Ls Sia Tar Fee 2 ae at ee er al Tldv yap 7d yuopevov €x Twos Kal eis TL TovetraL THY yéveoly, Kal dar > a“ WS age , oN a“ , , \ 3 , »* ‘ , apxns er apxyv, aro THs mpwTys Kwovons Kal éxovans Hdn Twa Piow n A - ért twa popdiy 7 Towdtov dAXo TéAos’ avOpwros yap avOpwrov Kat ‘ a ‘ > A \@ e ia WA A 4 > gurov yevva putov éx THs wept ExacTov UroKepevys VAyns. TO pev odv , , A UA: > lal s \ ‘ / a No be ‘ Xpovw Mpotépay THY VAHV avayKaiov civar Kal THY yéverw, TO Loy OE THY > , ‘ ‘ c , , ovolav Kal THV ExaoTOV Loppyv. ARISTOTLE. a CONTENTS CHAPTER I INTRODUCTORY CHAPTER II CELL-DIVISION AND GROWTH. 1. Cell-division 2. Growth CHAPTER III EXTERNAL FAcTORS. 1. Gravitation : 2. Mechanical agitation . 3. Electricity and magnetism . 4. Light 5. Heat. ‘ ; ‘ ; : : : ‘ 6. Atmospheric pressure. The respiration of the embryo. 7. Osmotic pressure. The role of water in growth . 8. The chemical composition of the medium 9. Summary . CHAPTER IV ’ Inrernat Factors. (1) The initial structure of the germ as a cause of differen- tiation. 1. The modern form of the preformationist doctrine . 2. Amphibia . : go. isces; “: . : 4. Amphioxus PAGE 22 58 78 89 91 93 97 109 115 126 155 158 163 178 179 Vill 13. CONTENTS . Coelenterata . Echinodermata . Nemertinea . Ctenophora . Chaetopoda and Rcthascx: . Ascidia ; ; . General considerations and cueliatone . The part played by the spermatozoon in the deters mination of egg-structure : The part played by the nucleus in differentiation . (2) The actions of the parts of the developing oem s on one another : ‘ : : : ‘ CHAPTER V Drixscu’s THEORIES OF DEVELOPMENT. GENERAL REFLEC- TIONS AND CONCLUSIONS APPENDIX A On the symmetry of the egg, the symmetry of segmentation, and the symmetry of the embryo in the Frog . APPENDIX B On the part played by the nucleus in differentiation InpEX oF AUTHORS °. INDEX OF SUBJECTS ADDENDA . PAGE 181 183 204 208 213 229 240 247 251 271 303 310 315 322 341 CHAPTER I INTRODUCTORY Tuat living creatures reproduce their kind is a fact which is _familiar to us all, but it is the peculiar privilege and province of the embryologist to observe and to reflect upon that marvellous series of changes whereby, out of a germ which is comparatively structureless and unformed, a new organism is developed which is, within the limits of variation, like the parents that gave it birth. Development is the production of specific form. From a particular kind of germ only a particular kind of individual will normally arise, though unusual conditions may lead to the formation of an abnormality or monstrosity. Thus, while the germ is the material basis, development is the mechanism of inheritance. The student of heredity seeks to express in terms which shall be as exact as possible, ultimately mathematically exact, the degree of similarity between the offspring on the one hand, and parents and more remote ancestors on the other. The embryologist has under his very eyes the process by which that similarity is brought about, and even when the resemblance shall have been stated with all possible precision, it will still remain for him to give an explanation of those changes whereby the inheritable peculiarities of the species are handed on from one generation to the next. Used in the widest sense of the word, development includes not merely the formation of a new individual from a single cell; whether fertilized or not, but also the phenomena of budding and regeneration. In a narrower sense, however, the term is restricted to the first of these processes, and a corresponding distinction is made, however artificially, between Experimental Embryology and Experimental Morphology, when the subject is treated from a physiological point of view. JENKINSON B 2 INTRODUCTORY I In development two factors are obviously involved. One is growth, or increase of volume, more correctly increase of mass ; the other is differentiation, or increase of structure; and, in multicellular organisms, both these factors are accompanied by division of the nucleus and the cell. Segmentation is the first sign, or almost the first sign, the developing ovum gives of its activity; and this cutting up of the egg-cell into parts, which marks the beginning, is also con- tinued during the later stages of ontogeny, and goes on as long . as the life of the organism endures. Growth is especially characteristic of the embryonic and of the adolescent organism. It occurs at different rates in the different cells, and indeed the growth of a group of cells is in itself often an act of differentiation. Growth may depend upon the absorption of water or the assimilation of other substances, ~and this may lead simply to an increase in the size of internal cavities, as in the blastula of Echinoderms or the Mammalian blastocyst ; to an increase in the volume of the living proto- plasm; or to the secretion of intracellular or intercellular substances, either organic (for example, the notochordal vacuoles, the matrix of cartilage and bone) or inorganic (the skeletal spicules of Echinoderm larvae, Sponges, and Coelenterata), This increase of mass is not only conditioned by the presence of food in the form of substances found in the environment, but depends on such external circumstances as temperature, atmospheric and osmotic pressure, and so forth. But while the embryo is dividing up its material—a material which is already to a certain extent heterogeneous, composed, for example, of protoplasm and deutoplasm or yolk—while it is increasing its mass, it is also undergoing a process of differentia- tion; and, as even a superficial acquaintance with embryology will inform us, one of the most characteristic features. of differentiation is that it occurs in a series of stages which follow upon one another in regular order and with increasing complexity. When segmentation has been accomplished—some- times, indeed, during segmentation—certain sets of cells, the germ-layers, become separated from one another. Each germ- layer contains the material for the formation of a definite set of I INTRODUCTORY 3 organs—the endoderm of a Vertebrate, for instance, contains the material for the alimentary tract and its derivatives—gill-slits, lungs, liver, bladder, and the like ; the germ-layers are therefore not ultimate but elementary organs, and elementary organs of the first order. In the next stage these primary organs become subdivided into secondary organs—as the archenteron of an Echinoderm becomes portioned into gut and coelom-sac, or the ectoderm of an Harthworm into epidermis, nervous system, and nephridia—and in subsequent stages these again become suc- eessively broken up into organs of the third and fourth orders * and so on, until finally the ultimate organs or tissues are formed, each with special histological characters of its own. This end is, however, not necessarily reached by all the tissues at the same time, Indeed, it is no uncommon thing for certain of them to attain their final structure while the others are yet in a rudimentary condition; thus, in some Sponges the scleroblasts begin to secrete spicules in the larval period, nematocysts may be formed in the Planula of the Coelenterates, notochordal tissue is differentiated in the newly hatched tadpole of the Frog; and, speaking generally, larval characters are developed at a very early stage. To this regular sequence of ontogenetic events Driesch has applied the term ‘rhythm’, the rhythm of development, The organs of the body are, however, by no means all formed of single tissues—bone, epithelium, blood, and the rest—but are com- pounded, frequently of very many tissues, and this ‘ composition ’, to quote a term of Driesch’s again, is another of the obvious features of organogeny. While, therefore, in the last resort all differentiation is histo- logical, that final result, the assumption by the cells of their - definitive form, is only achieved after many changes have taken place in the position of the parts relatively to one another while the organs are being compounded, and so its specific shape conferred upon the whole. body. It is possible to find a few general expressions for the manifold changes that take place in the relative positions of the parts. Several years ago, in 1874, His compared the various layers of the chick embryo to elastic plates and tubes; out of these he B 2 4 INTRODUCTORY I suggested that some of the principal organs might be moulded by mere local inequalities of growth—the ventricles of the brain, for instance, the alimentary canal, the heart—and he further succeeded in imitating the formation of these organs by folding, pinching, and cutting india-rubber tubes and plates in various ways. This analysis, however, deals only with the foldings of flat layers, and must be supplemented by a more exhaustive catalogue of the processes concerned in ontogeny, such as that more recently suggested by Davenport. Davenport resolves the changes in question into the movements of cells or cell aggre- gates, the latter being linear, superficial, or massive, and within ~ the limits of these categories the phenomena are susceptible of further classification. The catalogue proceeds as follows :— I, THe Movements or SINGLE CELLS. 1. Migration of nodal thickenings in a network of protoplasm: e. g. the migration of the ‘ cells’ to the surface of the Arthropod ovum to form a blastoderm, the movements of vitellophags, and yolk-nuclei (Fig. 1). OEE OF Fie. 1.—Sections of the egg of Geophilus ferrugineus showing two stages in the formation of the blastoderm: b/, blastoderm; dp, yolk pyramids ; gr, groups of blastoderm cells on what will be the dorsal side ; k, nuclei surrounded by masses of protoplasm. (After Sograff, from Korschelt and Heider.) I INTRODUCTORY 5 2. Migration of free amoeboid bodies: e.g. the mesenchyme eells in the Echinoderm gastrula, the lower layer cells of Elasmobranchs, the blastomeres eee the yolk-cells in Triclads and Salps. . 3. Aggregation of isolated sails a. Linear aggregates: e.g. the kidney of Lamellibranchs, the yolk-gland of Turbellaria, capillary blood-vessels. b. Superficial aggregates: e.g. the blastoderm of Arthro- pods, the formation of the imaginal gut-epithelium in some Insects. ce. Massive aggregates: e.g. the gemmule of Sponges, the spleen of Vertebrates. 4. Attachment of isolated cells to another body: e.g. the union of muscles to the shell in Mollusca and Arthropoda, of tendon to bone in Vertebrates, the application of skeletal cells to the notochord. 5. Investment and penetration by isolated cells: e.g. the follicle cells between the blastomeres in Tunicata, the muscles of the gut in various animals, the septa of the corpus luteum, the formative cells of the vitreous body of the Vertebrate eye, the immigration of the nephric cells in the Earthworm. 6. Transportation of bodies by wandering cells: e.g. of the buds in Doliolidae. 7. Absorption by wandering cells: e.g. phagocytosis in Insect pupae and in the tadpole’s tail. : 8. We may place here the frequent alterations in the shapes of cells, which do not apparently involve growth: e.g. when flat cells become columnar. II. Tur Movements or Ceti AGGREGATES. A. Linear Aggregates. 1. Growth in length: e.g. the growth of the roots and stems of plants, of the stolons and hydranths of Hydroids, the out- growth of nerves, of the necks of unicellular glands, the growth of the blood-vessels from the area vasculosa into the body of the Chick embryo, of blood-vessels towards a parasite, the growth of mesoblastic and other germ-bands in Annelids, the back-growth of the Vertebrate segmental duct, and the like. 6 INTRODUCTORY I 2. Splitting. a, At the end, that is, branching: e.g. of nerves, blood- vessels, kidney tubules, glands, tentacles. 8. Throughout the length: e.g. the segmental duct of Elasmobranchs, the truncus arteriosus of Mammalia. 3. Anastomoses: e.g. of the dorsal and ventral roots of the spinal nerves, of nerve plexuses, of capillaries, of bile capillaries, of the excretory tubules of Platyhelmia. 4. Fusion with other organs: e.g. of a nerve with its end- organ, of the vasa efferentia with mesonephric tubules, of nephridia with the coelom in Annelida. B. Superficial aggregates. i. Increase of area. - a. Growth of a sphere. 1, Equal in ali directions: e.g. the blastula of Echino- derms. 2. Unequal. a. Unequal in different axes: e.g. the conversion of a spherical blastula into an ellipsoid Planula in Coelenterata, or into an ellipsoid Sponge larva, or of the spherical into the ellipsoid blastocyst in Mammalia. B. Unequal at different poles: e.g. the formation of ovoid forms, such as Planulae, the club-shaped gland of Amphioxus, the auditory vesicle of Vertebrata. b. Growth of a plane surface. 1, Equal in all directions: e.g. the growth of the blastoderm over the yolk in Sauropsida, or Cephalopoda. 2. Unequal. a. When parts lying in one plane move out of that plane: e.g. invaginations and evaginations of all descriptions (Fig. 2). 8B. When parts—e. g. a row of cells—lying in one plane are moved in that plane: e.g. the germ-bands of Clepsine, by the growth of the epiblast (Fig. 3). ii, Alterations of thickness. a. Increase: thickenings: e. g. the formation of the central nervous system in Teleostei, the formation of gonads from the I _ INTRODUCTORY 7 coelomic epithelium, the development of hair follicles, the tropho- blast in the Mammalian placenta (Fig. 4). Ay 7a E ol olol oye [ele lelelolelelelolele]s[ejelelajeyo Fig. 2.—Three stages of an invagination or evagination. (After Korschelt and Heider.) ol ele [olde eleloleiaeklep lel vey EES uv a SOO! ofeleleie ee) RoSeeker’ (2; Fig. 3.—Displacement of a row of cells in an epithelium. (After Kor- schelt and Heider.) Fig.4.—Four stages in the formation of an epithelial thickening of many layers. (After Korschelt and Heider.) b. Decrease: thinnings: e.g. in the roof of the thalam- encephalon and medulla, the outer layer of the lens, the tropho- blast of the Mammalian blastocyst. 8 INTRODUCTORY I iii. Interruptions of continuity. a. By the atrophy of part of a layer: e.g. when the floor of the archenteron together with the underlying paraderm dis- appears in Amniota (Fig. 5). A pogoggoeouooooogoo0n0 B _ geleelerezs—~s-elele[e[elelelo ee elelalelelejelej9> elelelelelelelelo Fic. 5.—Three stages in the development of an interruption of con- tinuity perpendicular to the surface of an epithelium. (Perforation.) (After Korschelt and Heider.) 6. By the detachment of a part: e.g. of the medullary plate from the ectoderm in Amphiowus (Fig. 6), of the notochord from the roof of the archenteron in Urodela and Petromyzon. A AOOU MUU 0) 9)8)0)0)* le levee Ba PMS CPE, Pease D RRR Ee Co, Fie. 6.—Scheme of the formation of the medullary canal in Amphioxus. (After Korschelt and Heider.) fA INTRODUCTORY 9 iv. Concrescence of layers. a, By their margins: e.g. the edges of the ectoderm over the medullary plate, the edges of the embryonic ectoderm inside A Aslolefololee> selelelelelolele PB eJefelole] e)e)eexeielelelolololelele Zi s[oJele[efelejelefe[ejelelelelejefejelele Fie. 7.—Fusion of two cell plates by their margins. (After Korschelt and Heider.) the serosa of Sipunculus, the embryonic plate with the tropho- blast in some Mammals (Figs. 7, 9). b. By their surfaces (Figs. 8, 9, 10): e.g. when the stomo- daeum or proctodaeum open into the gut, when the medullary A (After Korschelt and Heider.) folds meet, when the edges of the peritoneal groove close to form the canal of the oviduct in Amphibia and Amniota. This concrescence is commonly followed by a communication of the cavities on opposite sides of the adherent layers, as when the stomodaeum opens into the gut, or the amnion-folds unite ; but not necessarily, as when the somatopleure fuses with the trophoblast, or the allantois with the somatopleure in Mammalia. v. Splitting of a layer into two: e.g. the inner wall of the pineal vesicle in Lacertilia (Fig. 11). 10 INTRODUCTORY I )) O) a = iW Fie. 9.—-Diagram to illustrate Fie. 10.— Scheme of the formation of the formation ofthefore-gut(stom- the medullary canal in a Vertebrate. odaeum) and its opening into the (After Korschelt and Heider.) mid-gut: ec, ectoderm ; md, mid- gut ; vd, stomodaeum. (After Kor- schelt and Heider.) plalylalolelalelgeleleleleslelelale ewENee CREE SOR EES Fie 11.