENTAL FISH EMBRYOLOGY Ull TRANSPLANTATION EXPERIMENTS BY DR. JANE OPPENHEIMER hl2 EXPERIMENTAL FISH EMBRYOLOGY GOLDEN X ALBINO = WILD When blno sword is mated w swordtail . BACK TO THEIR ANCESTRAL COLORS tlie fi,olden swordtails are liibred, tliey produce notliing but golden; when the al- tails are inbred, they produce nothing but albinos. When the golden swordtail itli the albino, tliey produce young all ol' whicli have the coloring of the wild U About 1921 WILD X WILD WILD WILD WILD golden WILD golden WILD WILD About 1954 WILD X WILD \ WILD WILD WILD albino WILD albino WILD WILD 2a golden X golden albino X albino 1 golden golden golden golden albino albino albino aibmo ? (Done in 1940) golden X albino WILD WILD WILD WILD X golden 1 Expec[«d Obiervfd WILD 60 68 golden 60 52 120 120 406 WILD WILD 4b WILD X WILD WILD WILD WILD albino X WILD 1 Expected Observed WILD 188 202 golden 63 65 albino 83 67 334 334 Expeaed Observed WILD 28 33 albino 28 23 56 56 HISTORY OF SEPARATION AND REUNION OF COLOR GENES IN THE SWORDTAIL This chart shows tlie independent origin ol' Llie golden and the albrno, and the results obtained when these mutations were crossed. The backcrosses ol" the "synthetic" wilds to golden and albinos show tlie genetic structure of these differences. Some of the dis- crepancies in the ratios in tlie oackcross and K., results may be accounted for by the dif- ferential viability of the three types of swordtails. The albino is the weakest, and the golden is definitely weaker than the wild tj-pe. The total of 334 Fo offspring is made up of the progeny of several females. Some Kj matlngs produced Fo youn^; In the ratio of 9:3:1 so closely as to be remarkable. EXPERIMENTAL FISH EMBRYOLOGY hl3 Stippled daughters Stal Stat Sl.'ppled sons it Cold Bonf) and daughters ■ I Cold platyfish — 5l8t Stippled sons and daughters 3 Stippled platyfishes - StSt Stippled sons and daughters This diagram Illustrates the way In which the gray or stipple pattern of the platy- fish is inherited. In the pair on the left, the father carries the dominant factor (StSt) for stippling while the mother is non-stippled (stst) . The parents are designated as P-,. The first filial generation (Fj) , obtained from this mating, are all stippled (Stst) like their father. Similar results are obtained when the mother (see pair on right) is the stippled parent (StSt) and the father is non-stippled. The offspring of this mating are also all stippled, this time like their mother (Stst). When the individuals of the first generation (Fj) are bred> together from either series, and the second generation (Fo) is produced, the following results are obtained: there are three stippled fishes (StSt) or (Stst) to every non-stlppled one (stst) . There are male and female representatives in each group. Tills is an example of simple mendelian inheritance of a single heritable character. The stippling is actually due to the presence in the skin of very small black cells, technically known as mlcromelanophores . w z Noll spoiled mother sp »\\ ^^^-'■■M L Z Sp Sp spotted father ^!g:>:i w z Spotted daughters sp Sp z z Sp sp Spoiled sons W 2 Non spotted daughters sp sp w z spoiled dauehlcrs sp Sp Spotted fiona Spoiled sons This diagram Illustrates the peculiar way in which the spotted (Sp) pattern is in- herited. The female Is represented by the formula W Z while the male is Z Z. The spotted factor is associated with sex. Wlien the spotted platy is the male parent (Zsp Zsp) and a non-spotted platy is the female parent (Wsp Zsp) , all the offspring of the mating, both the sons and daughters, are spotted like their father. When the spotted individuals of the first generation (Fj^) are mated together, brother to sister, t}ie ratio of three spot- ted platyfishes to one non-spotted is obtained in the second generation (Fo) . Up to this point the results of this mating resemble tlie inheritance of the stippling factor, but note the following significant fact: all the non-spotted fishes obtained in the second generation are females. And the spotted platyfishes contain both males and females, but there are two spotted sons to every non-spotted daughter. This is an example of the In- heritance of a sex-linked heritable factor. A similar type of Inheritance is found in birds and moths for certain characters. hlk EXPERIMENTAL FISH EMBRYOLOGY -4i.--. X Xiphophorua hdlcrii mother ■-^f Platypoecilus futhrr Gold platjr Red-finned platy-helleri hybrid W^-"''^ X Xii'hoplinnii h^llerii mother Xiphopliorus hcllerii mother X Platypoecilufl father Red platy ^. Platypoecilus father Black platy Black platy-helleri hybrid Xiphophonia hellerii mother X Plalypofrilu!! falhrr Stippled and Spotted platy Mottled platy-helleri hybrid The platyflslies and the Mexican swordtail will readily hybridize. Tlie hybrids are classified according to tlie original varieties of tlie species used in the cross. The gold platy and the swordtail produce the red finned platy-helleri )iybrid. The red platy and the swordtail produce the red platy-helleri hybrid. The black platy and the sword- tail produce tlie black platy-liel leri liybrid. The stippled and spotted platy and the swordtail produce ',he mottled platy-helleri hybrid. These liybrids frequently develop the neoplastic disease known as nelanosis. EXPERIMENTAL FISH EMBRYOLOGY i^l5 DISCUSSION: The foregoing sections on Fish indicate that this Class of Vertebrates Is coming in- to its own in the field of Experimental Emhryology, Most of the procedures outlined are of an operational nature. There are also the studies on hyhridlzation; on environmentally Induced teratologies ( Stockard, 1921); on the effect of x-lrradiation of one of the gametes (Solfcerg, I958); on the physiological response of the embryo to changes in the environment (V'aterman, 19^+0 and the various Japanese workers with Oryzias). There is an ever increasing amount of work of a cytological and cytochemical nature, all of which should be Included in an exhaustive treatise on experimental embryology of fish- The teratologlcal forms of fish embryos proiiuced bj Irradiating, germ cells. No. 1 represents a control embryo 4 days after fertilization. No. 2 shows slight reductions In the anterior and posterior regions. The succeeding stages (3, 4 and 5) sliow greater re- ductions of head and tall. Other parts are entirely lacking in these embryos. Deformi- ties of the heart are shown In drawings to the right of each emoryo. Embryos deformed as much as No. 5 often do not develop a Iieart. The heart deformities consist c)ilerii of an elongation, and Improper formation ol t.ie chambers, usually associated with an edema of the pericardial cavity. (FromSolberg I958: Jour. Exp. Zool. 28:'J-17) The fish are rapidly becoming a contestant for attention along with the amphibia. Possibly the work of Oppenheimer (1959) cam be cited as a bridge between these two classes of vertebrates, for she found tliat a large variety of fish anlagen differentiated quite normally when grafted into the amphibian hosts and that "fish epidermis and cartilage have been found morphologically continuous with coniparable structures formed by amphibian cells in grafts," and "the fish grafts are occasionally seemirigly Innervated by nerves originat- ing from the amphibian cranial ganglia; it is not known whether the apparent innervation is a functional one." (Jour. Sxp. Zool. 80:592) Ul6 EXPERIMENTAL FISH EMBRYOLOGY REFERENCES: Abramowltz, A. A., 1937 - "The role of the hypophyseal melanophore hormone in the chroma- tic physiology of Fundulua." Biol. Bull. 75:15'*. Adelmaiin, E. B. , I956 - "The problem of cyclopia." Quart. Bev. Biol. Il:l6l. Alexander, L. E,, 19'+2 - "The capacity of the eye cup of Pundulus heteroclitua for induc- tion and regeneration of lenses as studied in lenseless eyes." Jour. Exp. Zool. 91:111. Allee, W. C, A. J. Kinel, & W. H. Hoskins, 19^0 - "The growth of goldfish in homotypical- ly conditioned water; a population study in mass physiology." Jour. Exp. Zool. 8i*:l;17. Armstrong, P. B., 1938 - "Mechanism of hatching in Fundulua heteroclitus. " Biol. Bull. 72:511*. Aronaon, L. R., 191+5 - "Influence of the stimuli provided by the male cichlid fish, Tulapla macrocephala, on the spawning frequency of the female." Physiol. Zool. 18:U05. Bailey, R. J., 1953 " "The ovarian cycle in the viviparous teleost, Xiphophorous helleri." Biol. Bull. U:206. Baker, J. A. & M. S. Ferguson, 19i^2 - "Growth of platyfish free from bacteria and other microorganisms." Proc. Soc. Exp. Biol. & Med. 51:ll6. Baker, J. A. & W. K. Hagan, 19'4-2 - "Tuberculosis of the Mexican platyfish ( Platypoecilus maculatus)." Jour. Infectious Diseases. 70:21+8. Birnie, J. H. , I95U - "Regeneration of the tail fins of Pundulus embryos." Biol. Bull. 66:516. Bondie, von Cecil, 191+5 - "Stages in the development of the picked or spiny dogfish. Squalls acanthia." Biol. Bull. 88:220. Bonnet, D. D. I959 - "Mortality of the Cod egg in relation to temperature." Biol. Bull. 76:1+28. Brinley, F. J. & G. B. Jenkins, 1959 " "Studies on the antagonism between implant and host in fish embryos." Physiol. Zool. 12:31. Burger, J. W., 1939 - "Some experiments on the relation of the external environment to the spermatogenetic cycle of Fundulus heteroclitus." Biol. Bull. 77:9^ (see ibid. 80:51). Child, C. M., I9I+5 - "A further study of oxidation- reduction pattern In teleost develop- ment." Jour. Exp. Zool. 100:577. Daniel, R. J., I9I+7 - "Distribution of glycogen in the developing salmon (Salmo salar)." Jour. Exp. Biol. 2l+:125. Devillers, C, I9I+7 - "Explantations 'in vitro' de blastoderms de poissons (Salmo esox)." Experienta. 5:71. Dildine, G. C, I956 - "The effect of light and temperature on gonads of Lebistes." Anat. Rec. 67:suppl. 6I. Doudoroff, P., I9I+5 - "The resistance and acclimatization of marine fishes to temperature changes. II. Experiments with Fxindulus and Atherinops." Biol. Bull. 88:19!+. Eakln, R. M., 1959 - "Regional determination in the development of the trout." Arch. f. Ent. mech. 159:271+. Evana, G. , 1956 - "The relation between vitamins and the growth and survival of goldfishes in homotypically conditioned water." Jour. Exp. Zool. 7l+:l+l+9- Goodrich, H. B., 1927 - "A study of the development of Mendelian characters in Oryzias latipes." Jour. Exp. Zool. I+9. Goodrich, H. B. , R. L. Hine, & H. M. Lesher, I9I+7 - "The Interaction of genes in Lebistea reticulatua." Genetics. 52:555. Gordon, M., I9I+5 - "Feeding Platyfishes and Swordtails." Aquarium. 12:86. Gordon, M., I9I+5 - "Genetic studies of speciation in the Swordtail-Platyf lah group and of the experimentally produced hybrida." Trana. N. Y. Acad. Sci. 5:65. Gordon, M., I9I48 - "The Biology of Melanomaa." N. Y. Acad. Sci. Publ. Vol. h, p. 2l6. Grobatein, C, I9U7 - "Decline in regenerative capacity of the Platypoecilus maculatus gonopodiim during its morphogenesis." Jour. Morph. 80:ll+5 (see also I9I+7 Jour. Exp. Zool. 106). Guyenot, E. & W. Plattner, I959 - "EScherchea aur la Veaaie natatoire dea Poissons. II. Response a dea critique et valeur dee documents radiographiques." Rev. Suisse de Zool. U6:525. Easier, A. D. & W. M. Faber, I9I+I - "A tagging method for small fish." Copeia. 5:l62. EXPERIMENTAL FISH EMBRYOLOGY krj Hayes, F. E., D. A. Darcy, & C. M. Sullivan, 19^6 - "Changea in the inorganic constituents of developing salmon eggs." Jour. Biol. Qiem. 163:621. Hinrichs, M. A., 1938 - "The microscopic anatony of twins and douhle monsters in Fundulus heteroclitus." Physiol. Zool. 11:155. Hoadley, L., I928 - "On the localization of developmental potencies in the embryo of Fundulus heteroclitus." Jour. Exp, Zool. 52:?. Hooker, D., I932 - "Spinal cord regeneration in the young rainhow fish, Lehistes res- ticulatue." Jour. Comp. Neur. 56:277. Hoover, E. E. & H. E. Huhbard, 1937 - "Modification of the sexual cycle in trout hy con- trol of light." Copeia, k:206. Hooper, A. F., I'^k^ - "The early embryology of Platypoecilus maculatus." Copeia. 14^:218. Hubbs, C. L., I9I+O - "Speciation in fishes." Am. Nat. 7l+:198. Hyman, L., I92I - "The metabolic gradients of vertebrate embryos. I. Teleost embryos." Biol. Bull. 40:52, Ikeda, Y., 1937 - "Potassium accumulation in the egg of Oryzlas latlpes." Jour. Fac. Scl. Tokyo Imp. Univ. i*:313 (see ibid. U:307). Jones, H. W., 1939 - "Analysis of the development of fish embryos by means of the mitotic index. V. The processes of early differentiation of organs of Fundulus heteroclitus." Trans. Am. Mlcroscop. Soc. 58. Langrebe, F. W., I9I+I - "The role of the pituitary and the thyroid in the development of Teleosts." Jour. Exp. Biol. l8:l62. Lewis, W. H., 19^5 - "The role of the superficial gel layer in gastrulatlon of the zebra fish egg." Anat. Eec. 85:38. Lewis, W. H. & E. C. Eoosen-Bunge, I9U3 - "The formation of the blastodisc In the egg of the zebra fish, Brachydania rerio." Anat. Eec. 85:38. LI, M. H. & F. M. Baldwin, 19kk - "Testltcular tumors in the teleost receiving sesame oil-" Proc. Soc. Exp. Biol. & Med. 57:l65, Luther, M., 1957 - "Transplantations- und Defektversuche eun Organisations zentrum der Forellenkeimscheibe." Arch. f. Ent. mech. 157:1+0U. Lynn, W. G., 19^46 - "Situs inversus vlscerum in conjoined twins if the brook trout." Jour. Morph. 79:1. Marza, V. D. & E. E. Marza & M. J. Guthrie, 1957 - "Histochemistry of the ovary of Fundu- lus with special reference to the differentiating oocytes." Biol. Bull. 73:67. Mathews, S. A., I9U0 - "The effects of implanting adult hypophysis into sexually immature Fundulus." Biol. Bull. 79:207 (see ibid, 77:92). McEwen, E. S., I9U0 - "The early development of the swim bladder and certain adjacent parts In Hemlchromus blmaculata." Jour. Morph. 67:1. Moore, W. G., I9I+I - "Studies on the feeding habits of fishes." Ecology. 22:91. Nabrit, S. M. , I929 - "The role of the fin rays in the regeneration of the tall fins of fishes (Fundulus and Goldfish) ." Biol. Bull. 56:255. Nicholas, J. S., 1927 - "Application of experimental methods to the study of developing Fundulus embryos." Proc. Nat. Acad. Scl. 13:695. Nicholas, J. S. & J. M. Oppenhelmer, 19'+2 - "Eegulation and reconstitution in Fundulus." Jour. Exp. Zool. 90:127. Nigreli, E. F., I9U3 - "Causes of disease and death in fishes in captivity." Zoologica, 28:203. Noble, G. K., K. F. Kumpf, 8c V. N. Billings, I938 - "The induction of brooding in the Jewel fish." Endocrin. 23:353. Oppenhelmer, J. M., I936 - "Structures developed in anjihibians by implantation of living fish organizer." Proc. Soc. Exp. Biol. & Med. ^k-.kSl. Oppenhelmer, J. M., 1957 - "The normal stages of Fundulus heteroclitus." Anat. Eec. 68:1. Oppenhelmer, J. M., 19^+7 - "Orgajiizatlon of the teleost blastoderm." Quart. Eev. Biol. 22:105 (see this paper for her numerous other references). Parker, G. H. , 19'<-0 - "Neurohumors as chromatophore activators." Proc. Am. Acad. Arts & Scl. 73:165. Parker, J. B. , 19'+2 - "Some observations on the reproductive system of the yellow perch." Copeia. U:223. Paateels, J,. I936 - "Etudes sur la gastrrilation des vertebre's meroblastlques. " Arch, de Biol. 47:205. Pelluet, D. , V^kk - "Criteria for the recognition of developmental stages In the salmon (Salmo salar)." Jour. Morph. 7'+:395. Perelra, J. Jr. & D. M. Cardoso, 193'* - "^pophyse et ovulation chez les polssons." Comp. rendu. Soc. Biol. 116:1133- U18 EXPERIMENTAL FISH EMBRYOLOGY Phllipa, F. S., 19'>0 - "Oxygen consumption and its inhibition In the development of Fundi;ilu3 and various pelagic fish eggs." Blol. Bull. 78:256. Price, J. W., 19^5 - "A device for ohserving living fish embryos at controlled tempera- tursj." Ohio Jour. Sci. 1*5:83. Eegnler, Marie-Theresa, 1958 - "Contribution a 1' Etude de la Sexuallte des Qyprinodontes Vlvlparea (Xiphophorus hellerl, Lebistes retlculatus) ." Bull. Biol, de la France at de la Belglque. 72:585. Richards, A., 1935 - "Analysis of early development of fish embryos by means of the mito- tic index. I. The use of the mitotic index." Am. Jour. Anat. 56:355. Boblnson, E. J., & B. Hugh, 19't-5 - "The reproductive processes of the fish, Oryzias latlpes." Biol. Bull. 81+:115. Roosen-Runge, E. C, 1959 - "Karyokineee during cleavage of the zebra fish, Brachydania rerlo." Biol. Ball. 77:79 (see ibid, 75:119). Rusaell, A., I959 - "Pigment inheritance in the Fundulus-Scomber hybrid." Biol. Bull. 77:1+25. Scrimshaw, N. 3., 191+5 - "Embryonic development in Poeclllld fishes." Biol. Bull. 88:255. Smith, D. C. & G, M. Everett, igl*-? - "The effect of thyroid hormone on growth rate, time of sexual differentiation and oxygen consumption in the fish Lebistes retlculatus." Jour. Exp. Zool. 9U:229. Solberg, A. N., 1958 - "The susceptibility of the germ cells of Oryzias latlpes to x-radiation and recovery after treatment." Jour. Exp. Zool. 78:1+17. Solberg, A. N., I958 - "The developmsnt of a bony fish." Progressive Fish Culture #1+0. Spek, J., 1953 - "Die bipolare Differenzierung des Protoplasmus des Teleostei Eies und Ihre Enstelung." Protoplasma. l8:52. Stockard, C. R., I92I - "Developmental rate and structure expression; an experimental atudy of twins, double monsters, and single defonnltles, and the interaction among embryonic organs diiring their origin and development." Am. Jour. Anat. 28:115. Stone, L. S. & P. Sapir, I9I+O - "Experimental studies on the regeneration of the lens in the eye of anurans, urodeles, and fishes." Jour. Exp. Zool. 35:71. Tavolga, W. 8e E. Hugh, I9I+7 - "Development of the Platyfish, Platypoecilus maculatus." Zoologica. 32:1. Tchou-Su, M. & C. H. Chen, I956 - "Eecherches aur I'actlvabllite de la fecondablllte de I'oeuf du poisson osseux caraccius auratus." Chinese Jour. Exp. Biol. 1. TeVinkel, L., I9I+5 - "Observations on later phases of embryonic development in Squalus aoanthiae." Jour. Morph. 73:117. Tung, T. C, C. Y. Chang, & Y. F. Y. Tung, I9I+5 - "Experiments on the developmental potencies of blastoderms and fragments of teleostan eggs separated latltudinally." Proc . Zool . Soc . London, 115 : 175 • Turner, C. L. , 19^+7 - "The rate of morphogenesis and regeneration of the gonopodlum in normal and castrated males of Gambusia afflnis." Jour. Exp. Zool. 106:125 (see IQl+O, Jour. iMorph. 67:271). Vivien, J., 1939 - "Rose de I'hypophyse dans le determlame du cycle genital femelle d'un Teleosteen, Gabius paganelles." Comp. rendu. Acad. Sci. 208:9l+8. Waterman, A. J., I9I+0 - "Effect of colchicine on the development of the fish embryo, Oryziaa latlpes." Biol. Bull. 78:29. Wlnge, 0. & E. Detlersen, I9I+8 - "Colour Inheritance and sex determination in Lebistes retlculatus." Compt. rendu. Labor. Carlsberg, Ser. Physiol. 2l+:227. Wolf, L. E., 1931 - "The history of the germ cells in the viviparous teleost, Platypoecilus maculatus." Jour. Morph. & Physiol. 52:115. Yamamoto, T., I9I+0 - "The change in the volume of the fish egg at fertilization." Proc. Imp. Acad. Tokyo Univ. l6:l+82. "Aquiculture is as susceptible to scientific treatment us agriculture: ond the fisherman, who has been in the past too much tlie hunter, if not the devastat ing raider, must be- come m the future the settled farmer of the sea, if his harvest is to be less pr n car ious . H. A. Ilerdman EXPERIMENTAL CHICK EMBRYOLOGY No attempt will be made here to describe the most delicate of the transplantation operations (e.g., those by Willier and by Hamburger and their students) that have been performed on the chick embryo. The student is, however, directed to the work of these investigators, particularly to inter-specific transplantations. Hecent work of Spratt (l9'+7, 19'+8) has demonstrated that the extreme precautions of a dust-free operating room, ultra-violet lighting, masking, etc., are not necessary so long as reasonable precautions are taken to avoid the actual introduction of bacteria in- to the hen's egg. It is therefore safe to predict that the chick embryo will be used in- creasingly in courses in Experimental Embryology, and that it will supplement the work on the amphibia admirably well. The hen's egg is available during the months when amphibian material is scarce. Aside from the general procedures for handling the hen's egg in the laboratory, the experimental procedures to be described will include (1) Detenaination of morphogeneti c movements by means of vital dyes and charcoal particles, (2) Explantations or the cultur- Ing of Isolates on artificial media (5) Chorio-allantoic grafting and (ij-) Transplantations. This exercise has the single experimental object, the hen's egg. It involves four different procedures and in this respect this exercise cannot be weighed with the others of this Manual. It Is recommended that the material on the hen's egg be assigned to the second half of the second (Spring) semester, when the amphibian material is no longer available and after the student has had the benefit of some months of experimental and operational experience with amphibian material. THE PROCURING AND CARE OF LIVING MATERIAL Procuring of eggs: One must establish a reliable source of highly fertile eggs, which prove to be perfectly normal in development. There are the usual seasonal varia- tions in fertility with the low point during late Summer and Pkll, and the high point Just after the peak of the winter. With optimum conditions fertility may reach as high as 90^ but the low point may go to 2jja or even less. There is no evidence that any particular breed lends Itself better than others to operational procedures. However, certain flocks of hens give more viable eggs, particiilarly when they are provided with adequate sunlight Storing of eggs: If possible, eggs should be used shortly after being layed. How- ever, eggs may be stored at cool ten^eratures (10°C.) for as long as a week. The percen- tage of development will drop rapidly thereafter. Incubators : The small (Oakes) incubators will hold several dozen eggs and are quite satisfactory although they are not provided with as sensitive temperature and humidity controls, and forced-air draft for ventilation, as are other and larger models. The large (Buffalo) incubators are excellent for the incubation of larger numbers of eggs, and the physical factors are well controlled. Place the Incubator away from drafts and sunlight. Temperature control: Most incubators are provided with temperature control devices regulated close to the optimum range of 105° - 105°F. (about 57.5°C.). If the Incubator is not provided with forced draft, the temperature should be regulated at about 100°F. Since the heated air tends to rise, the hanging thermometer should have its mercury bulb at the level of the eggs. Each inch above the eggs the temperature may register at least a degree higher than at the egg level. Once controlled incubation has begun it should not be interrupted if normal development is desired. Bimidity control: A relative humidity of 60^ is best, although higher humidity, is not deleterious. In the smaller Incubators several finger bowls of water should be placed among the eggs. In the larger incubators there are generally large pans beneath the eggs which pans should be filled with sand and kept constantly moist. Dehydration is one of the commonest contributors to lethality. -4-19- 1+20 EXPERIMENTAL CHICK EMBRYOLOGY Botatlon of the esffa : The hen generally turns the eggs frequently. This is not necessary for the first day or two hut thereafter all eggs should be turned at least twice daily in order to prevent adhesion of the membranes, dotation of operated eggs will, of Course, be limited. The candling of egga : Incubation time is not an accurate criterion of ontogenetic age so that It is necessary to provide a device for sending light through the blastoderm so that its age can be approximated by direct observation. The mail order houses offer inexpensive candling equipment but it is very simple to make one. Fasten a light socket to a board; place around the socket a large tin funnel cut to fit, which acts as a reflec- tor; invert over the 100 watt bulb a small waste basket through the bottom of which is cut a circular hole; fasten beneath the hole a piece of coarse wire gauze, somewhat depressed, to cup an egg; cover the hole (outside of basket) with a heavy felt cloth (to cut out extraneous light) and cut a slit in its center slightly shorter than the length of the average egg. A switch may be provided for the light, fastened to the base board. The entire cost should be about 50 cents. The egg is placed over the slit in the black cloth, and will be caught by the wire screen, and a strong light will come up through the egg so that the blastoderm can be seen directly. It must be remembered that the 100 watt bulb gives off a great deal of heat so that the examination should not be extended for long. A dark room for the candling is best. The blastoderm can be seen by hS hours and the yolk-sac circulation by 60-72 hours. Thereafter, movements of the embryo and the interlacing extra-embryonic circulation be- come increasingly apparent. Embryos which die early generally show coagulation of blood in the sinus termlnalia (blood ring). Prom the seventh to the thirteenth days the chorio- allantoic circulation can be seen, but after the thirteenth day the embryos appear more and more opaque, accentuating the air space at the blunt end. Embryos which die during the latter half of Incubation show an indistinct air space demarcation. There are two peaks in the mortality curve of Incubated hen's eggs, one about the third or fourth day and the other Just before hatching, when the extra- embryonic membranes are drying up. Under the most ideal conditions even the best eggs show 5^ mortality on the fourth day, 15^ on the 19th day, and Tf> mortality during the balance of the incubation period of twenty-one days (Romanoff, 1951)' TECHNICAL AND OPERATING EQUIPMENT REQUIRED Provided by the Institution: Temperature-controlled incubator(3) with turning and hatching trays. Autoclave Dry sterilizing oven Heating plates or electrically- controlled stage warmers for warming eggs or embryos during operations. Candling equipment 100 cc. beakers with (soft) paraffin and small (paint-type) brushes Absorbent cotton Covered containers for discarded eggs Spencer microscope lamp, condenser and diaphragm. Dissecting microscope Round bottom (250 cc.) flask of water supported by double pinch-clamp, to ab- sorb heat from light source. Solutions: Glass distilled water Locke's (1907 Jour. Physiol. 56:208) solution NaCl - 0.9 gr. NaHCOj - 0.02 gr. KCl - O.OU gr. Diet, water - 100 cc. CaCU - O.O2U gr. (anhydrous) (Use at 58° - i+0°C.) Physiological saline (0.9^ NaCl) Phenol red (of known concentration) MS 222, made up l/5,000 in Locke's of physiological saline (anesthetic). Fixatives: Bouin, Klinenberg's Pi cro- sulphuric, Aceto-formalln. EXPERIMENTAL CHICK EMBRYOLOGY ^^21 Provided ty the Student: 12 petri dishes {h" diameter) 12 watch crystals (2" diameter) 2 regulation finger howla 2 Erlenmeyer flasks (500 cc.) 2 Erlenmeyer flasks (125 cc.) 1 graduated cylinder (100 cc.) 1 graduated cylinder (10 cc. ) 2 wide-mouthed pipettes, smooth edges ( inside diameter h mm. ) 2 fine pipettes (inside diameter 1 mm.) 12 regular medicine droppers 6 shell depression slides ^ oz. round cover glasses, #1 thickness (to cover depression) 1 small glass tumbler, cotton in hottom and partially filled with ^0'f> alcohol for sterilization of operating Instruments. Absorbent cotton Operating equipment: 1 hack-saw with extra blades (ampoule saw may be satisfactory) 2 watchmaker's forceps, #5 1 pair regulation forceps 2 steel needles, ground to fine points 1 pair coarse scissors (large) 1 pair fine scissors (small) PREPARATION OF EQUIPMENT All glassware and instruments should be thoroughly washed in non-caustic soapy water; rinsed in hot, running tap water; and put aside to dry on a deem cloth towel. The metal Instruments and culture dishes may be further sterilized in the dry sterilizing oven while the solutions should be autoclaved at 15 pounds for at least 15 minutes. Thereafter the operating equipment may be kept In 70^ alcohol to which a drop of iodine solution has been added. The greatest source of bacterial or mold infection is not from such pre-sterillzed equipment but rather from the hands and breath of the operator. One should work in a draft-free room, away from windows, and the embryos should be exposed to the air only when absolutely necessary. PRELIMINARY SUGGESTIONS 1. Become re-acqualnted with the normal morphogenesis of the chick egg. 2. Select eggs that are uniform in size, shape, and color for any one experiment. 5. Check the incubator temperature and humidity at least once each day. Temperatures above 105 °F. are more deleterious than below that level. k, Bemember that genetic factored may contribute to high mortality. It cannot be stressed too frequently that one must become thoroughly acquainted with the source of the eggs being used. 5- Do not wash or submerge the eggs for long In water because the embryo normally breathes air through its porous shell. Washing will remove a thin surface cuticle which protects the chick embryo against the Invasion of microorganisms. Prior to operations within the egg, sterilization of the shell and exposed shell membrane may be accomplished with a cotton swab soaked in 70^ alcohol, 1% iodine in alcohol, or chlorazene. The chlorazene is made up by adding 5 tablets to a quart of warm water. 