—Three stages in the development of an interruption of continuity parallel to the sur- face of an epithelium. (Dela- mination.) (After Korschelt -and Heider.) I INTRODUCTORY ll Cc: Massive aggregates. i. Changes in volume. a. Unequal in different axes: e.g. when the spherical larva becomes cylindrical in Dicyemidae. B. Unequal at different points : e.g. the outgrowth of limb- buds of Vertebrates and other forms, of the buds of plants. ii. Rearrangement of material. a. Simple rearrangement of cells: e.g. in the formation of the concentric corpuscles of the thymus, in the development of kidney tubules in the metanephric blastema of Amniota, in the grouping of the cells to form ectoderm, gut and atrium in the Salps. 6. Development of an internal cavity: e.g. segmentation cavities, lumina of ducts and blood-vessels, of the coelom and many generative organs. c. Dispersion of the elements of an aggregate: e.g. in gemmule formation in certain Sponges, in unipolar immigration in some Sponges and some Coelenterates, in the liberation of the germ-cells. iii. Division of masses. a. By constriction: e. g. the segmentation of the mesoderm and neural crest. b. By splitting: e.g. the nervous system from the ectoderm in Teleostei and many Invertebrates, the notochord from the roof of the archenteron. iv. Fusion of masses : e. g. of originally separate nerve ganglia (Vertebrates, Arthropods, Annelids), of myotomes, of somites in Arthropods. v. Attachment of one mass to another: e. g. of sclerotome to notochord. It will be seen that this résumé of the principal kinds of movement executed by the developing parts extends His’s principle of the local inequality of growth from flat layers to linear and massive aggregates and at the same time includes the movements of isolated cells. Davenport, however, is not content merely to give a simple classification of the phenomena ; he goes further, and endeavours to express them in terms of responses to stimuli, an idea due in the first instance to Herbst. 12 INTRODUCTORY I Thus he suggests that the migrations of vitellophags and mesenchyme cells, the thickenings, thinnings, and perforations of flat layers, the rearrangements of cells in a massive aggregate, their dispersion, the constriction, and splitting and fusion may be regarded as tactic responses, the growth in various ways of linear aggregates, the concrescence of layers and masses as so many tropic responses to stimuli which may be positive or negative and exerted by other organs or by agents in the world outside. Now it is clear that the analyses both of His and Davenport aim at something more than a mere description of ontogenetic events, for a serious attempt is here made to give a causal, if you will a mechanical, explanation of those events, and the sub- ject thereby raised at once from the level of mere morphology or morphography to a loftier, aetiological point of view. There are, indeed, two methods by which embryology, like any other branch of zoology, may be investigated. One is purely descriptive, anatomical, morphological. By this method, truly, great results have been achieved. The life-histories of members of all the most important groups of the animal king- dom have been worked out, and the science of Comparative Embryology has been built up. Nor has an explanation of the: process been lacking. For ontogeny is, the fundamental Bio- genetic Law assures us, a recapitulation of and therefore explicable in terms of phylogeny; and since on this principle the individual repeats in its development the ancestry of its race, embryology affords a means of tracing out the relationships of the organism and establishing the homologies of its parts. Unfortunately a more intimate acquaintance with the facts has made it abundantly clear that development is no mere repeti- tion of the ancestral series, that the organism has manifold ways of attaining its single end, that those resemblances in early stages which were held to constitute the most triumphant vindication of the Biogenetic Law bear no constant relation to the similarities of adult organization, that the attempt to find in pits aes an absolute criterion of homology is vain. The facts thus remain unexplained, as in truth it was only to be supposed that they would. A method, however comparative, which relies on mere observation, and is content to wait for 1 INTRODUCTORY 13 Nature’s own experiments, cannot hope to arrive at sound in- ductions, or to establish general laws of causation. There is, however, another way. Development, the production of form, may be regarded as one of the activities, one of the functions of the organism, to be investigated like any other function by the ordinary physiological method of experiment ; and the ideal of the experimental or physiological embryologist is to give a complete causal account, whether the causes are external or internal, of each stage, and so of the whole series of ontogenetic changes, his weapon, to borrow Roux’s splendid phrase, ‘die Geistesanatomie, das analytische causale Denken.’! This effort is, of course, no modern one. Speculation into the nature and essence of development begins, indeed, with the Greeks, and theories of fertilization and development are to be found in the writings of Aristotle.? In fertilization the male element, which, according to Aristotle, provides the formal and efficient causes in providing the necessary perceptive soul, acts upon the mere matter, endowed only with a nutritive soul, which is given by the female, in the same sort of way, to use his own illustration, as rennet coagulates milk. In the germ thus formed the parts of the embryo, which can only be said to pre-exist potentially, arise not simultaneously but in gradual succession, first the heart, then the blood, the veins from the heart and the various organs about the veins by a process of condensation and coagulation, the anterior parts of the body being built up first. This Aristotelian doctrine appears to have persisted through the Middle Ages; it reappears in the seventeenth century in the pages of Hieronymus Fabricius ab Aquapendente and his pupil William Harvey in essentially the same form, although both authors differ from Aristotle in certain matters of observational detail. Thus Fabricius? states that ‘ope generatricis facultatis pulli partes, quae prius non erant, produci atque ita ovum in pulli corpus migrare’, while Harvey * gives to development as thus conceived of the name of ‘ Epigenesin sive partium super- additionem’, though he believes that in some cases (Insects) the * Roux, 1885. ? Aristotle, De Gen., i. 20. 18; ii. 4.48; 5. 2, 3,10; 6. DeAn., ii. 4. 2, 15; 5; 6. ® Fabricius, 1. c., p. 22. * Harvey, l. c., Ex. 44. 14 INTRODUCTORY I process is one of ‘metamorphosis’ or the simultaneous origin of all parts. In generation properly so called, however—in the development of the Chick, for example—the process ‘a parte aliqua, tanquam ab origine, incipit ; eiusque ope reliqua membra adsciscuntur: atque haec per epigenesin fieri dicimus: sensim nempe, partem post partem’, and this ‘pars prima genitalis’ Harvey held, in opposition to Aristotle, to be the blood. But in spite of the exact observation and brilliant exposition of his followers, the teaching of Aristotle was destined to be overshadowed and eclipsed, temporarily at least, by a new hypo- thesis which, appearing: first towards the end of the seventeenth century, swept the schools and universities, and dominated biological speculation for a hundred years. This was the theory of Evolution or Preformation. According to it the future animal or plant is already present in miniature in the germ with all its parts complete, invisible or hardly visible it may be, but still there, and not merely ‘ potentid’; and in development there is no such thing as ‘ generation’, but only growth, whereby that which was before impalpable and invisible becomes tangible and manifest to our eyes. A further and logical] outcome of the hypothesis was the doctrine of ‘emboite- ment’, enthusiastically described by Bonnet as ‘une des plus belles victoires que l’entendement pur ait remporté sur les sens ’.? The organism present already in the germ, with all its parts complete, possesses of necessity the germs of the next generation, and so on in indefinite though not in infinite regress, for as Bonnet is careful to tell us, ‘ I] ne faut pas supposer un emboite- ment a V’infini, ce qui seroit absurde. La divisibilité de la matiére A l’infini par laquelle on prétendroit soutenir cet em- boitement est une vérité géométrique et une erreur physique.’? Swammerdam solved the difficulty in another way. All the germs of the human race must have been present in the bodies of our first parents, and ‘exhaustis his ovis humani generis finem adesse ’.* The theory became widely held. First put forward by Marcello 1 Harvey, 1. c., Ex. 50. 2 Bonnet, Cont. de la nat., 7™° partie, c. ix ; Ciuvres, vol. iv, p. 270. 5 Bonnet, Consid. sur les corps org., ¢. viii; Cuvres, vol. iii, p. 74. * Swammerdam, 1679, pp. 21, 22. I INTRODUCTORY 15 Malpighi in the memoir, ‘ De formatione pulli in ovo,’ which he presented to the Royal Society of London in 1673, it was not only adopted by biologists of prestige, by Swammerdam, Haller, who in his early days had been an advocate of Epigenesis, de Buffon, and Bonnet, but secured the adherence of philosophers of such eminence as Malebranche and Leibniz. In some cases it was accepted as a result of observation. Thus Malpighi,' in the treatise referred to, asserted that he had himself observed the chick in the unincubated egg, ‘inclusum foetum animadvertebam, cuius caput cum appensae carinae staminibus patenter emergebat, while de Buffon? expresses himself even more categorically. ‘J’ai ouvert,’ he says, ‘une grande quantité d’ceufs a différens temps, avant et aprés l’incubation, et je me suis convaincu par mes yeux que le poulet existe en entier dans le milieu de la cicatricule au moment qu’il sorte du corps de la poule.’ To others, however, it was rather a matter of theoretical necessity. Haller explains his conversion from the contrary opinion by asking the very pertinent question, ‘Cur vis ea essentialis quae sit unica tam diversas in animali partes semper eodem loco, semper ad eundem archetypum struit; si materies inorganica mutabilis et ad omnem figuram recipiendam apta est ? Cur absque ullo errore ex gallinae mista materie ea vis semper pullum, ex pavone pavonem fabricatur ?” ‘Nil nisi vis dilatans et progrediens recipitur. Ab ea nihil sperarem nisi vasorum rete tamdiu continuo amplius futurum quamdiu vis expandens resistentiae superandae par est. Cur loco eius retis cor, caput, cerebrum, ren struuntur? Cur in singulo animali suus ordo partium? Ad eas quaestiones nulla datur responsio,’ a charge which is, of course, perfectly just.® _ Bonnet’s argument is different. The heart of the chick, he points out, is already present in the egg; and since anatomy teaches that all the parts of an animal ‘doivent avoir toujours coexisté ensemble’, preformation follows as a matter of course.* The belief in preformation continued paramount till towards the end of the eighteenth century, nor was it till the publication 1 Malpighi, 1. c., p. 4. 2 de Buffon, 1. ¢., p. 351. ® Haller, 1778, VIII. i. 29, p. 121. ‘ * Bonnet, Cont. de la nat., 7™* partie, c. ix; Cuvres, vol. iv, p. 261. 16 INTRODUCTORY y 1 in 1774 of Caspar Friedrich Wolff’s Zheorta Generationis that the evolutionists were aroused from their dogmatic slumbers, Putting speculation on one side, Wolff returned to the method of Harvey, Fabricius, and may we not say also of Aristotle, the method of exact observation. He demonstrated the presence in the unincubated egg not of a complete organism, but of ‘globules’ ; ‘partes enim constitutivae, ex quibus omnes corporis animalis partes in primis initiis componuntur, sunt globuli,’ ! and described the epigenetic formation of the heart and blood-vessels, the central nervous system, the limbs and the ‘ Wolffian’ bodies from these primary elements. Development thus consists of the gradual production and organization of parts; ‘embryonis partes sensim produci, mea observata suadent,’* and again, ‘ suppeditari prius partem, deinde eam organisari intelligitur ’.? The ground was thus taken from beneath the feet of the’ preformationists, and Epigenesis restored to its former place of honour as the fundamental expression of developmental fact. ~ Tacitly accepted by all the great embryologists of the nine- teenth century—Pander, von Baer, Reichert, Bischoff, Remak, Kélliker, Kowalewsky, Haeckel—the epigenetic idea continued to control the progress of research. ‘These were men who set themselves to describe the sequence of changes that the embryo passes through with all possible accuracy, and over as wide a range as might be of animal form, They made Comparative Embryology. On the facts that they discovered new light was shed by the doctrine of descent with modification, or evolution in the wider sense of the word. Von Baer had pointed out that in any group of animals the embryos were more like one another than were the adult organisms, and this now became easily translated by Haeckel into the idea that the form which is in every group—ultimately in all groups—the common starting- point of individual development is representative of the common ancestor of the race. Ontogeny was thus not merely expressed but explained in phylogenetic terms. Now that, as we have already seen, the proposed explanation 1 Wolff, 1. c., Praemonenda, xviii. ? Id. ib. xxiii. 8 Id. ib., De Gen. An., § 240. a INTRODUCTORY 17 has very largely broken down, Epigenesis, taken by itself, remains, not a theory in terms of cause and effect, but a mere description of what occurs, and it is the crying need for such a theory that has given birth to modern experimental embryology. The new era opens with the publication by Wilhelm His in 1874, just a hundred years after the appearance of the Theoria Generationis, of a remarkable series of essays entitled Unsere Kérperform und das physiologische Problem threr Entstehung. In these essays His, who was already in revolt against the ‘ Biogenetic Law’, not only sought to give a mechanical explanation of differentiation, but also laid down his famous ‘Prinzip der organ- bildenden Keimbezirke.’ According to this principle of ‘germinal localization ’, every spot in the blastoderm corresponds to some future organ: ‘das Material zur Anlage ist schon in der ebenen Keimscheibe vorhanden, aber morphologisch nicht abgegliedert, und somit alssolches nicht ohne Weiteres erkennbar.’