6. The membranes should be observed Just prior to the time for hatching. When the extra-embryonic circulation begins to regress, the incubation temperature can be lowered and the humidity raised. Should the chick's beak happen to lie beneath the artificial window, this window should be removed on the 19th or 20th day. Frequently operated chicks must be assisted In the hatching process on ttoe 21st day. Do not attempt to remove the chick until the yolk sac is completely re- tracted into the chick mid-gut. U22 EXPERIMENTAL CHICK EMBRYOLOGY THE REMOVAL OF CHICK BLASTODERMS OR EMBRYOS Early Blastodenna: The chick ■blastoderm always floats around to the upper surface of the heavier yolk mass. Hold the egg in the palm of the hand for a minute or two and then crack the underside of the shell on the edge of a finger bowl 2/5 full of Locke's or saline solution, and (in the manner of cracking an egg for frying) allow the egg contents to flow out into the solution. The blastoderm will shortly move around to the most dorsal posi- tion. Grasp the chalaza or the yolk on one side with forceps held in the left hand (for right handed operators), then, with sharp-pointed scissors, quickly cut around the blas- toderm ^" from its border. This movement of the scissors can be aided by a contrary move- ment of the forceps, turning the entire egg mass with the forceps as the blastoderm is be- ing cut with the scissors. The cut Is made through the very thin and transparent vitel- line membrane, through the blastoderm proper, and into the underlying yolk. Grasp the cut edge (vitelline membrane, blastoderm, and yolk) and roll it back from the bulk of the yolk mass. Draw it away from the yolk mass. Holding the edge of the • vitelline membrane with forceps work a dissecting needle around its border, and use the needle to roll the blastoderm away from the membrane. Then, with a wide-mouthed pipette, remove the blastoderm to a Petri dish containing 20 cc. of Locke's or Saline solution. All of the yolky opaque area may now be trimmed off with scalpel or dissecting needles, beneath a dissecting microscope. The young blastoderm is extremely yolk-adherent. Some investigators inject warm Locke's solution beneath the blastoderm before excising it. This raises the blastoderm off of the yolk by providing an elevating vesicle of fluid beneath, separating it from the yolk. Other investigators find it simpler to gently auck the blastoderm (after re- ■ — SOMITE O —ENTODERM SR-SINUS RM0M80IDALIS' # —NEURAL CREST LP-LATERAL PLATE M —HEAD MESODERM ^-MEART ®- CMOftDA O-NEPHfiOS • - ERYTHROCYTES Prospe superficial rl^ht lialf. the boundar assumptions Itmlted to accurate ex Tills c based large work of Hiiii (ectodermal ctlve areas layer Is s}i The Interr y between th that Invagl material des perlmental v omposlte map ly on work o t (endoderm placodes) . In tlie definitive primitive streak blastoderm of the clilck. The own at the left while the invaglnated material is seen In the upted line on the left side of the anterior streak region marks e ectoderm and the still unlnvaglnated mesoderm. This Is on the nation Is as yet Incomplete and that future Invagination will be tlned to form the embryo proper. All mesodermal boundaries need erlflcatlon. Is drawn by Rudnlck (l'J44 Quart. Kev. md. 19:187) and Is f Pasteels. In addition, there have been contributions from the cells), Wolff (morpliogenesls of trunk and tall), and Ynteraa EXPERIMENTAL CHICK EMBRYOLOGY ^^25 moval of the vitelline memtrane ) in and out of a wide-mouthed pipette, thereby removing most of the adherent yolk. Still others play a gentle stream of medium on the surface of the inverted blastoderm, blowing the yoli: granules away. The early stages are particularly fragile and the less handling the better. In most instances the peripheral yolk may be trimmed away with scalpel, scissors, or even with needles, and the yolk adherent to the underside of the area pellucida is negligible. It la particularly difficult to handle the blastoderms of less than l8 hours without damage. Later Embryos: Chick embryos of 56 or more hours of incubation are rather simple to remove from their eggs. The entire egg mass is broken Into a finger bowl of Ballne solu- tion cracking the underside of the shell after allowing the egg to remain motionless for several minutes. The embryo, unless it Is so far advanced that it is heavy, will float to the upper surface. Excise the embryo as In the manner described above or. If the embryo Is well formed, grasp the yolk-sac umbilicus with forceps, cut It distally to the forceps, rupture other membranes, and draw the embryo away from its yolk. If the embryo Is young it may be pulled into a submerged watchglass and transferred to a fresh finger bowl of solution. If It is advanced. It may be transferred In an ordinary teaspoon whose bowl has been perforated with many small holes. EXAMINATION OF EXCISED CHICK BLASTODERMS The early blastoderms may be transferred to a watchglass In several drops of Locke's or saline medium (wanned to 58°C.), oriented with needles into the proper position and then the medium sucked off by means of a fine bore pipette, while encircling the blasto- derm. In this way the blastoderm will be flattened onto -the dry bottom of the watchglass. Immediately add fresh medlxim (at 58°C.) so that it flows beneath the blastoderm, lifting it up on the surface tension of the fluid. If 0.001^ phenol red (pH indicator) is added to the medium, the slightly purple tinge of the alkaline medium will provide an excellent background for greater clarity of the chick embryo structures, wltho^it adding any toxic factor. Such a watchglass may be placed on a wanning stage or on an electrically control- led heating stage and the living embryo examined for a considerable period of time. If one is interested In general morphology of these early stages it is advisable to mount embryos of the same age In the normal position, and also upside down, so that, in the latter Instance, one has a view directly into the intestinal portals. Such a mount may be made on a glass slide providing a out-out in filter paper Is made of just such size as to frame the area pellucida and mask out the area opaca. If such a blastoderm Is first Inverted, pulled up onto the slide (while the slide is submerged In solution), most of the medium drained off, and the filter paper frame added, the embryo will remain flat. The yolky margins of the blastoderm will adhere firmly to the filter paper. A cover glass may be added providing Its comers are elevated slightly by bits of Permoplast, to prevent crushing. The embryo may now be hydrated with the appropriate medium. It must be remembered, however, that the chick embryo cannot acquire sufficient oxygen from any aqueous medium directly, and that it can be "drowned", particularly if It has already developed its own circulatory system. The ideal environment is a closed space, completely humidified, with the blastoderm floating on the surface of a nutrient medium. The later stages may be examined in the manner of any vertebrate form. After about 5 days the embryo will take on a definite avian appearance, and It will shortly become possible to make a dissection to study the internal organs. This is so because the car- tilage and bone will not have developed. There is very little cartilage, even at 8 days. PERMANENT PREPARATIONS OF CHICK EMBRYOS The primitive streak stages can be best fixed while still on the egg, by dropping the fixative onto the blastoderm gently from above. Since fixation renders the blastodenn rather brittle, it is best to cut it out within a few minutes after fixation, and then transfer it to a Syracuse dish with fresh fixative for the requisite time. (Use instru- ments other than those for operating purposes.) ^+2^+ EXPERIMENTAL CHICK EMBRYOLOGY later blaatoderm and embryos may be excised in the manner described on preceding page and fixed either in Syracuse dishes or on slides (when they are held in place by the fil- ter paper rings). Within a minute of fixation gently wash the entire flat blastoderm off of the slide and into adecjuate (similar) fixative by a gentle stream of fixative from a pipette. Some of the yolk will remain adherent to the slide, and if the blastoderm is allowed to remain long on the slide, it is apt to be torn during subsequent removal. Still later stages (up to 8 or 9 days) may be fixed "in toto" in any of the standard fixatives. If it does not interfere with structures important in the examination, it is always well to slit the abdomen to allow fixative to penetrate to the viscera the more readily. HISTOLOGICAL PBOCEDUKES: Fixatives: Klelnenberg' s Picro-sulphuric, Bouln, Michealis' fluid, or 0-5^ acetic acid in 10^ formalin. (Given in order of preference.) Fixation should be for at least h hours for the earlier stages to U8 hours for the 9 day embryos. Decoloration: The picric acid of the fixatives leaves the embryo stained a brilliant yellow. This may be removed with lithium carbonate but more quickly and satisfactorily by adding about 5^ by volume of NBi4.0H to the 70^ alcohol during dehydration. The alkaline ammonia decolorizes the yellow picric acid. Bleaching of older stages may take 2h hours and several changes. Dehydration: Dehydration must be slow in order to avoid damage to the delicate blastoderms and to insure complete dehydration of the larger, later embryos. One hour periods for the early stages and as much as 12 hour periods for the older embryos, in each of the graded alcohols, is generally indicated. The alcohols should include 55^, 70^, 80^, 90^, 95^ and finally 100^. If the yellow picric is not entirely removed, a small amount of LlCOi may be added to each of the alcohols from 70^ to 90^- Make two changes in absolute alcohol. Clearing: This is beat accomplished by transferring the embryo from absolute alcohol to pure cedar oil for 2k hours or more (i.e., until translucent). Then transfer to xylol for JO minutes. Embedding: Embed in 56°C. paraffin to which has been added y^ Bayberiy Wax and 5^ Beeswax (measurements by weight). A total of 1 hour for the earliest stages to as much as 5 hours for the 9 day chick embryo la Indicated. For the large embryos with some cartilage a final embedding in a paraffin- rubber mixture Is suggested (5O-60 minutes). Sectioning: For cytological studies the sections should be no more than 10 microns in thickness. For study of organs the sections may be as thick as 20 microns. Boil some distilled water, cool, and to every 10 cc. add 1 drop of egg albumen, mix thoroughly. Place a few drops of this albumen-water on the slide, and float the ribbon on It. When the ribbon is properly oriented, draw off the excess fluid with pipette and filter paper, and dry on a warming plate at it-0°C. Staining: • 1. Sectioned material: a. Heidenhaln's Iron Haematoxylln: Excellent for chromosome studies. Mordant the sections for 12 hours in kio iron alum, rinse, stain for 6 or more hours in Heidenhaln's Haematoxylln, then destaln in 2% iron alum while observing it under the dissection microscope. Stop the destalnlng when the section has a uniform grayish appear- ance, by placing It in slowly rxinnlng tap water. EXPERIMENTAL CHICK EMBRYOLOGY U25 b. Conklln's Haematoxylin: Add 1 drop of ELinenterg' 3 Picro Sul- phuric to each 1 cc. of seasoned Delafleld's Haematoxylin. Stain sections for 6 to 10 minutes, rinse in (alkaline) tap water. Counterstain with 1 dip in 0.5^ eoaln in 951^ alcohol (counterstain not recommended for photographs ) . c. Harris' Haematoxylin: Stain for 6 minutes, blue in alkaline tap water, and counterstain if desired. 2. Whole mounts: These may he stained for from 6 hours to 2 days in either of the (above) Haematoxylins, depending upon the size and stage of the embryo. Large embryos (72 hours and older) may thus be stained in toto and later sectioned. If the stain does not penetrate adequately,, the sections may be further stained after mounting. LUJTOVALL TECHNIQUE FOB STAINIIC OF CHICK EMBKfO CABTILAGE: This technique may be used either on a chorio-allantoic graft which has developed cartilage (and bone) or it may be used with the whole embryo of 9 to 10 days Incubation age. The Spalteholz technique may be used even for later stages for complete trans- parencies. 1. Fix in Bouin's or ELlnenberg's Picro- sulphuric for 2k hours. 2. Transfer to 70^ alcohol containing 2^ NHi^OH to decolorize. Several changes over a period of several hours may be necessary. 5. Using forceps, remove skin, feathers, and all fatty tissue. k. Stain for 2 to 5 days in 0.25^ methylene blue (or toluidlne blue) made up in 70^ alcohol to which is added 3% HCl by volume. This will overstaln. 5. Destaln in several changes of 70^ alcohol for about k8 hours. 6. Dehydrate lor k hours in 95^ alcohol. The softer tissues will become destalned and somewhat transparent. 7. Transfer the embryo to Methyl Salicylate (oil of wlntergreen) to which has been added 2'y^ (by volume) of benzyl benzoate. In this the embryo will clear conplete- ly and may be stored. (See Lundvall, 190*1: Anat. Anzeiger 25 and I906, Anat. Anzelger 27. ) MODIFIED SPALTEHOLZ' METHOD FOB STAINING SKELETAL ELEMENTS: The following procedure is excellent for post-metamorphic amphibia and for chick embryos beyond the 10th day of Incubation. 1. Fix In 95^ alcohol two weeks to harden. 2. Transfer to 1^ KOH for 2h hours. 5. Transfer to tap water and, with forceps, pick off as much fleshy material as possible. k. Transfer to 95^ alcohol, change once in 6 hour period. 5. Transfer to ether for 1 to 2 hours to dissolve away any fat, or use acetone if there is little or no fat. 6. Transfer to 95^ alcohol for 6 hours, change once. 7. Transfer to 1^ KOH for 6 days. 8. Put In Alizarin red "S" for 12 hours. 9. Transfer to 1^ KOH for 2k hours. 10. Put in Moll's solution for 2k hours. 11. Store In 100^ glycerine. METHODS FOR OBSERVING THE DEVELOPMENT OF THE CHICK EMBRYO The hen's egg is fertilized at the upper end of the oviduct so that by the time It is layed the embryo has reached the stage of gastrulation, at least. The blastoderm of such an egg shows no visible structures at this time but after 18 hours of incubation the primitive streak may be discerned. Candling will not generally indicate einy development before the 53 hour stage. It Is now possible to replace a portion of the shell with a cover-glass window through which development can be observed from day to day. A second method is to remove h26 ■ EXPERIMENTAL CHICK EMBRYOLOGY the ahell over the air space (at the blunt end of the egg) and to provide a removable (shell) cover, so that the embryo can be watched at stated intervals for developmental changes. These procedures have two uses: First, it is not possible to maintain an excised embryo on culture media and to have it develop perfectly normally for more than several days (and at early stages only). By these methods the embryo may be observed under per- fectly normal conditions and morphogenesis can be studied, at least until the embryo as a whole becomes opaque from the development of its organs. Such embryos can be carried through to hatching. Second, when grafts are added to the chorio-allantois, they can be observed through the window, at least for a number of days. THE WINDOW METHOD: Secure an egg of less than 55 hours of incubation (7 or 8 day embryos may be similar- ly treated when making chorio-allantoic grafts) and place it in a bed of cotton in a finger bowl. Orient the smaller or pointed end of the egg to the right, and tilt it slightly below the horizontal so that the blastoderm will float slightly toward the blunt (air-space) end. Leave the egg in this position for a few minutes. Holding the egg in this exact position, remove it (without Jarring) to the candler and locate the blastoderm. Mark its position with pencil on the ahell above. Eetum the egg, in exactly the same position, to the cotton bed. With forceps apply a cotton swab, soaked in 95^ alcohol plus 1^ iodine (or chlorazene solution), to the upper surface of the egg, including the area of the blastoderm. Wipe dry with sterile cotton. Arrange the microscope lamp so that its light shines at an angle onto the egg surface but have the light far enough away so that its heat is barely felt on the back of the hand. Check the temperature of the light at the egg surface by a ther- mometer. It should be leas than 105°F. Secure a previously sterilized moujating ring of non-toxic material such as glass, celluloid, pliofilm, pyralin, or thin rubber washers with a maximum diameter of not more than Ji/h of an inch. Place the ring on the shell over the region of the blastoderm and, with a sharp and sterile needle, scratch the shell along the inner margin of the ring. Replace the ring in 95^ alcohol in a covered Stender, and brush away any shell particles with sterile cotton. A portion of the shell within the demarked area is now to be removed. Eemember that all instruments used muat be sterile, the hands must be clean, and the operator should avoid breathing into or allowing any dust to blow Into the egg while opened. There are various tricks to removing the egg shell, gained largely through experience. Aids to the removal, however, are a dental drill with circular disc; ordinary hack-saw blade; ampoule saws; or merely a sharp scalpel. The ahell is to be sawed through, without damage to the underlying shell membrane. Bectangular, triangular, square or circular open- ings have been used. Probably the aimpleat procedure la to make a square opening by saw- ing through the shell by a alight rotary motion of the saw, following the outer curvature of the shell, on each of the four sides but working along parallel sides of the square for the first two cuts. Avoid cutting through the shell membrane. Some workers leave the fourth side as a sort of hinge, but it does not generally break straight so that cuts along all edges are advised. Saw gently, and only through the shell, brushing away the shell particles as they are dislodged. When the four sides are sawed through, make the comer breaks with a needle or scalpel, and gently grasp the square piece of shell and remove it, intact. Before invading the shell membrane aee that there are no shell fragments lying on it. Moisten the shell membrane with one drop of sterile Locke's solution. Be-aterilize in- struments And the margins of the shell opening, if it seems desirable. (The host egg is left at this stage in chorio-allantoic grafting to await the preparation of the graft.) With a sharp (sterile) needle puncture the center of the shell membrane, directing the point of the needle under the membrane and away from you, and at right angles to the egg axle. If the needle is sharp, it can be brought upward and thus be used as a knife to cut a slit-like opening in the shell membrane. With sharp (sterile) scissors, cut away the shell membrane to the margins of the shell opening. EXPERIMENTAL CHICK EMBRYOLOGY 1+27 Remove the mounting ring from the 95^ alcohol, let It air-dry hriefly, dip it into 1+5°C. melted paraffin, and place it on the egg so that -"t encircles the shell opening. With a small water-color paint "brush paint melted paraffin onto the outer margins of the mounting ring so that it is thoroi^hly sealed to the shell and no air can pass beneath. Do not allow any paraffin to get into the egg. Secure a circular coverslip, previously cleaned, and holding it with forceps pass it through a gas flame, exposing "both sui-faces. The coverslip should Just fit the mounting ring. While still warm, "bring the coverslip into position on the mounting ring and gently press it into place. The paraffin adherent to the mounting ring should melt and fasten the coverslip tightly to it. Paint a ring of paraffin on the outer edge of the coverslip, further sealing it to the egg shell. The entire operation consists simply of providing a sealed window in the place of a limited amount of egg shell. Eetum the egg to the incu- bator. In the same position, for the first 2k hours. Thereafter the egg may be rotated somewhat, but the embryo and its membranes should be kept away from the window. Two modifications of the above procedure have been practiced, "but neither is neces- sary. One is to puncture the air space so that it will be deflated and the embryo will be further depressed away from the upper shell membrane. If this is done, simply cover the puncture with paraffin or scotch tape. The second modification consists of adding egg-albumen from a second egg, to fill up the space between the embryo and the glass win- dow. While albumen is bacteriolytic, this practice Is ill advised, because it tends to add to the infection hazzard through handling, and it generally clouds up the window. Of course, a glass window is not the only useful type. Pliofilm, cellophane, and even Scotch tape have been used. If there is apt to be considerable delay between the preparation of the host, and the graft tissues, it i.s advisable to protect the embryo with a temporary Scotch tape covering of the shell opening. Square and round coversllps may be used without a mounting ring, but paraffin sealing is the more dlffj.cult. The embryo may be examined from time to time but it must be remembered that with each removal of the shell cap there is opportunity for bacterial infection and Increased evapo- ration. Embryos may be carried through to hatching. THE SHELL CAP METHOD: This is the method of Price and Fowler (I9I+0). It consists of using a shell from the blunt end of one egg to cover the exposed (blunt) end of an egg from which the shell has been removed. This cover may be removed at periodic Intervals, and the embryo observed through a much larger aperture than in the case of the cover glass window. There is, of course, added danger of Infection. Instead of the shell cap, a fitted glass cap, the edges of which are fastened to the shell, can be used and need not be removed to observe the chick development within. This type of shell cap has the one disadvantage of not allowing free transfer of respiratory gases. Save the blunt half of egg shells from any unincubated eggs should be left in position, and the shells may be sterilized in alcohol, to which a little iodine is added. The cut edges of the shell caps may be made smooth and less brittle by dipping them in melted paraf- fin. Candle a fertilized egg to mark the margin of the air space at the blunt end. Place the fertilized egg in a Syracuse dish with Permoplast base so that the blunt end is uppermost. Clean off the entire blunt end with iodized 95')^ alcohol, and wipe it dry with sterile cotton. With needle, lancet, or forceps make a small hole in the center of the blunt end and then gradually pick away the shell. The shell membrane EXTRA SHELL CAP SPACE AT ^-'^ ~~" BLUNT END, SHELL C»P / / , ' ' ' ' ^ :?< \ EXPOSED f^ / '/^ ^ r-EMBRTO -YOLK MASS ! \ 1 \ 1 r *->^ ^CHALSZA ALBUMEN 1^28 EXPERIMENTAL CHICK EMBRYOLOGY maJtlng an ever-increaslngly large circular hole. It will help to limit the shell cracking If a small hack saw is used to cut a shallow ring around the shell, Just within the limit of the air space. The shell should be picked away to within about ^ inch of the inner shell membrane and embryo. Pick away all of the outer shell membrane, but remember that this membrane extends completely around the embryo. Avoid ripping or tearing this tough membretne . Place a sterile shell cap over the exposed embryo, see that the egg is securely held in the Permoplast of the Syracuse dish, and return the egg to the incubator. The humidity of the incubator must be increased to above 6o^ for these eggs, since there is a much greater exposed surface for evaporation than in the other method. 'B^ the 17th day the egg shell may be sprinkled twice daily with a small amount of sterile water at the incubator temperature, but do not immerse the egg in water. The embryo can be drowned within the shell. MORPHOGENETIC MOVEMENTS AS DETERMINED BY VITAL STAINING AND CHARCOAL PARTICLES Before beginning the following study of morphogenetlc movements the student should re-acquaint himself with the various descriptions of the processes of normal development from the earliest primitive streak stage (about l6 hours of incubation) to at least U2 hours of Incubation when the heart- starts to beat. (See Lillie: "The Development of the Chick".) The pre-gastrulation stages are not available, since they occur within the ovi- duct and every fertilized egg is at least in the primitive streak stage when layed. A condensed survey la given below. The primitive streak, considered by many as homologous to the blastoporal lips of the amphibia, consists of a longitudinal thicken- ing of ectoderm extending through from almost the anterior liriit throi;igh about 2/5 of the length of the area pellucida. The primitive groove is probably formed as a result of mesodermal outgrowth, and beneath it all is a thin layer of endoderm lying on the yolk and attached to the streak only at the level of Hensen's node. The bulk of the embryonic tissues arise from material of this streak as it becomes telescoped posteriorly in favor of an anteriorly elongating embryo. The mar- gins of the area pellucida and the area opaca together fonn the extra-embryonic structures. PRIMITIVE STR£«K Presumptive areas of the chick primitive streak, modified from Wllller & Rawles 1935: Proc. Soc. Exp. Biol. & Med. 32:1293. At about 20 hours of incubation a head process appears anterior to the primitive streak, and this consists of the anterior limit of the notochord and overlying medullary plate ectoderm. There is definite cephallzation (precocious development of the anterior structures) but as the embryo lengthens the primitive streak shortens (with the recession of its anterior end, or Hensen's node). As the neural folds of the future brain region become approximated, the medullary plate is lengthened posteriorly and the somites begin to appear, formed out of mesenchyme which was derived (by migration) from the sides of the primitive streak. The first pair of somites appear at about 21 hours of incubation and will be located at a position Just posterior to the future otic vesicles. The first four somite pairs appear during the first day, all to be Incorporated in the head (occipital) musculature. The primitive streak is therefore not to be considered as part of the embryo proper, but rather as a remnant of the blastoporal lips out of which are derived the tis- sues of the embryo. Wetzel (1929) and Pasteels (1957) have mapped out the prospective organ-forming areas of the primitive streak stage of the chick embryo, in a manner similar to that used by Vogt (1926) eind others on the amphibia, by the xxae of vital dyes. In fact, these investi- gators have found that there is surfirlslng similarity in amphibian and avian morphogenetlc EXPERIMENTAL CHICK EMBRYOLOGY k29 movements, and that the three major movements of convergence, invagination, and elongation are found in both. The notochord and the floor of the neural tube arise from the material of Hensen's node, as shown by deep vital dye staining. The anterior end of the primitive streak (exclusive of Hansen's node) gives rise to the lateral walls and roof of the neural tube, and to the somites. The anterior part of the head arises from prenodal materials. Vital staining and charcoal marking are useful procedures in determining the prospective significance of the various areas within the whole, normal, developing embryo. (Isolation of these same areas onto culture media indicates that their prospective potencies are even greater. ) THE METHOD OF VITAL STAINING: Chick embryo areas are not as simple to stain with vital dyes as are the comparable areas of amphibian embryos. The fat globules and yolk often absorb some of the stain and then disintegrate. Beyond a certain concentration, the living cells of the chick embryo seem unable to tolerate the vital dyes. However, the staining must be fairly deep to be significant for the primitive streak embryo is tridermic. In all instances the vitally stained embryo must be removed to a watchglass, cleaned of all excess and adherent yolk, and examined with transmitted light within 2h to hQ hours of the staining. Procedure: 1. Prepare agar chips by soaking beaded agar in Nile Blue Sulphate (l/lO,000) and also in Neutral Red (l/l0,000) and drying on glass plates. The agar beads will take up the dye and, when dry, may be further cut down to any appropriate size under a dissection microscope. Another method is to prepare some 2$ agar in distilled water, add the dye (above concentration), pour the hot agar onto glass plates and, when cool and dry, peel off the agar in thin and narrow strips by means of a scalpel. These strips may then be cut to any sj,ze or shape. 2. Wash instruments in warm soap and water, rinse, air dry and then place them In 80^ alcohol for sterilization. The instruments include watchmaker's (#5) for- ceps, fine scissors, sharp scalpel, and hack saw blade. 5. Locate the blastoderm of an 18-20 hour incubated egg, outline It on the shell with pencil, and then place the egg (in the same position) in a finger bowl or #2 Stender on abundant cotton. Orient the egg with the blunt end to the left. U, Following the procedure described above, make a window in the shell measuring from 12 to 15 nm. in diameter. Do not injure the shell membrane. The window should be within the circumscribed area, in the uppermost part of the shell. 5. Place a drop of sterile Locke's (or saline) solution on the shell membrane, and, when moist, rapture the membrane and remove it to the margins of the shell opening. Avoid touching the underlying blastoderm and, if necessary, remove any excess albumen with sterile pipette. 6. With watchmaker's forceps place a small piece of blue (Nile Blue Sulphate) agar (or even a particle of Nile Blue Sulphate powder) on the central region of the blastoderm. The blastoderm, at this stage, is not readily visible but will be- come apparent with blue staining. Eemove the blue agar after a few minutes, i.e., after some of the dye has diffused into the embryo through the vitelline membrane. (If powdered dye is used, this will be more difficult to remove.) Do not overs tain. 7. Place a Neutral Red granule or red agar on the blastoderm in an anterior posi- tion, i.e., anterior to Hensen's node, for a few minutes. Remove with forceps. If this proves difficult, add a drop of sterile saline solution to float the granule upwards, and then remove with watchmaker's forceps. 8. Make a sketch of the blastoderm, any identifiable structures of the embryo, and the positions of the colored areas. If a temporary window is placed over the shell opening (coverslip) the sketch may be made during a brief candling. 9. With melted, and soft paraffin seal a circular coverglass onto the egg surface, over the window. This need not be made with the care of transplantations for the duration of the experiment is short. 1+50 EXPERIMENTAL CHICK EMBRYOLOGY 20 HOURS 22 HOURS ^"'i k"*^ 1 1 V ; 1 JX1 ^H ^^^V \ 1 J[. K^JIIf^ ^^^m' ImzshSjrS WHOLB CHICK EMBRYOS iS-33 HOUR STAGE EXPERIMENTAL CHICK EMBRYOLOGY 1^1 10. After 2k hours, crack the underside of the shell on the side of a finger "bowl ahout 2/3 full of Locke's (or saline) solution, cut out the entire blastoderm, transfer it with a wide-mouthed pipette to a watchglass of saline solution, and examine by reflected and transmitted light on a warming stage, to determine the changes in the size and positions of the colored areas. Variations in the procedure: 18-20 hour incubation stage. 1. Attempt to stain Hensen's Node specifically, and 2l4- hours later excise the blastoderm, split the embryo lengthwise through the neural tube, invert the blastoderm, and locate the stain. If the Node was stained, the notochord and ventral neural tube should be stained. 2. Stain the Primitive Streak posterior to Hensen's Node, and 2k hours later ex- cise and examine the blastoderm for the position of the stain. 3. Stain a spot directly anterior to Hensen's Node with Nile Blue Sulphate and a second spot to either side of the midline, at the same level as the Nile Blue, but use Neutral Red. In this way the movements of the Pre-Nodal areas may be determined 2k hours later. k. Stain the Primitive Streak stage with several spots in the two colors, excise quickly after 2k hours, and fix in an attempt to preserve the vital dye in posi- tion (see section on Morphogenetic Movements for specific directions, based on Detwiler's paper, for preserving vital dyes). THE METHOD OF CABBON- MARKING IN VITRO: (Spratt, I9U7, I9I48) Ejy this method the entire blastoderm of an 18 to 20 hour incubated chick embryo is excised, placed on albumen medium and c\iltivated in vitro for 2k or more hours (at 105°F, ) and the morphogenetic movements are determined by movement of adherent particles of blood charcoal. 1. To prepare the albumen medium separate the yolk from the albumen of one unincubated egg and add the albumen to 50 cc. of chick Binger's solu- , tlon (0.9^ NaCl, 0.0^2^ KCl, and 0.02'+^ CaCl2 made up in glass distilled water) contained in a 500 cc. Erlenmeyer flask. Stopper and shake the flask vigorously for 1 minute. 2. Excise the blastoderm from an I8 to 20 hour incubated egg and place it in chick Binger's Carbon-marking In vitro: at 103°?., in a watchglass, and remove all ex- chick Olastoderm Primi- oeas yolk. tlve Streak Stage. 5. With wide-mouthed pipette, remove the blastoderm in minimum of medium, and transfer it to the surface of the (above) albumen-Blnger' 3 in a watchglass at 103°F. (on a warming stage). With mlcropipette, remove all excess medium from the surface of the blastoderm so that it floats freely on the surface film of the culture medium. k. Place carbon (blood charcoal) particles on the Hensen's Node, directly in front of it, and at one or more levels of the Primitive Streak (see figures above). Indicate by parallel charcoal marks on the medium exactly where these particles are placed. These marks to be used as reference marks later. Watchmaker's forceps may be used for this marking. The forceps may be dipped into the char- coal, shaken of all excess particles, and then touched to the relatively dry upper surface of the blastoderm. There is sufficient moisture to provide a sticky surface so that the carbon particles will come off the forceps very readily, and will remain adherent to the blastoderm. • 5 . After 2k to k8 hours examine the blastoderm, cultured in vitro, for any change in the position of the carbon particles. It will, of course, be necessary (as always) to make sketches at the beginning and at the termination of the experi- ment. U52 EXPERIMENTAL CHICK EMBRYOLOGY EXPLANTING AND CULTIVATING EARLY CHICK EMBRYOS IN VITRO* Spratt (19't7, 19^) has shovm that the bacteriolytic property of egg albiimen used In hla various culture media makes it unnecesaary to include the elaborate sterilization pro- cedures used in the classical tissue culture methods. Tap water can be used in the place of distilled water when egg-albumen is included In the medium. When the egg-albumen is not in- cluded, the glassware and the instruments are dry sterilized and the solutions are autoclaved. rl'v''.'-' ■"■"."'■;... Preparation of the equipment: 1. Wash, rinse (in hot running water), and set aside the glassware and in- struments to dry on a clean towel. 2. Prepare moist-chamber culture dishes as follows: Place a moist cotton ring in a Petri dish, set a watch crystal with concave side up in the center of the petri dish, and replace the cover of the dish. All parts must have been previously sterilized. (Method of Fell and Eoblson, I929 and Waddington, 1952.) Preparation of the culture media: Physiologically balanced "Ringer's" for chick embryos: A. Unbuffered isotonic salt (Spratt, 1947) NaCl 0.9 grams KCl 0.01+2 grams CaClg O.O2I+ grams Doubly distilled water 100.0 cc. B. Buffered isotonic salt (Bomanoff, 191+5) , NaCl! 0.86^ KCl [ 0.051^ CaCl ! 0 . 02^ MgClg • 6^0. 0 . 01^ K^PO^ 0.02^ Na2KP01+'12B^0 0.08^ Glucose 0.2^ Made up in glass distilled water. ■■■';>■ -:-^ ■:.■■.-■ ■^|\ ■ ■ ■• ^ \ V '^ :£■?:. ; ^:- ■ Vv* %\%Vi^v i.Vv . . Mil ■■»■ . ..,••• ....■.; mr' va? Camera luci hour-old, 1 terlor port blastoderm 0.4 ram. pes result, was verbatim th sallne-agar tie practlc late many s symmetrical morphogenes da drawing o iving explan ion of a sho which was tr terlor to th obtained aft e method out albumen med e, the stude imilar cases and essentl is. f a typical 10- t of the an- rt head-process ansected about e node. This er carrying out lined for la. With a lit- nt can accumu- of beautifully ally normal From Spratt l^k^ Science. 106:U52 (Note: Romanoff found that this medium respiration of all embryonic tie change. ) could sustain a nearly constant rate of sues tested, without providing any pH 1, Ringer -albumen -Agar medium: This medium is made up from 2 components, as fol- lows. A. Ringer- albumen component: 1. Separate the yolk from the white (albumen) of a fresh, unincubated egg. 2. Add the albumen to 50 cc. of ordinary chick Ringer's in a 500 cc. Erlen- meyer flask, previously sterilized. 5. Stopper the flask and shake the contents vigorously for 1 minute. * The author is Indebted to Dr. N. T. Spratt, Jr. for these procedures, and for helpful suggestions in the organization of this exercise on the chick embryo. EXPERIMENTAL CHICK EMBRYOLOGY l4^ k. Add 0.5 ng- of phenol red. This will cause the medium to hecome slightly purple, when the alhumen is present, so that it will be the easier to see the white explaxits . B. Binder- Agar component: 1. Place from O.I5 to 0.15 grams of powdered (USP Xl) Agar in a small Erlen- meyer flask along with 50 cc. of chick Ringer's solution. The Agar may be increased to 0.2 or 0.**- grams, in which case a much firmer medium that is more easily handled, will result. 2. Bring the Blnger-Agar mixture to a slow boil over a small flame of the Bunsen burner (or in a water bath). Agitate constantly to prevent the Agar from sticking or charring. After the Agar Is completely dissolved, cool the mixture slowly down to kO° to 45°C. 5- Add to this Ringer-Agar 20 cc. of the El nger- Albumen mixture. Exclude the toamy portion. Gently shake to mix the two media. C. The medium: When the two components are mixed the medium Is completed. Po\ir ap- proximately 2 cc. of this mixture into each previously prepared, sterile, watch crystal which is supported In a cotton moat in a Petri dish. Cover the Petri dish ajid allow the medium to gel (about 50 minutes to 1 hour) be- fore moving the dishes. 2. Saline-agar "A": a. Make up 100 cc. of unbuffered chick "Ringer's" ("A" on preceding page). b. Add 0.25 grams of powdered Agar (USP XI). c. Sterilize by boiling (gently) or autoclaving. Avoid charring the Agar. d. Make up a stock solution of 1^ NaHCO^, sterilize by filtration (if possi- ble) through a Berkefeld filter, and then saturate with CO^. e. Cool the saline solution down to 4o°C. and add 1 to 2 cc. of the sterile bicarbonate solution. f . With sterile pipette place 2 cc. of the medium in culture dishes, where it will slowly set to form a soft gel. 5. Sallne-Agar "B" : (See White, 19if6) a. Add the following salts to 100 cc. of double distilled water. NaCl 0.85 grams KCl 0.057 grams Ca(N05)2'%0 0.021 grams MgSOij^ 0.027 grams Fe(N0,),-9B20 O.OOOli^ b. Add 0.25 grams of Agar, gently boll to dissolve Agar (avoid charring). c. Prepare the buffer separately, and sterilize it before adding to the medium. Nag HPOj^ -12320 0.01^5 grams K^POi^ 0.0026 grams NaHCOj 0.055 d. When the saline-agar is cooled to Uo°C. add the buffer. e. It is advisable to add 0.001 gram percent of phenol red. The pH range is generally between 7-5 and 8.9 • k. Saline-agar plus yolk-albumen extract: a. Mix the entire contents of a fresh, unincubated egg with 50 cc. of saline- agar "A" (above). Shake well In an Erlenmeyer flask. b. Centrifuge the mixture at 2000 R.P.M. for 50 minutes to 1 hour. c. Draw off some of the supernatant fluid and mix with an equal volume of saline-agar "A" kept at 55° to Uo°C., on a warming stage. d. Place 2 cc. of this mixture In each of the previously prepared culture (watch crystal) dishes. (The amounts of yolk and of albumen may be varied to determine their relative value in growth. ) 1*5*^ EXPERIMENTAL CHICK EMBRYOLOGY 5- Sallne-A^ar plus yolk extract (omitting all albumen): a. Separate the yolk from the albumen in about 50 to 100 cc. of chick Ringer's, without rupturing the vitelline membrane. Pull off the chalaza and such albumen as you can grasp with the forceps. With glass rod roll the yolk over to remove any adherent albumen. Pass the yolk through several changes of Einger'a, In each removing more of the albumen. b. When fully denuded of its albumen, pour off all the Ringer's, puncture the vitelline membrane with sharp forceps, grasp it, and allow the yolk to flow out into a sterile beaker. c. Add about 10 cc. of the albumen- free yolk to 20 to UO cc. of saline-Agar "A", shake vigorously to mix. (Note: The unbuffered, pure yolk has a pH range of k.^ to 6.0, hence this fac- tor must be considered in relation to the results. It would be Instruc- tive to compare this with buffered saline "B".) 6. Saline-Agar plus pure albumen (omitting all yolk): a. I'llx 10 cc. of pure egg albumen with from 15 to Uo cc. of chick Ringer's. Shake thoroughly, and centrifuge (as above). b. Mix 1 cc. of the above (supernatant) mixture with 1 cc. of saline-Agar "A" to make up the substrate. (This may be made up with the buffered "B" or the non- buffered "A" saline solutions. ) 7. Saline-Agar (or blood plasma) plus embryonic extract: a. Place 5 embryos, ages from 5 to 8 days, in 20 cc. of Panne tt-Compt on saline and thoroughly crush. b. Centrifuge the embryonic mash and draw off the clear supernatant fluid. c. Mix the embryonic extract with 1. Buffered saline-Agar "B" at i+0°C. or 2. Blood plasma. NOTE: It is impractical for the average graduate student to test all of the above 7 media. It is therefore recommended that they be used in the sequence given (above) as far as time and facilities will allow. Preparation of the explant : (1) Incubate fertile eggs for 20 to 2*+ hours at 58°C. (2) Open an incubated egg into a finger bowl containing 100 cc. of sterile chick Ringer's solution. Simply break the egg open as though it were to be fried, taking care not to break the yolk. (5) With forceps, grab the chalaza or the yolk, and cut (with sharp scissors) through the vitelline membrane, making a circle around the blastoderm about ^ inch away from its border. By cutting in one direction and rotating the yolk mass (with forceps) in the opposite direction, the blastoderm can be quickly encircled. (k) Grasp the margin of the blastoderm with the forceps, and roll it back and away from the yolk. When the blastoderm is entirely freed from the yolk mass, and can be clearly seen, work a blunt dissecting needle between the blastoderm and the thin, transparent vitelline membrane. Now transfer the blastoderm, by means of a wide-mouthed pipette, to a Petri dish containing about 20 cc. of sterile chick Ringer's solution. (5) With fine dissecting (glass) needles trim away all of the yolky-opaque area. The early embryo is now ready to be cultured, in whole or in part. Embryonic parts to be used as explants : Stages to be used: a. Definitive primitive streak b. Head-process stage c. Head-fold stage d. One to 6 somite stage EXPERIMENTAL CHICK EMBRYOLOGY 435 THE DIFFERENTIATION OF CHICK EXPLANTS ON VARIOUS TYPES OF MEDIA SALINE- AGAR SALINE- AGAR YOLK- ALBUMCN (EMTIRE E&C CONTtMTS) SALINE - AGAR + YOLK EXTRACT (TRACE OF ALeUMEN) g:^ GENERALIZED RESULTS lOt HOURS LATER 20; HOURS LATER NON- BUFFERED SALINE- AGAR + PURE YOLK EXTRACT |no albumen] YOLK EXTRACT (TRACE OF albumen) 8UFFERE0 SAUNE- AGAR + PURE YOLK EXTRACT NO ALBUMEN) SALINE- AG A R + PURE ALBUMEN EXTRACT [NO yolk) (T) 101 HOURS LATER 20 « HOURS LATCR Development on various media. The sketches are based on camera luclda drawings of the living explants. All are drawn approxi- mately to the same scale. The numbers in each block refer to the number of explants. i.e., 20 + hours after explantation. Only the albumen which adheres to the yolk after the ordinary method of separating the 2. The yolk was not washed. From Spratt 19^*7: Jour. Exp. Zool. 106:5l4-5. Cuts to "be made : (See figure, below) a. Tranaversely (I.e., at right angles to the embryonic axis) about 0.2 to 0.7 mm. posterior to the primitive pit, using both halves. h. Transversely through the primitive pit level, using both anterior and posterior halves. c. Transversely anterior to the primitive pit (i.e., through the early notochord) and simultaneously through the middle of the primitive streak. This gives three pieces of blastoderm, each potentially very different. d. Use also whole blastoderms. A /" A c J / D B \ i » / E Primitive Strea k Stage showing levels of tran- sections. Procedure for culturing: (1) Transfer the embryo, or parts, to be cultivated by means of a wide-mouthed pipette to the surface of a culture medium, orient as desired, and flatten it out by sucking away all excess superficial medium with a fine pipette. The embryo (or part) should float on a few drops (2 cc. ) of culture medium. (2) Cover the Petri dish and return it carefully to the incubator. kj,6 EXPERIMENTAL CHICK EMBRYOLOGY Varlationa In the procedure: (1) Glucoae medium: Some interesting effects can be seen if 100 to 800 mg. per- cent of glucose la added to the Binger-Agar medium as a substitute for the albumen. Such a medium should be buffered to pH 7-8 to 8. 5* (Sterilize the medium before adding the sterile buffer.) Spratt (19'+8, p. 6h) gives the following formula for his "minimal medium": a. Add 0.10 to 0.15 grams powdered Agar (U.S. P. XI) to 56 cc. of chick Singer's, shake, add 0.5'<- grama of glucose, and 0.001 gram percent of phenol red. This mixture is autoclaved in a small Erlenmeyer flask for 8 to 10 minutes. b. Cool the mixture to Uo°C. c. Add 2 cc. of 0.290 grams percent of Nag HPOjj. -12320, and 0.052 grams per- cent of KE^POjj. (previously mixed). d. Add 2 cc. of 1.10 grams percent of NaffiOj. e. Saturate the mixture with CC^. f. Immediately place 2 cc. of the prepared medium in each watch crystal. (2) Medium with other sugars : a. Sugars which serve as exogenous energy supply: mannose, fructose, mal- tose. b. Sugars which do not serve as an exogenous energy source: lactose, suc- rose. (5) Medium with amino acids added: See Spratt (19^) for list of amino acids which can be tested for nutrient value. {k) Cultivation of chick structures on plasma-embryonic extract: The classic method of explant culturlng has been on plasma clot or on a mixture of chick Elnger's and embryonic extracts. It is suggested that the student follow this procedure to compare its benefits with those described above . 1. Place whole chick embryos (5 to 8 days) in a sterile beaker and cover with an equivalent volume of sterile lyrode's solution, or sterile chick Ringer's. The embryos must be finely chopped. 2. Centrifuge the embryonic mash at 25OO r.p.m. for I5 minutes. 5. '<^lth sterile pipette, remove the supematent fluid and, without dilu- tion, place in culture dishes. About 1 to 2 cc. per dish. h. The culture dishes may be of several types, all dry sterilized. a. Depression slides which are to be covered with coversllp, ringed with vaseline. b. Coverslips ringed with paraffin (to limit the spread of the culture medium) inverted over a depression slide (hanging drop method) which acts as a moist chamber. 5. With sterile pipette transfer the excised anlage* to the culture medium, seal, and Incubate for from 1 to 8 days. In the case of the coversllp (hanging drop) method of culturlng, add the culture medium (within the paraffin ring), transfer the anlage to the medium, ring the margin of a depression slide with white vaseline, and invert the depression slide and bring it down over the coversllp so that it covers the explant. Gently press the depression slide into place, and transfer (without righting the depression slide) the whole to the Incubator at 58'-'C. After the explant has been in the incubator for 6 to 8 hours, the depression slide and coveralip may be quickly inverted, whereupon the culture becomes a hanging drop. During the preliminary Interval the cells of the explant will have had an opportunity to become adherent to the coversllp, and the whole explant will be visible under the microscope through the thickness of the coversllp. If the explant Is transferred to fresh, sterile medium every 3 to 'i- days. It may be carried for longer periods (e.g., 18 days). EXPERIMENTAL CHICK EMBRYOLOGY ^ SKETCHES OR PHOTOGRAPHS OF CULTUKED EXTLAMTS U58 EXPERIMENTAL CHICK EMBRYOLOGY For such in vitro studies it is suggested that the whole or parts of the eye of embryos ranging from 1 to 7 days be used (Dorris, 1958). The eyes may be carefully dissected out in sterile chick Singer's using fine glass needles. When whole eyes are transplanted, remove as much of the adherent mesenchyme as possible, leaving only the ectoderm im- mediately covering the optic cup. When parts of the optic cup are ex- planted, it is first necessary to puncture the border of the cup with a glass needle, impale the lens on the needle, and pull it out. With the cup In position, the needle can be inserted between the two layers at the choriold f Issues, thus isolating the retina which can be removed as a firm cup- shaped structure. In the older eyes the lens can be removed by dissecting away the overlying ectoderm, trimming the edge of the cup with fine scissors, and then separating the two layers with fine glass needles. The cup, or parts of either layer, can then be cut into smaller pieces for explantatlon. This type of study provides Information relative to the self- differentiating capacities of whole eyes, and (in explantatlon of pieces) to the capacity for independent differentiation of the parts of the eye. DISCUSSION: The technique of tissue culture was first used by Dr. Eoss Harrison in I907. Most of the work that has been done since with this technique has been with Isolated cells of the relatively older {k to 8 days) chick embryos. Only recently has Spratt (19'*-7, 19'*6) re- vitalized interest in this technique by finding that the entire blastoderm of the early (18 to 2k hour incubated embryo) stages could be cultured in vitro, either in toto or in transected parts. /•"v •v I k Camera lucida drawings of the anterior portion of a living 4-somite blastoderm explanted to a non-nutrient saline-agar medium: a, at the time of explantatlon; b, after 20+ hours' cultivation. Note the failure of development and the loss of organization which had been attained at the time the embryo was removed from its normal food supply - the yolk and albiimen. Note also that presence of part of the opaque area has not pre- vented the characteristic "degenerative" changes. Camera lucida drawings of a living explant to a yolk-albumen extract sallne-agar medium. a, at the time of explantatlon. b, after 55 hours' Incubation. Note the remarkably normal morphogenesis, differen- tiation, and increase in size of the ex- plant. The heart was beating rhythmical- ly when the drawing was made. From Spratt 191*^7: Jour, Exp. Zool. 106:514-5 From Spratt 191+8: Jour. Exp. Zool. 107:59 Spratt (19'+7) sets up a group of criteria for the adequacy of the cultvire medium used in support of development, as follows: EXPERIMENTAL CHICK EMBRYOLOGY k39 1. Begression of the primitive streak, formation and elongation of the notochord. 2. Formation and closing of the neural folds. 5. Development of the hrain and spinal cord. k. Formation of somites. 5. Development of optic vesicles and otocysts. 6. Morphogenesis and pulsation of the heart. 7. Formation and extension of a "tail" from the cut edge of the anterior piece. He states: "A medium which meets all of these requirements is considered adequate." From the works of 'Spratt it seems evident that the early tlastoderm does not have adequate endogenous food supply (as does the fish emhryo) hut depends upon some exogenous source for nutrition. The best medium proved to he the entire egg (yolk and albumen) ex- tracted and added to a saline-agar base. The albumen seema not only to be bacteriolytic but also seems to help maintain the proper pH of the blastodermic environment. A "com- plete" synthetic medium was finally devised, containing saline-agar, glucose, amino acids, vitamins, etc. on which explants xinderwent essentially normal morphogenesis, differentia- tion, and some growth. However, Spratt subsequently found that the glucose component was the only absolutely essential exogenous source of energy for morphogenesis and differen- tiation of the explanted early blastoderm. These developmental processes occurred in the almost total absence of growth. Mannose, fructose, and maltose were apparently Just as efficient as was glucose. In a study by Rawles (I956) followed by Eudnlck (1958) it has been possible, by ex- plantation and chorio-allantoic grafting, to map the organ forming areas of the early chick blastoderm. Eawles found that the developmental potencies within each area diminish peripherally from the center, and that there is superior developmental capacity of the left side of the blastoderm over the right. 07M.M.i 03MM^ 07MM' REGIONS OF ORGAN ANLAGE IN EARLY CHICK BLASTODERM This chart is made from the combined data of Willier and Bawles. kko EXPERIMENTAL CHICK EMBRYOLOGY I \:q Summarj' diagrams cased on a study of over 50 Dlastoderms growing in ovo and stained as shown in (a) with a 1:1 mixture of 0.5% Nile blue sulfate and 0.5'?J Neutral red. In (b) is shown the position of the stained band of cells 6 hours later. In (c) (drawn to a somewhat smaller scale than a and b) Is shown the pattern of stained cells 14 hours later (20 hours after the original marking as in a) . If (c) be compared with (b) it is to be noted that the end-bud and the neural tube have secondarily acquired the stain and keep it in the oxidized (blue) state. In the region just anterior and lateral to the bases of the omphalo- mesenteric arteries no stain can be seen (presumably it is in the reduced (color- less) state) . The stained regions lateral and parallel to the neural tube are in the lateral plate mesoderm (cf. Pasteels, 1937, fig. 18) . No exceptions to this re- sult liave been found. Diagrams posterio region ( vaginati the pres the node sion of left to more rap primitiv lying la illustrating r "stretching" neural plate) , on through the ence of invagi during the ea the streak. T right in the d id and extensi e streak relat teral to it. the pronouned antero- of the pre-nodal the absence of in- primitive pit, and nation Just behind rly stages of regres- ime increases from iagraras. Note the ve regression of the ive to marked areas Two above diagrams from Spratt 19't-7: Jour. Exp. Zool. 10k -.69 A -LIVER ^-MEART ©-CHORDA 6-THYROIO ©-NEPMROS X -INTESTINE • -ERYTHROCYTES' T-MELANOPHORES ■♦■ SKELETAL MUSCLE DISTRIBUTION OF POTENCIES IN THE DEFINITIVE PRIMITIVE STREAK BLASTODERM, TESTED UNDER VARIOUS EXPERIMENTAL CONDITIONS Ectodermal potencies shown on left, mesodermal and en- todermal on right; tliese have not been tested separate- ly. Posterior and lateral extent of mesodermal poten- cies has not been specified. From Rudnlck igl^lj-: Quart. Rev. Biol. 19:18? EXPERIMENTAL CHICK EMBRYOLOGY i;l+l Rudnick, on the other hand, hy isolation culture methods on plasma clots, found that two primary germ layers the ectoderm and endoderm seem to function more-or-less indepen- dently, and that the notochord and axial mesodermal structures apparently depend on the deginltive organization of the streak and node, or the head process, for realization in vitro. Below is presented a figure from her paper showing the recognizable differentia- tions from various regions of the early blastoderm when explanted in vitro. MED PLATE SS'C SENS EP BOOmALL MESENCHYME ERYTH 50% [MED PL ATE J HEART ? MESENCnrME ERYTH. 100% SENS EP CHORDA ? BODY WALL MESENCHYME ERYTH SO'/, MEDPLATE aS% SENS- EP BODY WALL MESENCHYME ERYTH .' MED PLATE HEART HEART soil, ERYTH 100% MED PLATE SOV. MESENCHYME CHORDA' HEART' ERYTH 13 % \ HE ART SO '/. Operating diagrams, showing structures differen- tiating from each piece. Approxlnrnte percentages are given In cases where they are thought of significance. From Rudnick I958: Jour. Exp. Zool. 75:599. THE METHOD OF CHOR lO-ALLANTOIC GRAFTING The grafting of early chick embryo parts to the chorio-allantois of an older (8^ to 10 day incubated) embryo has long since been perfected ( Headley, 192^, Willier, I92U) , The highly vascularized chorio-allantois of the avian embryo is located close to the shell membrane of an 8-day embryo, so that the shell over it can be removed and the explant can be placed upon it as a substrate for nutrition and growth. If the shell is replaced and sealed into place with melted paraffin, the explant will generally become vascularized and will develop for 9 to 10 days and may be re- ^^ ^^ ^^^^ ^^ covered before the natural breakdown of the ^^K ^^^^S^^^r ^^^ extra-embryonic membranes on the 19th day of ^^B. ^^Hitf^lr / fj/ incubation. Since there is complete isolation of the explant, there are no persistent inductive influences. However, the explant does come under the hormonal and other influences ema- nating from the blood of the host, and these must be considered. Further, there is a space limitation (within the host environment) Chick egg with window above the develop- ing embryo. Black washer Is sealed to the egg shell and to tlie covering cover- glass with paraffin. Irregular margin of torn shell membrane visible through the window. Embryo can develop and hatch. Ul;? EXPERIMENTAL CHICK EMBRYOLOGY CHORIO-ALIAKIOIC MEHBR»ME AHH I ON HP OF SHELL LBUNEM LAYERS OF THE SHELL SHELL HEHDRAKE CHORIO-ALLAHTOIS PIPETTE USED TO TRANSFER THE GRAFT THROUGH THE SHELL OPENING ALLANTOIC BLOOD VESSEL Diagram illustrat liijd, tlie metliod of chorio-allantoic grafting. Host embryo is generally incuoated for 8 to J days, the groft may be any anlage (e.g., the limb bud of 72 hour stage). Tlie graft is generally recovered at about tlie I'Jth day, just before the time of hatching of Xhe host when the mem- oranes begin to dry up. and the further possibility that the graft may develop a parasitic relationship to the host, thereby utilizing some of the nutritional requisities of the host and Indirectly alter the normality of the host environment. Nevertheless, the chorio-allantois is proving of value in the study of mammalian iso- lates (Nicholas and Eudnick, 1955) and in the culturlng of bacteria and viruses (Good- pasture, 1958). Experience has Indicated that the h8 hour eye and the 72 hour limb are the most satisfactory anlage to transplant. THE PBOCEDUBE: 1. Preparation of the hosts: Begin incubation of fertile host eggs, properly marked, about 8 days before the operation. Turn the eggs twice daily. 2. Preparation of the donors: Begin Incubation of donors on the 5th or 6th day of host incubation, so that the donors will be either 1+8 or 72 hours along at the time the hosts are 8 days incubated. Mark the eggs, and rotate twice daily. 5. Preparation of the host for the graft: (Work fast under the most sterile con- ditions) a. Candle the host eggs, select and mark the regions most suitable for a graft. The forked Junction of large blood vessels at some distance from the embryo should be indicated by a pencil drawing on the shell. b. Prepare a window in the shell, under aseptic conditions (see directions for this above). Bemove the shell in one piece. Moisten the shell mem- brane with several drops of sterile Locke's or chick Einger'a solution. This Is necessary in order to avoid Injury to the underlying and aome- tlmes adherent chorio-allantoic membrane, and consequent mpturing of the blood vessels. Cover the aperture with a sterile cover glass, or replace the shell, and return the (host) egg to the incubator while preparing the graft. k. Preparation of the transplant: (l^e - 1+8 hours, limb - 72 or more hours) a. Candle the donor to determine the approximate age. Crack the underside of the shell on the side of a finger bowl containing about 100 cc. of sterile chick Ringer's solution. When the blastoderm has moved around the dorsal position, quickly excise it, separate it from the vitelline membrane, and transfer it in a wide-mouthed pipette to a sterile watch- glass under a dissection microscope. Confirm the age and stage of development. EXPERIMENTAL CHICK EMBRYOLOGY ^ t Quickly, and with sterile needles and watchmaker's forceps, excise the anlage to he studied and transfer it in a drop of the sterile medium to the chorio-allantois of the prepared host. Replace the shell, and seal it with a ring of melted paraffin. Beplace the host egg in the incubator without shifting its gravitational axis, and leave it unmoved for 6 to « hours. After this interval, treat the egg as any regularly Incubated egg. The eye: The eye graft may be taken from any stage after about the 35 hour stage. The retina, pigmented layer, and lens may be easily distinguished in the graft, upon recovery. It is, of course possible to study the independent differentiation of parts of the early optic vesicle, cup, etc. Final study may be made after clearing with oil of wintergreen, when the pigment and retina show up well, and then after sectioning and staining. The limb bud: Under the dissecting microscope, locate the wing and leg buds (72 hours or older). Make transverse cuts with a sharp needle through the embryo, one through the neck level and the other between the wing and the leg buds. Then dis- sect out each appendage bud in the following manner: Cut parallel to the body at the base of the bud, but include some of the body somites. Then cut (at right angles to the first cut) at the anterior and the posterior limits of the bud. Finally make the cut parallel to the first, beyond the outer limit of the bud, thus excising the bud in a rectangu- lar piece of tissue, Eemove all yolk and loose tissue ad- herent to the bud. A single donor may provide several ap- pendage buds, and occasionally a single host may survive the Implantation of several buds. c. Make a complete record, including sketches, of the condition of the chorio-allantois and the donor (explant) at the time of the operation. 5. Becovery of the graft: , . v v ^ir, a. The graft must be removed before the host extra- embryonic membranes begin to diy up. This is generally by the l8th day of incubation, or about 9 to 10 days after the transplantation of the graft is made. b. Candle the host to attempt to locate the graft. This is generally diffi- cult because of the opacity of the host. However, if the graft has "taken" it will be found close to the original window. Make a cut through the shell about 1 centimeter outside of the original window, and remove the shell carefully, without rupturing the underlying chorio- allantois. Examine the underside of the shell to see if the explant may be adherent to it. The graft will generally appear as a fluid-filled vesicle, opaque, and without discernible structure. Particularly in the case of the appendage anlage, the detailed structure will not be seen until the tissues are histologically cleared. c. Use the Lundvall technique (see above) for quick staining of cartilage, etc. of differentiated limbs. The eyes should be sectioned after normal fixation (Bouin or Klinenberg' a) and stained with Conklin' s Haematoxylin. 6. Other embryonic areas to be tested by chorjo-allantolc grafting: It is sugges- ted that the following additional structures be given the opportunity to develop on the chorio-allantois. a. Anterior half of the primitive streak, Including Eensen s node. b. Neural crests and nerve cord, from embryos with 5 to 10 somites. c. Somite blocks. d. Heart anlage of h2 hour stage. e. The entire blastoderm of primitive streak stage. (Note: For further instructions the student ^^is advised to consult Hamburger's: "A Manual of Experimental Embryology" p. 1^J>-) kkk EXPERIMENTAL CHICK EMBRYOLOGY a: ffi Z UJ z X C5 2 Q. a: L o a. z < a: m z 111 z X en X CO q: O o tn (U o z 2 a CD a UJ (E I- to -I a < UJ UJ (O If o o * P CO i i 4 z k EXPERIMENTAL CHICK EMBRYOLOGY kk^ UJ z < X cn (1. o z a. t- V) g tij ^ z o < x^ o z e o X j- Si' V) t lli 5 " V OQ s UJ CO z z i o o u m o S^ § UJ 2 gi £ Q- ^ O O z o o 22 q: q: o a UJ o 2 < a: Q UJ 5 o 1+1+6 EXPERIMENTAL CHICK EMBRYOLOGY DEALINGS OE PHOTOGRAPE OF CHOBIO-ALLAJiTOIC GRAFTS EXPERIMENTAL CHICK EMBRYOLOGY khj INTRA-EMBRYONIC TRANSPLANTATIONS The most productive and successful Investigators in this field are Wlllier and Ham- hurger, and the student is directed to their papers. In some instances the graft (whether limt, eye, or neural crest) is merely Inserted into the coelom through a slit in the soma- topleure and in other instances the transplant is placed in a more solid (flank) region where it may he incorporated in an otherwise normal orgeua. llieae latter transplantations are the more difficult, but poasihly the more significant. The coelom of the 3-day embryo allows freer expansive growth than does the chorio- allantols, and it is an ideal nutritive environment. However, the graft may attach itself to any of a number of surfaces within the coelom, such as mesenteries, coelomlc epithelium, or the surface of the gonad and mesonephrlc primordia. Such grafts cannot be properly in- nervated, and limbs may develop morphologically but without movement. Hamburger and Waugh (19'<-0) found excellent histological differentiation which rapidly regressed without such Innervation. The method of candling, excising the blastoderm, trimming away the yolk and non- essential parts, and the final dissecting out of the organ anlage are all described (above) and are so well known that they will not be repeated here. The student must be reminded, however, of the increased necessity of absolute aseptic conditions, since trans- plants are to be Introduced directly into the tissues of the (5 day) host. Limb primordia grafted into the coelom: 1. Under aseptic conditions, expose a 5-day embryo through a shell aperture, and apply a small strip of sterile Neutral Bed stained Agar to the flank region, cover the opening of the shell, and return the egg to the Incubator for 10 minutes. It is best to rupture the vitelline membrane with #5 watchmaker's forceps so that the dye can penetrate the faster. 2. Under aseptic conditions excise the wing or leg bud (72 hours or older) of an- other embryo, clean it of all excess yolk and membranes, and place the watch- glass containing the donor tissue on a wanning plate at 58°C. 5. Prepare the host by opening the shell, adding a drop of sterile chick Blnger's solution, and removing the dyed agar with forceps. Should the blastoderm ad- here to the shell or window, shake it gently to separate it from the attach- ment. h. If the amino and chorion have grown over the flank region. Insert a (sterile) glass needle into the amnion, parallel with the body, and with an upward (cut- ting) movement, make a slit in the extra-embryonic membrane directly above the flank region where the graft Is to be Inserted. The membranes will heal, par- ticularly if there has been no hemorrhage. 5. Make a longitudinal slit posterior to the forelimb bud. Just above the entrance of the vitelline veins into the body. Avoid injury to any blood vessels. The posterior cardinal veins lie ventral to the lateral edges of the somites, and the lower splanchnopleure is highly vascular. Make the slit long enough for the insertion of the transplant. 6. Suck the transplant Into the end of a micro-pipette and, under good lighting and a dissecting (binocular) microscope with high power objectives, drop the transplant onto the embryo as near the slit as possible. With (sterile) glass needles orient the transplant and insert It into the coelom. It may be further oriented after the insertion, by using a blunter needle. Add a few drops of sterile chick Ringer's. 7. Replace the shell and seal the window with melted paraffin. Return the egg to the Incubator in the same position for 2*1 hours. 