! Conversely, every organ is represented by some region in the blastoderm, and ‘wenn wir consequent sein wollen’ in the fertilized, or even unfertilized, egg. In other words, although the parts of the embryo cannot be said to be preformed in the germ, the materials for those parts are already there, prelocalized, arranged roughly, at least, as the parts themselves will be later on. In this material unequal growth produces the form of the parts, and so of the whole body. Whether there is a strict causal connexion between each material rudiment and the organ which arises from it, whether these rudiments could be interchanged without prejudice to the normality of subsequent development, is a ques- tion which is not touched upon by His. It was reserved for another anatomist, Wilhelm Roux, to raise what in His’s hands had been merely a principle to the rank of a theory, the ‘ Mosaik- theorie’, or theory of self-differentiation. For Roux, no doubt, the ‘Mosaik-theorie’ was in part the outcome of the theoretical necessity of explaining the specific nature of development ; but it rests also upon a basis of observa- tion and experiment. The coincidence in a majority of Frogs’ eggs of the first furrow with the sagittal plane, the production of local defects in the embryo by local injuries to the egg, the 1 His, le. §ii. JENKINSON C 18 INTRODUCTORY - occurrence of certain natural monsters (Hemitheria anteriora, for example) in which one half of the body is normally de- veloped, the other entirely suppressed, and the experimental demonstration of the formation of a half-embryo from one of the first two blastomeres of the Frog’s egg when its fellow had been killed, all led Roux to regard the development of the whole and of each part as essentially a process of self-differentiation, a process, that is to say, of which the causes reside wholly within the fertilized ovum and within each part as it is formed, though allowance was made for the possible formative influence of the parts on one another in later stages. External conditions, though they may be necessary in the same sense as they are generally necessary to the maintenance of life, are yet of no importance for differentiation regarded as a specific activity of the organism. In the meantime, an experiment of Pfliiger’s had apparently shown that, however obviously each part of the egg-cell might be related to the production of a particular organ, the relation was no necessary one, but that, on the contrary, the parts were all equivalent and the ovum ‘isotropic’. Pfliiger demonstrated that in a Frog’s egg which had been prevented from assuming its normal position with the axis vertical, the planes of the * segmentation furrows bore no constant relation to the original ege-axis, that is to say to the structure of the egg, though they exhibited the same relation to the vertical as when de- veloping in the normal position. Further, in such forcibly upturned eggs the plane which included the original egg-axis and the present vertical axis became the median plane of the embryo, whose axes were disposed with regard to the vertical as in normal cases. Any part of the egg, therefore, might give rise to any part of the embryo, according to the extent to which and the direction in which the egg-axis had been diverted out of its original vertical position, and hence the egg-substance was ‘isotropic’; the planes of segmentation and the embryonic axes . being determined by gravity. In fact, Pfliiger went so far as to say that an egg only becomes what it does become because it is always. placed under the same external conditions. Nor was this conception of the isotropy of the ovum invali- dated by Born’s proof that in these eggs there is a redistribution I INTRODUCTORY 19 of yolk and protoplasm owing to the sinking of the former to the lower, the rising of the latter to the upper side of the egg. For though the egg thus comes to acquire a secondary structure about an axis which is vertical, still the arrangement of the parts of the supposed rudiments of the organs must have been dis- turbed. Yet from such ova normal tadpoles are developed. It became necessary, therefore, to locate the self-differentiating substance, the ‘idioplasma’, mainly, at any rate, in the nucleus ; and this idioplasma was imagined as composed of dissimilar determinant units, each representative of some part or character of the organism and arranged according to a plan or architecture which corresponds in some way with the architecture in the embryo of the organs represented. All that is necessary, then, and all that happens, at least in the early stages of development, is the gradual sundering of these units from one another by suc- cessive qualitative divisions of the nucleus and their distribution to the cytoplasm, where each determines the assumption by the cell to which it is allocated of that character which it represents. Roux’s ‘ Mosaik-theorie’ and Weismann’s very similar but more elaborate hypothesis of the constitution and behaviour of the germ-plasm both frankly involve the belief that every separately inheritable quality of the body has its own repre- sentative in the germ, with the difference, however, that this preformation, extended by Weismann to the adult characters, is limited by Roux to those of the embryo. The renewed inquiry into the nature and essence of development has thus simply resulted in the resuscitation of the eighteenth-century doctrine of evolution, though in a far more subtle form. Once again we find our- selves face to face with the old alternative, Preformation or Epigenesis ; and it is to the desire of solving this problem that a very considerable proportion of modern experimental research is attributable. Though much of this has been directed against and been destructive of the ‘Mosaik-theorie’, which as far as the nucleus is concerned has now been abandoned by Roux himself,! renewed investigation has proved the existence in many cases of definite and necessary organ-forming substances in the cytoplasm, while the necessity for finding a causal explanation 1 Roux, 1903. C2 20 INTRODUCTORY I of what is obviously in some sense a predetermined process, without presupposing the preformation in the germ of morpho- logical units representing every possible inheritable character, has issued in Herbst’s and Driesch’s conception of the events of ontogeny as so many responses to stimuli exerted by the develop- ing parts on one another. At the same time the need for inquiry into the external conditions and the part they may play in growth and differentiation has not been forgotten. Thus though it would be vain to pretend that the ideal of a complete causal explanation has yet been realized, still some material has been gathered for an answer to each of the two main questions: what are the internal, what the external con- ditions that determine the course: of development? These questions we shall discuss in the following pages. It will then only remain to inquire whether a causal explanation—in the accepted sense of the phrase—a mechanics of ontogeny which resolves the single occurrences first into general physiological laws, and these in the last resort into the generalizations of physics and chemistry, can ever afford a theory which may be said to be complete either from a scientific or from a philo- sophical point of view. Should the mechanical explanation prove to be scientifically insufficient, it may be necessary, with Driesch and the neo-vitalists, to invoke a consciousness of the end to be realized to guide and govern the merely material elements ; but even were this not so it would still be incumbent upon us to consider whether the end itself—not the conscious- ness of it—is not the final, and yet none the less the principal determining cause of the whole process. LITERATURE ARISTOTLE. De generatione animalium, ed. Bekker, Oxford, 1837. De partibus animalium, ed. Bekker, Oxford, 1837. De anima, ed. Trendelenburg, Berlin, 1877. C. Bonner. Céuvres, Neuchatel, 1781. Contemplation de la nature, vii™e partie; Considérations sur les corps organisés; Mémoire sur les germes; Palingénésie philosophique. G. Born. Ueber den Einfluss der Schwere auf das Froschei, Arch. mikr. Anat. xxiv, 1885. ey INTRODUCTORY 2] L, pE Burron. Histoire naturelle, générale et particuliére, vol. ii, Paris, 1749. C. B. DAVENPORT. Studies-in morphogenesis: iv. A preliminary catalogue of the processes concerned in ontogeny, Bull, Harvard Mus, xxvii, 1896. H. Drreson. Analytische Theorie der organischen Entwicklung, Leipzig, 1894. H. Fasrictus AB AQUAPENDENTE. De formatione ovi et pulli. Opera omnia, Lipsiae, 1687. A. HALLER. Hermanni Boerhaave praelectiones academicae: edidit et notas addidit Albertus Haller, v, Gottingae, 1744. Primae lineae physiologiae, Gottingae, 1747. Elementa physiologiae corporis humani, Lausannae, 1778. W. Harvey. Exercitationes de generatione animalium, London, 1651. C. Hergst. Ueber die Bedeutung der Reizphysiologie fiir die causale Auffassung von Vorgiingen in der thierischen Ontogenese, Biol. Centralbl. xiv, xv, 1894, 1895. W. His. Unsere Kérperform und das physiologische Problem ihrer Entstehung, Leipzig, 1874. G. W. Lereniz. CEuvres philosophiques, ed. by P. Janet, Paris, 1900. Systéme nouveau de la nature, 1695. Principes de Ja nature et de la grace, 1714. Essais de Théodicée. Préface, 1710. N. MALEBRANCHE. Recherche de la vérité, Simon’s edition, Paris, 1846. M. Matrieut. De formatione pulli in ovo, London, 1673. E. Priitger. Ueber den Einfluss der Schwerkraft auf die Theilung der Zellen und auf die Entwicklung des Embryo, Pfliiger’s Arch. xxxi, xxxii, 1883. _ W.Rovx. Einleitung zu den ‘Beitriigen zur Entwicklungsmechanik des Embryo’, Zeitschr. Biol. xxi, 1885; also Ges. Abh. 13, Leipzig, 1895. W. Roux. Ueber die kiinstliche Hervorbringung ‘halber’ Embry- onen, Virchow's Arch., 1888; also Ges. Abh. 22. W. Roux. Ueber Mosaikarbeit und neuere Entwicklungshypothesen; Anat. Hefte, 1893 ; also Ges. Abh. 27. W. Roux. Einleitung, Arch. Ent. Mech. i, 1895. W. Roux. Fiir unser Programm und seine Verwirklichung, Arch. Ent, Mech. v, 1897. W. Roux. Ueber die Ursachen der Bestimmung der Hauptricht- ungen des Embryo im Froschei, Anat. Anz. xxiii, 1903. W. Roux. Vortrige und Aufsitze tiber Entwicklungsmechanik der Organismen, Leipzig, 1905. J. SWAMMERDAM. Miraculum naturae sive uteri muliebris fabrica, Lugdunum Batavorum, 1679. Histoire générale des Insectes, Utrecht, — 1682, C.F. Wotrr. Theoria generationis, Halle a. d. Saale, 1774. CHAPTER II CELL-DIVISION AND GROWTH 1, CELL-DIVISION In a future chapter we shall see that there is no necessary connexion between segmentation and differentiation. Never- theless, since cell-division is the first sign, or almost the first sign, that a developing organism gives of its activity; since, moreover, cell-division accompanies the later processes of growth and differentiation, we may briefly discuss what is known of those factors which determine the direction of division in general, and in particular the pattern of segmentation. We shall first presume that segmenting ova may be grouped under several distinct types, as follows :— 1. The radial type. Here the first division is meridional, the second meridional and at right angles to the first, the third equatorial—or more often latitudinal—and at right angles to both the preceding, the fourth meridional and at forty-five degrees to the first two, the fifth latitudinal. What is charac- teristic above all of this type is, first, that four surfaces of contact between cells meet in one line; for example, the four surfaces between the first four blastomeres meet in the egg-axis, while each pair of animal cells lies exactly over each pair of vegetative cells after the third division; and secondly, the blastomeres are radially arranged about the axis. This type has been observed in Sponges (Sycon! (Schulze)), in Coelenterates, in Crinoids, Holothurians, and Echinoids (Fig. 12) amongst the Echinoderms, in Ectoproctous Polyzoa, in Amphiowus and the Vertebrates, and in some Crustacea—Celochilus (Grobben), Lucifer (Brooks), Cyclops (Hicker), Branchipus (Brauer) and some Cirrhipedes. Certain of these cases present special peculiarities. In Echinoids micromeres are formed at the vegetative pole by the division of the fourth phase (Fig. 12, /). 1 In Sycon the third cleavage is meridional, the fourth latitudinal. Fre. 12.—Normal development of the sea-urchin Strongylocentrotus lividus. (After Boveri, 1901.) The animal pole is uppermost in all cases, and in the first two figures the jelly with the canal (micropyle) is shown. a, primary oocyte, the pigment is uniformly peripheral. b, ovum after extrusion of polar bodies. The pigment now forms a subequatorial band. The nucleus is ex-axial. ce, d, first division (meridional). e, 8 cells, the pigment almost wholly in the vegetative blastomeres. f, formation of mesomeres (animal cells) by meridional division: the vegetative cells have divided into macromeres and micromeres. g, blastula. h, mesenchyme blastula. 7, j, k, invagination of the pigmented cells to form the archenteron of the gastrula. In j the primary mesenchyme is separated into two groups, in each of which, in k, a spicule has been secreted. In & the secondary, pigmented mesenchyme is being budded off from the inner end of the archenteron. 24 CELL-DIVISION AND GROWTH Il. 1 In Asteroids and Ophiuroids the division is at first tetrahedral, and to be classed, therefore, with those of the following type; after the second furrow, however, the blastomeres are rearranged, and division thenceforward is radial. In Vertebrata segmentation is altered in megalecithal eggs by the amount of yolk present. It becomes meroblastic ; still the radial type is preserved, though the sequence of the furrows is often altered, the third, for instance, being frequently meridional, and the fourth latitudinal. Amongst the Ascidians Pyrosoma has a large-yolked, telolecithal, and radially segmenting egg. In the Placental Mammals the first two divisions may conform to this type; but segmentation soon becomes irregular. The accumulation of yolk in the Arthropod egg has resulted in a totally different type of meroblastic segmentation. The yolk is here uniformly distributed about the central protoplasm. In the latter is placed the segmentation nucleus, and this central mass divides into a nunber of cells, which subsequently migrate to the surface and form a blastoderm; the egg is then centro- lecithal (Fig. 1). The stages of the development of this modifica- tion may be seen in the Crustacea. In certain forms—those alluded to above (with the exception of the Cirrhipedes)— division is holoblastic and radial. In Gammarus, Branchipus (Brauer), Peltogaster (Smith) segmentation is at first total, but the inner yolk-containing ends of the cells subsequently fuse. In Crangon, Moina, Daphnella, Daphnia, Orchestia segmentation is superficial. In Isopods and in Decapods segmentation is internal. In all cases the result in the end is the same, a peripheral blasto- derm, a central yolk. But the blastoderm is not always, though it is often, formed simultaneously over the whole surface. There are cases in which it appears first on the ventral side, and by what may be described as a still more precocious formation of the blastoderm, segmentation may begin at this, the future ventral, point, as in M/ysis and Oniscus. In these cases the egg is telolecithal. In the Insects, Arachnids, Myriapods, and Pertpatus novae- zealandiae, the segmentation is meroblastic and the egg comes to be centrolecithal. In Peripatus capensis and in some other species it would appear that the yolk has been secondarily lost. II. 1 CELL-DIVISION 25 2. The second type is the so-called spiral form of cleavage (Fig. 13). This is especially characteristic of the eggs of Poly- clads, Nemertines, Molluses (except Cephalopods), Annelids, and Sipunculoids (Phascolosoma). The peculiarity of this mode of division is that after the four-celled stage the blastomeres — usually known as the macromeres—give off ‘quartettes’ of micromeres towards the animal pole, the first quartette being given off dexiotropically (except in cases of reversed cleavage), 2.B. 2.0. Fie. 13.—Diagram of a ‘spiratiy’ segmenting egg in the 16-cell stage. 2 a-2 D macromeres ; 2 a-2d micromeres of second quartette ; 1 a1, 1 a 2- 141, 1d2 micromeres of first quartette. the second laeotropically, and so on in regular alternation, until four quartettes have been produced. The cells of each quartette divide meanwhile in conformity with the same law of alterna- tion of direction of cleavage. The direction of division is thus always oblique to the egg-axis, and this obliquity can be observed in the division of the first two blastomeres, the result of which is that of the two sister cells A and B A is nearer to the animal pole than B, while in the other pair C is nearer that pole than D; A being to the left of B and C to the left of D (to an observer standing in the axis with his head to the animal pole), the division is laeotropic. The arrangement of cells approaches the tetrahedral, especially when, as occurs very frequently, A and C are united by a cross, or polar, furrow above, B and D by a polar furrow at the vegetative pole, as in Nereis, Ichnochiton, Limax, Planorbis, Lepidonotus, Discocelis, and others. In Unio, however, it is B and D that are in contact at the animal, A and C at the vegetative, pole. In other cases 26 CELL-DIVISION AND GROWTH II. 1 (Ilyanassa, Capitella, Umbrella, Crepidula, Amphitrite, Arenicola, for instance) the furrows are parallel, the same two blastomeres, B and D, being in contact at both poles. In Zrochus both the ‘parallel furrows’ and the ‘crossed furrows’ conditions are found. A similar disposition is to be observed amongst the -micromeres of the first quartette. These micromeres, also, alter- nate with the macromeres. Not more than three contact surfaces between blastomeres, therefore, meet in one line. The eggs of certain Lamellibranchs—TZeredo, Cyclas—in which the ‘spiral’ arrangement is obscured by the large size of the D macromere, and possibly the ova of the Rotifera, are to be referred to this type. The tetrahedral arrangement of the first four cells is con- spicuous in Asteroids and Ophiuroids, where the planes of division of the first two cells are at right angles to one another. Before the next division, however, the cells shift their positions and come to lie in one plane, in which, however, the sister cells are not adjacent, but opposite, to one another. The eggs of Amphiowus sometimes segment spirally (Wilson). After the completion of the spiral period of division, seg- mentation becomes radial and then bilateral. 