8. Allow the host to continue development for about 9 days, and recover the trans- plant at about 12 days of incubation of the host. The recovery may be diffi- cult since the transplant may be hidden by some of the host tissues, and it may require an exploratory dissection of the host. 9. Make drawings, gross and histological analysis of the development of the trans- plant. 1^1*8 EXPERIMENTAL CHICK EMBRYOLOGY Limb prlmordia grafted Into the flank: (See Hamburger, 1938, 1959) 1. Follow the preliminary directions of the preceding exercise. 2. Preparation of the transplant: When excising the limb buds leave extensions of tissue from both the anterior and the posterior limits of the bud. These will be used to tuck the bud into the slit. 5. Preparation of the host: Make a longitudinal slit between the wing and leg bud of the 5 -day host embryo. The slit should be no longer than the actual bud, and should be close to the somites so that during growth it may pick up some of the developing muscles. Avoid hemorrhages. h. Implantation: Drop the bud onto the host embryo in the vicinity of the silt, and work the anterior end into the slit by means of a (sterile) glass needle. Then work the posterior end into the slit and, finally, using a blxmter needle, force the bud itself into the slit leaving the bulge of the bud exposed. It may be necessary to lengthen the slit slightly during this process. The slit will close sufficiently to hold the bud in place. It must not be allowed to slip into the coelom. 5. Gently transfer the host egg (after sealing, etc.), without jarring, to the in- cubator and leave undisturbed for UQ hours. Ejye prlmordia grafted into the flank region: (See Gayer, 19^2) 1. Prepare hosts of about 6o to .72 hours incubation and donors of about 30 to 56 hours of incubation. Donor should have from 10 to 12 somites. 2. With Neutral Eed or Nile Blue Sulphate agar stain the right wing bud of the host. 3. Quickly dissect out the right optic vesicle of the donor (in sterile, chick Blnger's) by making a transverse cut at the level of the Infundibulum (just be- hind the optic vesicle) and then a median cut anteriorly. The piece will in- clude the entire right side of the prosencephalon. (The left side may be used for a second host.) h. Make a longitudinal slit at the base of the wing bud of the 72 hour host, at about the level of the 20th somite. 5. Drop the excised optic cup onto the host embryo, in a minimum of sterile medium, and with the tip of a glass needle (not too sharp) tuck the cut surface of the brain into the slit. This will leave the optic vesicle exposed at the surface. 6. Since the eye Is essentially complete by about 10 days of incubation, the host may be sacrificed at that time and the transplant recovered. It should be com- pared with the eye at the donor age, the eye of any 10-day Incubated embryo, and the eye of the hatching chick. The graft may be fixed in ELlnenberg's (or Bouin's), dehydrated, and cleared in oil of wlntergreen for gross study after which it may be sectioned. (There is no particular point in carrying the host to hatching although this may be attempted. If successful it would indicate the achievement of a very delicate operation, but generally the eye would be resorbed.) Interspecific transplantation of neural crests: The neural crest of vertebrates gives rise, not only to the dorsal root ganglia, and to the sympathetic system, but also to the pigmented cells known as melanophores, wherever they are found, (see exercise on "Origin of Amphibian Pigment"). Since their ultimate location is often far removed from the neural crest, these cells exhibit extensive powers of migration and of proliferative capacity. They are found in the skin of amphibia, the feathers of birds, and in the mesenteries of the body cavity. Wlllier and Eawles have successfully transplanted neural crests between bird species which exhibit radically different color patterns, and some which are white. They found that the color pattern, in all cases, was due to the intrinsic genetic factors of the specific melanophores concerned, rather than to the feather structure. There was found to be some slight modification of pigment expression of the melanophores by the host feather germs. (See Wlllier I9I1I: Amer. Nat. 75:156 for review.) EXPERIMENTAL CHICK EMBRYOLOGY '^'^9 SKETCHES Airo. PHOTOGRAPHS OF CHICK GRAFTS 1+50 EXPERIMENTAL CHICK EMBRYOLOGY If the student has had success with transplantation of limb primordia, he might carry out the following procedure which has as its aim the study of neural crests transplanted from dark to light hreeds of fowl. 1. Donors consist of any dark breed (e.g., Brown Leghorn, Barred Bock, New Hamp- shire Bed). They should be from 50 to kQ hours incubated (less than 15 somites) at the time of the excision of the neural crest. 2. Hosts should be about 60 hours incubated. They should be White Leghorns. 5. Under the usual aseptic conditions, make a longitudinal slit in the chorion, the amnion, and finally at the base of the wing bud. Betum the covered host to the incubator. k. With a piece of Nile Blue Stained agar, stain for 10 minutes the head, anterior to the otocyst. 5. Quickly excise the donor embryo from Its yolk, remove all yolk, and strip a piece of akin from the dorsal and dorso-lateral surface of the head (where it is stained). The neural crest cells will be adherent to the undersurface of this piece of skin. 6. With pipette and sterile chick Elnger's transfer this neural crest material, to the previously prepared slit at the base of the wing bud of the host. Gently push it deeply into the opening and make certain that it sticks. 7. Seal the window, iand incubate the egg (without rotation for 1+8 hours) for at least two weeks. If development seems normal, allow some hosts to go to hatch- ing. ldii 2 days 3davs «t days 5 days 6 days ,JJ^''^ , (0.0002 g:am) f 0.003 gtam) lO.OZqum^ (O.OSgiaml (0|3guml (0.29gtaml (0.57 gxim) adays 9 days lOdays 11 days 12 da>(s '~i'*^^^ , ,Jl^^'^^ , (1.15 gumO M.Sigjimsl (2.26gtartis> (3(.8etimsl f5.07 gumil (7.37exims^ O.TtgtiniO 16 days n days 18 days 19 days 20 dais 21 days (I200gumi^ (IS.SrgumO (Ig'sfgtamil (2r83 gzamO (25 (,2 gxams) OO.eigiamil (Hatched) DAILY CHANGES IN THE WEIGHT AND THE FORM OF THE DEVELOPING CHICK EMBRYO (WHITE LEGHORN) Reprinted by permission of N. Y. State College of Agriculture from Bulletin #205, Bomanoff, 195 1. EXPERIMENTAL CHICK EMBRYOLOGY ^31 BEFERENCES: Alexander, L. E,, 1957 - "An experimental study of the role of optic cup and overlying ectoderm In lens formation in the chick emhryo." Joiir. Exp. Zool. 75:'+l» Baker, L, E., I936 - "Artificial media for the cultivation of flhroblaata, epithelial cells and monocytes." Science. 85:605. Barry, A., 19^+1 - "Effect of exsangulnatlon on the heart of the emhryonic chick." Jour. Exp. Zool. 88:1. Beverldge, W. I. B. , 19^+7 - "Simplified techniques for Inocxilatlng chick emhryos and a means of avoiding egg white in vaccines." Science. 106:52't. Burt, A., 19^+5 - "Growth of spinal ganglia in plasma from vitamin B deficient chickens." Jour. Cell. & Comp. Physiol. 22:205. Butler, E., 1955 - "The developmental capacity of regions of the incuhated chick hlasto- derm as tested In chorlo-allantolc grafts." Jour. Exp. Zool. 70:557. Canzanelli, A., M. Greenblatt, G. A. Rogers, & D. Rapport, I959 - "The effects of pH changes on the In-vltro Oq consumption of tissues." Am. Jour. Physiol. 127:290. Carpenter, E., 19'<-2 - "Differentiation of chick embryo thyroids in tissue culture." Jour. Exp. Zool. 89:1+07. Carrel, A. & L. E. Baker, I926 - "The chemical nature of suhstancee required for cell mul- tiplication." Jour. Exp. Med. kk-.'^O'i. Catlzone, 0. & P. Gray, I9l<-1 - "Experiments on chemical Interference with the early morpho- genesis of the chick. II. The effects of lead on the central nervous system." Jour. Exp. Zool. 87:71. Cohn, A. E. & A. E. Mlrskl, I929 - "Physiological ontogeny. A chicken embryo. XIX. The hydrogen Ion concentration of the blood of chicken embryos as a function of time." Jour. Gen. Physiol. 12:1*65. Davis, J. 0., 19kk - "Photochemical spectral analysis of neural tube formation." Biol. Bull. 87:75. Dorrls, F., 19!*^! - "The behavior of chick neural crest in grafts to the chorlo-allantolc membrane." Jour. Exp, Zool. 86:205 (See Ibid I958, Vol. 78:585) (See Ibid 1959, Vol. 80:515) DuShane, G. P., I9I+I+ - "The embryology of vertebrate pigment cells. II. Birds." Quart. Eev. Biol. 19:98. Eaatllck, H. L., I9I+5 - "Studies on transplanted embryonic limbs of the chick. I. The development of muscle In nerveless and in Innervated grafts." Jour. Exp. Zool. 95:27. Fell, H. B. & R. Eoblson, I929 - "The growth, development, and phosphate activity of embryonic avian femora and limb buds cultivated In vitro." Bioch. Jour. 25:767 (See ibid. I95O, Vol. 2l+:1905). Frapes, R. M., M. W. Olsen, & B. H. Neher, 1914-2 - "Forced ovulation in normal ovarian follicles in the domestic fowl." Proc. Soc. Exp. Biol. & Med. 50:508. Fugo, N. W., 1914-0 - "Effects of hypophyaectony in the chick embryo." Jour. Exp. Zool. 85:271. Gayer, H. K., I9I42 - "A study of coloboma and other abnormalities In transplants of eye prlmordia from normal and Creeper chick embryos." Jour. Exp. Zool. 89:105. Glueckaohn-Schoenheimer, S., I9I4I - "The development of early mouse embryos in the extra- embryonic coelom of the chick." Science. 95:502. Goodpasture, E. W., I958 - "Some uses of the chick embryo for the study of infection and immunity." Amer. Jour, ^glene. 28:111. Green, T. W. & J. M. Blrkeland, I9I42 - "Use of the chick embryo in evaluating disinfec- tants." Proc. Soc. Exp. Biol. & Med. 51:55- Hamburger, V. C, I9I+I - "Transplantation of limb prlmordia of homozygous and heterozygous chondrodys trophic (Creeper) chick embryos." Physiol. Zool. ll4-:555. Hamburger, V. & M. Waugh, 1914-0 - "The primary developnent of the skeleton In nerveless and poorly Innervated limb transplants of chick embryos." Physiol. Zool. 15:567. Hamburger, V. C. & E. Keefe, 19l4-l4 - "The effects of peripheral factors on the prolifera- tion and differentiation In the spinal cord of chick embryoa." Jour. Exp. Zool. 96:225. Hanks, J. H. , I9I+8 - "The longevity of chick tissue cultures without renewal of medium." Jour. Cell. & Comp. Phye. 51. Harrison, E. G. , I907 - "Observations on the living developing nerve fiber." Proc. Soc. Exp. Biol. 8e Med. l4:lUo. Harrison, R. G. , W. T. Aatbury, & K. M. Rudall, 1914-0 - "An attempt at an x-ray analysis of embryonic processes." Jour. Exp. Zool. 85:559. 1*52 EXPERIMENTAL CHICK EMBRYOLOGY Hetherlngton, D. C, 19't-^ - "Frozen-dried serum as a medium constituent for tissue cul- tures." Proc. Soc. Exp. Biol. & Med, 57:196, Hlllemann, H. H. , 19^2 - "The design ana use of microelectrodes for the production of lesions in the pituitary rudiment of chick embryos." Anat. Bee. Qki'^h^, Hoadley, L. , I929 - "Differentiation versus cleavage in chorlo- allantoic grafts." Arch. f. Ent. mech. 116:278. Hunnn, Frances D., I9I+2 - "The growth and migration of cultured melanophores from the neu- ral crest when grafted into the embryo." Jour. Exp. Zool. 90:101. Hunt, E. A., 1952 - "The differentiation of chick limb buds in chorio-allantolc grafts with special reference to the muscles." Jour. Exp. Zool. 62:57. flyman, L. H., I927 - "The metabolic gradients of vertebrate embryos. III. The chick." Biol. Bull. 52:1. Jacobson, W., 1958 - "The early development of the avian embryo. II. Mesoderm formation and the distribution of presumptive embryonic material." Jour. Morph. 62:h-k^, Joy, E. A., 1939 - "Intra-coelomic grafts of the eye primordium of the chick." Anat. Rec. 71+: 1+61. Kirby. D. B., 1927 - "The cultivation of lens epithelium in vitro." Jour. Exp. Med. 1+5:1009. Kosin, I. L., 19'+'+ - "Macro- and microscopic methods of detecting fertility in unincubated hen's eggs." Poultry Science 23:266 (See also 191+5 Anat. Bee. 91:2i+5). Kume, Matozo, 1935 - "The differentiating capacity of various regions of the heart rudi- ment of the chick as studied in chorio-allantolc grafts." Physiol. Zool. 8:73- Landauer, W. & L. Baumann, 19^+3 - "Rumplessness of chicken embryos produced by mechanical shaking of eggs prior to incubation." Jour. Exp. Zool. 93:51- Lewis, M. E., 1922 - "The importance of dextrose in the medium of tissue cultures." Jour. Exp. Med. 35:517- Lewis, M. R. & W. H. Lewis, I9II - "The cultivation of tissues from chick embryos in solu- tion of NaCl CaClg, KCl, and NaHCOj." Anat. Bee. 5:277. Lillie, F. B., 1942 - "On the development of feathers." Biol. Bev- 17:2l*-7 (See also Lillie & Wang:19l+1+ Physiol. Zool. 17:1). Meyer, H. , I936 - "Zuchtung du Retina dee Huhnes in vitro." Zeltschr. f. Mikroskop. Anat. Pbrsch. 59:151. Moog,' F., 19'+'+ - "Localization of alkaline and acid phosphatases in the early embryogene- sie of the chick." Biol. Bull. 86:51. Morgan, V. F. & D. E. Chichester, 1955 - "Properties of the blood of the domestic fowl." Jour. Biol. Chem. 110:285. Murray, P. D. F., 1932 - "The development in vitro of the blood of the early chick embryo." Proc. Roy. Soc. London B. 111:1+97 (Also ibid, I938, 12k:k21). Neher, B. H. & R. M. Fraps, I9I+6 - "Fertility and hatch abiJity of the prematurely ovulated hen's egg." Jour. Exp. Zool. 101:83. Nicholas, J. S. & D. Rudnick, 1935 - "The development of embryonic rat tissues upon the chick chorio-allantois." Jour. Exp. Zool. 66:193. Pasteels, J., 1937 - "Etudes sur la gastnilation des Vertebres Meeoblastiques. III. Oiseaux." Arch, de Biol. i+8:382. Olaen, M. W., 19^+2 - "Maturation, fertilization, and early cleavage in the hen's egg." Jour, Morph. 70:515- Philips, F. S., 191+2 - "Comparison of the respiratory rates of different regions of the chick blastoderm during early stages of development." Jour. Exp. Zool. 90:83. Plckels, E. G,, 191+2 - "Apparatus for rapid, sterile removal of chick embryos from eggs." Proc. Soc. Exp. Biol. Se Med. 50:22l+. Price, J. W. & E. V. Fowler, I9I+0 - "Ifeg shell cap method of incubating chick embryos." Science. 91:271. Rawles, M. E. , I936 - "A study of the localization of organ-forming areas in the chick blastoderm of the head-process stage." Jour. Exp. Zool, 72:271- Rawlee, M. E. , I9I+3 - "The heart-forming areas of the early chick blastoderm." Physiol. Zool. 16:22 (See ibid I9U6, Vol. 18:1). Romanoff, A. L., I9I+3 - "Cultivation of the early chick embryo in vitro." Anat. Bee. 87:365. Romanoff, A. L., I9I+3 - "Differentiation in respiratory activity of isolated embryonic tissues." Jour. Exp. Zool. 95:1- Romanoff, A. L.. I9I+5 - "^drogen-lon concentration of albumen and yolk of the d'-voloping avian egg.'* Biol. Bull. 87:223- EXPERIMENTAL CHICK EMBRYOLOGY 1+53 Bomanoff^ A. L., G. Bump^ & E, Holm, 1938 - "Artificial Incutatlon of some upland gam© tird eggs." Bulletin #2, N. Y. State Conservation Department. Eudnick, D., 1938 - "Differentiation in culture of pieces of the early chick ■blastoderm." Jour. Exp. Zool. 79:399. Eudnick, D., l^kk - "Early history and mechanics of the chick hlastoderm - a review." Quart. Eev. Biol. 19:187. Sandstrom, C. J.^ 19^0 - "Heteroplastic transplantation and species specificity. I. A com- parison of the effects of reciprocal chorio- allantoic transplants of macerated and unnHcerated duck and chick kidney tissue." Biol. Bull. 79:329- Schechtmann, A. M., 19'4-7 - "Antigens of early developmental stages of the chick." Jour. Exp. Zool. 105:329. Schmidt, G., 1937 - "On the growth stimulating effect of egg white and its importance for embryonic development." Enzymologia. '+:'4-0. Spratt, N. T. Jr., 19^1 - "Development in vitro of the early chick blastoderm explanted on yolk and albumen extract saline-agar substrata." JoviC. Exp. Zool. 106:J)k^. Spratt, N. T. Jr., 191*7 - "A simple method for explanting and cultivating early chick embryos in vitro." Science. 106:452. Spratt, N. T. Jr., 191*8 - "Development of the early chick blastoderm on synthetic media." Jour. Exp. Zool. 107:39. Stiles, K. A. & E. W. Watterson, I938 - "The effects of Jarring upon the embryogeny of chick embryos." Anat. Bee. 70:7- Stilwell, E. F., 191*7 - "The influence of temperature variation upon the occurrence of multipolar mitoses in embryonic cells grown in vitro." Anat. Bee. 99: Strangeways, T. S. P. & H. B. Fell, I926 - "Experimental .studies on the differentiation of embryonic tissues growing in vlvi and in vitro. II. The development of the isolated early embryonic eye of the fowl when cultivated in vitro." Proc. Boy. Soc. 100:275 (See ibid I927, Vol. 102:9). Swezy, 0., I915 - "Efeg albumen as a culture medium for chick tissues." Biol. Bull. 28:1*7. Tompkins, E. B., B. B. Cunningham, & P. L. Kirk, 191*7 -"mtosis, cell size and 'growth' in culture of embryonic chick heart." Jour. Cell. & Comp. Physiol. 30: Vollmer, H. , 1935 - "Eine Methode zur Beobachung der Entwicklung dee Bihn-embryo in vitro." Ztschr. Zellforsch. mikr. Anat. 23:566. Waddington, C. H. , 1932 - "Experiments on the development of chick and duck embryos, culti- vated in vitro." Phil. Trans. Boy. Soc. London B. 221:179. Waterman, A. J., I9UI* - "Viability of embryonic chick tissues following storage at low temperatures." Growth. 8:175. Weiss, P., 19li-l* - "In vitro transformation of spindle cells of neural origin into macro- phages." Anat. Bee. 88:205. Weiss, P. & E. Amprlno, 191*0 - "The effect of mechanical stress on the differentiation of scleral cartilage in vitro and in the embryo." Growth. l+:2l*5. Wetzel, P., 1925 - "Untersuchungen zm Huhnerkeim. I. Uber die Untersuchung des Lebenden Kelms mit Neueun Methode, Besonders der Vogtschen Vltalen Farbmarkierung. " Arch. f. Ent. mech. 106:1*65 (See ibid I929, Vol. 119:118 and 1931:Engebn. d Anat-u-Entw'gesch, 29:1). White, P. B., 191*6 - "Cultivation of animal tissues In vitro in nutrients of precisely known constitution." Growth. 10:251. Willler, B. H. , 19l*2 - "The control of hair and feather pigmentation as revealed by graft- ing melanophores in the embryo." Annals of Surgery. 116:598. Willler, B. H. & M. E. Eawles, 1931 - "The relation of Hensen's node to differentiating capacity of whole chick blastoderms as studied in chorio-allantoic grafts." Jour. Exp. Zool. 59:1*29 (See ibid 191*1*, Genetics 29:309). Willler, B. H. & M. E. Eawles, I938 - "Feather characterization as studied In host-graft combination between chick embryos of different breeds." Proc. Nat. Acad. Scl. 2l*:l*l*6. Wright, G. P., 1926 - "Presence of a growth-stimulating substance in the yolk of Incubated hen's eggs." Proc. Soc. Exp. Biol. & Med. 23:603. Zwilling, E., 19l*5 - "Production of tall abnormalities in chick embryos by transecting the body during the latter part of the second day of incubation." Jour. Exp. Zool. 98:257. GLOSSARY Many of the following terms were used for the first time within the last decade. The definitions are derived from numerous current writings of Experimental Embryologlsts and the author has attempted to incor- porate the various shades of meaning Into succinct statements. *********** ABORTION - termination of pregnancy at a non-viable stage of the foetus. ACHONDEOPLASTIC - refers to miniature adult skeletal condition of some midgets. ACIDOPHIL - oxiphll; cell constituents stained with acid dyes, often used to designate an entire cell type. (See basophil) ACTION, DYNAMIC - Weiss' variation 6f t;he field theory or concept. ACTIVATION - stimulation of spermatozoon to accelerated activity, generally by chemical means (e.g., fer- tllizin); process of initiating development in the egg; the liberation of naturally occurring evocators from an inactive combination. ACTIVATION CENTER - one of the two organization centers in the insect egg. ACTIVITY, FUNCTIONAL - stimulation or inhibition of a developing organ by environmental variables (e.g., hypertrophy of the urodele gills in an oxygen deficient medium). ADAPTATION - functional and correlative change, however brought about. ADAPTATION, FUNCTIONAL - an organ will adapt itself structurally to an alteration, qualitative or quantita- tive, of function (Boux). ADNEXA - extra embryonic structures discarded before the adult condition is attained. AESTIVATION - reduced activity during the summer period by some animals, term opposed to hibernation. AFFINITAT - tendency to contact; may be positive, resulting in maximal contact or may be negative, resulting in minimal contact (Holtfreter, 1939) • Not thigmotaxis. AFFINITAT, INTERFAZIALE - tendency to contact between marginal surfaces or interfaces of different blaa- tomeres ( Lehmann) . AFFINITAT, INTEABLASTEMATISCHE - tendency to contact between cells of the same blastema (Lehmann). AFFINITY - tendency of cells and tissues of the early embryo to cling together when removed from their normal environment. Equivalent to the cytarme of Boux. AGENESIS - developmental failure of a primordium (e.g., absence of arm or kidney). AGGLUTINATION - cluster formation; a spontaneously reversible reaction of spermatozoa to the fertllizin of egg-water. AGGREGATION - coming together of cells (e.g., spermatozoa) without sticking, a non-reversible response com- parable to chemotropism. AGNATHUS - absence of lower Jaw. AKINETIC - without a kinetochores (e.g., in a chromosome). ALIMENTAHY CASTBATION - prolonged starvation (Adams, 1930). ALLANTOIN - nitrogenous portion of allantoic fluid. ALLAKTOIS - an extra embryonic sac-like extension of the hindgut of amniotes, having the dual function of excretion and respiration. ALLO-HAPLOID - androgenetio haploid. ALLOMETRY - study of the relative sizes of parts of animals at different absolute sizes, ages, weights, or chemical compositions. Term now used in place of heterogony by Huxley and Teissier (1956). ALLOMOEPHOSIS - the physical or chemical relation of parts of an organism at some early stage to either the whole or part of a later stage, (e.g., the egg size compared with the adult size or weight). ALLOPHORES - red pigment in solution. ALLO- POLYPLOID - polyploid species hybrid. AMBOCEPTOR - a synonym used for fertillzln in suggesting its double combination with the sperm and egg receptors in the process of fertilization. This double receptor may also receive blood inhibitors, or antl -fertllizin. AMELUS - failure of the extremities to develop, remaining as mere stubs. AMNION - thin, double membrane enclosing the embryos of some invertebrates and of reptiles, birds, and mammals. It Is derived from the somatopleure in vertebrates. AMiilOTIC BANDS - fibrous bands from the amnion to the embryo due to local necrosis of foetal tissues. AMNIOTIC RAPHE - point of Junction of the amniotic folds as they encircle the embryo, synonymous with sero-amniotic or chorio-amniotic Junction. AMPHIBLASTIC - complete but unequal segmentation in teloleclthal eggs. AMPHITOKY - parthenogenetic reproduction of both males and females. AMPLEXUS - the sexual embrace of female ty the male amphibian. This may or may not occur at the time of ovi position. AMPHOTEROKY - production of both sexes in a- single parthenogenetic brood. ANALOGOUS - structures said to have the same function but different embryologloal and/or evolutionary origin. Opposed to homologous. ANDBOGAMONES - the antl-fertllizins of Llllle, so named Ijy Hartmann. An acidic protein of low molecular weight. ANDBOGENESIS - development of the egg with paternal (sperm) chronosomes only, accomplished by removing the egg nucleus after activation by the spermatozoon but before syngany (Wilson, 1925). May also be accomplished by irradiation damage of egg nucleus. -U5U- GLOSSARY 1+55 .ANDEO-MEBOGONES - egg fragmenta developing with the sperm nucleus only, achieved either through surgical removal of the egg nucleus and some cytoplasm; by constricting the pro-nuclei apart prior to syngamy; or by centrlfuging the pro-nuclei apart. ANEUPLOIDY - deviation from normal diploidy but involving partial sets of chromosomes (Tackhohn, 1922). ANEUPLOIDY, MULTIFOBM - complex chromosomal mosaics, possibly the result of multipolar mitoses (Book, 194U) ANEUBOGENIC - used in relation to organs developed vithout proper components of the central nervous system (e.g., limb buds in embryos without spinal cords). ANEMTEFION - formation and constriction of archenteron by evagination instead of invagination, following the application of heat (Driesch, I895). ANGENESIS - regeneration of tissues. ANLAGE - a rudiment; a group of cells which indicate a prospective development into a part or organ. Syn., ebauch^ or primordium. ANIMALIZATION - changing by physical or chemical means the presumptive fate of embryonic areas which normally would have become endodermal. Syn., ectodermlzation, or animal! si erxuig of Lindahl. ANOEMOGENESE - a course of development which deviates in a typical manner from the normal (Lehmann). ANTEEIOE - toward the head; head end. Syn., cephalic, cranial, rostral. APEOSOMUS - featureless face due to the arrest of development, the skin covering normal but lacking in eyes, nose, and mouth. AECHENCEPHALON - anterior portion of the brain which gives rise to the telencephalon and the dlencephalon; pre-chordal brain. AECHIPLASM - specific material which gives rise to the asters and spindle (Boveri). AEEA - a morphogenetic cell group representing one of the constituent regions of a fate-map, generally of a blastula stage or later. ABEAL - first an invisible, then a sharply differentiated region of the blastema, out of which develops a primitive organ; the organ arising from the blastema through segregation, ( organogenetlsches of Lehmann) . AREA OPACA - the marginal ring or extra-embryonic blaBtoderm of the chick embryo around the area pellu- cida, opaque because of direct contact with the underlying yolk. AEEA PELLUCIDA - the central portion of the chick blastoderm from which the embryo is developed, pellucid because it la lifted off of the underlying yolk, providing a space beneath the blastoderm through which light can be transmitted. AEEA VASCULOSA - the portion of the area opaca of the chick blastoderm in which the extra- embryonic blood vessels will develop. AEEA VITELLINA - the portion of the area opaca of the chick blastoderm peripheral to the area vasculosa. ARHHENOKAEYOTIC - refers to a blastomere of the normally fertilized egg where there has been a separation of the nuclear components; or in cases of dispemy, where the haploid chromosomes from the single sperm are isolated in the blastomere. AERHENOTOKY - parthenogenetic production of males, exclusively. ASSIMILATION - process of determinative incorporation of a foreign blastema into the functional status of the host blastema. ASTEE - the "atar-shaped structure" aurroundlng the centrosome ( Fol, 1877); lines radiating in all direc- tions from the centrosome during mitosis. ASTCMUS - complete lack or atresia of the mouth. ASYNTAXIA DOESALIS - failure of the neural tube to close. ATELIOTIC - arrested development of the skeleton due to non-union of the epiphyses, characteristic of some dwarfs . ATOKUS - without offspring. ATTRACTION, NON-SPECIFIC - attraction of nerve fibers toward any structure in the vicinity, (e.g., graft rudiments of chick embryos such as brain tissue, if placed on the chorlo-allantois, will often send out nerve fibers toward the nearby muscle segments, a situation that would not occur under normal conditions) . AUFLAGERUNG - the placing of competent or responsive ectoderm on a dead inductor in order to test the in- ductive power of the latter. AUTOGAMY - self-fertilization. AUTOPARTHENOGENESIS - parthenogenetic stimulation of eggs to develop by materials from other eggs. AUXESIS - growth by cell expansion but without cell division. AXIS - central or median line. The egg axis takes into account the concentration of deutoplasm, cyto- plasm, and the position of the nucleus, so that the egg axis and egg polarity are essentially the same. AXIS OF THE CELL - a line passing through the centrosome and nucleus of the cell. AXIS OF THE IMBFYO - a line representing the antero-posterlor axis of the future embiyo. BAHNUTJG - competence or labile determination (Vogt, 1928). BALANCER - cylindrical and paired projections of ectoderm with mesenchymatous cores, used as tactile and balancing organs by some urodeles in the place of (anuran) suckers. (Rudimentary or absent in A. tigrlnxm. ) BALFOUE'S LAW - the intervals between cleavages are longer the more yolk a cell contains in proportion to its protoplasm. "The velocity of aegmentation in any part of the ovum is, roughly speaking, propor- tional to the concentration of the protoplasm there; and the size of the segments is inversely pro- portional to the concentration of the protoplasm." (Balfour - "Comparative Embryology). U56 GLOSSARY BAEFUTH'S EULE - when oblique cuts are made on amphibian tails, the axis of the regenerated tall will be at first perpendicular to the cut surface (Barfuth, I89I). BATESON'S RILE - (a) The long axes of reduplicated structures lie In the same plane. (b) Two reduplicated limbs are mirror images of each other about a plane which bisects the angle between the long axes of the members, and which Is at right angles to the plane of these axes. BASOPHIL - cell constituents having an affinity for basic dyes, often used as an adjective for an entire cell. (See acidophil) BAUCHSTUCK - that portion of the amphibian gastrula (i.e., the ventral half) from which all the organizer area has been removed, thus preventing the formation of any neural axis. BEDEUTUNGSFKEKDE SELBSTDIFFERENZIEEUNG - self-differentiation independent of the original presiimptive fate or the presumed fate. Implied by the new environment. Neither selfwise nor neighborwise. BIDDER'S OKJAM - anterior portion of the gonad which is ovarian in character, developing from part of the rudiment conaistlng wholly of cortex. A structure indicating failure of medullary substance to dif- fuse to the anterior extremity of the gonad rudiment, found most frequently in male toads. BIO-ELECTKIC CURRENT - an electrical potential characteristic of life, disappearing upon death, associated with activities of muscle, nerve, secretion, and early embryos. BIOGENKTIC LAW - ontogeny is a recapitulation of the early development of ancestral phylogeny. Embryos of higher forms resemble the embryos of lower forms in certain respects but they are never like the adults of the lower (or ancestral) forms. Not to be confused with the recapitulation theory. BIOLOGICAL MiMOHY - ontogenetic unfolding of anlagen phyletlcally accumulated. BIOLOGICAL INTEGRATION - correlation of parts through neural or humoral (or both) Influences, acquired during development. BIOLOGICAL ORDER - fundamental basis of experimental studies, the conformity of biological processes to causal postulates. BIOBGAN - an organ in the physiological rather than the morphological sense. BIOTONUS - the ratio between assimilation and dissimilation, A/D ratio (Verwom). BLASTEMA - an Indifferent group of cells about to be organized into definite tissues, kept together by the ectoplasmic matrix of the constituent cells. Considered to be primitive, embryonic, relatively undifferentiated regenerating cell masses. Thought by some to be produced by reserve cells which were arrested during earlier embryonic development. BLASTEMFELD - unitary field-like structure (or functional state) without an anlage. Primordial fields present in the egg stage, and other fields activated only by the processes of induction during development (Lehmann). BLASTOCOEL - cavity of the blastula. Syn., segmentation or subgermlnal cavity. BLASTODERM - "Because the embryo chooses this as its seat and its domicile, contributing much to its configuration out of its own substance, therefore, in the future, we shall call it blastoderm." (Pander, I8l7). BLASTOKINESIS - a reversal of the cephalo-caudal axis in an egg, often accomplished by movement during early development (e.g., insects). Syn., revolution. BLASTOMEMl - one of the cells of the early cleavage of an egg. When there is a discrepancy in size the smaller blaetomere is a micromere; the intermediate one is a mesomere; and the larger one is a macro- mere, but all are blastomeres . BLASTOPORE - the opening of the archenteron (gaatrocoel) to the exterior, occluded by the yolk plug in amphibian embryos; consisting of a slit-like space between the elevated margin of the blastoderm and the underlying yolk of the chick blastoderm; and represented In the amnlota as the primitive streak. Approximate region of the future anus. BLASTOPORE, DOBSAL LIP OF - the region of initial Involution of cells in the amphibian or chick gastrula; general area of the "organizer"; original grey crescent area of many amphibia; the cells which turn in bene'ath the potential central nervous system (Amphioxus) and form the roof of the archenteron (urodela). Syn., germ ring or marginal zone. BLASTOPORE, VENTRAL LIP OF - region of the germ ring opposite the dorsal lip after involution has reached this point; region which gives rise to the peristomlal mesoderm of the frog. Syn., germ ring. (Note: The lips of the blastopore are continuous and represent the involuted germ ring.) BLASTOTOMY - separation of cells or groups of cells of the blastula, by any means. BLASTULA - a stage in embryonic development between the appearance of distinct blastomeres and the end of cleavage (i.e., the beginning of gastrulation) ; a stage generally possessing a primary embryonic cavity or vesicle known as the blastocoel; invariably monodermlc, although the roof may be multi- layered. BLOOD ISLANDS - pre-vaacular groups of mesodermal cells found in the spleinchnopleure, from which arise the blood vessels and corpuscles. Generally extra-embryonic (chick). BCiTLE CELLS - long-necked, cylindrical cells of the blastoporal lips (amphibia) whose function may be purely morphogenetlc and related to the involutionary processes of gastrulation, (Holtfreter, 19'*5)- Rufflnl (1925) showed that flask-shaped cells appear wherever Infoldings occur, as in the formation of the neural tube, eye vesicle, nasal placode, atomodeum, proctodeum, etc. These cells are held together by strands of surface coating. BBADYAUXESIS - negative heterogony ( Needham & Lemer, 19l*0), the part grows more slowly than the whole. BRADYGENESIS - lengthening of certain stages in development. BBAMCHIAL - having to do with respiration (e.g., branchial vessel In gill). BBAMCHlOMEHif - type of metamerism exemplified in the visceral arches. BEYSHTHALMIA - eyes that are too large, may be due to oversized lenses. BUD - an undeveloped branch, generally'an anlage of an appendage (e.g., limb or wing bud). BUPHTHALMIA - eyes that are .too large (Harrison, 1929)- GLOSSARY 1*57 CACOGENESIS - Inability to hybridize; means "bad descent" (kakogenesis) . CAENOGENETIC - term for new stages in ontogeny which have been intercalated as an adaptation to some in- evitable condition which the mode of life of the young animal Imposed ( Baeckel) . CALCIUM- RELEASE THEORf - theory of Heilbrunn that the activating agent in parthenogenetic stimulation releases calcium from calcium proteinate in the cell cortex, and the free calcium then brings about a protoplasmic clotting necessary to the initiation of development. CABCINCGEN - a chemical substance which is capable of causing living cells to become cancer-like In growtl. and behavior. CABYOLYSIS - solution or dissolution of the nucleus. CASYOEHEXIS - breaking up of the nucleus, or its rupture. CELL - protoplasmic territory under the control of a single nucleus, whether or not the territory is bounded by a discrete membrane. ^ this definition a syncitium is made up of many cells with physio- logical rather than morphological boundaries. CELL CHAIN THEORY - theory of neurogenesis wherein the peripheral nerve is of pluricellular origin; op- posed to the outgrowth theory. CELL-CONE - a sub-system of an ordered class of cells; a single cell (other than a zygote) and all cells derived from it in a division heirarchy. CELL LINEAGE - the study of the origin and fate of specific cells ( blastomeres ) in early embryonic development. Syn., cytogeny. CELLULATION - development of cytoplasmic areas around normal (syncltial) nuclei or by nuclei migrating from living blastomeres as in the chick blastoderm. CELL THEORY - the body of any living organism is either a structural and functional unit or Is composed of a nucleus and its sphere of influence, whether or not that sphere Is bounded by a norphologlcal entity. "Omnis cellula e cellula." Vlrchow CENTRIOLE - the granular core of the centrosome, the radiating area comprising the centrosphere. Appears within the centrosome during mitosis, (Conklln). CENTROSOME - the dynamic center Involved in mitosis, including the central granule (centriole) and the surrounding sphere of rays (centrosphere). It is the center of the aster which outlasts the astral rays. Double centrosome called diploaome. CENTROSOME, HETEROOTNAMIC - Ziegler's hypothesis that centrosomes may have different powers, thereby causing unequal division of the blastomeres such as occurs In many molluscs (e.g., Crepidula) . No evidence of this although there are occasionally size differences In asters of the same spindle complex. CEPHALO- THORACOPAGUS - fusion of the head and chest regions in conjoined twins. CERVICAL CYST - imperfect occlusion of a branchial (2nd) cleft. Syn., branchial cyst. CERVICAL FISTULA - incomplete closure of the branchial cleft. CHALONES - Internal secretions with depressing effects, opposed to hormones. CHEWO- NEUROTROPISM - chemical attraction of degenerating nerve upon regenerating nerve fibers. The chemical nature of nerve orientation (growth and connectlona) depending upon diffusing substances which seem to attract nerve fibers. CHIMEEA - compound embryo derived by grafting together major portions of two embryos, generally of dif- ferent species; exchange of parts too great to be called a transplant. From Greek nythology: fore- part a lion, middle a goat, and hlndpart a dragon. CHORDA-MESODERM - region of the dorsal lip of the blastopore, arising from the grey crescent area, destined to give rise to notochord and mesoderm In the amphibia. CHORIO-ALLANTOIS - a common membrane formed by the fusion of the inner wall of the chorion and the outer wall of the allantois (chick), consisting of outer ectoderm, intermediate fused mesoderm, and inner endoderm. CHORIO-ALLANTOIC GRAFT - graft from various sources which, by virtue of Its weight and other factors, provides local irritation of the chorio-allantoic membrane of the chick so that the graft becomes vascularized and surrounded by indifferent tissue, offering the graft excellent conditions for sur- vival, growth, and differentiation. Graft not incorporated by the host from which it receives nutrition for growth. CHORION - an embryonic membrane developed in the chick as a corollary to the amnion; encloses both the amnion and the allantois. Never maternal in mammals. CHROMATID - longitudinal half of an anaphase. Interphase, or prophase chromosome at mitosis. One of four strands (in melosls) involved in crossing over and visible after pachytene. Becomes a chromosome at metaphase of the second (reduction) division. CHROMATIN - deeply staining substance of the nuclear network and the chromosomes, consisting of nucleln; gives Feulgen reaction and stains with basic dyes. CHROMATOBLASTS - potential pigment cells which, upon proper extrinsic stimulation, will exhibit pigmen- tation. CHROMATOPHORE - pigment bearing cell frequently capable of changing size, shape, and color; responsible for superficial color changes in many animals (e.g., squid £ind chameleon), under the Influence of the sympathetic nervous system and/or the neurohumors. CHROMIDIA - extra-nuclear granules of chromatin. CHROMONEMA - optically single thread within the chromosome, a purely descriptive term without func- tional implications. CHROMONUCLEIC ACID - one of the two types of nucleic acid detected in chromatin only (Polllster & Mirsky, 19'+'^). Syn., desoxyribose nucleoproteln, thymonuclelc acid. (See plasmonuclelc acid.) CHROMOPHOBE - cells whose constituents are non-stalnable; no affinity for dyes. 1*58 GLOSSARY CHEOMOSOME - the chromatic or deeply staining bodies derived from nuclear network, which are conspicuous during mitotic cell division and which are represented in all of the somatic cells of an organism In a number characteristic for the species; bearers of the genes. CHROMOSOME ABEREATION - an Irregularity in the constitution or the number of chromosomes which may pro- duce modifications in the normal course of development. CHEOMOSOMIN - acidic protein, present In nuclei, considered an essential part of the chromosomes (Stedman, 19''5). CLEAVAGE - the mitotic division of an egg resulting in blastomeres. Syn., segmsntation. 'CLEAVAGE, ACCESSOEY - cleavages in peripheral or deeper portions of the (chick) germinal disc caused by supernumerary sperm nuclei following (normal) polysperny. CLEAVAGE, ASYMMETRICAL - extremely unequal divisions of the egg aa In Ctenophores. CLEAVAGE, BILATERAL - cleavage in which the egg substances are distributed symmetrically with respect to the median plane of the future embryo. CLEAVAGE, DETERMINATE - cleavage in which certain parts of the future embryo may be circumscribed In cer- tain specific (early) blastomeres; cleavage which produces blastomeres that are not qualitatively equlpotential, (i.e., when such blastomeres are isolated they will not give rise to entire embryos). The early embryo is a mosaic of qualitatively different blastomeres with respect to further ontogeny. CLEAVAGE, DEXIOTROPIC - cleavage resulting In a right-handed production of daughter bla8tomere(s), as In some cases of spiral cleavage. CLEAVAGE, DISCOIDAL - cell division restricted to a more-or-less circular disc of protoplasm to one side of a relatively enormous mass of yolk (e.g., chick egg). Syn., meroblastic cleavage. CLEAVAGE, EQUATORIAL - cleavage at right angles to the egg axis, opposed to vertical or meridional. Often the third cleavage plane. Syn., latitudinal or horizontal cleavage. CLEAVAGE, HOLOBLASTIC - complete division of the egg into blastomeres, generally equal in size (Asterlas, Arbacia) although not necessarily so (Amphloxus, Frog). Syn., total cleavage. CLEAVAGE, HORIZONTAL - (See cleavage, equatorial.) CLEAVAGE, INDETERMINATE - cleavage resulting in qualitatively equl -potential blastomeres in the early stages of development. When such blastomeres are Isolated from each other they tend to give rise to complete embryos. Opposed to mosaic development. Syn., regulatory cleavage, or development. CLEAVAGE, LATITUDINAL - (See cleavage, equatorial.) CLEAVAGE LAWS - (See specific laws under names of Balfour, Hertwlg, and Sachs.) CLEAVAGE, LEVOTEOPIC - cleavage resulting In left-handed or counter-clockwise production of daughter bla3tomere( 3) as In some cases of spiral cleavage. CLEAVAGE, MERIDIONAL - cleavage along the egg axis, opposed to equatorial. Generally the first two cleavages of any egg. Syn., vertical cleavage. CLEAVAGE, MEROBLASTIC - (See discoldal cleavage.) CLEAVAGE NUCLEUS - the nucleus which controls cleavage. This may be the syngamlc nucleus of normal fertilization; the egg nucleus of parthenogenetlc or gynogenetlc eggs; or the sperm nucleus of androgenetic development. CLEAVAGE PATH - path taken by the syngamlc nucleus to the position awaiting the first division. CLEAVAGE, RADIAL - holoblastlc cleavage which results in (two) super-imposed tiers of cells as early as the 8-oell stage. Opposed to spiral cleavage. CLEAVAGE, SPIRAL - cleavage at an oblique angle with respect to the egg axis so that the resulting blas- tomeres (generally upper mlcromeres at the 8-cell stage) lie in an interlocking fashion within the furrows of the original blastomeres. The shift In expected position is due to intrinsic genetic factors rather than external pressure, (e.g., Mollsuca.) Opposed to radial cleavage. CLEAVAGE, SUPERFICIAL - cleavage around the periphery of centrolecithal eggs. Syn., peripheral cleavage. CLEIDOIC - refers to eggs that are more-or-less closed off from their environment (e.g.. Chick). CLINOSTAT - apparatus for keeping objects In constant rotation. COADAPTATION - correlated variation in two mutually dependent organs. COELOBLASTULA - spherical ball of cells (blastomeres) developing in early cleavage as a result of segmen- tation, provided with a large central cavity (blastocoel) . (e.g., Echlnodermata. ) COELCM - mesodermal body cavity of chordates, from the walls of which develop the gonads. It Is sub- divided in higher forms Into pericardial, pleural, and peritoneal cavities. Extended as the exocoel or extra embryonic body cavity of chick embryo. COENOBLAST - the layer which will give rise to the endoderm and mesoderm (obsolete). COLLOID - dispersed substance whose particles are not smaller than lu nor larger than 100)i, approximately. Physical state of protoplasm. COLORLESS PIGMENT CELL - same as dependent or potential pigment cell of DuShane and Hamilton. Syn., farbloae plgmentzellen. COMPETENCE - state of reactivity, of dis-equlllbrium In a complex system of reactanta. Possessing labile determination (Reaklonafahlg) or having reaction possibility (Raven). Competencies may appear simultaneously or \n sequence within a given area, some to disappear later even without function. Embryonic competence seems to be lost in all adult tissues but may be reclaimed in a blastema. It is a name for the state of the cell area at or before the time when irritability is resolved and a developmental path is chosen. The word supercedes the older words of "potence", "potency", or "potentiality" ' COMPRESSION - either the acceleration of development or the extension of a certain (e.g., pre-hatchlng) period, resulting in the completion (or omission) of certain (larval) stages, in an unbalanced time schedule. GLOSSARY 1*59 CONCBESCENCZ - the coming together of previously separate parts (cell areas) of the embryo, generally re- sulting In a piling up of parts. One of the corollaries of gastrulation where a bottle-neck of cell movements occurs at the lips of the blastopore. Original meaning (His, IST"*) referred to presumed pre-formed parts of the fish germ ring. (See confluence.) CONE, EXUDATION - (See cone fertilization.) Term used by Fol, 1879. CONE, FEETILIZATION - a conical projection of the cytoplasm from the surface of the egg to meet the spermatozoon which is to invade the egg cortex. The cone makes contact and then draws the sperm in- to the egg. Not universally demonstrated but seen in the starfish (Chambers). Syn., exudation cone. CONES OF GBOWTH - the enlarged outgrowth of the neuroblast forms the axis cylinder or axone of the nerve fiber and is termed the cone of growth because the growth processes by which the axone Increases in length are supposed to be located there. CONFLUENCE - similar to concrescence except that this term refers specifically to the "flow" of cells (or cell areas) together, without presumption of any organ preformation. Said areas have certain potentialities in unaltered normal development. (See concrescence.) CONSTBICTION - gradual closure of the blastopore (germ ring) over the yolk toward the vegetal pole. May be due to stretching of the marginal zone, to a pull or tension of the dorsal lip, or even to the narrowing of the marginal zone. Syn., convergence of Jordan or Konzentrlsches Urmundschluss of Vogt. CONVEEGENCE, DORSAL - material of the marginal zone moves toward the dorsal mid-line as it involutes and invaglnates during gastrulation, resulting in a compensatory ventral divergence. Syn., confluence of Smith or dorsal Eaffung of Vogt. CORDS, MEDULLARY - structures which give rise to the urogenital connections and take part in the forma- tion of the seminiferous tubules, and are derived from the blastema of the mesonephrlc cords (amphibia). CORDS, SEX - strands of somatic cells and primordial germ cells growing from the cortex toward the medulla of the gonad prlmordium. Best seen in early phases of testes development. CORRELATION COEFFICIENT - correlation of growth rates of different parts (of embryo). COETICIN - sex differentiating substances spread in some amphibia by the blood stream and in other forms by diffusion, acting as a hormone. (See medullarln. ) CRANIAL - relative to the head; "cranlad" means toward the head. Syn., rostral, cephalad. CEANIOPAGUS - cranial union in conjoined twins. CRANIOSCHISIS - open- roofed skull associated with undeveloped brain. Syn., acranla. CRESCENT, GREY - crescentic area between the original animal and vegetal hemispheres on the surface of the (frog) egg, grey In color because of the migration of black pigment away from the area and to- ward the sperm entrance point (Eoux, 1888) which Is therefore opposite; region of the presumptive chorda-mesoderm, the future blastopore and anus. CRESCENT, YELLOW - crescentic area on the surface of the (Aecldian) egg, yellow In color. Gives rise to the mesoderm of such embryos. CREST, NEURAL - paired cell masses derived from ectoderm cells along the edge of the former neural plate, and wedged Into the space between the dorso-lateral wall of the closed neural tube and the Integu- ment. Gives rise to spinal ganglia after segmentation. CREST SEGMENT - the original neural crests which become divided Into segments, with the aid of the somites, from which develop the spinal and possibly also some cranial ganglia. CROSS- FERTILIZATION - union of gametes produced by different individuals which, if they are of different species, may produce hybrids with variable viability. CYCLOPIA - failure of the eyes to separate; median fusion of the eyes which may be due to suppression of the rostral block of tissue which ordinarily separates the eyes; exaggeration of the vegetatlviza- tlon tendencies. CYTARME - flattening of previously rounded blastomerea against each other following the coii5)letlon of cleavage. CYTASTERS - asters arising Independently of the nucleus in the cytoplasm. May contain centrosomes, and achromatic figure with attraction sphere and astral rays and may divide and even cause the cyto- plasm around them to divide. Activity and structure unrelated to chromosomal material. CYTE - a suffix meaning "cell" as oo-cyte (egg forming cell), spermato-cyte (sperm forming cell), or osteo-cyte (bone forming cell). (See specific definitions.) CYTOCHORISMUS - apparent partial separation of the blastomeres at the flat, previously continuous sur- face (Eoux). CYTOCHRCME - an oxldlsable pigment found in nearly all cells exhibiting definite spectral bands in re- duced form, discovered by Kellin (1925). Insoluble in water, poisoned by HON, COg, and 1^3. CYTOLISTHESIS - tendency of embryonic cells to aggregate and to fill up disruptions of their union even in the absence of a common surface membrane, due to surface tension and selective adhesiveness (Roux, I89I*). Moving of cells over one another by sliding, rotation, or both processes. CYTOLYSIS - breakdown of the cell, indicated by dispersal of formed components. CYTOSOME - cytoplasmic mass exclusive of the nucleus. CYTOTAXIS - coming together of (amphibian) blastula cells after being teased apart in salt solution (positive cytoteucls). Crawling, amoeboid movement similar to chemotaxis (Roux). May also Include repulsive movements of cell groups (negative cytotaxis). CYTOLEOSIS - process by which a cell, already irreversibly differentiated, proceeds to its final specialization (Hoadley). CYTOTROPISM - inevitable movement of a cell in response to external forces (Rhumbler, 1899). U60 GLOSSARY DABK-FIELD RING - an orange-yellow colored Illuminated ring aa opposed to the silvery white surface of the sea urchin egg as described by Ruunstrom (1928) under dark field Illumination. Not related to echlno- chrome. ttey be the area which Is Invaglnated during gastrulatlon. DEDIFFEREnroiATION - process of giving up specialized characters and returning to the more primitive (embryonic) conditions, supposedly regaining the original and wider range of potencies. Manifestation of powers of cell adaptation to an abnormal environment such aa in tissue culture, not normally found in tie living organism except possibly in the blastema of regenerating tissue. The existence of thla change in cell structure and function now questioned. Syn., catachony and einschmelzung. DEFLECTION - when dedifferentiated cells remain unable to redifferentlate, lying outside the area deter- mined by the term modulation. Cells turned away from the line of normal ontogenesis (Kasahara, 1955). DEGHOWTH - actuql reduction in mass subsequent to prolonged period of growth, probably indicating greater catabolic than anabolic processes. Follows inanition. DELAMINATION - separation of cell layers by splitting, a process of mesoderm formation. DETERMINANT - a Weismanlan concept of a corpuscular unit which determines the qualities and actions of cells In which it is contained. Determinants possess powers of growth and propagation and together constitute the germ plasm. This concept suggests that histological differentiation is brought about by differential division until a single determinant is left within the cell. DETERMINATION - a process of development Indicated when a tissue, whether treated as an isolate or a trans- plant, still develops In the originally predicted manner; the fixing of fates or final assignments of parts of the embryo at definite ontogenetic time; the firm capacity of a tissue for self-differentia- tion from which it cannot be deterred, no matter what its environment, within viable limits. An embryologlcal rather than a genetic concept. (Harrison, 1953' Am. Nat. 67:506) DETEJMINATION, DYNAMIC - opposed to induction and refers, for example, to the tendency of the marginal zone to Invaglnate even when transplanted. (Vogt, I925.) Formative movements. DETEMINATION, FIELD - state of organization within an embryonic areas probably Independent of the sub- strate; field of action ( Wirkungafeld) or province of action (Wirkungskreis) of Weiss (I925). (See Field.) DETEBMINATION, LABILE - definite but not irrevocably fixed ability of tissue exposed to inductive influ- ences to continue development in the induced direction even though isolated as fragments. Syn., com- petence, or Bahnung. DETERMINATION, MATERIAL - formative movements which result in histological differentiation. DETERMINATION, NEGATIVE - lack of certain essential ingredients within the blastomere necessary to the formation of a complex embryo (e.g., blastomere "D" in Dentalium and Tublfex). DETERMINATION, PROGRESSIVE - determination in time rather than in space, advancing from the more general to the more specific. DETERMINATIONSGESCHEHEN - all of the Invisible processes In a blastema (and its vicinity) which determine the morphogenesis of the region. These processes may Involve two phases, those of self -organization and those of segregation. (Lehmann, 19^*2.) Syn., determinatorsystem or reallsatorayatem ( Lehmann, 191+2 ) . DEUTENCEPHALON - caudal region of the brain which later forma the mesencephalon, metencephalon and myelencephalon. DEUTEROKY - reproduction of both sexes from parthenogenetic eggs (see arrhenotoky and thelytoky). DEUTOPLASM - yolk or secondary food substance of the egg cytoplasm, non-living. DEVELOPMENT - gradual transformation of dependent differentiation Into self-differentiation; trans- formation of invisible multiplicity Into a visible moaalc; elaboration of components In successive spatial hierarchies. DEVELOPMENT, MOSAIC - "all the single primordla stand aide by side, separate from each other like the stones of a mosaic work, and develop independently, although in perfect harmony with each other, into the finished organism." (Spemann, I958). Some believe there Is pre-locallzation of embryonic potencies within the egg, the teat for which would be aelf-differentiatlon. DEVELOPMENT, REGULATIVE - type of development requiring organizer or inductor Influences since each of the early blastomeres could develop into whole embryos. Structxirea are progressively determined through the action of evocators. DIAPAUSE - a normal state of dormancy in the development of some anlmala (e.g.. Insects) not to be con- fused with hibernation because this condition is Independent of any environinental factors. DICEPHALUS TETRABRACHINUS - condition attained when the first furrow of the amphibian egg coincides with the sagittal plane and the constriction Is exaggerated, resulting In duplications of the chorda, auditory vesicles, and fore-limbs. DICHIRUS - partial duplication of digits in hand or foot, possibly inherited. A type of Polydactyly. DICHOTCMY, DIFFERENTIAL - embryonic segregation; capacity of embryonic cells for self-differentiation becomes itself differentiated (Llllie, 1929). DIFFERENTIATING CENTER - area responsible for the localization and determination of various regions of the embryo, resulting In harmonious proportioning of parts. DIFFERENTIATION - acquisition of specialized features which distinguish areas from each other; progres- sive increase in complexity and organization, visible and invisible; elaboration of diversity through determination leading to histogenesis; production of morphogenetlc heterogeneity. Syn., dlfferenzlerung. DIFFERENTIATION, AXIAL - variations In density of chemical and often indefinable Inclusions in the direction of one diameter of the egg, called the egg axis (see gradient). GLOSSARY 461 DIFFERENTIATION, CELLULAE - the process which results In specialization of a cell as measured by its dis- tinctive, actual, and potential functions (Bloom, 1937) • DIFFERENTIATION CENTER - one of the true organization centers in the developing insect egg. Syn., dif- ferenzier^ingszentrum (of Lehmann, 19'*'2). DIFFERENTIATION, CORPORATIVE - differentiation resulting from the physiological functioning of parts. DIFFERENTIATION, DEPENDENT - all differentiation that la not self-differentiation; the development of parts of the organism under mutual Influences, such Influences being activating, limiting, or in- hibiting. Inability of parts of the organism to develop Independently of other parts. Such a period in ontogeny always precedes that of Irreversible determination. "Experimental embryology is a study of the differentiations which are dependent, causally effected." (Roux, 1912). Syn., correlative differentiation, Abhflnglge Dlfferenzlerung, Differentiation provoquee. DIFFERENTIATION, FITNCTIONAL - differentiation of tissues resulting from forces associated Vlth functions (stresses and strains) which they are performing. DIFFERENTIATION, INDIYIDUATIVE - differentiation due to the action of morphogenetlc fields rather than to physiological functioning of parts; opposed to corporative differentiation. DIFFERENTIATION POTENCY - the total repertoire of tllfferentiations, cytological and histological, avail- able to a given cell. Wider significance than prospective fate. DIFFERENTIATION, REGIONAL - refers to fact that different parts of the organizer will induce different end organ formation; also refers to organ districts In a (limb) field. DIFFERENTIATION, SELF - the perseverance in a definite course of development of a part of an embryo, re- gardless of its altered surroundings (Roux, 1912). Syn., differentiation spontanee. DIKENETIC - dicentric, having two klnetochores. DIMEGALY - possessing spermatozoa of two sizes. DIPHYGENIC - having two types of development. DIPLICHRCMOSOIE - two identical chromosomes, held together at the kinetochore and originated by doubling of chromosomes without separation of daughter chromosomes. DIPLOID - normal number of chromosomes, double the gametic or haploid; complete set of paired chromosomes as in the fertilized egg or somatic cell. DIPYGUS - (See dupllcatus inferior.) DISSOGENY - having two sexually mature periods, one as a larva and one as an adult. DISTRICT - a portion of a morphogenetlc field with certain specific determinations. Syn., terrltolre. DIVERGENCE, VEMTEAL - divergence of material from the mid-ventral line, compensatory to the process of dorsal convergence in gastrulation (Vogt). DIVISION HEIRAECHY - "four dimensional array of cells of which one and only one member (the zygote) is before all other members In time, and Is the only one to which every other term stands in a relation which is some power of D (i.e., the relation is Dpo)." (Bertelanffy & Woodger, 1953.) DOMINANCE - in embryology this term refers to parts of a system which have greater growth momentum and also which gather strength from the rest, such as the dorsal blaetoporal lip. DOWNAN EQUILIBRIUM - distribution of ions on two sides of a semi -permeable membrane with diffusion until concentration of diffusible ions on the two sides of the membrane is the same, involving ionic rather than molecular balance. DOPA - 3:lt:dloxyphenylalanin, ein intermediate oxidation product of tyrosine and one that appears as a precursor of melanin pigment in mammals (Black, 1917). DOESO-VEMTRAL - orientation of a graft or transplant so that the original dorsal-ventral axis is Inverted in relation to the host field. D-QUADRANT - one of the four early blastomeres of the annelid embryo which has the prospective function of giving rise chiefly to mesoderm. DOUBLE ASSURANCE - cases where inductions usually occur but are not absolutely necessary; two processes working together, either one of which would be sufficient to accomplish the end result. Ability to bring about a morphogenetlc process by means other than the usual one, (e.g., 'removal of the eye cup in R. esculenta and the overlying ectoderm will form a lens anyway, without the normal Inducing Influences of the eye cup). Term used by Rhumbler (1897) in connection with cell division and by H. Braus (1906) in development. Syn., doppelte sicherung. DUPLICITAS CRUCIATA - double monsters, obtained by grafting or by inversion of the 2-cell amphibian embryo. DUPLICITAS INFERIOR - conjoined twins fused anteriorly, having two rumps. Syn., dipygus. DYSMEROGENESIS - cleavage resulting in unlike parts. DYSPLASTIC TREATMENT - introduction of a transplant from organism of a different phylum. (E.g., frog to mammal or vice versa. ) DYSTELEOLOGY - apparent lack of purpose in organic processes or structures although they may ultimately be shown to be teleological. ECDYSIS - process of moulting a cuticular layer, shedding of epithelium by amphibia. ECHINOCHROME - red pigment of Echlnoderm eggs which probably has respiratory function. ECTODERM - the outermost layer of a didermic embryo (gastrula). Syn., epiblast. ECTOPIC - out of Its normal position, used in connection with transplants. ECTOPLASM - external layer of protoplasm of the (egg) cell, the layer immediately beneath the cell mem- brane. Seat of Lillle's fertilizin and of all developmental processes, according to Just. Syn., egg cortex plasmalemma. EGG, ALECITHAL - eggs with little or no yolk. EGG, CLEIDOIC - egg which is covered by a protective shell (e.g., eggs of reptiles, birds, and oviparous mammals ) . U62 GLOSSARY EGG, ECTOLECITHAL - egg having the formative protoplasm surrounded by yolk. EGG ENVELOPE - material enveloping the egg but not necessarily a part of the egg, generally derived from the ovary (vitelline membrane or chorion of fish) or from the oviducts (Jelly or albumen). BtXJ, GIANT - abnormal polyploid condition where chromosome complexes are multiplied, resulting In giant cells and embryos. EGG, HOMOLECITHAL - egg which has little yolk scattered evenly throughout the cytoplasm (e.g., sea urchin, mammal). Syn., Isoleclthal egg and obsolete term, aleclthal egg. EGG JELLY - the mucin covering deposited on the amphibian egg as It passes through the oviduct. EGG, MACEOLECITHAL - egg with large amount of yolk, generally teloleclthal. EGG MEMBRANES - Includes all egg coverings such as vitelline membrane, chorion, and the tertiary coverings from the oviduct. EGG, MICROLECITHAL - egg with small amount of yolk. Syn., meloleclthal, ollgoleclthal. EGG RECEPTOR - part of Llllle'a scheme picturing parts that go Into the fertilization reaction Involving fertlllzln. ifeg receptor plus amboceptor plus sperm receptor gives fertilization. EGG, TELOLECITHAL - egg with large amount of yolk concentrated at one pole. EGG WATER - watery extract of materials diffusing from living (Echlnoderm) eggs, presumably the "fertlllzln" of Llllle. Syn., egg water extract. EIDOGEN - a chemical substance possessing the power to modify an embryonic organ otherwise induced; force In regional differentiation, possibly including Inductors of the second-grade level. EINSTECKUNG - method of testing the power of induction by implanting a tissue, living or dead, or a chemical substance, into the blastocoel of a living gastrula. ELECTRODYNAMIC THEORY OF DETOLOPMENT - theory that cell mitoses establish a definite differential poten- tial capable of orienting growing nerve roots (axis cylinders) and thereby directing them (e.g., to- ward the brain) . EMANCIPATION - dynamic segregation from "autonomisatlon" (Weiss, 1935); establishment of local autonony within embryonic areas. IMBRYO - a stage in. the ontogeny of the fertilized egg limited to the period before the Intake of food. EMBRYOMA - (See teratoma) EMBRYONIC FIELD - region of formative processes within the embryo, larger th£m the area of ultimate realization of structures concerned (Gurwitsch, 1922). EMBRYONIC SHIELD - a thickened, shield-like region of the blastoderm which will give rise to the body of the (fish) embryo. EMBBYOTHOPHY - the means or the actual nourishment of the embryo. ENCAPSIS - superordinate system within the embryo. Processes may be purposeful for a subordinate system and yet destroy another system to which it itself Is subordinate. These relations are called encapsla ( Heidenhain) . ENCHYLEMA - the liquid phase of the endoplasm in which are suspended yolk granules and mitochondria (Monne) . ENDODEHM - the Innermost layer of the dldermlc gastrula. Arises from the vegetal hemisphere of amphibia. Syn., entoderm. ENDODERMISATION - shifting of an animal-vegetal gradient of an egg toward the vegetal gradient, causing hyper-development of the endodermal structures. Can be brought about by physical, chemical, or surgical means. ENDOPLASM - Inner medullary substance of the egg cell which Is generally granular, soft, watery, and less refractory than the ectoplasm. Surrounded by ectoplasm. ENDYSIS - the development of a new cuticular covering, opposed to ecdysis. ENTELECHY - Driesch's theory of harmonious, equlpotentlal system suggested an agent controlling develop- ment which he termed "elan vital". An Intensive manifoldnesa; the intangible controlling order of developnent ("Intensive Mannigfaltigkelt" of Driesch). ENTOMESOBLAST - cell which will give rise to the trunk mesoderm in the determinate type of cleavage characteristic of annelids. ENTO-MESODESM - refers to that portion of the invaglnatlng blastoporal lips which will induce the forma- tion of medullary fields in the amphibia. ENTOPIC - in the normal position, opposed to ectopic (referring to transplsints) . ENTRANCE CONE - the ten^oraiy depression on the surface of the egg following the entrance of the spermatozoon. ENTRANCE PATH - (See path, entrance or penetration) ENTWICKLUNGSMECHAHIK - causal embryology (Roux); the seat and effective duration of the morphogenetic forces which are explored, by microsurgical means, and which seem to be responsible for the develop- ment of embryonic .segregation (Lehmann, 19'+2). ENTWICKLUNGSPOTENZEN - the total accomplishment of a blastema, experimentally determined (Raven). EPIBOLY - growing, spreading, or flowing over; surrounding of inner masses (yolk and/or cells) by over- growing ectoderm; process by which the rapidly dividing animal pole cells (often mlcromeres) grow over and enclose the vegetal hemisphere material. Increase in areal extent of the ectoderm. (Simile: rubber cbp being pulled down over grapefruit.) EPIGAMIC - tending to attract the opposite sex. EPIGENESIS - developing of systems starting with primitive, homogeneous, lowly organized condition and achieving great diversification. Term coined by Harvey, the eLntlthesis of preformation. EPIGNATHUS - union upon the Jaw of parasitic growth. EPIMOEPHOSIS - proliferation of material precedes the development of new parts. ERGASTOPLASM - basophilic parts of the cytoplasm, mitochondria of cytologlats. GLOSSARY k63 EBEEBA'S LAW - "a cellular membrane at the moment of its formation tends to assume the form which would be assumed under the same conditions by an elastic membrane destitute of weight" (Gray, 1931). ESTEOGEN - secretion product of the ovary which controls oestrus and endometrial growth. ESTBOUS CYCLE - the periodic series of changes which occur in the mammalian uterus, related to the prepara- tion of the uterus for implantation of the ovum, and to repair. ESTBUS - period of the reproductive cycle of the mammal when the uterus is prepared for implantation of the ovum. ETHEOGENESIS - development of the spermatozoSn without fertilization; male parthenogeneaia. EUCHEOMATIN - the part of the regular chromatic structure of the nucleus which Is rich in thymonucleic acid, and presumably the genes, alternating (in the chromosomes) with achromatic regions. It is in the form of discs, and takes methyl green stain. EUPLOIDY - deviation from the normal diploid condition but Involving complete sets of chromosomes (Tackholm, 1922). EUTELY - constancy of cell numbers in the various organs of plants and animals, more dependable in animals than in plants (Wettstein, 1927, Heilbom, 1954). EVAGINATION - the growth from any surface outward. EVOCATION - mere calling forth of potentialities through contact; non-assimilative induction, no organiza- tion except that which is present within the host material; that portion of the inductive response which can be achieved by killed, crushed, or narcotized organizers of any level. EVOCATOE - a chemical substance which has the power of calling forth the latent potentialities of an embiyonic area; a morphogenetic stimulus. EXCLUSIVITY - discreteness of the differentiation process (Weiss). EXGENITO-VIVIPAEITY - the embryo in a stage of development which corresponds to the egg stage of ovo- viviparous forms, obtains its nourishment by means of a trophamnion, trophoserosa or trophochorlon. Development occurs in the haemocoele, not in the uterus (e.g., Strepsyatera). EXOGASTEULA - gaatrulation modified experimentally by abnormal conditions so that invagination is partial- ly hindered and there remains some mesendoderm not enclosed by ectoderm; evaglnatlon of the primary intestinal cavity, or archenteron. (See vegetativisatlon. ) EXOGENOUS - originating from without the organism. "EX OVO OMNIA" - all life comes from the egg (Harvey, l65T). EXPEEIMENTAL ^4ETH0D - concerted, organized, and scientific analysis of the causes, forces, and factors operating in any (embryologlcal) system. EXPEEIKENTUM CEUCIS - the final, concluding experiment when a control is no longer needed; presumably a method of final and- triumphal demonstration when all pioneer work has come to a successful ending. EXPLANATION - culturing of isolated blastema or tissues in vitro. EXPBESSIVITY - the degree to which a group of organisms is affected by the presence of a particular gene (see penetrance). EXTENSION - process of gastrulatlon; elongation of central cells of the marginal zone, and then the more peripheral cells, toward the groove of the blastopore. Syn., elongation, aelf-stretohlng (Schechtman), Strekung, Staffelung (Vogt). EXTEA-OVATE - extrusion of a portion of the egg substance beyond the cell boundary, achieved in hypotonic solutions. Syn., exovate. EYE, ANTEEIOE CHAKBEE OF - one of the best sites for observation of transplanted tissues, which tissues can be seen through the transparent cornea. The aqueous humor often is not as species specific as a nutrient environment as may be other tissues of the same organism. FAEBLOSE PIGMENTZET.T.KH - colorless pigment cell in dermal and subcutaneous tissues (e.g., in axolotl). (Schuberg, 1903 •) FATE MAP - a map of a blastula or early gastrula stage which indicates the prospective significance of the various surface areas, based upon previously established studies of normal development aided by means of vital dye markings. FATE, PEOSPECTTVE - destination towards which we know, from previous experience, that a given part would develop under normal conditions; lineage of each part of the egg through its cell descendants into a definite region or portion of the adult organism. FEEDING, MAXIMAL - procedure whereby the organism is provided with all the food that it can possibly con- sume. FETUS PAFYEACEUS - compressed fetus, abnormal: "paper-doll fetus". FEETILIZATION - activation of the egg by a spermatozoon and syngany of the two pronuclei; union of male and female gamete nuclei; amphimixis. FEETILIZATION CONE - conical projection of the egg cortex to meet the spermatozoon destined to invade the egg, generally engulfing the spermatozoon. Seen in annelids, molluscus, echinoderms, and ascidians. FEETILIZATION, DRY - placing milt (concentrated sperm) of aquatic forme directly over practically iry eggs, the procedure allowing greater concentration of sperm before flooding the eggs with water. FEETILIZATION, FEACTIONAL - fertilization following partial removal of sperm by centrifugation after partial penetration (Llllie, 1912). Elongated heads of some sperm (e.g.. Nereis) are easily frag- mented during penetration by centrifugation. FEETILIZATION MEMBRANE - a non-living membrane seen to be distinct from the egg shortly after fertiliza- tion, very probably the vitelline membrane elevated off of the egg (or from which the egg has shriinken away by exosmosis). (See Gostello, 1939:Phy3. Zool. 12.) k6k GLOSSARY FERTILIZATION, PARTIAL - oases where sperm head, after entering the egg cortex, does not move fast enough toward the egg nucleus to arrive before cleavage seta In, although the sperm aster may have reached the egg nucleus and given rise to the segmentation spindle. FERTILIZATION, SELECTIVE - physiological block to some combinations of sperm and egg, such as in cases of self-aterlllty (e.g., Clona). May Indicate differential fertilizing powers of spermatozoa even from a common source. FERTILIZIN - chemical substance In the egg cortex of mature (Echlnoderm) eggs, called "sperm laoagglutlnln" (Llllle, 1916) since its presence Is necessary for fertilization of certain forms. Supposedly possesses two side chains, one apermophlle and the other ovophlle. Soluble colloidal substance ( agglutinin) produced by eggs to attract sperm. FERTILIZIN, ANTI - "ifegs contain In their interior a substance capable of combining with the agglutinat- ing group of the fertllizln, but which is separate from it as long as the egg is inactive" (Lilllel FEULGEN REACTION - Schlff's aldehyde test accomplished by hydrolysis of thymo-nuclelc acid to yield the aldehyde which reacts with fuchsln giving a brilliant violet or pink color, a specific test for the thymo-nuclelc acid of chromosomes. FIBRILLATION - process of formation of (collagenous) fibers by the aggregation of ultramicrona whose axes are nearly parallel. May be the method of axis formation in limb rudiments (Harrison). FIELD - mosaic of spatio-temporal activities within the developing organism constitute fields; areas of instability with positional relations to the whole organism, within which specific differentiations are about to take place (e.g., heart or limb fields). Etynamlc system of interrelated parts In perfect equilibrium In the undifferentiated organism. Not a definite circumscribed area (like a stone in a mosaic) but a center of differentiation with Intensity diminishing with the distance from the center, and with different fields overlapping (Harrison, I918). A system of patterned conditions In a self-sustaining configuration (Weiss, I926). Has a material substratum which may be reduced without fundamentally altering the original field pattern. Field is both heteroaxlal and hetero- polar. "Morphe concept" of Gurwltsch (1911+). FIELD, DISTRICT - a district whose activities show field character although none of Its elements can be identified with any particular component of the field. FIELD, GRADIENT - the direction along which the field Intensity changes most rapidly. FIELD, HETEROAXIAL - field in which developing structures vary along three coordinates in space. FIELD, HBPEROPOLAR - the effects within a field differ in two opposing senses along the same axis. FIELD, INDIVIDUATION - fields are under the control of the organizing forces of the host whose differentia- tion leads toward the realization of a conjlete Individual. FIELD LAWS - (1) When material Is split off from a field bearing system, that portion remaining contains the field In its typical distribution and structure. (2) When unorganized but labile material enters the field it is Included within the field. Any field spreads over the whole of the material at its disposal, preserving its initial structure even though somewhat enlarged. (3) Fields have the tendency of taking up and including within themselves any equivalent fields from contiguous environment (e.g., whole embryos formed from two fused eggs). (See Schotte, 19'+0: Growth suppl. p. 6't.) FIELD, MORPHOGENBTIC - embryonic area out of which specific structures will develop; fields which determine the development of form in a unitary structure (Gurwltsch, 1930). FIELD, ORGAN - area in which a specific organ of the embryo will develop (e.g., eye field). FIELD, TACTIC - field governing the displacement of cells (e.g., grouping of cells in the cartilaginous prlmordium of the amphibian skeleton - Anlkln, 1929). FIELD, VEGETATIVE - early differentiated part of the Echlnoderm embryo; presumptive endoderm. FOLLICLE - a cellular sac within which the egg generally goes through the maturation stages from oogonium to ovum; made up of follicle cells, theca Interna and externa. FOVEA GERMINATIVA - pigment-free spot of the animal hemisphere where the amphibian germinal vesicle gives off its polar bodies. FRAMBOISIA - protrusion of cells following treatment of the embryo with anlaotonic solutions (Eoux). FREEMARTIN - mammalian Intersex due to masculinzatlon of a female by its male partner when the foetal circulations are continuous and the sex hormones are Intermingled, as In parabiosis. FUNCTION, HOMOLOGOUS - synchronous behaviour (e.g., when supernumerary limbs are grafted near the control limb, they may acquire innervation from the plexus of the control and will thereafter contract syn- chronously and with the same degree of intensity as the control) (Weiss, 1936: Biol. Rev. 2 - Resonance Theory of Reflex Activity.) FURCHUNG - division of the egg cell into blastomeres by mitosis. CALVANO- NEUROTROPISM - differences in electrical potential responsible for growth and connections of developing nerves. Galvanic forces in neurogenesis. GAMETE - a differentiated (matui*e) germ cell, capable of functioning in fertilization, (e.g., sperma- tozoon, oviun. ) Syn., germ cell. GAMETOGENESIS - the process of developing and maturing germ cells. GASTROSCHISIS - improper closure of the body wall along the mid-ventral line. GASTRULA - the didermlc or double- layered embryo, possessing a newly formed cavity known as the gastrocoel or archonteron. The two layers are ectoderm (external) and endoderm (internal) with only positional significance when first formed. GLOSSARY 465 GASTHULATION - dynamic prooeeaeB Involving cell movements which change the embryo from a monodermlc to a di- or trl-dermlc form, generally involving Inward movement of cells to form the enteric endoderm. Process varies In detail in different forms, but may include epiboly, concrescence, confluence, in- volution, invagination, extension, convergence - all of which are descriptive terms for morphogene- I, tic movements. GEFALLE - a continuous, quantitative gradation of a definite condition within a cell colony. (See gradient) GEL - a system in which there is a reduction in the amount of solvent relative to the amount of solid sub- stance, thereby causing the whole to become viscous (e.g., asters). GENE - self-producing molecule transmitted by the chromosome which determines the development of the characters of the individual, some of which may be solely embryonic. GENETIC LIMITATION - each cell must react exclusively in accordance with the standards of the species which it represents. GENOME - haplold gene coii5>lex; minimum (haploid) number of chromosomes with their genes derived from a gamete. GENOTYPE - the actual genetic make-up of an individual, regardless of its appearance (opposed to phenotype) GEOTONUS - position correct in respect to gravity. GEEM - the egg throughout its development, or at any stage. GEHM BAND - distinguishable bands of material in the (Molluscan) egg which will give rise respectively to ectoderm, endoderm, and mesoderm of the embryo. GEPM CELL - a cell capable of sharing In the reproductive process, in contrast with the somatic cell. (E.g., spermatozoon or ovum.) Syn. gamete. GEHM LAYKR - a more-or-less artificial spatial and histogenic distinction of cell groups beginning in the gastrula stage, consisting of ectoderm, endoderm and mesodermal layers. No permanent or clear cut distinctions, as shown by transplantation experiments. GEEM RING - ring of cells which show accelerated mitotic activity, generally a synonym for the marginal zone which becomes the lips of the blastopore. The rapidly advancing cells in epiboly. (Syn., marginal zone) GERM WALL - advancing boundary of the (chick) blastoderm including syncltia and the zone of Junction. GERMINAL LOCALIZATION - every area of the blastoderm (or of the unfertilized egg) corresponds to some future organ. Unequal growth produces the differentiation of parts (His, iS?**). This concept led to the Mosaic Theory of Roux. (See fate map.) GERMINAL VESICLE - the pre-maturatlon nucleus of the egg. GERONTOMORPHOSIS - phylogenetlc effects produced by modifying characters which are present In the line of adults. GESTALTEN - a system of configurations consisting of a ladder of levels; electron, atom, molecule, cell, tissue, organ, and organism, each one of which exhibits specifically new modes of action that cannot be understood as mere additive phenomena of the previous levels. With each higher level new con- cepts become necessary. The parts of a cell cannot exist Independently, hence the cell is more than a mere aggregation of its parts, it is a patterned whole. Coherent unit reaching a final configura- tion in space (W. Kohler). Gestaltung means formation. GESTATION - period of carrying the young (mammal) within the uterus. GIBBS-THOMPSON LAW - solidification from the accumulation of surface-acting substances by the lower sur- face tension at the surface of a drop (or cells), causing the potential energy of the combined system (liquid drop immersed within another liquid drop with which It is not mlscible) to drop to a minimum. GONOCHORISM - development or history of sex differentiation (Haeckel). Opposed to hermaphroditism. GONOMEHY - continued separation during cleavage of the chromosome sets from sperm and from egg in hybrid crosses. Theory that the maternal and paternal chromosomes remain apart throughout development. GRADIENT - gradual variation along an axis, scaled regions of preference, two-dimensional pattern. (gefalle of Boveri.) (See writings of Child.) GRADIENT, ACTrVITV - gradient established with appearance of the grey crescent in the amphibian egg, extending dorso-ventrally across the equator. GRADIENT, AXIAL - metabolic gradient determined by differences in electrical potential or by experiments demonstrating differential susceptibility (e.g., to KCN) . GRADIENT CONCEPT - Idea of physiological polarity indicated when an individual (e.g., Planarla) is trans- sected and each fragment reproduces the missing portions while retaining the original polarity; any two-dimensional concentration gradient as shown, for Instance, by anlmalizing or vegetalizing factors In early morphogenesis (Ruunatrom). GRADIENT, INHIBITION - refers to the balance of animal and vegetal hemisphere gradients In the ( sea urchin) egg (Ruimstrom) which gradients are actually antagonistic to each other and yet both are necessary for normal (balanced) development. GRADIENT, PIGMENT - when pignent Is present it is generally concentrated at the centers of greatest meta- bolic activity. GRAFT - a portion of one embryo removed and placed either among the tissues (a transplant) or the membranes (e.g., chorlo-allantoic graft) of another embryo. GRAFT, CHOHIO-ALLANTOIC - method of growing a graft on the extra-embryonic membranes of the chick, the membranes reacting to the local irritation of a (foreign) graft In such a manner as to surround It with a richly vascular tunic of indifferent tissue, rich in the requisites for survival, growth, and differentiation of the graft. The graft is never incorporated as a transplant by the host itself. Graft on the chick chorlo-allantols. GRAFT KfBBID - organism formed from host and graft, showing characteristics of both stocks. 1+66 GLOSSARY GECMTH - cell proliferation; a developmental (synthetic) Increase In total mass of protoplasm at the expense of raw materials; an embryonic process generally following differentiation (see heterogony) . GBOWTH, ACCRBTIONABY - growth Involving Increase In non-living structural matter. GBOWTH, AUXETIC - growth Involving Increase In cell size alone. GBOWTH CIECUMSTAMTIAIS - factors not responsible for the characteristics but for the realization of growth. GBOWTH COEFFICIENT - growth rate of a part relative to the growth rate of the whole (organism) depending on factors Inherent In the tissues concerned (see heterogonlc growth). GBCWTH, DiTSHAEMOBIC - heterogonlc growth to an extreme, relative growth rates becone extremely unbalanced (Champy, I92U). GROWTH EQUILIBErUM - regulation of growth of part In respect to the organism as a whole. GBCWTH GRADIENT - quantitative grading of growth variables In such a way that the body appears to be a field system of Interconnected metabolic areas. GROWTH, HETEROGONIC - different rates of growth In different regions of the embryo, or In transplant as compared with host control organ. (See heterauxesls. ) GBCWTH, ISOGONIC - similar rates of growth In different regions of the embryo. GBCWTH, MOLTIPLICATIVE - growth involving Increase In the number of nuclei and of cells. Syn. , merlstlc growth. GBCWTH, PARTITION COEFFICIENTS OF - inherent growth rates (e.g., in limb rudiments) involving changes In proportions. GBOWTH POTENTIALS - capabilities or predispositions for growth. GBCWTH REGULATION - a substance (R) postulated by Harrison, distinct from nutritional factors, present In the circulating medium of the organism, which controls growth. GUANOPHORES - pigmented cells found in lateral line organs and in pericardium, having yellow guanln crystals which give a highly refractive metallic luster to the cells. GYNAWDRCWORPH - condition where part of an animal may be male and another part female, not to be confused with hermaphroditism which is concerned primarily with the gonads. GYNOGAMONES - highly acidic, polysaccharide, containing protein of low nitrogen content, and elongate, gel-forming molecular structure. Possibly the fertlllzlns of Lillle, but so naned by Hartmann. GYNOGENESIS - development of an egg with the egg nucleus alone. This may be brought about by rendering the sperm nucleus functionless for syngany by irradiation or other neans, or by surgical removal. Opposed to androgenesis. HAEMOTROPHE - the nutritive substances supplied to the embryo from the maternal blood stream of vivi- parous animals. HAPLOID - having a single complete set of chromosomes, none of which appear in pairs, the condition in the gametic nucleus. Opposed to diploid, or twice the haplold, where the chromosomes appear as pairs (e.g., as in somatic cells). HARMONIOUS-EQUIParENTIAL SYSTEM - an embryonic system in which all parts are equally ready to respond to the (organism as a) whole. The segmenting egg is a system of equivalent parts subdividing harmoniously, according to inherent tendencies, Into smaller systems until the proper role in development has been assigned to each part of the embryo (Driesch). Isolated blastomeres tend to give congjlete but smaller embryos. HARRISON'S RULE OF MINOR SYMMETRY - (1) If the antero-posterior axis of a limb-bud Is reversed in a graft, the resulting limb will have the asymmetry proper to the opposite side of the body from that on which it is placed (I.e., it becomes dlsharmonlc, whether originally taken from the same or the opposite side). (2) If the antero-posterior axis is not reversed in grafting, the resulting limb will have the asymmetry proper to the side on which It is placed (i.e., it becomes dlsharmonlc, whether originally taken from the same or the opposite side). (5) If double limbs arise, the original member (I.e., the first to begin development) will have Its asymmetry fixed with rule (1) or (2) depending upon the orientation of the graft, while the secondary member will be che mirror Image of the first. HATCHING - the beginning of the larval life of the amphibian, accomplished by temporarily secreted hatching enzymes which aid the embryo to escape its gelatinous capsule; the process of emergence of the chick embiyo from its shell, involving critical changes In structure and functions. HEDONIC - reptilian skin glands which secrete musk and are active during the breeding season. HEMIBLASTULA - half-blastula derived by cauterizing one blastonere of the 2-cell stage (Boux). HSWIGONY - one-half egg fragment (Delage, I899). HQUKAHYOTIC - haplold. In merogony, hemikaryotic arrhenokaryotlc androgenetlc or in artificial par- thenogenesis, hemikaryotic thelykaryotl c gynogenetlc. HEMIMEUJS - failure of distal portion of appendages to develop. HENSEN'S NODE - einterior end of the primitive streak of the chick embryo, corresponding to the region of the dorsEil lip of the amphibian egg; region of future midbrain (position). HENSEN'S THEOBY - nerve fibers are formed out of protoplasmic bridges which exist throughout the embryonic body, protoplasmic bridge theory. HEBMAPHBODITE - an Individual capable of producing both spermatozoa and ova. HEBTWIG'S LAW - the nucleus tends to place itself In the center of its sphere of activity; the longitudinal axis of the mitotic spindle tends to lie in the longitudinal axis of the yolk-free cytoplasm of the cell. GLOSSARY ^61 HETEROAGGLUTININ - agglutinin (fertillzin) of eggs which acta on sperm of different species, substance extractable from egg water which causes irreversible agglutination of foreign sperm. HETERAUXESIS - the relation of the growth rate of a part either to another part of or to the whole organism. May include comparison of organisms of different sizes and ages, but of the same grouD. (See growth, heterogonic; isauxesia, bradyauxesls, tachyauxesis. ) HETEBOCHBOMATIN - part of the chromatic structure which seems to be related to the formation of the nucleolus. Takes a violet stain after methyl green but is digested away by ribonuclease. Probably represents both thymo- and ri bo-nucleic acids. HETEBOCHBONY - alteration and reversal of the sequence of stages in ontogeny. HETEBOGONY - constant differential growth ratios (Pezard, I9I8). HETEBOGONY, NEGATIVE - when the growth coefficient is below unity. HETEBOGONY, POSITIVE - when the growth coefficient is above unity, the parts increasing in relative size. HETEBOMOEPHOSIS - differential morphological differentiation under varying environmental conditions wherein the major animal gradient la flattened; appearance of an embryonic organ inappropriate to its site; regenerated part different from that which was lost. Bateson's homoeoals or Goethe's metamorphy. HETEROPLASIA - development of a tissue from one of a different kind. HETEBOPLEUBAL - transplant to the other of bilateral sides. HETEEOPLOIDY - any deviation from the normal diploid number of chromosomes (Winkler, 19l6)- HETEBOPYCNOSIS - condensation of some (sex) chromosomes in gametogenesis. HETEBOTOPIC - transplant to same side but different region from the original. HETEBOTBOPHIC - acquiring nourishment from without the orgeinism. HIBERNATE - to spend the cold (winter) period in a state of reduced activity (n., hibernation). Opposed to aestlvate, HISTOGENESIS - the appearance, during embryonic development, of histological differentiation; the develop- ment of tissue differentiation. HISTOLYSIS - the destruction of tissues. HISTOPELEOSIS - process by which a cell-line, already Irreversibly differentiated, proceeds to Its final histological specialization (Hoadley). HISTOMEBE THEOBY - ontogenetic division of histological systems resulting in the synthesis of vi (higher) organ ( Heidenhain) . HISTOTBOPHE - the nutritive substances supplied to the embryos of viviparous forms from sources other than the maternal blood stream (e.g., from uterine glands). HOLOENTOBLASTIA - blastula almost entirely composed of endoderm used by Herbst for sea urchin larvae with nearly complete suppression of ectoderm by ll\.hlum salts. HOLOMORPHOSIS - entire lost part replaced at once or later. HOLTFBETER'S SOLUTION - now designated (by Holtfreter's request) Standard Solution. NaCl - 5-5 gr. , KCl - 0.05 gr., CaClg - 0.1 gr., NaHCO} - 0.2 gr., %0 - 1 liter. HOMOIOTHEBMAL - refers to condition where the temperature of the body of the organism is under the con- trol of an internal mechanism; the body temperature is regulated under any environmental conditions. Opposed to poikllothermal. Syn., warm blooded (animals). HOMOIOTBANSPLAHTATION - transplantation between different but related individuals. HOMOLOGOUS - organs having the same embryonic development and/or evolutionary origin, but not necessarily the same function. HOMOMOBPHOSIS - new part like the part removed ( Drlesoh) . HOMOPLEURAL - transplant to some same as that from which It was renoved. HOBIZOKTAL - an unsatisfactory term sometimes used synonymously with frontal, longitudinal, and even sagittal plane or section. Actually means across the lines of gravitational force. HOBMONE - a secretion of a ductless gland which can stimulate or inhibit the activity of a distant part of the biological system already formed. HORMONE, MOBPHOGENETIC - term used by Neeiham to refer to Inductors which manifest distant effects. HUMOBAl' SYSTEM - body fluids carrying specific chemical substances which may circulate in formed channels (blood vessels or lymphatics) or diffuse freely in the body cavities or tissue spaces, (e.g., neuro- humors of Parker which act on the pigmentary system) . HYBBID - a successful cross between different species, although organism may be sterile (e.g., mule). HYBRIDIZATION - fertilization of an egg by sperm of a different species. HYDBODYNAMICS - process by which the detailed architecture of the blood vessels is derived, such details as size, angles or branching, courses to be followed, etc. The internal water pressure may be the cause of specific developmental procedure. HYALOPLASM - ground substance of the cell apart from the contained bodies. HYPEBINNEEVATION - supplying an organ with more than a single (normal) nerve fiber. HYPERMETAMORPHOSIS - protracted and complete metamorphosis. HYPEEMORPHOSIS - overstepping previous ontogenies, though harmonious. HYPERPLASIA - overgrowth; abnormal or unusual Increase in elements composing a part. HYPEBTBOPHY - Increase in size due to increase in demands upon the part concerned. HYPEBTROPHY COMPENSATORY - Increase In size of part or a whole organ due to the loss or removal of part or the'whole of an organ (generally hypertrophy in one member of the pair of organs). HYPOMORPHIC - cells or tissues which are subordinate to formative processes (Heidenhain). HYPOMOBPHOSIS - harmonious underdevelopment. ^ . ^ ^^ HYPOPHYSIS - an ectodermally derived solid (amphibia) or tubular (chick) structure arising anterior to the stomodeum and growing inwardly toward the infundlbulum to give rise to the anterior and intermediate parts of the pituitary gland. Syn"., Rathke's pocket (chick). 