3. The third type of cleavage is the bilateral. The first two divisions intersect in the axis; the next may be equatorial, as in Ascidians. In this case the bilaterality becomes evident in the succeeding phase, in which the divisions in two adjacent cells of the animal hemisphere meet the first furrow, while in the other two they meet the second. The bilaterality is marked in the reverse way in the vegetative half of the egg. The egg is thus divided into what will be anterior and posterior, dorsal and ventral, and right and left halves. In future divisions the bilateral symmetry is retained. The egg of Amphiowus may divide in this fashion (Wilson), and this is the normal method, according to Roux, in Rana esculenta, In the Teleostei and some Ganoids (Lepidosteus) the bila- terality becomes evident in the third division, which is parallel to the first, the fourth being parallel to the second division. The egg is in fact iso-bilateral. The Ctenophore egg also possesses two planes of symmetry, Il. CELL-DIVISION 27 for the third division is meridional and unequal in such a manner that the next stage—eight cells—is composed of two opposite pairs of small and two opposite pairs of large cells. The mesomeres in the sixteen-celled stage of Echinoids are bilaterally arranged. — In the Cephalopoda the egg is large-yolked, and segmentation consequently meroblastic. After the first two meridional divisions V. © Le FE Fig. 14.—Three segmentation stages in the blastoderm of Sepia offi- cinalis; the segmentation is of the bilateral type. 7, left; 7, right; I-V, first to fifth cleavages. The top sides of the figures are anterior. (After Vialleton, from Korschelt and Heider.) the bilateral disposition sets in, for the furrows of the third phase are unequally inclined to the first furrow in two halves— the future anterior and posterior halves—of the egg (Fig. 14). The egg of Ascaris megalocephala also exhibits a bilateral cleavage, but not on the plan just described. The first division is equatorial. Then the animal cell divides meridionally, and, as it will prove, transversely, the vegetative cell latitudinally. 28 CELL-DIVISION AND GROWTH i Before the next division the most vegetative cell (P,) slips round to what will be the posterior side. All four cells are bi- laterally arranged about the plane in which they all lie, and this will become the sagittal plane of the embryo. The anterior and posterior ends, and therewith the right and left sides, are likewise now determined. The bilateral symmetry is preserved in future divisions, at least in the vegetative hemisphere ; in the animal part of the egg the blastomeres of the left side become tilted forwards, those of the right side backwards (Fig. 155, p. 255). 4. In the Triclad Turbellarians, in Trematoda and Cestoda, segmentation is irregular, the blastomeres separate from one another and lie amongst the yolk-cells. The same phenomenon may be witnessed in the Salps, and the separation and sub- sequent reunion of the blastomeres has also been described in " Coelenterates and in Asteroids, Although these types of segmentation are distinct enough from one another, intermediate conditions are readily found. The radial easily passes into the spiral type for example, for in many eggs of the former kind the ‘cross furrows’ have been observed at either one or both poles, while the animal blas- tomeres may be rotated slightly on the vegetative, and so lie not over, but in between, them. The radial symmetry. again may become bilateral, as when the meridional furrows of the fourth phase, instead of passing through the animal pole, meet the first or second furrow, symmetrically on either side of one of these divisions ; this occurs as a variation in Rana fusca and (normally (Roux)) in Rana esculenta, In Ophiuroids and Asteroids the tetrahedral arrangement is lost, and the egg segments radially. In Amphiowus all three types oceur. All three forms may therefore have been derived from one, though what that one was we do not know. In any case, however, one feature is common to them all; in all cases suc- cessive divisions are at right angles to one another. This is the law formulated by Sachs long since for the divisions of the cells of plants. It holds good for the segmenting animal ovum, though exceptions may, of course, be found. The alternation of dexiotropic and laeotropic divisions, for instance, in spirally seg- menting ova continues for a long period with striking regularity, II. 1 CELL-DIVISION 29 and it is comparatively rare for a cell to disobey the rule. The rule is, however, no universal law of cell-division. Every em- bryologist will recollect the continued division of a teloblast in the same direction to form a germ-band, which is such a con- _ spicuous fact in the development of Molluses, Annelids, and Arthropods. The four polar nuclei of Insect eggs, lying in one straight line, may also be cited. The direction of division and the size of the blastomeres are not, however, the only factors which determine the actual pattern of segmentation. The cells can, and do, shift their positions on one another. This is of common occurrence, and a few examples will suffice. The rearrangement of the tetrahedrally disposed cells in Asteroids and Ophiuroids has been noticed already. In many ‘spiral’ ova the micromeres have been observed to rotate on the macromeres, or one quartette to be pushed out of position by the cells of another. In Ascaris the cell P, slips to one side. Further, cells change their shape. Two factors are therefore involved in the production of the pattern of cleavage, the direction of division, and the movements of the cells, and these factors in their turn demand explanation. T’o these must be added the shape, the size, and the rate of division of the cells. The two latter depend very largely upon the amount of yolk present in the egg; yolk-cells are large, the yolk divides slowly, or not at all. This was expressed long since by Balfour in the formula, ‘The velocity of segmentation in _ Fic. 15.—Segmentation of the t of th OES Frog’s egg under the influence any pare of the ovum Is, rougnty of a centrifugal force (from Kor- speaking, proportional to the con- schelt and Heider, after O. Hert- centration of the protoplasm there; Deed ie need yolk and the size of the segments is (yolk-syncytium) : /h, blastocoel ; inversely proportional to the con- ”” Y olk-nuclei ; d, yolk. centration of the protoplasm.’! The rule has been vindicated by O. Hertwig experimentally. If the egg of the Frog be centri- 1 Comp. Emb. i. c. 3. 30 CELL-DIVISION AND GROWTH Il. 1 fugalized with sufficient force the yolk is driven still more to- wards the vegetative pole, while the protoplasm is accumulated in the animal half of the egg. Such eggs segment meroblastically, a cap of cells or blastoderm being formed lying on the surface of a nucleated but undivided yolk. The yolk-nuclei, moreover, are enlarged, as in megalecithal fish eggs (Fig. 15). The rule is, of course, only applicable to telolecithal eggs, and for many of these it holds good, notably for Vertebrates. In other classes there are, however, exceptions, which are best known in those whose segmentation has been most carefully studied, the ‘spiral’ eggs of Turbellarians, Annelids, and Molluscs. Large cells, in these ova, often divide more quickly than small ones; the second quartette of micromeres, for instance, is formed before the first quartette divides in Crepidula, Unio, Limax, Trochus, Aplysia depilans, Discocelis, and the cells of the third quartette before the first products of division have had time to divide again in Limax, Umbrella, and Aplysia limacina. 4d is often formed before the corresponding cells in the other quadrants (in Unio, for example), but in Crepidula this is in accordance with the rule, since 4a, 40, and 4 contain more yolk than 4d. In Arenicola, though the yolk is uniformly distributed, the cells are still unequal. Other exceptions are to be found in the continued unequal division of teloblasts, in the formation of the micromeres in Kchinoids, and in the unequal division of the blastomeres in the third and fourth phases in Ctenophors. According to Ziegler the formation of the micromeres in Ctenophors cannot be due to the presence of yolk, since they are still formed when part of the vegetative hemisphere is removed, as Driesch and Morgan have also found. Ziegler indeed puts forward another hypothesis to account for unequal division; he supposes that the centrosomes are hetero- dynamic. So far there appears to be little evidence in support of this view. It is quite true that in many cases of unequal division the asters—not the centrosomes—-vary in size with the size of the cells. This occurs, for instance, in the division of the first micro- meres and of the first somatoblast in Nereis, in the formation of the first and second quartettes, and in the division of the first somatoblast in Unio, in the division of the cell CD in Asplanchua, IL 1 CELL-DIVISION 31 and in the division of the pole cells of Annelids (Wilson and Vejdovsky). It is doubtful, however, whether it is not the inequality in the cells. that is responsible for the inequality of the asters, there being more room in a large cell for the out- growth of the astral rays. At any rate, there are many cases of unequal cleavage—in polar body formation—where the asters are of the same size. Until evidence is brought forward of a difference in the size of the centrosomes the hypothesis is no more than a conjecture. Before quitting this subject we should refer to a rule which Zur Strassen has found to hold good for the rate of segmentation of Ascaris megalocephala. The cells do not all divide at the same rate, but in certain groups of cells division is found to occur simultaneously. These cells are related, descended from some one cell, and the more nearly related the cells are, the more nearly together do they divide. Coincidence in time of division depends therefore on the degree of cell-relationship. The direction of division of the cell depends upon that of the nucleus, since, speaking generally, the division occurs in the equatorial plane of the spindle, or, in other words, the plane of division is at right angles to the direction of elongation of the spindle or separation of the centrosomes. The latter again depends on the relation between the nuclear spindle and centro- somes on the one hand, and on the other the cytoplasm and its contents, more particularly the yolk. The relation between the (resting) nucleus and the cytoplasm has been expressed by O. Hertwig in the following empirical rule: ‘The nucleus always seeks to place itself in the centre of its sphere of activity.’ The sphere of its activity being not the inert yolk but the cytoplasm, we find, in accordance with this rule, that the nucleus places itself in the centre of the ege where the yolk is uniformly distributed (isolecithal), nearer the animal pole but still in the axis where the yolk is on one side (telolecithal). Examples of the former condition are to be found in Echinoids (the fertiliza- tion nucleus is nearly, but not quite, central) and large-yolked Arthropod ova, of the latter 1 in the eggs of Vertebrates, Molluscs, and many others. The nucleus, however, may wander from this position, as occurs, 32 CELL-DIVISION AND GROWTH Il.1 for instance, in the egg of Echinoids after the expulsion of the polar bodies and before fertilization. Apart from such excep- tions, due very likely to some temporary alteration in the relations of yolk and cytoplasm, the rule is a reliable one. The relation between the dividing nucleus, the spindle and centrosomes and the cytoplasm has been stated by O. Hertwig in his second empirical rule ‘that the two poles of the division figure come to lie in the direction of the greatest protoplasmic mass’, by Pfliiger in the formula, ‘ the dividing nucleus elongates in the direction of least resistance.’ The objection that has been urged to this latter expression, that in a fluid the pressure is equal in all directions, may be set aside. For though the cytoplasm is fluid it is an extremely viscid fluid, and the presence of the suspended yolk granules Fie. 16.—Diagram of the segmentation of the Frog’s egg (after O. Hertwig, from Korschelt and Heider). A, first (meridional); B, third (latitudinal) phase of segmentation; p, superficial pigment of animal hemisphere; pr, protoplasm ; y, yolk; sp, spindle. must certainly offer a greater resistance than the fluid itself, and greater in proportion to their number and size. Pfliiger’s formula, therefore, if not merely a truism, resolves itself into a restatement of Hertwig’s rule. This rule certainly holds good for a large number of cases, for it explains, for instance, the two first meridional divisions of all spherical telolecithal and radially segmenting eggs, the third, latitudinal (in small- yolked eggs), and possibly also subsequent meridional and lati- tudinal divisions (Fig. 16). It will not, however, in the present state of our knowledge, explain the obliquity of the spindles to the egg-axis in spirally dividing ova, nor cases of bilateral division ; 1 In Sycon the third is meridional, the fourth latitudinal. iz ja AS CELL-DIVISION 33 here, it is evident, other factors must come into play, in the second case probably a bilateral symmetry in the constitution of the cytoplasm. These exceptions may, however, ultimately prove to be special cases of Hertwig’s rule. A very striking confirmation of the rule is to be found in the division of the egg of Ascaris nigrovenosa (Figs. 17, 18). The Fie. 17.— Four stages in the fertilization of the egg of Ascaris nigrovenosa. (After Auerbach, from Korschelt and Heider.) egg of this worm is ellipsoid.. At one end (that turned towards the upper end of the ovary) the polar bodies are extruded, and here the female pronucleus is placed. The spermatozoon enters at the Fia..18.