1*68 GLOSSARY HYPOPLASIA - undergrowth or deficiency In the elements composing a part. ffifPOTHESIS - a complemental supposition; a presumption based on fragmentary but suggestive data offered to bridge a gap in incomplete knowledge of the facte. May even be offered as an explanation of facts un- proven, to be used as a basis of expectations to be subject to verification or disproof. EfPOTHESIS, WORKING - an attempt to find an answer to sone feature of a conjilete biological situation by utilizing accepted physical and chemical principles. BJfSTEBOTELy - formation of a structure is relatively delayed. IDIOPLASM - equivalent to germ plasm of Welsmann. Dissimilar determinant imits of self-differentiating capacity (genes) each representing some part or character of the orgeinism arranged in some plan comparable to the future arrangement of organic parts (Welsmann). IMPLANT - tissue or organ removed to an abnormal position; graft. IMPLANTATION - process of adding, superimposing, or placing a graft (or a chemical fraction thereof) with- in a host without removal of anything from the host. Implants may be into the body cavity or into the orbital or anterior eye chamber cavities. INCOMPATIBILITY - opposed to affinity; tendency of cells or cell groups to repel each other when removed from their normal environmsnt. May be expressed in terms of cytolysis or histolysis of one of the cells or groups of cells. INDIVIDUATION - assimilative induction concerned with regional character of the structure derived in response to (living) organizer activity; opposed to evocational responses. Refers to process in different regions as affected by the organizer, not by a single chemical substance such as an evocator. Regional nature affected by host environment. INIUCTION - causing cells to form an embryonic structure which neither the Inductor nor the reacting cells would form if not combined; the calling forth of a morphogenetic functional state in a competent blastema as a result of contact. In contrast with evocation. Induction is successive, and purposeful In the sense that one structure leads to another. Sometimes loosely used to Include evocator influ- ences from non-living materials. Originally meant diversion of development from epidermis toward medullary plate (Marx, 1925). INDUCTION, ASSIMILATIVE - transformation of one presumptive area into a different direction under the influence of inductive forces (Spemann). INDUCTION, AUTONOMOUS - if the inducing Implant and the host do not cooperate to form an harmonious whole, the material of the Implant may not be used although the inductive forces are uninjialred. The in- ductor takes no part in the inducted structure (e.g., all chemical inductions). Opposed to comple- mentary Induction. INDUCTION CAPACITY - organizational capacity; acquired with age and subsequently lost. INDUCTION, COMPLEMENTARY - when the inductor, using some of its own material, cotqiletee Itself out of the reacting system (host material); (e.g., when presumptive epidermis is transplanted to presumptive brain region and the embryo completes itself out of the transplanted material). Opposed to autonomous induction. INDUCTION, DIRECT - case where a chemical compound acts in a manner similar to the naturally occurring inductor to produce a new neural axis in competent ventral ectoderm. INDUCTION, HETEROGENBTIC - when an organizer induces something other than Itself, such as secondary organizer optic vesicle inducing lens formation. INDUCTION, HOMOIOGENBIIC - where embryonic part induces its like (e.g., medullary plate induces medullary plate) . INDUCTION, INDIRECT - Induction by a chemical compound in ventral ectoderm of a new neural axis by the liberation of a masked evocator in the reacting tissue. INDUCTION, PALISADE - induction of neural-like tissue but without tube formation; cells arranged in palisade manner around an inductor. INDUCTOR - a loose word which Includes both organizer and evocator (Needham). Generally means a piece of living tissue which brings about differentiations within otherwise indifferent tissue. INDUCTOR, NUCLEAR - a ncrphogenetic stimulating substance which is derived from the nucleus and there- fore bears hereditary Influences, but generally operating within the cell in question. The influ- ence may be diffusible. INFECTION - the acquisition of inductive power by a group of cells not normally possessing such power, but acquiring it by diffusion from temporarily contiguous organizer material. Syn., Wecfcung. INFUNDIBULUM - funnel-like evaginatlon of the floor of the diencephalon which, along with the hypophysis, will give rise to the pituitary gland of the adult. INGRESSION - inward movenent of the yolk endoderm of the amphibian blastula. (Nicholas, 19'*5. ) INHIBITION - restraint or nullification of a tendency to differentiate. INHIBITION, DIFFERENTIAL - restraint in a gradient field where toxic agents inhibit regeneration in the most active regions. INHIBITION, TROPHIC - functional inhibition, contrasted with morphogenetic. INSTINCT - "the overt behavior of the organism as a whole" "which is in physiological condition to act according to its genetically determined neuromuscular structure when adequate internal and ex- ternal stimuli act upon it." ( Hartmann, 19'*2, Psychosomatic Med. U:206.) INSTITUTION - labile determination or competence of early germ (Graeper). INTERSEX - an individual without typical sexual differentiation. Not hermaphrodite. INVAGINATION - movement by in-sinking (Elnstulpung of Vogt) of the egg surface and forward migration (Vordringen) involving displacement of inner materials. The folding or Inpushing of a layer of (vegetal hemisphere) cells into a preformed cavity (blastocoel) as one of the methods of gaatrula- tlon. Not to be confused with involution. GLOSSARY U69 INVOLUTION - rotation of a sheet of cella upon Itself; movement directed toward the Interior of an egg; the rolling Inward or turning in of cells over a rim. One of the movements of gastrulatlon (e.g., chiok) . Syn., embolic Invagination (Jordan); einrollung, or umachlag (Vogt). lEIDlOCYTES - inorganic salt crystals. ISAUXESIS - relative growth comparisons in which the rate of the part Is the same as that of the whole. (Syn., isogony.) (Needham, igltO.) ISO- AGGLUTININ - (Syn., for fertilizln.) ISO-ELECTRIC POINT - set of conditions under which the protein tends to give off hydrogen ions Just suffi- cient to balance the tendency to give off hydroxyl ions; a state where the Ionization of the protein Is balanced. ISOGONY - proportionate growth of parts so that growth coefficient is unity and there are constant relative size differences. Equivalent relative growth rate. ISOLATION - removal of a part of a developing organism and Its maintenance In the living condition as in tissue cultures. Physiological Isolation may be achieved by Interposing a mass of Inert material (e.g., yolk) between two regions. The bifurcations of regenerating limbs or the production of double hearts by interposing an Inert barrier or one which is not subject to assimilative induction. ISCMBTEY - study of relative sizes of parts of animals of the same age. ISOTROPIC - synonym for pluripotent (Lillie, I929). ISOTROPy - originally used (Pfluger, 1883) to mean absence of predetermined axes within the egg; now means condition of egg where any part can give rise to any part of the embryo (i.e., equivalence of all parte of the egg protoplasm) . JANICEPS - Janus monster, face to face union of conjoined twins. JANUS EMBRYO - double monster with faces turned in opposite directions. Syn., duplicitas cruclata typlca. JELLY - mucin covering of (amphibian) egg, derived from the oviduct and applied to the outside of the vitelline membrane. In Frog, apparently necessary for successful fertilization. KEEN-PLASMA RELATION - ratio of the amount of nuclear and of cytoplasmic materials present In the cell. It seems to be a function of cleavage to restore the kem-plaema relation from the unbalanced condi- tion of the ovum (with its excessive yolk and cytoplasm) to the gastrular or the somatic cell. KINETOCHORE - spindle fiber attachment region. Syn., centromere. LAEOTROPIC - turned, colled, inclined to the left or counter-clockwise. Syn., lelotroplc. LAMP-BRUSH EFFECT - the side branches and loops from the chromosomes of young oScytea give such an ap- pearance. Syn., "Bursten" effect of Ruckert and Camoy. LARVA - stage in development when the organism has emerged from its membranes and Is able to lead em independent existence, but may not have completed its development. Except for neotony and paedo- genesis, larvae cannot reproduce themselves. LARVAL CHARACTERS - characters seen In the larva which may be dominant or recessive (as indicated when hybrid crosses are reversed) but which are not dependent upon an Fg to determine the status. E^ cytoplasm Is dominant over sperm influences In early development of hybrids. Larval skeletal dif- ferences seen In Echlnoderm larvae of different combinations. LATERAL LINE SYSTEM - a line of sensory structures along the side of the body of fishes and larval amphibia, generally embedded in the skin and Innervated by a branch from the vagus ganglion. Pre- sumably concerned with the recognition of low vibrations in the water. LEAST SURFACE PRINCIPLE OF PLATEAU - homogenous system of fluid lamellae so arrange themselves that the individual lamellae adopt a curvature such that the sum of the (external) forces of all Is, under the specific conditions, at a minimum. LECITHIN - organismlc fat which Is phosphorlzed in the form of phosphatides. LETHAL DEFECT - the suppression of a vital organ or of some vital function by a local defect. LIESEGANG'S FIGURES - process of stratification as of formative substances in the egg. LIMICOLA CELL TYPE - the movement of Isolated embryonic cells resembles that of Amoeba limlcola (Rhumoler, I898) having balloon-like pseudopodla. LIPIN - fats and fatty substances such as oil and yolk (e.g., lecithin) in eggs. Important as water holding device in cells as well as Insuring cell immisciblllty with surrounding media. (E.g., cholesterol, ergosterol.) LIPOGENESIS - omission of certain stages in ontogeny. LIPOPHORES - pigmented cells In the dermis and epidermis, derived from neural crests and characterised by having diffuse yellow (llpochrome) pigment in solution. LIPOSOMES - droplets of yellow oil which may be formed by the coalescence of droplets of broken down lipochondria (Holtfreter, I9U6). LITHOPEDION - mummified or calcified fetuses; "stone-child". LOBSTER CLAW - missing digits in hands or feet, or split hand or foot; probably inherited. LOCALIZATION - cytologlcal separation of parts of the mosaic egg, each of which has a known specific subsequent differentiation. There is often a substratum associated with these areas, made up of pigmented granules, but It Is the cytoplasm rather than the pigmented elements in which localiza- tion occurs. 1*70 GLOSSARY LUNAE PERIODICITY - maturation and ovl position during certain phases of the lunar cycle (e.g.. Nereis llmbata sheds its gametes in the period from the full moon to the new moon in June to September). MACERATIOM - to swell by soaking. In water the connective tissue between cells is loosened and the cells tend to separate. MACEOCEPHALUS - abnormally large head due to abnormal development of the cranium. Often the brain Is swollen with cerebrospinal fluid. Syn., hydrocephalus. MACBOMERE - larger of the blastomeres where there is a conspicuous size difference, generally the yolk- laden endoderm forming cells. Opposed to mlcromere. MACB06QMIA - gigantism, enlarged skeleton due to disturbed function of the pituitary and possibly also the thyroid glands. MACFOSTCMIS - failure of the primitive mouth slit to reduce normally. MARGINAL BELT - ring of presumptive mesoderm of the amphibian blastula, essentially similar to the grey crescent of the undivided egg. MATRIX - ground substance surrounding the chromonemata, usually less chromatic and mfiking up the body of the chronoeome. Syn., kalymma or hyalonema. MATRIX, INTERCELLULAR - the cytoplasmic wall substance of cells in a whole blastema which forms an Integrated foam structure and, because of its continuity, shows a very definite syncitlal character. (Moore) „ MATURATION - the process of transforming a primordial germ cell (spermatogonium or oogonium) into a functionally mature germ cell, the process involving two special divisions, one of which is always meiotlc or reductlonal. MAUTHEH'S FIBHK - two highly differentiated, giant neurones found in the medulla of teleost fishes and amphibia and possessing extensive dendritic connections; axones extend from VIII cranial ganglion through the spinal cord. The fibers are functional particularly in maintaining the sense of eq^uillbrlum and are Indlspensible for sustained rhythmic motor reflexes. MECHANICS, DEVELOPMENTAL - "analysis of the first found results of the experimental study of development of the egg." (Morgan) MECHANISM - assumption that biological processes do not violate physical and chemical laws but that they are more than the mere functioning of a machine because material taken into the organism becomes an Integral part of the organism, through chemical changes. Syn., the scientific attitude. MEDIAN PLANE - "middle" plane (of the embryo). May be median saglltal or median frontal. MEDULLARIN - a sex differentiating substance spread in some amphibia by the blood stream as a hormone, and in other forms by diffusion (see cortlcln). MELANOBLAST - prospective pigment cell which will bear melanin (Ehrmann, I896) but confused by some authors to Include any pigment synthesizing cell. May be present and yet unable to develop pig- ment (e.g., white axolotl). MELANOKINS - stimuli which act upon melanophores, such as temperature, humidity, light, hormones, and certain pharmacological agents (Bytlnski-Salz, 1958). MELANOPHORES - cell with brown or black (melanin) pigment granules or rods, found in every class of vertebrates. Derived from the neural crests and migrating throughout the body. MELANOPHOEE, ADEPIDERMAL - dermal melanophore. MELANOPHORES, DEPENDENT - dermal melanophores (e.g., In white axolotl) which will develop pigment only under the influence of overlying transplanted pigmented epidermis. ( Du Shane, 19'*3.) MEMBRANOUS CELL TYPE - fan-like protuberances of Isolated embryonic cells, having serrated pseudopodla (Holtfreter, 19't5). MEMBRANE, DESEMET'S - thinned out ectoderm of the cornea which occurs in response to the contact of the developing optic cup. MEMBRANE, FERTILIZATION - a membrane representing either the elevated vitelline membrane or a newly formed membrane found at the surface of an egg Immediately upon fertilization or following arti- ficial parthenogenetlc stimulation (activation); generally considered an adequate criterion of successful activation of the egg. First seen by Fol (I876) on the starfish egg. MERISIS - growth by cell multiplication (in plants). MEROGON - an egg fragment, generally with incomplete nuclear components. MEROGONY - development of fertilized but enucleated egg fragments (Delage, 1899). MEEOGONY, ANDRO - development of an egg fragment which contains the sperm nucleus only (Batalllon & Tchou-Su, 195'*). This may be accomplished By surgical removal of the egg nucleus (as it is giving off its polar body) or by irradiation damage of the egg nucleus (e.g., androgenesls) . MEBOGONY, DIPLOID - fragment of an egg developing under the influence of the normal diploid nucleus. MEROGONY, DOUBLE - cases where both halves of an egg develop following fertilization, one with a diploid fusion nucleus and the other with an haploid sperm nucleus (Dalcq, 1932). MEROGONY, GYNO - the development of a fragment of a fertilized egg which fragment contains the egg nucleus only. MEROGONY, PARTHENOGENETIC - development of a fragment of an unfertilized egg containing the egg nucleus and activated by artificial means (E. B. Harvey, 1935). MEBOGONY PABTHENOGENETIC GYNO - fragment of an egg conteilning the egg nucleus only, stimulated to develop by artificial means. MEROMORPHOSIS - the new part regenerated is less than the part removed. MESENDODERM - newly formed layer of (Urodele) .gastrula before there has been separation of endoderm and mesoderm, group of cells lying posteriorly to the lip of the blastopore, Invaginated during gae- trulatlon. Syn., mesentoblaat, ento-mesoblast. GLOSSARY 1*71 MESENCHyWE - the form of embryonic meaoderm or meeotlast in which migrating cells unite secondarily to form a syncitium or netvork having nuclei In thickened nodes between Intercellular spaces filled with fluid. Often derived from mesothelium. MESIAL - Syn., median, medial, middle. MESODEHM - primary germ layer which arises from the marginal zone to take up Its assigned position between the outer ectoderm and the inner endoderm. MESOMERE - cells of intermediate size when there are cells of various sizes (macromeres and micromeres be- ing the largest and the smallest, respectively). Also used as synonym for intermediate cell mass which gives rise to the nephrlc system. METABOLISM - the sum total of chemical changes occurring in the life of an organism. METABOLISM, ANIMAL - metabolism which brings about or is associated with the differentiation in the animal (ectodermal) direction (e.g., sea urchin eggs in sulphate ions) characterised by increased oxygen consumption. Can be checked by lithium. (See animallzatlon. ) METABOLISM, VEGETAL - metabolism which brings about or is associated with the differentiation In the vegetal (endodermal) direction (e.g., sea urchin eggs In lithium chloride) characterized ty a break- down of proteins and checked by an absence of sulfate ions. (See vegetativisatlon. ) METAMORPHOSIS - the end of the larval period of amphibia when growth is temporarily suspended. The change is from the larval (aquatic) to the adult (terrestrial) form. There Is autolysis and resorption of old tissues and organs such as gills, and the development of new structures such as eyelids and limbs; changes In structure correlated with changes in habitat from one that is aquatic to one that is terrestrial; change in structiu-e without retention of original form, as in the change from spermatid to spermatozoon. METAMORPHOSIS, ANUEAN - loss of tall, larval mouth, and gills; reduction In the gut; developnent of limbs. Period ends with the appearance of the tympanum. METAMORPHOSIS, URODELE - period of gill reduction, shedding of skin and the development of eyelids. METAPLASIA - permanent and Irreversible change in both type and character of cells; transformation of potencies of an embryonic tissue Into several directions, generally an Indication of a pathological condition (e.g., bone formation In the lung). It Is thought that some differentiated tissue may become undifferentiated and then undergo a new differentiation In a different direction. METATHETELY - the appearance of early embryonic structures at a stage later than normal (e.g., the reten- tion of larval organs by Insect pupae). Opposed to prothetely. MICROCEPHALUS - small or pin-headed; a condition due to the arrested development of the cranium and the brain, accompanied by reduced mentality. MICHOGNATHUS - retarding of lower Jaw In the new bom. MICEQMERE - smaller of the cells when there Is variation In the size of blastomeres. MICSCMBTRY - measurement of a microscopic object, using an ocular micrometer. MICROPHTHALMIA - eyes that are too small, often due to undersized lenses (Harrison, 1929). MICROPYLE - an aperture In the egg covering (e.g., fish eggs) through which spermatozoa may enter. Generally the only possible point of fertilization in eggs bearing mlcropyles. MICROSOMIA - dwarfism, reduced skeleton, due possibly to disturbed function of the pituitary and thyroid glands. MICROSTOMUS - small mouth; excessive closure of the mouth. MICROSURGERY - procedures described by Spemann, Chambers, Harrison and others where steel and glass In- struments of microscopic dimensions are used to operate on small embryos. MILIEU - term used to Include all of the physico-chemical and biological factors surrounding a living system (e.g., external or Internal melleu). MITOCHONDRIA - small, permanent cytoplasmic granules which stain with Janus Green B, Janus Red, Janus Blue, Janus Black 1, Ehodamln B, Dletheylsafranin, dilute methylene blue, and which have powers of growth and division and are probably lipoid In nature, and may contain proteins, nucleic acids, and even ertzymes. Syn., plastens. MITOGENETIC RAYS - rays of short wave-length emanating from a growing point (e.g., onion root tip - Gurwitsch, 1926) which rays excite cell division when they encounter tissues capable of prolifera- tion. Such rays come from disintegrating, dead tissues of regenerating tails (e.g., axolotls - Blacher, 1950). MITOTIC INDEX - the number of cells. In each thousand, which are In active mitosis at any one time and place In an organism (Minot, I908); the percentage of actively dividing cells. Often considered as a measure of growth activity. MODULATION - physiological fluctuation of a cell in response to environmental conditions, indicating latitude of cell adaptation; cellular changes reversible without residue (Weiss, 1939); temporary reactions of cells to new environmental conditions without loss of original potential functions (e.g., reversible histological differentiation at the end of ontogeny). MODULATOR - specific Inducing substance which goes beyond basic evocation and will Induce a specific kind of tissue characteristic of a definite region (e.g., neural tube of mid-body level - Waddlngton) . MOLTING - periodic shedding of the upper, comlfled epidermis, common among amphibia and reptiles, and possibly associated with breeding activity. MONOSPERMY - fertilization accomplished ty only one sperm. Opposed to polyspermy. MONSTER, AUTOSITE- PARASITE - double embryos with great size discrepancy so that the smaller one bears a parasitic relationship to the larger; variously produced. MONSTER, DICEPHALUS - double-headed abnormality, produced by any means. MONSTER, ISCHIOPAGUS - double embryos, widely separated except at the tail; produced by any means. 1»72 GLOSSARY MOBPHOGENESIS - all of the topogenetlc processes which result In structure formation; the origin of char- acteristic structure (form) In an organ or In an organism compounded of organs. MORPHOGENETIC MOVEMEMTS - cell or cell area movements concerned with the formation of germ layer (e.g., during gaatrulatlon) or of organ prlmordia. Syn., G«staltungsbewegungen. MOHPHOGENETIC POTENTIAL - product of a reaction between the cortex and the yolk Just sufficient to bring about response in a competent area; threshold value (Dalcq and Pasteels). MORPHALLAXIS - an old part transformed directly into a new part or whole organism, a type of regeneration, resulting in a whole from a part (e.g., each piece of a dissected Planarla or Tubifex becomes a com- plete organism) . MOSAIC - a type of egg or development In which the fate of all parts are fixed at an early stage, possibly even at the time of fertilization. Local Injury or excisions generally result In the loss of specific oiigans in the developing embryo. Such eggs or embryos react by recovery to such experimental proce- dures as blastomere separation, parabiosis or merogony. Opposed to regulative development. MOVEMEOT, FOBMATIVE - localized changes in cell areas resulting in the formation of specifically recogniz- able embryonic regions (Vogt). MOVmEOT, HCMOLOGOUS - movement of homologous muscles in transplanted limbs, the synchronous contraction of muscles. NACHBAHSCHAFT - morphogenetlc effects produced by contact with other tissues or structures of a develop- ing organ; contiguity effects. NECBOHOHMONES - the chemical substances produced by degenerating nuclei which cause the premature and incomplete divisions of oocytes in sexually mature mammals and in the formation of oligopyrene spermatozoa in Mollusca. NECBOSIS - local death of a cell or group of cells, not the whole body. NEIGHBOBWISE - the reaction of a transplant appropriate to its new environment, indicating its plas- ticity, pluri potency, or lack of determination. Syn., Artsgemass. NEMAMEEE - one of the physical units composing a gene-string or genonema, which carries the genes. May be composed of several genes, or a single gene may extend over several nemameres. Governs bio- physical reactions of the gene-string. NECMOBPHOSIS - new part not only different from part removed but also like an organ belonging to another part of the body; or unlike any organ of the body. NEOPLASM - a new growth, generally a tumor. Histologically and structurally an atypical new formation. NEOTONY - sexual maturity in the larval stage; a condition of many urodeles (e.g., Necturus, Azolotl) and of experimentally produced thyroidless anuran embryos where the larval period Is extended or retained, i.e., the larvae fall to go through metamorphosis. Appearance of larval conditions In the adult. NEURAL CREST - a continuous cord of ectodermally derived cells lying on each side in the angle between the neural tube and the body ectoderm, separated from the ectoderm at the time of closure of the neural tube and extending from the extreme anterior to the posterior end of the embryo; material out of which the spinal and possibly some of the cranial ganglia develop, and related to the develop- ment of the sympathetic ganglia and parts of the adrenal gland by cell migration. rffiUBOBIOTAXIS - concentration of nervous tissue takes place in the region of greatest stimulation. NEUEOGEN - an evocator which causes neural Induction In vertebrates. May include the organizer, chemical substances, carcinogens, oestrogens, etc. NEUBOGENESIS, MECHANICAL HYPOTHESIS OF - mechanical tension of plasma medium in any definite direction is said to orient and aggregate the fibrin micellae in a corresponding direction. NEUROHUMOBS - hormone-like chemical substance produced by neroua tissue, particularly the ends of develop- ing nerves which consequently act as stimulating agents. NEURULA - stage in embryonic development which follows gaatrulatlon and during which the neural axis is forned and histogenesis proceeds rapidly. The notochord and neural plate are already differentiated, and the basic vertebrate pattern is indicated. NEUTRAL MEDIUM - an environmental medium for the embryo which is free from any chemical or physical in- ductors, and is physiologically isotonic. NORMALIZING - formative action anchored in the organization associated with the determination of develop- ment, not super-material entelechy but an Integral part of the organism Itself. Integrating and balancing tendencies. mjCLEAL REACTION - sections of tissue hydrolyzed with HCl before treating with Schlff's reagent may give a characteristic red or purple color known as the nucleal reaction. (See Feulgen reaction.) NUCLEAR MEDIUM - calcium free but otherwise balanced and isotonic salt medium in which the Isolated germinal vesicle can survive for some time. NUCLEOFUGAL - refers to outgrowth In two or nore directions from the nuclear region as a center, such as in the formation of nyelln around a nerve fiber, starting at the sheath cell nucleus as a center and growing in two directions. NUSSBAUM'S LAW - the course of the nerve within the muscle may be taken as the Index of the direction in which that particular muscle has grown. OEDBMA - excessive accumulation of water (lymph) in the tissues and cavities of the body; niay be sub- cutaneous and/or intracellular. Due to a block in drainage channels and generally associated with cardiac Inefficiency. GLOSSARY U73 OMNIPOTENT - used in connection with a cell which could, under various conditions, assume every histological character known to the species, or which, by division, could give rise to such varied differentiations. ONTOGETTf - developmental history of an organism; the sequence of stages in the early development of an organism. "OMNE VIVUM E VIVO" - all life is derived from pre-existing life (Pasteur). "OMNIS CELLUTJl E CELLULA" - all cells come from pre-existing cells (Virchow). OOPLASM - cytoplasmic substances connected with building rather than reserve materials utilized in the developmental process . OPTICO-OCULAE APPARATUS - includes all the structures related to the eye: optic vesicles, optic stalks, and primaiy optic chiasma, which develop from the simple median anlage precociously found in the medullary plate (LePlat, I919). OEGAN-FOBMING SUBSTANCE - substances which, by chemo-differentiatlon and segregation are localized in different blastomeres bringing about a mosaic of development. OEGAN, HUDIMENTABY - organ which is present but without any detectable physiological manifestation. OBGANIC POINTS THEORY - discarded theory of Bonnet and yet much like chemo-differentiation. The preformed determinants are xuiequally distributed between blastomeres during early cleavage. OBGANICISM - laws of biological systems to which the ingredient parts are processes are subordinate; idea of organism as a whole (Loeb). OEGAMIZATION - indicated by the inter- dependence of parts and the whole. "When elements of a certain degree of complexity become organized into an entity belonging to a higher level of organization" says Waddlngton, "we must suppose that the coherence of the higher level depends on properties which the isolated elements Indeed possessed but which could not be exhibited until the elements entered into certain relations with one another." Relations beyond mere chemical equations; border- ing on the philosophical idea. Process of differentiation or specialization which takes place according to a definite pattern in space and time, not chaotically in the direction of haphazard distribution (see Gestalten). OEGAHIZEE - the chorda-mesodermal field of the amphibian embryo; a living tissue area which has the power of organizing indifferent tissue into a neural axis. Organizer is more than an evocator or in- ductor because definite axial structures are caused to develop. Term first used by Spemann to describe a "dorsal quality" qualitatively different from vegetal hemisphere material. Term organizer now used for graded inductions such as primary or first grade organizer (dorsal lip; Induces neural axis) secondary or second grade organizer (optic cup induces lens); and tertiary or third grade (tinnulus tympanicua Induced tympanic membrane formation). OEGAHIZEE, NUCLEOLAE - localized region of a particular set of chromosomes where the nucleolus is found, each nucleolus being associated with a set of chromosomes. OEGANOGENESIS - emancipation of parts from the whole; appearance or origin of morphological differentia- tion. OETHOTOPIC - transplant to homologous region. OSMOTIC PEESSUEE - P equals kCT; P is the force under which water tends to pass through a membrane into a substance that cannot diffuse through this same membrane (e.g., sugar and collodion membrane) and this force Is directly proportional (k) to the molecular concentration (C) of the substance (sugar) and to the absolute temperature (T). The terms isotonic, hypertonic, and hypotonic are used to express osmotic pressure relations such as exist between the cell contents and Its environment. OTOCEPHALY - tendency to fusion or approximation of ears, accompanying cyclopia. OUTGEOWTH NEUEONE THEOEY - the cells found along the course of a nerve fiber, the fiber developing as a protoplasmic outgrowth (extension) from a single ganglion cell. OVOPHILE - presumed receptor portion of amboceptor suitable to receive the egg receptor, antl-fertlllzin, or blood inhibitors. In the fertilizln reaction (Lillie). OVOPOSITIOM - the process of egg laying. OVOVrVIPAEITY' - condition in which egg contains enough yolk to carry the embryo to hatching. After this stage the larva is liberated from the maternal organism without receiving further nourishment. OVULATION - the release of eggs from the ovaiy, not necessarily from the body. PAEDOGENESIS - relative retardation of the development of body structures as compared with the reproduc- tive organs; reproduction during lar'^al stage; precocious sex development. PAEDCMOEPHOSIS - introduction of youthful characters Into the line of adults. PALINGENBIIC - term used for repeated or recapitulated stages which reflect the history of the race (Haeckel). PARABIOSIS - lateral fusion of embryos by injuring their mirror surfaces and approximating them so that they grow together (see teloblosis). PAETHENOGENESIS - development of the egg without benefit of spermatozoa; development stimulated by arti- ficial means. PARTHENOGENESIS, ARTIFICIAL - activation of an egg by chemical or physical means (e.g., butyric acid, hypertonic solutions, irradiation, needle prick, etc.) PAETHENOGENESIS, FACULTATIVE - eggs normally fertilized before development may, on occasion, develop when fertilization is delayed before sperm penetration. PAETHENOGENESIS, NATURAL - maturation of the egg leads to development without the aid of spermatozoa (e.g., some insects). PARTITION- COEFFICIENT - the factor which determines the size of any part at any time by parcelling out materials; relative capacity for various parts of the embryo to absorb food from a common supply at different times. Such coefficients are expressions of intrinsic growth potentials, so balanced In normal development that no single structure can monopolize the nutriment to the detriment of other structures. hjk GLOSSARY PAETHENOGENETIC CLEAVAGE - fragmentation of protoplasm of old and unfertilized chick eggs, originally thought to be true cleavage. PATH, COPULATION - path along which the pronuclei approach each other, the aperm of the amphlhia general- ly leaving a trail of pigment taken In from the surface coat. PATH, PENETRATION - the path of the sperm as It enters the egg hefore It veers Into the copulation path. PATHFINDERS - pioneering nerve fibers which assume the task of growing Into the uninvaded peripheral tissues (Weiss). PENOTRANCE - the degree to which a group of organisms expresses the presence of a gene. (See Expressivity.) PERIBLAST, CENTRAL - cells of ayncltlal nature beneath and separate from the blastoderm of fish. PERIBLAST, MARGINAL - cells of