—Three diagrams of the rotation of the fertilization spindle in the egg of Ascaris nigrovenosa. e, s, the directions in which the female and male pronuclei approached one another in A; 1, 2, 3, successive positions of the spindle. (From Korschelt and Heider, after O. Hertwig.) JENKINSON D 34 CELL-DIVISION AND GROWTH Il. 1 opposite end. The line of union of the two pronuclei therefore lies in the long axis of the egg. Nevertheless the fertilization spindle is not formed in the minor axis of the ellipsoid as one might expect. The two pronuclei rotate together through 90°, the spindle is developed, as usual, in a direction at right angles to their line of union, that is to say the axis of the spindle lies in the major axis of the egg, and the rule is confirmed. There is a similar rotation of the fertilization spindle in the egg of another Nematode, Diplogaster (Ziegler), and in the Rotifer Asplanchna the spindle, at first oblique, becomes later coincident with the long axis of the ovum (Jennings), Curiously enough, this rotation of the pronuclei does not occur in another ellipsoid egg, that of the Rotifer Cal/idina, According to Zelinka, the polar body is formed at one end of the long axis, but the fertilization spindle lies in the minor axis, the first division includes the major axis, and the law is disobeyed. After the division, however, the cells rotate, and the plane of contact is then, as in Ascaris nigrovenosa, transverse. Again, all polar divisions violate the rule, as also does the first division of the fertilized egg of Ascaris megalocephala, and the division of the cells of the germ-bands of Crustacea parallel to their length (Bergh). On the other hand, Hertwig has brought forward experimental evidence in support of his generalization. In the eggs of the Frog the directions of some of the divisions were altered by com- pression between glass plates. The eggs were just allowed to assume their normal position with the axis vertical. They were then placed between glass plates and compressed. In the first series of experiments the plates were horizontal. In such eggs the first furrow was meridional and vertical, the second meridional and vertical and at right angles to the first. So far, therefore, division was as in the normal egg. In the third division the furrows were, however, not latitudinal and horizontal, but nearly vertical, being parallel to the first furrow above, to the second furrow below. The surface of contact, therefore, formed by the furrows of this phase must pass through a meridional position in the interior of the egg. The fourth furrows are latitudinal. Born has repeated the experiment and confirmed this result (Fig. 19). He adds, however, that the furrows of the third division pass 1 CELL-DIVISION 35 towards the vegetative pole below, or may even remain parallel to the first furrow throughout. The fourth furrows, Born says, are parallel to the second. I have myself observed that this division may be either parallel to the second, or latitudinal, even in different — Ah ; ia Bop Fie. 19.—Segmentation of ie Frog’s egg under pressure. The compression is in the direction of the axis. A, view of the egg between horizontal plates; the animal part is shaded. B, C, D, first (1), second (2), third (3), and fourth (4) divisions as seen from the animal pole. (After Born, from Korschelt and Heider.) Fria. 20.—The first four divisions (J, IJ, III, IV) in a Frog’s egg com- pressed between horizontal plates in the direction of the axis. The third furrow is more or less meridional and vertical in three quadrants, horizontal in the fourth, and this a smaller quadrant. The fourth furrow is meridional in this quadrant, horizontal in the remaining three. quadrants of the same egg (Fig. 20). It will be observed that _ the quadrant in which the third furrow is latitudinal is smaller than the others. It is of great interest to observe the striking similarity between the direction of the third and fourth furrows in these eggs and the corresponding divisions in the Teleostean ego where the blastodisc is compressed by the chorion. In the second series of experiments made by Hertwig the glass D 2 36 CELL-DIVISION AND GROWTH IL. 1 plates were vertical, the eggs, therefore, compressed not, as before, in, but at right angles to, the axis. The first furrow was meridional, and therefore vertical, and at right angles to the plates. The second was latitudinal and hori- zontal, and also at right angles to the plates. The furrows of the third phase were parallel to the first, those of the fourth, in the four upper animal cells, parallel to the plates. Born again MN 2B A D Wii | 3 3 \ it ti i / f 7 7 if 7 i | : p 4 F : : | | oo 7 4 : Fie. 21.—Segmentation of the Frog’s egg under pressure. The pressure is at right angles to the axis. A, view of the compressed egg. The pigmented animal portion is shaded. B, C, D, views of the egg from the animal pole after the first (1), the second (2), and the third (3) divisions. E, F, G, views of the egg from the compressed side after the first (1), the second (2), the third (3), and the fourth (4) divisions. The first furrow may pass as (1’) in E. (From Korschelt and Heider, after Born.) confirms this account (Fig. 21). The direction of the furrows of the third phase is, however, variable ; it may be not parallel to the first, but perpendicular to it. In this case it may be parallel to the second, or so oblique to it as to become nearly parallel to the glass plates. The direction of the fourth division depends on that of the third, to which it is at right angles. It may, therefore, be either oblique and nearly parallel to the plates, as described by Hertwig, or parallel to the second furrow and perpendicular to the plates. In a third series of experiments Hertwig placed the plates Il. CELL-DIVISION 37 obliquely, at 45°. In these eggs the yolk sinks slightly from the upper to the lower side, while the cytoplasm rises in the opposite direction ; in other words, a bilateral symmetry is con- ferred upon the egg by the combined action of pressure and gravity. The plane of this symmetry is midway between and parallel to the plates. The first furrow is at right angles to the plates and to the plane of symmetry. We are indebted to Driesch for a gimilar series of experiments on Echinoderm eggs. Driesch compressed the eggs of Echinus under a cover-glass supported by a bristle. The direction of the egg-axis with regard to the pressure was not known, but the es Fie, 22.—Echinus: segmentation under pressure. . a@, preparation for third division (radial); b, preparation for fourth division (tangential); b’, after fourth division; c, another form of the 8-cell stage (third division parallel to first); d, the same after removal of the pressure. (After Driesch, 1893.) Echinoid egg is nearly isolecithal. When the egg membrane remained intact the first two furrows were vertical, that is, in the direction of the pressure, since the slide and cover-glass were horizontal, and at right angles to one another. The spindles for the next division are again horizontal, and usually tangential, sometimes, however, radial. The eight-celled stage consists, therefore, of a flat plate of cells. At the next division the formation of micromeres—which would ordinarily occur at this moment—is suppressed ; the spindles are horizontal and radial, the furrows, therefore, vertical and tangential (Fig. 22 a, 0, 4). In certain cases cell-formation is wholly or partially sup- pressed. When the pressure is less (in those eggs which lie nearer the bristle) the micromeres may be, but generally are not, formed. The spindles are no longer horizontal. Similar results are obtained when the eggs are released from strong compression. In another experiment the eggs were first deprived of their membranes. The first and second furrows’ are vertical and generally at right angles to one another. Sometimes, however, 38 CELL-DIVISION AND GROWTH IL.1 the second is parallel to the first, or one blastomere may lie apart from the other three. Should the eggs be now released from the pressure, each blastomere becomes rounded off, and—after two more cleavages—the sixteen-celled stage consists of two plates of eight cells lying over one another. But if the pressure is maintained, the spindles are horizontal and the blastomeres lie all, or nearly all, in one plane (Fig. 22¢, d). Fig. 23.—Segmentation of the egg of Echinus microtuberculatus under pressure. (After Ziegler, 1894.) a, 8 cells in one plane; b, 16 cells, the last division having been tangential ; c, d, 16-32 cells: the direction of the spindles in c is shown by the line: it is in the greatest length of each cell; ¢, 64 cells: a cross signifies a vertical or oblique division, a line a horizontal division. i Ziegler has followed the segmentation of the compressed eggs a step further (Fig. 23). As the figures show, the first two divisions are at right angles to one another, while the furrows of the next two phases are, roughly, parallel to the first and second. In the next division—sixteen to thirty-two cells—the outer cells divide radially, the inner more or less tangentially, these divisions being, like the previous ones, at right angles to the compressing plates. In the following phase, some cells (those marked with a line) still divide in the same direction as before; but in others (distinguished by a cross) the spindle is perpendicular to the II. 1 CELL-DIVISION 39 plates and the division horizontal. Ziegler points out that, in the former cases, the cells have greater dimensions in the hori- zontal plane than in the latter. This, however, may be the effect, not the cause, of the direction of the spindle-axis. Two other pressure experiments may be mentioned here. In Nereis Wilson produced a flat plate of eight equal cells by applying pressure in the direction of the axis. The formation of the first quartette of micromeres was thus suppressed. On relieving the pressure eight micromeres were formed. For the Ctenophora (Lerée) Ziegler has shown that the normal inequality of the third and fourth divisions is not altered by pressure.? The foregoing experiments all agree in demonstrating the perfectly definite effect produced by pressure upon the segmenting egg. The nuclear spindles place themselves at right angles to the direction of pressure, the divisions fall at right angles to the compressing plates. This holds good for the first three or four divisions, at least, and sometimes for Jater phases still. In all these cases, therefore, the nuclear spindle elongates in a direction of least resistance, and, in the normal] uncompressed egg, we may argue, with Hertwig, the least resistance is offered by the greatest protoplasmic mass. Even in the compressed eggs, however, the greatest extension of the protoplasm, or the least extent of the yolk, is a factor which must in some cases come into play. When the egg of the Frog is compressed between vertical plates, the nuclear spindle does not elongate in any direction at right angles to the pres- sure, but in one only, a horizontal ; and this is the direction of the greatest protoplasmic mass, since the egg-axis is vertical. Speaking generally, therefore, experiment has upheld Hert- wig’s contention that the direction of nuclear division, and therefore of cell-division, is determined by the relation between the nucleus with its centrosomes and the cytoplasm with its yolk. There are one or two experiments which do not support Hertwig’s view. Boveri stretched the eggs of the sea-urchin Strongylocentrotus in the direction of the axis. The fertilization spindle lay in the usual equatorial position, occupied, that is, the minor axis of the ellipsoid. 1 I have recently had occasion to notice that when the egg of Antedon is compressed in the direction of the axis the third division is meridional instead of latitudinal. 40 CELL-DIVISION AND GROWTII iN. Again, Roux observed that Frogs’ eggs sucked up into a tube with a narrow bore became elongated either parallel or trans- verse to the length of the tube, the axis of the egg lying in each case lengthways. In the first case the division was at right angles to, in the second usually parallel to, the tube in accord- ance with the rule; but exceptionally, in the transversely stretched eggs, the division was not perpendicular to, but co- incided with, the extent of the greatest protoplasmic mass. However important a factor the disposition of the yolk may thus be in deciding the direction of cell-division, it is certainly not the only factor. In the eggs pressed between horizontal plates there are many—an infinite number—of directions of least resistance. In one of these the segmentation spindle elon- gates, and at right angles to this the first furrow falls. This is probably determined—as it is determined in the normal Frog and Sea-urchin egg—by the point of entrance of the spermato- zoon, or at least by the direction of the sperm-path in the egg. The second division is at right angles to the first, and here the direction may very possibly be decided on Hertwig’s principle. But why, in the next phase, should the furrows be at right angles to the second rather than to the first, for the extent of the protoplasmic mass is as great in each of the four cells, in a direc- tion parallel to the first as to the second furrow? Here, it is clear, some other reason must be found for this succession of divisions at right angles to one another. The cause is probably to be sought for in the direction of division of the centrosomes ; for these divide—frequently soon after the telophase—at right angles to the axis of the previous figure. We thus gain a new expression for Sachs’ Law. The original direction of divergence of the centrosomes is, however, by no means always the ultimate one, for the growing spindle may be twisted out of its original position. Conklin has made a careful study of this phenomenon in Crepidu/a, in which egg he finds that vortical movements are set up in the cytoplasm by the escape of nuclear sap at the beginning of mitosis. The move- ments are in opposite directions in sister cells, centre in the spindle poles, and often carry both nucleus and spindle into a fresh place. These currents, which had been noticed previously by other observers (by Mark in Limaw and by Iijima in Nephe/is), may IL. 1 CELL-DIVISION 41. thus play an important part in the production of the cell pattern. We shall see elsewhere that they, and other protoplasmic move- ments, are also of the very greatest significance in differentiation. There remains now to be noticed another principle, which is especially applicable to plant-cells with fixed walls, though it may possibly be used for the phenomena of animal segmentation as well. Berthold has pointed out that when a newly formed cell-wall places itself perpendicular to the previously existing walls it is—at least in a good many instances—simply obeying the laws of capillarity, it merely conforms to the principle of least surfaces formulated by Plateau. This principle is as follows: ‘ Homogeneous systems of fluid lamellae so arrange themselves, the individual.lamellae adopt a curvature such that the sum of the (external) surfaces of all is under the given conditions a minimum.’ A fluid lamella, of soap solution, for example, placed across the imterior of a hollow, rigid cylinder, or parallelepiped, or cube, is, with the film coating the internal surface of the vessel in which it lies, a special case of such a system of lamellae, and, in obedience to the principle, the lamella places itself at right angles to the walls of the cavity and transverse to the long axis. In the case of the plant-cell, the cell-plate, formed by solidifi- cation of the spindle fibres in the equator of the mitotic figure, represents the soap-laimella, and like the latter in its parallele- piped, the cell-plate, or new cell-wall, places itself perpendicular to the old one, and transverse to its length. There are very numerous cases in which the law is obeyed, but if is not so in all. Under certain conditions the lamella should be not at right angles, but oblique to the wall of the chamber across which it is stretched. If, to take a concrete case, the lamella be made to move (by abstracting air) towards one end of its receptacle (a cube or parallelepiped), it will reach a critical position in which the principle of least surface can only be satisfied by its occupying an oblique position. The point at which this occurs is when the lamella is distant = from the end, where a is the length of the side of the cube (short side of the parallelepiped). The lamella now forms the fifth side to a wedge-shaped space (quadrant of a cylinder, whose radius ‘ 4 are P ; is ¢ = 9”) but as more air is abstracted, and it moves still 42 CELL-DIVISION AND GROWTH II. 1 further toward the end, it comes to another critical position when it must lie across one corner, forming so the base of a pyramid, or octant of a sphere. This position is defined by the equation 7, = 4, where 7, is the radius of this sphere. It is impossible, therefore, for a very flat cell, or short cylinder, to be divided in conformity with the principle parallel to its longest side, and yet this occurs, as, for instance, in cambium cells. It will also be noticed that this principle does not explain why one particular direction is selected when many are apparently equally possible. We turn now to a consideration of the remaining factor which assists in determining the shape of the cells and so the geo- metrical pattern of segmentation ; this is the movement of the cells upon one another. That such movement does occur we have already seen; the question which immediately suggests itself is whether in taking up their new positions the cells obey the laws of capillarity as enunciated for systems of fluid lamellae such as soap-bubbles by Plateau in his principle of least surfaces. This principle, as we have seen, demands that the sum of the external surfaces should be, under the conditions, a minimum, or, expressed in physical rather than in geometrical language, that the total surface energy should be minimal. In accordance with this doctrine of minimal surface energy a drop of fluid floating in a fluid medium assumes, as need hardly be said, the form of a sphere. In a system of drops contact surfaces will be formed between the drops, provided that each possesses a coating film which has a positive energy with the media it separates ; a film, that is, of such a nature that the total surface energy would be diminished by apposition, without, however, involving the disappearance of the separating film and fusion of the drops. In other words, the film must be insoluble in both the external and the internal media. A simple example of this is afforded by the behaviour of the spheres of jelly covering the eggs of the Frog, when taken from water and floated between chloroform and benzole. Two or more such drops of jelly cohere by their coating films, and form systems of lamellae—the films, that is, at the external surfaces and between the opposed surfaces of the II. 1 CELL-DIVISION 43 drops—in which the principle of least surfaces is obeyed. Soap- bubbles form similar systems. But where this condition is not fulfilled, as in oil-drops floated, for instance, between alcohol and water, the drops either unite or separate, each retaining its spherical form. The geometrical analysis of such systems given by Plateau is as follows. In a system of two bubbles the curvature of the surface of contact is given by the equation 7 = : : >; where 7 is the radius of that surface, p, p’ the radii of the larger and smaller bubbles. Since the pressure varies inversely with the radius, the surface of contact is convex towards the larger bubble. When p = p’, 7 = a, and this surface is plane. Since there is equilibrium the external surfaces of the bubbles and their common surface meet at angles of 120°. In a system of three bubbles there are three contact surfaces ; these meet in one line and make angles of 120° with one another. When there are four bubbles, however, the four con- tact surfaces cannot meet in one line except for an inappreciable instant; they immediately shift their positions in such a way that two opposite bubbles meet and separate the other two from one another. There are thus five surfaces of contact, and these make angles of 120° with one another as before. This is the arrangement when four bubbles—whether equal or unequal is no matter—are placed side by side in the same plane. When, however, one bubble is placed in a different plane to the re- maining three, four surfaces are formed and disposed in such a manner that the four lines, each formed by the intersection of three of these surfaces, meet in one point, making with one another angles of 109° 28’ 16”, the angles at the centre of a tetrahedron. In short, the four are now tetrahedrally arranged. The systems of drops of jelly alluded to above arrange them- selves as do soap-bubbles under similar cireumstances. What holds good of four holds good of an assemblage of any number of bubbles. The size of the bubbles is a matter of indifference, except to the curvature of the surfaces of contact, and, to a certain extent, to the arrangement. Thus, if four equal bubbles be placed in a plane, they will form together five surfaces of contact, one of which will be between two opposite 44, CELL-DIVISION AND GROWTH i..2 bubbles. If these two be now diminished, or the opposite two enlarged, the surface of contact will be between the opposite pair of larger bubbles. On the other hand, it is possible to bring smaller opposite bubbles into contact, while the larger ones remain apart. Again, on four bubbles lying in one plane, four small ones may be superimposed in such a fashion that while two lie at either end of the surface of contact, the other two lie over between the two opposite large bubbles below. If now the two latter small bubbles be enlarged, they will displace the other two until all four come to lie not over but between the . Fre, 24.—Diagrams of systems of soap-bubbles. A-C, four small bubbles superimposed on four large ones. In A and B the bubbles are not compressed; in ¢ the lower bubbles have been circumscribed by a cylindrical vessel. In B the upper bubbles are small enough to show the surfaces of contact between each and the two adjacent large bubbles below. These surfaces are invisible in A and c. D is a system of eight bubbles in one plane, four forming a cross in the centre. In all figures notice the fifth contact surface or ‘polar furrow’. ‘fe : er) bubbles below, the usual arrangement when four are super- imposed on four (Fig. 24 A-C). The final disposition must depend, therefore, not merely on the principles of least surfaces, but also, provided that the conditions of that principle are fulfilled, on the’ sizes and initial arrange- ment of the bubbles. It will hardly need pointing out that very many ova adopt the form which presents the least external surface, that of a Il. CELL-DIVISION 45 sphere, when placed in a fluid medium, and it is also a familiar fact that after the first (and subsequent) divisions the blasto- meres are flattened against one another (Cytarme, to use Roux’s term), and that whether they are compressed. by an egg membrane or not (examples of the second alternative are to be found in Unio, Dreissensia, Umbrella, Crepidula, Aplysia lima- cina, Asterias), though the surface of contact is not always eurved when the cells are unequal. The two cells, however, often become rounded off and partially separated from one another prior to the next division. Such a separation (Cyto- chorismus) has also been observed by Roux in the case of cells of the Frog’s egg, which, having been isolated in albumen or salt solution, have subsequently reunited. That the cells flatten against instead of apelin one another, as free oil-drops would do, suggests that they, like soap-bubbles, are provided with an insoluble coating-film, while their subse- quent separation may be provisionally explained. by supposing that this coating-film becomes temporarily dissolved under the action of some substance formed in the cell. _This idea is borne out by a striking experiment of Herbst’s, who found that in sea-water deprived of its calcium the blastomeres of the sea- urchin egg came apart and resumed their spherical shape. . At the same time the surface membrane underwent a visible altera- tion, becoming radially striated. It seems reasonable to conclude that there is a membrane by which contact is normally effected, and that this is soluble in sea-water devoid of calcium. On the addition of calcium the cells cohere again. It may be mentioned that when systems of drops of jelly, floating in a medium of oil and united by their coating-films of water, are removed to alcohol, in. which both oil and water are soluble, the films disappear and the drops separate. In the next stage (four cells) the type of segmentation in which the laws of capillarity are most strictly obeyed is obviously that which we have distinguished above as the spiral or tetra- hedral type, and Robert has been able to show that successful imitations of the four-, eight-, twelve-, and sixteen-celled stages of the egg of Zrochus may be made with soap-bubbles. Four equal bubbles were placed in a porcelain cup, which held them together in the same way that the actual cells are held 46 CELL-DIVISION AND GROWTH VI together by the vitelline membrane. Five surfaces of contact were formed, that between two opposite bubbles representing the cross furrow or polar furrow in the egg. In the Zrochus egg, however, the polar furrows need not be parallel at the animal and vegetative pole; they may be at right angles to one another, and this tetrahedral arrangement of crossed polar furrows may be imitated by lifting up one of the bubbles and bringing it into contact with its opposite, one pair of bubbles being now in contact below, the other pair above. This arrange- ment is, however, unstable while the four bubbles remain in one plane, the two bubbles soon coming into contact both above and below. When the bubbles are not confined within a cup the instability of the ‘ crossed-furrow’ condition is extreme. By reducing the volume of the bubbles that are in contact the other two may be brought together; as the polar furrow changes positions there is at least a temporary condition when they are crossed. As we have already pointed out, both conditions—the ‘ parallel furrows’ and the ‘ crossed furrows’—are met with in the eggs at the four-celled stage of Molluscs, Annelids, and marine Turbellarians. Whether both opposite pairs or only one opposite pair of blastomeres are in contact does not, however, appear to depend upon whether the vitelline membrane is close to and compresses the egg or not. In most cases of crossed furrows the membrane fits, it is true, quite closely (Nereis, Ichnochiton, Podarke, Lepidonotus, Discocelis, Physa, and possibly Limax and Planorbis, if there is in these two, as in Physa, a very fine mem- brane between the albumen and the ovum); so also, speaking generally, where the furrows are parallel the membrane is absent (Umbrella, Aplysia, Dreissensia, Crepidula), but in Amphitrite and Clymenella it is lightly applied to the egg. It is remarkable that when the furrows are crossed, it is the A and C cells which meet at the animal pole, the B and D cells at the vegetative (except only in Unio), and this must depend on other properties of the cells than their surface tensions. But it may be very plausibly suggested that the explanation of the fact that it is the cells B and D which meet to make the ‘ parallel’ furrows is to be looked for simply in the large size of D. Robert has indeed shown that by simply altering the sizes of II. 1 CELL-DIVISION 47 the bubbles the conditions observed in the four-celled stage of other types—Nereis, Arenicola, Unio, Aplysia, Discocelis—may be faithfully copied. It only remains to be added that the contact surfaces of the cells, like those of the bubbles, make angles of 120° with one another. Robert has also imitated the eight-celled stage (the four micromeres alternating with the four macromeres), the stage of twelve cells (division of the micromeres), and that of sixteen cells (second quartette formed). The bubbles of the second quartette may be made to slide in between the macromeres and so rotate the whole first quartette, as happens in the egg. The division of the micromeres in the egg results in the arrangement of four cells crosswise in the centre, four others occupying the spaces between the arms of the cross. The bubbles behave in the same manner. In the eight-celled stage the micromeres alternate with the macromeres. In the case of the bubbles this is not necessarily so; the two sets of bubbles may be superposed if the ‘ polar furrow’ in one tier is at right angles to that in the other, or if, as pointed out above, the upper bubbles are small, Otherwise superposition is a very unstable condition. It would appear then that many of the patterns exhibited by eggs with a spiral cleavage are explicable by reference to the laws of surface tension. The principle of least surfaces may be extended to other cases. The first four blastomeres of Ophiuroids and Asteroids form a perfect tetrahedron, though this arrangement is subsequently discarded for one which could not be imitated with soap-bubbles (we may notice in passing that in the first case the egg is tightly invested by its membrane, in the second it is perfectly free). In Ascaris megalocephala the four cells come to lie, as do four bubbles, in one plane, and polar furrows have been seen in many eggs which belong to another type of segmentation (in Coelenterates (//ydractinia), Sponges (Spongilla), Crustacea (Branchipus, Lucifer, Orchestia), Vertebrates (Petromyzon, Rana), Ascidians, and Amphiozus). The principle of least surfaces—not more than three surfaces meeting in a line, not more than four lines meeting in a point— is, however, not of itself sufficient to explain the whole of the phenomena even in this most favourable tetrahedral type ; 48 CELL-DIVISION AND GROWTH IL. 1 other factors must intervene, just as other factors intervene in a mass of soap-bubbles—their size and initial arrangement— in the determination of the actual pattern. These other factors are the direction of cell-, that is of nuclear, division, and the. magnitude of the cells; and these, as-we have seen, in turn depend upon the relation between the nucleus and the cytoplasm with its included yolk. Thus it is the direction of the spindles - which determines whether the micromeres of the first quartette shall be given off laeotropically or dexiotropically ; the direction of division, oblique to the egg-axis, again determines that the micromeres shall alternate with the macromeres and not be superimposed upon them; the size of the cells and the direction Fig. 25.—Mitotic division with elongation of the cell-body in a proto- zoon, Acanthocystis aculeata. (After Schaudinn, from Korschelt and Heider.) of division may determine the position of the polar furrow, while the rate of division will also not be without effect, since the whole arrangement at any stage depends in part on the disposition at the stage before. There is one other point that is worthy of notice. The mitotic spindle possesses considerable rigidity, and is able as it elongates to materially alter the shape of the cell. This may be seen in many cases in Annelid, Molluscan and other eggs—the division of the first micromeres in Nereis is an instance—and in the Protozoa (Fig. 25). Another interesting case is the Rotifer Asplanchaa, where, preparatory to the fourth division, the shortest axis of the cells—in which the spindles are placed—becomes by the elongation of the spindles the longest. This alteration of shape is itself an important factor in deciding the positions to be taken up by the daughter cells. © Il. 1 CELL-DIVISION 49 In the other types—radial and bilateral—the principle of least surfaces is obviously disobeyed, for here four or more surfaces meet in one line and at angles other than 120°. Roux (1897) has, however, shown that if a certain condition be imposed on the system of lamellae, figures may be produced which very closely resemble the patterns presented by radially and bilaterally segmenting ova. This indispensable condition is that the system shall be surrounded by a rigid boundary, as the eggs themselves are by a membrane. Roux’s system was made by dividing into two, four, and eight a drop of paraffin oil suspended in a closely fitting cylindrical vessel between alcohol and water. To this medium was added calcium acetate to prevent the drops reuniting. The drop was divided with a glass rod. DOE Sic Fig. 26.—Roux’s oil-drops. A and B, the drop divided equally; C and D, unequally. Each of the two equal drops divided equally in £, unequally in F. (From Korschelt and Heider, after Roux.) When the two drops formed by the first division were equal the surface of contact was flat, when unequal convex towards the larger one, in accordance with the rule (Fig. 26 A—D). When the second was also equal, four drops were formed with four surfaces of contact meeting in one line, or enclosing between them a small ‘segmentation’ cavity. If the division of the two equal drops was unequal, and the smaller cells adjacent, they pushed into the larger ones; the result, in fact, was the same JENKINSON E 50 CELL-DIVISION AND GROWTH If.+1 as would have been produced by an equal following on an unequal division, the four surfaces meeting in one line as before (Fig. 26 E, F). The appearance presented is like a side view of a radially “a ei ts Fig. 27.—Arrangement of four oil-drops produced by unequal division of two equal drops, the small and large drops alternating. The first division is shown by J: the second (IZ) may pass as in a or in J, but the result is always as in c, the two large drops meeting in a polar furrow and excluding the small drops from the centre; the system is symmetrical (iso-bilateral) about the dotted lines in c. (After Roux, from Korschelt and Heider.) segmenting ege@ after the third division. When, however, the smaller drops were not adjacent, but opposite, five surfaces of con- tact were formed, a polar furrow appearing between the two larger and joining the centres of mass of the two smaller drops, whether these are unequal or not. The direction in oR which the division I J of the drops is per- formed isirrelevant; Als the final result is always the same. 7 Should two adjacent Fie. 28.— A and B are diagrams of an oil-drop drops be equal, the divided into four and eight to explain Roux’s Siig Seat GAL notation. Cisa figure of the oil-drop divided into POT turrow 1s stl eight equal parts. (From Korschelt and Heider.) formed by the union of those two which have together the larger mass (Fig. 27). Fist CELL-DIVISION 51 The length of the polar furrow varies directly with the size of the drops which unite to form it; its direction makes an angle with the plane separating the first two, which varies Yaa Ss ZB 2a, (12, (Skea Fig. 29.—Arrangements of six oil-drops. In all cases A= B=a=b. In A, a’ =a", 0 =b". In Ba >a", >b’. Ind, @ 40 % = —— _ Chest-girth. , paees Stature. ———— «Weight. 20 %- 10 %- ee a. ~~ -~ Years, 1 23 4.5 6 7 8 9 101112131415161718 1920 Fie. 40.—Curves showing the alteration during the first twenty years of life of the rate of growth as measured by weight, stature, and chest- girth in the human being (males). (Constructed from Quetelet’s data.) The abscissae are years, the ordinates percentage increments. (The percentage increment of weight for the first year could not be included in the figure. It is given in Fig. 37.) There are nevertheless few cases in which the exact difference in rate has been ascertained. From those few cases, however, it appears that the individual parts, though they do not grow with equal rapidity, still obey the same law as the whole. Thus human stature exhibits the same loss of growth-power as is shown by the weight of the whole body, with this II. 2 GROWTH 69 difference, however, that the rate is not so high in early stages, the descent in later stages less abrupt. This will be seen in Table IV, in which such figures as are obtainable for the pre- natal growth-rate are given, and in Fig. 40, in which the curve of change of growth-rate in human stature has been con- structed from Quetelet’s data (male Belgians). The percentage 50 % | 1 1 40 % | Head-Length. ------- Leg-Length. Stature. ------- Vertebral Column. 30% - 20 %- 10 % | SE; al ae or = i eet A: ee ie eS ee A — Years. 123 45 6 7 8 9 10111213141516171819 20 Fie. 41.—Curves showing the alteration during the first twenty years of life of the rate of growth of stature, length of head, length of vertebral column, and length of leg in the human being (males). (Constructed from Quetelet’s data.) Ordinates, percentage increments ; abscissae, years. increment in the first year is only 39-6 as against 190.3 for weight, in the second year 13-3 as against 22-2 for weight. Thenceforward the rate slowly declines, until at the fortieth year it is zero, and after the fiftieth year increasingly negative. The rate of increase of stature is always slightly less than that of 70 CELL-DIVISION AND GROWTH II. 2 weight. Quetelet’s figures do not show the rise in rate about the time of puberty, but this phenonienon is apparent in the data furnished by Bowditch, Boas, and Roberts (see Fig. 42). The change in the growth-rate is practically the same in women as in men. As with weight, the rise of rate at the time of puberty is earlier. The decline in the growth-rate of chest-girth is shown in the same figure (Fig. 40). It will be noticed at once that in this case the drop in the first year is very great indeed, from nearly 50 % to nearly 5 %, and that the rate is only diminished by another 2-4 % in the next nineteen years. The weight will depend upon the volume and the volume on both stature and girth; in fact a rough weight-curve might be constructed from the measure- ments of stature and girth. It is evident that the sudden loss in the rate of total growth after the first year is due to the very rapid decrease in the percentage increment of girth. It may be mentioned that other measurements of girth—girth by the sternum, the waist, the hips, the neck, the biceps, the thigh—show the same exceedingly abrupt decrease, almost to the minimum rate, between the first and second years. In ‘other cases—distance between the eyes, width of mouth, length of hand, length of foot, arm-length, leg-length, length and breadth of head, distance from the crown of the head to the first vertebra, length of the vertebral column—the change is more gradual ; the rate of change, however, differs in different cases. As an instance of this let us consider the measurements— from the crown to the first vertebra, the length of the vertebral column, and the leg-length—which together make up the total stature. The growth-curves of these three and of the whole stature are presented in the figure (Fig. 41), from which it will be seen that the growth of the leg is faster than that of the verte- bral column (until the eighteenth year), and this than that of the head. Increase in stature takes place at nearly the same rate as that of the vertebral column, but is on the whole a little faster. There are few cases—besides man—in which we possess in- formation as to the growth of the parts. In the sea-urchin, Strongylocentrotus, Vernon has shown that the growth-rate of the oral and aboral arms of the Pluteus diminishes rapidly from II. 2 GROWTH 71 the third to the fifth days, more slowly from the fifth to the eighth days. After this the rate becomes negative, as the skeleton of the Pluteus is used up by the developing urchin. The eurves of change of rate of growth—as constructed from Vernon’s figures—are shown in the chart (Fig. 38). In Careinus moenas a gradual decrease in the growth-rate of the frontal breadth can be ascertained from Weldon’s data. We have next to consider another feature of growth, the alteration of variability. The facts at our command are derived from a study of Echinoid larvae (Vernon), Duck embryos (Fischel), Guinea-pigs (Minot), the Periwinkle (Bumpus), the Crab, Carcinus (Weldon), and the human being (Bowditch, Pearson, Roberts, and Boas). Vernon has shown that in the Pluteus of Strongylocentrotus the variability of the body-length increases regularly up to the fifth day, and then decreases regularly again to the sixteenth day. So Fischel’s measurements of Duck embryos seem to establish a greater variability in younger than in older stages. This is true of the whole length, the head (as far back as the first somite), the hand and trunk together, and the total length exclusive of the primitive streak. The data are, however, too few to be treated statistically; the variability can only be roughly estimated from the extent of the limits within which the part varies at each stage. Minot, who expresses the variability of guinea-pigs by the difference between the mean weight and the mean weight of the individuals above, and of those below, the mean, likewise finds that the range in variation diminishes with age, and further that, in the case of the males, there is a period—from about the fourth to the ninth months—when the variability is very much less than at any other time. No such sudden fall is observed in the female, only a steady diminution. A more satisfactory calculation of the alteration of variability may be made from the measurements taken by Bumpus of the ‘ventricosity’ (ratio of breadth to length) of the shell of the Periwinkle, Littorina littorea. The series of observations is very large, and includes both British and American forms. In the accompanying table (Table V) the coefficients of variability (the standard deviation expressed as a percentage of the mean) are 72 CELL-DIVISION AND GROWTH IL. 2 given for each age, as determined by length of shell, for the English and American periwinkles separately and also for the complete series. It will be seen that the variability increases slightly and then diminishes again. ‘This is the case also in the American examples, where the fall at the end of growth is greater still, but in the British specimens there is only a slight fall, at 20-21 mm., followed by a considerable rise. The possible significance of this difference in the behaviour of the same species on the two sides of the Atlantic we shall discuss in a moment. TABLE V Showing the alteration in the variability of the ventricosity of the shell of the Periwinkle (Littorina littorea) during growth. Coefficient of variability ( ax 100). Length in mm. British. American. All. -15 2.77 3-27 3-25 16-17 2-94 3-41 3-34 18-19 3-02 3-39 3°35 20-21 2-93 3-03 3-13 22 3-25 2-84 3-02 In the meantime let us consider another case, the Crab, Carcinus moenas, the variability of the frontal breadth of which was examined by the late Professor Weldon. Weldon found that the variability, as measured by the quartile error (Q), first increased and then suddenly diminished with age (as deter- mined by carapace length). If the variability is measured by the coefficient of variability (easily calculated from Weldon’s data) the result is the same. This will appear from the table (Table VI). TABLE VI Showing the change in the variability of the frontal breadth with age in Carcinus moenas, (After Weldon.) Carapace Length h o engl ras : & x 100. 7-5 9.42 1-64 85 9-83 1-76 9.5 9.51 1-73 10-5 9.58 1-78 11-5 10-25 1.93 12-5 10-79 2-06 13-5 10-09 1-95 For the calculation of the variability at different ages in man data have been provided by Roberts, Bowditch, and Boas. Some II. 2 GROWTH 73 of these results are collected in the following table (Table VII), from which it may be gathered that the variability diminishes at first, then rises until it attains a maximum at about the time of puberty, and then diminishes again, reaching finally a value which is lower than the original. The values for the coefficient obtained by the different investigators are fairly similar, and agree very well with those first given by Pearson (for male new-born infants, weight 15-66, stature 6-50; for male adults, weight 10-83, stature 3-66). It may be seen from the values for the new-born that the variability has already undergone a diminu- tion before the age at which the other observations begin. TABLE VII Showing the change in the value of the coefficient of variability in the male Human being during growth. Coefficient of variability (3 i * 100 ). Weight. Stature. Years. Boston Worcester, English Boston Toronto Worcester, (Bowditch). Mass. Artisans | (Bowditch). (Boas). Mass, (Boas). (Roberts). (Boas.) 4 14-00 5 11-56 11.48 4.76 4.82 6 10-28 12-04 10-08 4.60 4.34 5-40 7 11-08 11-87 10-29 4.42 4.35 4.24 8 9-92 11-83 10-78 4.49 4.58 4.32 9 11-04 12-29 10-85 4.40 4.41 4.30 10 11-60 12-92 11-06 4.55 4.68 4.44 11 11-76 14-45 11-90 4.70 4.53 4.51 12 13-72 15-56 11-48 4-90 4-85 4.49 13 13-60 18-07 11-76 5-47 5-36 5-21 14 16-80 16-80 12-74 5-79 5-64 5-43 15 15-32 18-28 14.00 5-57 5-71 5-19 16 13-28 13-95 12-95 4.50 3-92 17 12-96 11-23 11-55 4.55 3-32 18 10-40 12-18 3-69 19 10-29 20 9-03 10-50 10-92 12-04 Further, the variability does not merely diminish as the animal grows older. Its diminution accompanies the diminu- tion in the rate of growth, and when—as at the time of puberty in man—that rate increases, the variability increases too. The variability of such parts as have been examined for the 74 CELL-DIVISION AND GROWTH II. 2 purpose alters in the same way as that of the whole body. Besides weight and stature Boas has recorded measurements of height sitting, head-length, head-width, length of fore-arm, and hand-width. Though the evidence, it must be admitted, is scanty, it is none the less a remarkable fact that in all the cases we have examined the variability, whether of the whole organism or of its parts, decreases with the decrease in the rate of growth. We seem to be in the presence of a phenomenon of general occurrence, though what the significance of the phenomenon is is not at present clear. As is well known, Weldon has argued that the decline in the variability of the older crabs is due to a selective death-rate, an argument which is supported by the same author’s observations on the snail C/ausiia, since in this form the variability of the adult was found to be the same as the variability of the same individuals when young, but less than the variability of the general population of young. It is possible that the marked decrease in the variability of the American as compared with the British periwinkles may also be attributable to the same cause, since this animal has only recently been introduced into America, and may, therefore, be subjected to a more severe struggle for existence in its new environment. It is doubtful, however, whether this explanation will fit all cases. Vernon has suggested that at periods of rapid growth the effect produced upon the organism by a change in its environ- ment must be much greater than at other times, and, since he has further shown that. one of the effects of an adverse change of circumstances is an increased variability, he argues that an increase in variability would naturally accompany a high growth- rate. Lastly, Boas points out that the rate of growth is itself a variable magnitude, and this ‘ variation in period’ may, with other causes, be a factor in producing the actual variation at each stage. Should that be so, the variability would necessarily increase and diminish with an increasing and diminishing growth- rate, since those that are above the mean would tend to remove II. 2 GROWTH 75 themselves further from the mean than those that are below. could approach it, and the more so the faster they were growing, and conversely. We have finally to consider very briefly what little is known of the alteration with growth in the value of the correlation between various organs. Such data as we have indicate that, like variability, this value rises and falls with the growth-rate. Boas has ascertained the correlation coefficient (p) in man between weight, stature, height sitting, length and width of head at different ages. Some of these results are tabulated below (Table VIII) ; from this table it will be evident that the value of p decreases, increases, and decreases again. The values are for girls, and the period of increase is earlier than that found for boys. In the chart (Fig. 42) are given the successive values of growth-rate (stature), variability (height), and correlation co- efficient (height sitting and head-length) for boys; the three magnitudes rise at about the time of puberty, and subsequently decline together. Boas urges that if the actual variability is in part the effect of variation in period, this effect will be greater during periods of rapid development. It follows from this that if the various organs of the body are equally affected by a change in the growth-rate, correlations would be closer during periods of rapid growth than at other periods. TABLE VIII Values of the correlation coefficient, p, during growth for four different correlations. Girls, Worcester, Mass. (Boas). Years. Stature and Stature and Stature and Stature and Weight. Height sitting. Length of Head. Width of Head. 7 ‘73 -74 +30 21 8 ‘76 “79 -36 “15 9 80 82 *35 16 10 83 83 37 -16 11 81 84 37 25 12 77 82 38 27 13 73 83 38 37 14 -67 82 30 25 15 -65 -79 26 22 16 -60 74 25 -10 It will be noticed that the value of p for the different organs is different, being greater between axial organs—stature and height sitting, stature and length of head—than between longi- 76 CELL-DIVISION AND GROWTH IL 2 tudinal and transverse parts, such as stature and width of head. The correlation between stature and weight is high. To whatever cause it may be due this diminution of correlation with age is of the greatest interest, since it points to an in- creasing power of self-differentiation in the parts of the body. From other sources also there is evidence of a progressive loss 40 4 Correlation (p)- 30 > Height sitting and length of -2074 head. 67 5 a Variability 4 jeer ae o ©6100.) (F : ) 3- 97 15) 5% - Rate of Growth 4% (Percentage 8% 7 increments of S stature). ? oy al ty A “esr T T aaa | T T f 1 Voara. 