ayncltlal nature bounding the central blastoderm of the fish or chick. PERMEABILITY - property of a membrane indicated by the rate at which substances pass through, the phenomenon involving four attributea of maaa, area, time, and concentration aa well as the nature of the environment. PFLUGER'S LAW - the dividing nucleus elongates in the direction of the least resistance. pH - method of stating the measure of the hydrogen ion concentration, expressed as the log of the recipro- cal of the hydrogen ion concentration in gram-mols per liter. The negative value of the power of 10 equivalent to the concentration of hydrogen iona In gram-mDlecules per liter. The neutral solution (neither acidic nor basic) has a pH value of J: pH values less than 7 are acid and those more than 7 are alkaline. PHENOCOPY - the Imitation of a particular genetype by response to physiological factors In the environ- ment, but cariylng no hereditary implication. PHENOCRITICAL PERIOD - the period in the development of ein organism when a particular gene effect can be most easily influenced by environmental factors. PHENOTYPE - the expressed genetic Influences. PHOCCMELUS - failure of proximal portion of appendages to develop, diatal parts may be normal. PHYLOGEIiy - series of stages in the history of the race; the origin of phyla. PLACODE - plate or button- like thickening of ectoderm from which will arise sensory or nervous structures (e.g., olfactory placode). PLANE - (See "section".) PLASM - a dlatinguiahable region of mosaic eggs which gives rise to later and specific organ development. PLASMATiT'WMA - the outermost, thin, vlacous layer of the ectoplasm in the fertilized egg which does not change by centrifugation. PLASMAL REACTION - related to the presence of fat and aldehydes in the cytoplasm (Feulgen and Voit, 192'*). It is not specific, however, as positive reactions are given by certain alkalla, aliphatic ketones, some unsaturated compounds (e.g., oleic acid), weak salts of strong bases (e.g., acetates and phos- phates), some amino oxldea and certain catalytic oxidizing aystems. PLASMODEMS - fine protoplasmic threads (presumably) connecting cells mitotically derived from a parent cell; used in connection with marginal cells in the blaatodiac of fishes and birds. PLASMODESMATA - protoplasmic bridgea claimed ( Paton, 1907) to be the means of nerve fiber growth; plas- modeamata supposedly incorporated into the substance of the axone during its origin. PLASMONUCLEIC ACID - one of the two types of nucleic acid, this one occurring in the cytoplasm, , in the plasmosome (nucleolus), and possibly in minute quantities in the chromatin (Pollister & Mlrsky, I9I1IH Nature 155:711). (See chromonuclelc acid.) PLASTENS - (See mitochondria.) PLASTICITY - the ability of early cell areas (tissues) to conform to environ mental Influences, such plasticity disappearing at the end of gaatrulatlon. Syn. , plurlpotency . PLASTIN - thread-like structural elements of the cytoplasm which form a gel framework by net formation. (Frey-Wysaling) PLATEAU'S LAWS - not more than three (3) planes can meet at any one edge and not more than four edges can meet at any one point. Reference is made to cleavage planes. PLEICTROPISM - multiple effects of a single gene due to effects upon metabolism. PLURIPOTENT - condition \rtiere cell or embryonic area la amenable to several courses of differentiation. An undetermined state. POIKILOPLOID - variable chromosome number. POUCILCTHEHMOUS - cold-blooded; animals which depend upon the environment to regulate their body tempera- ture. Animals lack temperature regulating mechaniama. (E.g., amphibia, fish). Opposed to homol othermous . POLAR FURROW - space between blastomeres of Uo cell stage due to shifting of the mitotic axes in each of the blastomeres, generally associated with spiral cleavage. POLARITY - strateficatlon; axial distribution; assumption that behind any visible differences in the egg (cell or embryo) there is an invisible arrangement of some (imagined?) basic material. The type of polarity may be Inherent, predetermined, while the direction of polarity may be conditioned by the environment. Related to the animal-vegetal and anterior-posterios axes. (See gradient.) Syn., Schl cktungspolari tat . POLAR LOBE - lobe which remains attached to one blastomere into which it is periodically withdrawn during the Intervals between mitoses, and which gives rise to the entomesoblaat and hence to mesoderm. Also "yolk lobe," although this lobe may actually to devoid of yolk. POLAR PLASM - In detemdnflte cleavage (e.g., annelid and mollusc eggs) some of the vegetative pole proto- plasm may be identified in early blastomeraa by Ita particular conaistency. This may be the material of the polar pole. GLOSSARY 1*75 POLE, ANIMAL - the protoplasmic portion of a telolecithal egg from which the polar bodies are given off, in «hlch the germinal vesicle is found, and which has the highest metabolism and gives rise to the princi- pal parts of the nervous system and sense organs. Eegion of least yolk concentration. Syn., apical pole or hemisphere (See anlmalization) . POLE, VEGETAL - region of the egg opposite the animal pole; region of lowest metabolic rate; pole with greatest density of yolk in telolecithal eggs, generally the endoderm forming portion of the early egg. (See vegetativisation. ) POLYDACTYLY - extra digits in hands or feet; in man probably inherited. POLYMBRYONY - natural isolation of blastomeres leading to the production of multiple embryos; develop- ment of several embryos from a single zygote. POLYHYDRAMNIOS - condition where the amniotic fluid exceeds two liters. POLYPLOID - possessing a multiple number of chromosomes, such as triploid (3 times the haplold number) tetraploid (k times the haploid), etc. Always more than the normal diploid number of the typical zygote. (Winkler, 19 l6.) POLYPLOIDOGEN - a chemical substance which brings about the polyploid condition, usually by inhibiting certain phases of nuclear division. POLYSPERMY - entrance into the egg of more than a single sperm, normally (e.g., chick and urodele) or under pathological conditions (e.g., Anura, Echinodermata, Mollusca, etc.) (Hertwig, 1887; Boveri, 1907; Herlant, I9II). Normal polyspemy is sometimes called "physiological polyspemy" while the abnormal is pathological, brought about by chemical or physical conditions (see Clark, I936. Bio. Bull.). POST-GENERATION - regeneration out of newly formed rather than already differentiated tissues; restora- tion of parts of the embryo by utilization of materials (unused) from an injured (cauterized) blas- tomere (Eoux). POTENCY - ability to develop embryologically; capacity for completing destiny; ability to perform an action; "future development verbally transformed to an earlier stage (Waddlngton) . The test of potency is actual realization in development. It is not the same as competence. It is an explana- tory rather than a descriptive term (Boux, I892) for developmental possibility." "A piece of an embryo has the possibility of a certain fate before detennination, and the power to pursue* it afterwards." (Needham, 191+2.) POTENCY, ACTIVE - cases of self-differentiation lAere potencies are realized in isolation even without inductive forces (Bautzmann, 1929)- POTENCY, PASSIVE - potencies formed in the presence of Inductive forces only (Bautzmann, 1929) • POTENCY, PROSPECTIVE - the sum total of developmental possibilities, the full range of developmental performance of which a given area (or germ) is capable. Somehow more than, and inclusive of, prospective fate and prospective value. (See these terms.) Connotes possibility, not power. Not to be confused with coiqietence. POTENTIAL, MORPHOGENETIC - the strong or weak ability to develop into specific structures (Dalcq and Pasteels, I958). PEEFOEMATIONISM - arrangement of parts of the future embryo are spatially identical in the egg (ovist) or in the homunculus of sperm (spermist); anlagen of all parts of the organism are already present in the egg (or sperm). PEERTNCTIONAL PERIOD - period during which the morphological and histological differentiations proceed to prepare the organs for functioning (Eoux). PRESUMPTIVE - the expected (e.g., the fate of a part in question) based on previous fate-map studies. PRIMOBDIA, PRESUMPTIVE - place and extent of prospective values of early gastrular surface as regards its realization into specific organ areas in the normal process of development. Not necessarily checked by self-differentiating technique. PRIMORDIUM - the beginning or earliest discernible indication of an organ. Syn., rudiment, anlage. PRONUCLEUS - either of the gametic nuclei in the egg after fertilization and before syngamy; female pronucleus is the mature egg nucleus after the elimination of the polar bodies, distinct from the germinal vesicle which is the pre-maturatlon nucleus. PROSPECTIVE SIGNIFICANCE - the normal fate of any part of an embryo at the beginning of development. Syn., prospective Bedeutung, Potentlalite reelle. PROTANDROUS - hermaphroditism in which the male elements mature prior to the female. PROTHETELY - the appearance of structures at an early stage of development which normally appear later (e.g., pupal organs in larval insects). Opposed to metathely. (Schultze) PBOTOGYNOUS - hermaphroditism in which the female elements mature prior to the male. PROTOPLASMIC BRIDGE THEORY - (See Hensen's theory and plasmadesmata. ) PYCNOSIS - increase in density of the nucleus (or the cytoplasm) which may be hyperchromatlc. I>ycnotlc cells in the central nervous system are called chromophile cells. Such cells have an Increased affinity for haematoxylin and methylene blue. PYGOPAOUS - rump union in conjoined twins. RACHISCHISIS - cleft spine, due to failure to close completely. RANDZONE - term (German) for marginal zone, the line between the animal and the vegetal hemispheres of amphibian eggs or the region of initial involution for gaetrulatlon. RATE-GENES - one and the same gene may lead to different rates of formation of specific materials such as melanin. U76 GLOSSARr REALISATOBSYSTEM - pertaining to the non-apeolflc complexity of the netabollsa-apparatiis which guarantees the normal course of determination ahd topogenetlc transformations In a blastema. (Lehmann.) (See determination. ) EEALIZATION FACTOE - factor Involved In the achievement of a certain end organ production, often associ- ated with the establishment of a gradient. EECOHSTITUTION - an aspect of regeneration where a new organ Is formed within old tissues rather than by regeneration from a cut surface. A re-arrangement of parts to give new form, particularly In hydrold experiments (see blastema). EgCOVEKf, DIFFERENTIAL - differential acclimatization In a gradient system where a low concentration of depressants Indicates that regions of highest activity show greatest powers of adjustnent. RECUPEEATION - the reappearance of competence at a late stage In development (e.g., limb or tall blastema cells). BEDIFFEEENTIATION - secondary differentiation within the area delimited by the term modulation. (Kaaahara, 1955: Arch. f. Exp. Zexi. 18). A return to a position of greater specialization In actual and potential functlonu (Bloom, 193?: Physiol. Eev. 17). REDUPLICATION - double or even treble growths (e.g., limbs) connected with one another at some point along their length, the reduplicated member being (usually) a mirror Image of the original (see Bateson's Rule). REGENE3?ATI0N - repair or replacement of lost part or parts by growth and differentiation past the phase of primordial development. The vast organizing potencies of the different regions of the early embryo are lost after the completion of development and there remain only certain regions of the body which are said to be capable of regeneration. Regenerative powers are more extensive among embryos and adults of phyletlcally low forms. REGENERATION, BIAXIAL - regeneration which leads to two apical or two basal regions, accomplished In a form like Planaria by cutting off the head and splitting the body from the anterior cut surface, or from the posterior. The latter procedure will often give rise to a crotch head. REGENERATION, PHYSIOLOGICAL - changes which occur as a part of the life cycle of the organism. REGENERATION, RESTORATIVE - changes occurring in regular fashion after an accident, bringing about a re- placement of lost or damaged parts. REGENERATION, WOLFFIAN - appearance of a new_ lens in the eye after removal of the former lens, due to possible regeneration from the upper margin of the iris. REGENERATIVE CAPACITY - the ability to replace lost parts, the ability which varies (generally) inverse- ly with the scale of degree of development. REGION, PRESUMPTIVE - regions of the blastula which, by previous experimentation, have been demonstrated to develop In certain specific directions luider normal ontogenetic conditions (e.g., presumptive notochord or lens). Not as definite as anlage. REGULATION - a reorganization toward the whole; the power of pre-gastrula embryos to utilize materials remaining, after partial excision, to bring about normal conditions in respect to the relation of parts; somewhat comparable to regeneration of later stages, but more flexible and more extensive in early development. Ability to adjust to a strange environment and yet to develop along lines of normal development. REINTEGRATION - the restoration to the organism, after the period of self differentiation and through the action of hormonal and neural factors, of control by its Individuation field. RESONANCE THEORY OF REFLEX ACTIVITY - the central nervous system can emit different forms of excitation and a specific muscle will respond only to that excitation appropriate to it. Rather than differ- ent conducting pathways for the central nervous system and peripheral end organs (e.g., limbs), all components of an excitation are transmitted to all muscles, but only that muscle, for which a specific component is contained, will respond. Each muscle has motor neurones which act as selec- tive transmitters. This Is the explanation (Weiss, 1936: Biol. Rev. 2:h6h) for the simultaneous movement of homologous muscles even though transplanted limbs may be supplied by non- homologous nerves. (See function, homologous.) RESPONSE, HOMOLOGOUS - an extra (transplanted) muscle is made to act by the central nervous system to- gether with the normal muscle of the same name. REUNITION - reassembling of parts of an organism Into a functional whole (e.g., sponges as Microclona) after separation of component parts. RICHTUNGSPOLARITAT - (German) polarity of direction, orientation of particles toward the animal or the vegetal pole, but found throughout the ovum. ROHON-BEARD CELLS - giant ganglion cells in the spinal cord, derived from the trunk neural folds, and which form the sensory pathway Including that of the peripheral sensory nerves. They have large rounded nuclei and a considerable amount of cytoplasm which stains differentially with Heidenhain's modification of Mallory. They are never found in the ventral part of the cord. They are associated with that sensory area which is functional during the flexure of the tall following tactile stimula- tion. BHYTHM, METACHRONAL - sequential contraction (cilia or muscle). EHYTB1, SYNCHRONAL - simultaneous contraction (cilia or muscle). SACH'S LAW - successive divisions tend to occur at right angles to each other, due to the position of the previously formed centrosomes. (See Hertwlg's Laws.) SECTION - thin (microscopic) slice In any of several possible planes, achieved with a microtome or razor blade. GLOSSARY U77 SECTION CBCSS - cut made at right angles to the long axla of the embryo. Syn. , tranaverse section. SECTION, FFONTAL - cut made parallel to the longitudinal axis of the embryo and separating the more dorsal from the more ventral. Syn., horizontal section. SECTION, SAGITTAL - out made parallel to the longitudinal axis of the embryo but separating the right from the left portions. Term often confused with "median" or "longitudinal" which really mean no more than "axial," hence could also be "frontal". SECTIONS, SEEIAL - thin (microscopic) slices of an embryo laid on the slide in sequence (generally from left to right, as one reads) so that the beginning of the embryo is at one side (left) and the end of the embryo at the opposite side (right) of the slide. SEGMENTATION - term used synonymously with cleavage. Also means serial repetition of embryonic rudinenta (structural patterns) in successive levels of regular spacing, as In the case of somites, and spinal nerves. Syn., cleavage. SEGREGATION - the separation of self-differentiating embryonic rudiments; the organizational process of embryogeny; autonomlzlng (Weiss); the aggregation of various spatial systems Independent of each other and leading to self-differentiating potentialities. Originally used (Ray Lankaater) in discussing the gastrea theory to mean a separation of the physiological molecules that are going to form ecto- and endoderm. SEGREGATION, EMBRYONIC - progressive restriction of original potencies In the embryo; the process of step by step repartltlonlng of the originally homogeneous zygote Into the separate parts of the presump- tive embryo. SEGREGATION, PRECOCIOUS - segregation found In mosaic eggs where local differences arise even before cleavage and a minimum of modification in response to any Internal environmental factors occurs In subsequent development (Lankester, 1877). SELBSTOEGANIZATION - invisible process of construction and reconstruction of a normal blastema, with Its quantitative organization gradient which is itself the basis for the segregation from qualitatively differing organ- forming regions. SELF-DIFFERENTIATING CAPACITY - the capacity of a part of a developing system to pursue a specific course. The characters of that course are determined by intrinsic properties of the part (Roux, I88I). There can be no self-differentiation without prior induction. (See differentiation, self.) SELF- ORGANIZATION - obsolete term which meant the alleged appearance of a lens without the stimulus normal- ly coming from the optic cup (see double assurance). SELFWISE - behavior of a transplant in a manner expected in its original environment. In accordance with its normal prospective significance. SENESCENCE - the progressive loss of growth power; old age. SENSITIZATION THEORY - calcium Is the true activating agent in artificial parthenogenesis and other sub- stances increase the permeability of the egg cortex to calcium (Pasteels). SENSORY LOAD - determined by the number of receptor organs associated with a specific nerve. SEX, HETERODYNAMIC - the sex in which the gametes are of two klnda with respect to the possession of specific sex influencing chromosomes, such as the X-chromosome in Drosophila. The frog and human male are presumably heterogametic. SIGNIFICANCE, PROSPECTIVE - actual fate of any part of the original egg. Syn., Drlesch's "prospektive Bedeutung" . SITUS INVERSUS - an inversion of the bilateral symmetry; reversal of right and left symmetry. SITUS INVERSUS VISCERUM - twisting of the digestive tract and sometimes the heart, occurring naturally (rarely) or as a result of shifting of embryonic parts (Spemann, I906) as In reversing a square piece of presumptive neural plate and archenteron of the early gastrula. SOL - a colloidal system In which the particles of a solid or of a second liquid are suspended In a con- tinuous phase of a liquid, the particles or their aggregates being too large to go through animal membranes rapidly or at all. SOMATIC DOUBLING - doubling of the initial number of chromosomes with which the egg begins development, occurring (probably In most cases) at the first or early mitotic divisions (cleavages) of the egg, after fertilization. SOMATOBLAST - blastomere with specific germ layer predisposition such as actodermal somatoblasts. SPALTUNG - (German) fusion of posterior neural axes in a twin embryo, simulating an induction. SPECIFICITY - the summation of the cytochemlcal characteristics of different protoplasms (Humphrey and Burns, 1939)- SPERMOPHILE GROUP - portion of the amboceptor in Lillie's fertlllzln hypothesis into which sperm recep- tors fit in the fertilization reaction. SPERM RECEPTOR - chemical group associated with the spermatozoa, reacting with fertlllzln (amboceptor) in Lillie's side chain hypothesis of the fertllizin reaction. SPINA BIFIDA - split tall caused by a variety of abnormal environmental conditions such as heat, cold, lack of oxygen, centrlfugation, any of which may prevent the proper closure of the blastopore which leads to this split-tailed condition. STEPWISE INHIBITION - successive inhibitions of organic processes by successively stronger applications of external agents. STEREOBLASTULA - solid blastula experimentally produced by subjecting (Echlnoderm) eggs to alkaloids; normal blastocoel filled with solid mass of cells (e.g., Crepldula). STERILITY, SELF - Inability of eggs and sperm of the same (hermaphroditic) Individual to fuse and give rise to an embryo (e.g., Ciona Intestlnalls, an Ascidian) . STERNOPAGUS - sternal union of conjoined twins. STICOTHOPISM - faculty of acquiring and losing clavlform shape of the bottle cells of the blastoporal lip during gastrulatlon (Ruffini, 1925). U78 GLOSSARY STIMULATION, DIFFERENTIAL - varying responses of a gradient system to favorable conditions, as when an optimally high ten^wrature is applied to a regenerating Planarlan and a bigger and better head re- sults than under normal (temperature) conditions, (See inhibition, differential.) STIMULUS, FOIMATIVE - concentration of (chemical) substance In the dorsal lip of the blastopore leading to the formation and demarcation of embiyonic fields. STIMULUS, OXYGENOTACTIC - differential stimulation of a developing organism by exposure to oxygen. Pre- sumably a factor in the spreading of the blastoderm (chick) oyer the yolk. Syn., oxygenotaxi s . STOKE' S LAW - formula for determining viscosity V = ^'^^ ^g'n'^^ (formula generally omits c and q) where V is the speed at which granules travel through cytoplasm under a centrifugal force of cq absolute units; g is the gravity constant; cT is the specific gravity of the granules; p is the specific gravity of the cytoplasm; a is the radius if the granules; n is the coefficient of viscosity of the cytoplasm; q is a factor which allows for the fact that there are many granules plus the displacement of cytoplasm in granule movement. SUBSTRATE - the substance which is acted upon by an enzyme. SUCKER - adhesive, cementing organ of the oral region of anuran larvae. SUSCEPTIBILIIY, DIFFERENTIAL - evidence of non-homogeneity when diffusely applied injurious agent brings about varying local reactions on the embryo. SYMPODIA - fusion, 'to varying degrees, of the legs (e.g., mermaid or siren condltlonl). SYNCYTIUM - propogation of nuclei with cytoplasmic growth but without cytoplasmic division so that there results a mass of protoplasm with many and scattered nuclei but with inadequate cell boundaries (e.g., chick marginal periblast and adult Nematodes). SYNDACTYLY - either bony fusion or fleshy webbing of the digits, generally the second and third digits being Involved. Probably inherited in man. SYNERESIS - a segregation of the colloidal phases, a corollary of ageing. SYNGAMY - fusion of gametes, applied specifically to the merging of sperm and egg nuclei. SYNOPHTHALMIA - fusion of the eyes as in cyclopia. SYNTONIC FACTOR - some regulating force which enables a particular cell to live harmoniously with other cells of the same type so that an organ will develop, not found in tissue cultures of cells Isolated prior to differentiation, present during organogenesis. SYNTONY - Indwelling integration of parts ( Heidenhaln) ; a natural force within and between cells develop- ing from the specific organization of living matter. TACBYAUXESIS - positive heterogony (Needham, igltO). TACHYGENESIS - speeding up and compression of ancestral stages in development. TACTILE DISPLACEMENTS - movements of parts of the embryo relative to each other, resulting in definite formations and distributions of the germinal material; evidence of organizational influences. TELOBIOSIS - terminal fusion of embryos through operative procedures (see parabiosis). TENDENZEN - (German) autonomous abilities of a germ layer to reach developmental capacities as such without the influence of inductive effects (Lehmann, Raven). (See neighborwise and selfwlse.) TERATOGENETIC - abnormality producing. TERATOLOGY - study of the causes of monster and abnormality formation. TERATOMA - structure which results from random differentiations; malignant assembly of tissues, often well differentiated histologically, generally embedded in an otherwise healthy organ. Some use term embryoma to refer to histological differentiation and teratoma to mean both histological and mor- phological differentiation of the abnormal growth. TETRAD - precocious splitting of the chromosomes in anticipation of both maturation divisions. THIXOTROPY - isothermal reversible sol-gel transformations (Fremdllch'a monograph). A thixotropic gel will liquefy if shaken or stirred, later to return to its previous consistency. THORACO-GASTROSCHISIS - failure of the body wall to close along the mid-ventral line, including the thoracic region. THORACOPAGUS - thoracic union of conjoined twins. TISSUE CULTURE - condition where an explant is able to survive and manifest vital activity; in vitro as opposed to in vivo culturlng of excised tissues or organs (see isolation culture). TOGOGENESIS - all of the processes of novement which result in structure formation. TOTIPOTENCY - related to theory that the isolated blastomere is capable of producing a complete organism. Roux (1912) Included several faculties such as (1) for self-differentiation; (2) for influencing dif- ferentiation or induction of other parts; (5) for specific reaction to differentiating Influences as in dependent differentiation. TRANSPLANT - an embryonic area (cell or tissue) removed to a different environment. Syn., graft. TBANSPLAOTATION - transfer of an embryonic blastema from one region to another of from one germinal layer to another. Incorporation of an isolated fragment by a living organism, not merely the sticking on of a graft. TRANSPLANT, AUTOPLASTIC - exchange of different parts within the same organism. TRANSPLANT, HETEROPLASTIC - exchange of parts between individuals of different species but within the same genus (e.g., from Amblystoma punctatum to A. tigrinum) . TRANSPLANT, HETEROTOPIC - graft location different from graft source; exchange made to a non-ljomologous region of the host; transplantation to a new site. TRANSPLANT, HOMOPLASTIC - grafts exchanged between members of the same species. Syn., homoloplaetlc transplant. GLOSSARY U79 TEANSPLANT, HOMOTOPIC - graft location the same aa the graft source; transplant to the identical site or homologous region. Syn., orthotopic. TRANSPLANT, XENOPLASTIC - graft between organiama of different genera or those still further resolved phylogenetically. Graded series would be autoplastic-heteroplastic - xenoplastic. TBIASTER - abnormal mitotic figure possessing three asters generally cauaing irregular distribution of chromosomes and abnormal cleavages. Other multiple aster conditions noted, (e.g., tetraster, etc.). TRIGGER REACTION - condition where the character, pattern, vigor, progress and speed of a response are in no way related to the releasing event. TBITOGENY - one-third of a. fragment (see merogony) . TROPHIC - the action of the nervous system in the absence of which the muscle tonus faila and in conse- quence, regeneration is impossible. TEOPHOCEBOMATIN - nutritive chromatin of the nucleus. THUE KNOT - slipping of the fetus through a looped umbilical cord to produce a true knot, distinguished from looped blood vessels which cause external bulgings called false knots. TWINS, IDEHTICAL - true twina, from a single egg and having common membranes and umbilicus. TWINS, ORDINARY - pleural pregnancy resulting from the fertilization of separate ova simultaneously liberated from individual follicles. Separate development, implantation, decidua capaularism, and fetal membranes . UMHULLUNG - (German) the proceas of wrapping an inductor in sheets of con5)etent ectoderm to teat its in- ductive power. UNIPOTENT - attribute of certain cells which can give rise to only one simple type of differentiation; presumptive fate and presumptive potency are identical. VALUE, PROSPECTIVE - the realization value of a part of the aum total represented by the proapective potencies. VEGETATIVISATION - shifting of the presuii5)tlve fate of normal ectoderm to become endoderm. Syn., vegeta- lization, endoderminzatlon. Opposed to animallzatlon. VERMIFORM CELL TYPE - elongated form of Isolated embryonic cells with flnger-llke protuberances at the antipole of the coated side ( Holtfreter) . VESICLE, GERMINAL - nucleus of the egg while it is a distinct entity and before the elimination of either of the polar bodies. VISCOSITY - measur'e of inner molecular frlctfon (see Stoke 'a Law). VITAL STAINIMJ - localized staining of embryonic areas with vital, non-toxic dyes, for ptirpoaes of study- ing morphogenetlc movementa (method of Eoux). VITALISM - a philosophical approach to biological phenomena which bases its proof on the proaent inability of scientists to explain all the phenomena of development. Idea that biological activities are directed by forces neither physical nor chemical, but which must be eupra-sclentlflc or super-natural. Effective guidance in development by some non-material agency ( aee mechanism). VITELLIN - egg-yolk phospho-protein. VITELLINE - adj., pertains to yolk, vein, or membrane. VITELLINE MEMBRANE - delicate, outer, non-living and non-cellular egg membrane derived (while In the ovary) probably by the Joint action of the egg and its follicle cells. It is probably the same membrane that is lifted off of the egg at fertilization and is subsequently known as the fertilization membrane. Syn., zona radiata (mammals). WEEEE'S-LAW - the degree of sensitivity to a stimulus in any reacting system is not constant but depends, not alone on the nature of the stimulus, but upon the period of life and the strength of an already existing stimulus. A stimulus therefore represents a change, but a reacting system takes Into ac- count any pre-existing stimulus upon which this change is built. Theory that equal relative differ- ences between stimuli of the same kind are equally perceptible. WOLF SNOUT - projecting of the premaxllla beyond the surface of the face, accoii5>einylng double (hare) lip and sometimes a cleft palate. XANTHOLEUCOPHORES - crystals and soluble yellow pigment; cells bearing such. XANTHOPHORES - yellow pigment in solution; cells bearing this yellow pigment. XIPHOPAGUS - xiphoid fusion of conjoined twins; sometimes the skin alone. YOLK LOBE - lobe of early developing mojluace embryo in which there la actually almost no yolk, but it appears hemla-llke from one of the early blastomeres, diaappearing between cleavages and capable, when Isolated, of giving rise to a dwarfed larva. Syn., antipolar or basal lobe. Opposed to polar lobe. YOLK NUCLEI - bodies responsible for precocious digestion of yolk, derived from nucleoli which break up and pass out through the nuclear membrane. Centers of yolk organization during the growth period of oogenesis. k80 GLOSSARY YOI£ PLUG - large yolk cells which are too large and sluggish to be Innnadlately Incorporated in the floor of the archenteron, hence are found protruding slightly from the mouth of the blastopore to form a plug vhich la distinct hy color from the surrounding pigmented marginal zone (amphihla). Term used as identification of a late stage in gastrulatlon. ZONE, MAEGINAL - presumptive chorda-mesodermal-endodermal complex at the Junction of the roof and the floor of the early gastrula. Syn., germ ring. HOTE: A glossary of some 350 terms in morphological embryology may be found In the author's "Laboratory Manual of Vertebrate Embryology". "On the other handt some very fundamental advances are, upon critical examination, found to rest upon the establishment of a j udic ious defini- tion, A notable instance of this is the enunc iat ion of the pr inc iple of the survival of the fittest, which is essent ially of the nature of a defini- tion, since the fit is that which survive s . " Lotka 1925 'OMNE VIVUM EX OVO" ■OMNIS CELLULA E CELLULA' Virchow