7 8 '9 10. 11 42-15. 14 15-16 Fre. 42.—Figure to show how the rate of growth (percentage incre- ments of stature), the variability (of stature) and the correlation coefficient (between height sitting and length of head) rise together at the time of puberty in man and then fall together. (Constructed from the tables of Boas.) of totipotentiality of the parts, of an increasing independence of the parts, of a tendency to be increasingly governed in their development by factors that reside wholly within themselves. But this evidence must be discussed elsewhere. II. 2 GROWTH 77 LITERATURE F. Boas. The growth of Toronto children, U.S.A. Report of the Commissioner of Education, ii, 1897. F, Boasand C. WIssLER. Statistics of growth, United States Education Commission, i, 1904. H. P. Bowprtcu. The growth of children, Massachusetts State Board of Health, 1877. H. C. Bumpus. The variations and mutations of the introduced Littorina, Zool. Bull. i, 1898. C. B. DAvENPoRT. The réle of water in growth, Proc. Boston Soc. Nat. Hist. xxviii, 1899 (1). C. B. DAavENPORT. Experimental morphology, New York, 1899 (2). A, Fiscnen. Ueber Variabilitit und Wachsthum des embryonalen Kérpers, Morph. Jahrb. xxiv, 1896. G. Kraus. Ueber die Wasservertheilung in der Pflanze, Festschr. Feier hundertjdhr. Best. Naturf. Ges., Halle, 1879. C.S. Minor. Senescence and rejuvenation, Jowrn. Phys. xii, 1891, C.S. Minor. The problem of age, growth and death, Pop. Sci. Monthly, 1907. K. Pearson. Data for the problem of evolution in man. III. On the magnitude of certain coefficients of correlation in man. Proc. Roy. Soe. lxvi, 1900. W. PREYER. Spezielle Physiologie des Embryo. VIII. Das em- bryonale Wachsthum. Leipzig, 1885. A. QUETELET, Anthropométrie, Bruxelles, 1870. C. Roperts. Manual of anthropometry, London, 1878. K, Semper. Animal Life, 5th ed., London, 1906. H. M. Vernon. The effect of environment on the development of Echinoderm larvae : an experimental enquiry into the causes of variation, Phil. Trans. Roy. Soc. clxxxvi, B, 1895. H. M. Vernon. Variation, London, 1898. W.F.R. Wetpon. An attempt to measure the death-rate due to the selective destruction of Carcinus moenas with respect to a particular dimension, Proc. Roy. Soc. lvii, 1894-5. W.F.R. Wetpon. A first study of natural selection in Clausilia laminata, Biometrika, i, 1901-2. CHAPTER II EXTERNAL FACTORS 1. GRAVITATION In the large majority of cases there is no definite relation between the vertical and either the axis of the egg, the planes of its segmentation furrows, or the position of the development of the embryo in it. Thus the eggs of insects are laid with the axis making any angle with the vertical, and the same may be said of Crustacean ova. In such eggs as develop freely in the sea (some Mollusca, for example, Polychaeta, Coelenterata, Cteno- phora) the axis and the planes of segmentation undergo a perpetual change of position, and Oscar Hertwig has shown that in the eggs of Echinoderms there is no necessary fixed relation between the direction of the planes of segmentation and the vertical. In these cases it is clear that the features of development referred to cannot depend upon the force of gravitation. There are, however, instances in which it seems possible that the directions of the planes of segmentation—bearing as they do a constant relation to the axis of the egg—may depend upon gravity, since the axis is normally vertical. It was Emil Pfliiger who in 1888 first brought forward experimental evidence to show that this was indeed the case. It is well known that the yolk of the hen’s egg always turns over so that the germinal disc is uppermost, and the egg of the Frog, free to rotate inside its jelly membrane, invariably takes up a position with the black pole uppermost, the white pole below. This property of the Frog’s ovum is exhibited alike by the ovarian, the coelomic, the uterine, and the freshly laid egg, by the living egg and the dead egg, by the whole egg, and by portions that contain both kinds of egg-substance, the yolk and the cytoplasm, as Roux showed by floating eggs or fragments in a medium of the same specific gravity. It is simply due to the fact that in the spherical telolecithal egg the heavier yolk ie 8 aa Ill. 1 GRAVITATION 79 is placed mainly on one side, while on the other the lighter protoplasm is more abundant, the yolk granules far smaller and more sparse. The distribution of these two substances determines indeed the axis about which the egg has a ‘ rotation structure’ or is radially symmetrical. The symmetry is further marked by the disposition of the pigment and the position of the nucleus. The pigment is placed in a thick superficial layer in the proto- plasmic portion, it extends over rather more, sometimes con- siderably more, than a hemisphere, for there is much variation in this respect, and its boundary is a circle whose plane is at right angles to the egg-axis—the line which passes through the centre of the egg, the centre of the pigmented portion or animal pole and the centre of the unpigmented portion or vegetative pole. There is also an axial, less-deeply pigmented plug in the animal hemisphere. The nucleus is placed axially, but excentri- eally, very much nearer the animal than the vegetative pole, in a pigment-free spot or ‘ fovea germinativa ’. The egg is invested with a layer of jelly (mucin), inside which it becomes eventually free to rotate. This, however, is not possible when the egg is first laid, for the jelly is at that time closely adherent to it. In water, however, the jelly swells up, and a narrow cavity is formed in about three hours between it and the egg, and the egg then turns over until its axis is vertical. The formation of the cavity is much more rapid if fertilization (insemination) has taken place; in this case the egg turns over in half an hour. The rapid formation of the perivitelline fluid is the first effect of insemination, and is due to some substance secreted by or accompanying the sperm, since the spermatozoon does not reach the egg for another quarter of an hour (O. Hertwig). A second effect is an alteration in the viscidity or cohesion of the egg-contents ; for while in the ovarian or uterine egg no alteration occurs apparently in the disposition of yolk, cytoplasm, and pigment, although the egg-axis may make any angle with the vertical, such an alteration is undoubtedly pro- duced by gravitation (see below) after fertilization has occurred. Another effect noted by Roux is that fertilized (not merely inseminated) eggs turn over more rapidly in a medium of like specific gravity than do unfertilized. 80 EXTERNAL FACTORS pO The most important first result of fertilization is, however, the replacement of the radial by a bilateral symmetry. About two or three hours after insemination a certain portion of the - border of the pigmented area, crescentic in shape and extending over about half its periphery, becomes grey by retreat of the pigment into the interior (Roux). The egg can now be divided into similar halves by only one plane, the plane of bilateral symmetry, which includes the axis and the middle of this grey crescent (Fig. 43). C D Fie, 43.—Formation of the grey crescent in the Frog's egg (R. tem- poraria). A,B from the side; c, D from the vegetative pole. In a,c there is no crescent, in B, D a part of the border of the pigmented area has become grey. The middle of the grey crescent is always diametrically opposite to the point of entry of the sperm (Roux and Schulze) ; the crescent has hence been held by Roux to be directly caused by that entrance. The plane of symmetry, as we shall see in another connexion, becomes in most cases the sagittal plane of the embryo, since the dorsal lip of the blastopore arises in the region of the grey Ill. x GRAVITATION 8] erescent. This side becomes the dorsal side of the embryo, while the animal pole marks, approximately, the anterior end. By complete disappearance of the pigment the grey crescent becomes added to the white vegetative area of the egg. The foregoing account applies in particular to Rana temporaria and 2. fusca; R. palustris appears to be similar, but in R. eseu- lenta it is stated that the egg-axis is eventually not vertical but oblique (Fig. 44). It seems, however, doubtful whether this obliquity is not rather apparent than real. The grey crescent has apparently not been recognized as such—the pigment is not so deep as in the other species—but included, nevertheless, in the white area, with the result that the centre of this, the definitive m. Fie. 44.—Egg of Rana esculenta after fertilization, in its normal ' position with the axis oblique (?). A, from the side; B, from above ; aa’, egg-axis ; mm, plane of first furrow. (After Korschelt and Heider.) white area, has been confounded with the centre of the original unpigmented area or vegetative pole of the vertical egg-axis. (Compare Fig. 44 A with Fig. 43 B.) As is well known, the planes of division during the first few regular phases of segmentation bear a perfectly definite relation to the axis. The first two, at right angles to one another, are meridional and therefore also vertical, the third furrows are parallel to the equator, therefore also horizontal; the furrows of the fourth phase are again meridional, and hence vertical, those of the fifth once more latitudinal and horizontal. It is this obvious relation of the planes of cleavage to the direction of gravity which has raised the question whether there is not a causal con- nexion between the two, the question which Pfliiger attempted JENKINSON G 82 EXTERNAL FACTORS III. 1 to answer by experiments, performed, however, not on the eggs of the Frog but on those of a Toad, Bombinator igneus. The close adherence of the unlaid egg to the glutinous jelly, which in its turn could be easily fixed to some object, provided a simple method of keeping the egg in any desired position. The eggs were removed from the uterus, attached with the axis at various angles to the vertical to watch glasses and fertilized with just enough sperm-water to allow of development, but not enough to permit of the formation of the perivitelline space and rotation of the egg into the normal position with the axis vertical. In these forcibly inverted eggs it was found that the furrows of segmentation bore the same relation to the vertical as in the normal egg; that is to say, the first was vertical, the second vertical and at right angles to the first, the third hori- zontal and nearer the upper pole, whatever the inclination of the egg-axis to the vertical, except in the extreme case where the white pole was exactly uppermost (180° of inversion), when segmentation did not occur at all. There was, however, no definite relation between the plane of the first (and therefore of subsequent) furrows and the original axis of the egg; the angle between this axis and the plane of the first furrow, as also that between the first furrow and the plane including the original and the actual vertical axes of the egg, might, it was found, have any value. Except in a few cases, and where the white area was nearly exactly uppermost, these eggs gave rise to normal embryos. The upper smaller cells divided more rapidly than the lower ones, whether pigmented or unpigmented, and the blastula stage was reached ; the dorsal lip of the blastopore appeared on one side a little below the (actual) equator, and the lower surface was covered over by the blastoporic fold in the ordinary way. Only in the failure of the whole egg to rotate after the closure of the blastopore (owing to the close adherence of the jelly) and in the irregular pigmentation (according to the original degree of inclination) did these embryos differ from the normal. One other point is worthy of notice. In the majority of cases the dorsal lip of the blastopore, marking the sagittal plane, appeared on the unpigmented side and lay in the plane including the III. 1 » GRAVITATION 83 original, now inclined, and the actual axis, or vertical line of intersection of the first two furrows. While, therefore, the cleavage planes are definitely related to the vertical but not to the original axis of the egg, the median plane of the embryo appears to be jointly determined by both. From these experiments Pfliiger drew remarkable and far- reaching conclusions. Heconceived of the eggas being meridionally polarized, composed of a large number of rows of molecules placed meridionally with regard to the original egg-axis. Each row is equivalent developmentally to every other row, but within the limits of each the molecules are of different value, since one end, for example, is anterior, the other posterior. Which of these equipotential rows shall lie in the sagittal plane of the embryo is decided by gravity, and by gravity alone. Similarly the vertical direction of the first two furrows, the horizontal direction of the third, is due to the operation of some general, though at present unknown, law, in accordance with which ‘ die Schwerkraft die Organisation beherrscht ’. } The original structure of the egg, on the other hand, has no definite relation either to segmentation or to the symmetry of the embryo, except, of course, in so far as the original axis together with the actual vertical axis determines its sagittal plane, the white side its dorsal side. ‘,..das befruchtete Ei gar keine wesentliche Beziehung zu der spateren Organisation des Thieres besitzt, sowenig als die Schneeflocke in einer wesentlichen Beziehung zu der Grdsse und Gestalt der Lawine steht, die unter Umstinden aus ihr sich entwickelt. Dass aus dem Keime immer dasselbe entsteht, kommt daher, dass er immer unter dieselben dusseren Beding- ungen gebracht ist.’ 2 It will certainly be agreed that so sweeping and revolutionary a dogma as this is in need of very substantial support; and though the facts as stated by Pfliiger are incontrovertible, as the repetition of his experiments has shown, it is unfortunate that he did not also take into consideration the internal changes occurring in his forcibly inverted eggs. The deficiency has been made good by Born. Like Pfliiger, Born found that in the 1 1, c. infra, xxxii. p, 24. 2 1. c. infra, xxxii. p. 64, G2 84 EXTERNAL FACTORS Ill. 1 forcibly inverted eggs the cleavage planes had the normal relation to the vertical, but not to the egg-axis; he observed, however, that the first furrow was usually in or at right angles to the streaming meridian, the plane, that is, including the original and the secondary vertical axes. The recent examination of 215 cases by the writer has shown that the first furrow tends to lie either in, or at right angles to, or at an angle of 45° to this plane. Subsequent development was normal, and the sagittal plane coincided with the streaming meridian. The dorsal lip appeared on the white side, which is thus anterior (anterodorsal). The examination of sections, however, showed that in these Fic. 45,—Sections through forcibly inverted Frog’s eggs. In A the egg has just been inverted, in B the streaming of protoplasm upwards and yolk downwards has begun. Both sections are in the streaming meridian or gravitation symmetry plane, including both the axis (aa’) and the vertical. 6D, pigmented animal protoplasm ; wD, unpigmented vegeta- tive yolk. a, animal pole; a’, vegetative pole; i, pigment-free clear area ; k, nucleus; p, superficial pigment. (After Born, from Korschelt and Heider.) inverted eggs there had been a redistribution of the contents, the heavy yolk sinking to the lower side, the lighter protoplasm, with the pigment and the nucleus, and the spermatozoon rising to the upper side (Fig. 45). The movement is rotatory, the cyto- plasm and yolk ascending and descending in opposite directions ; and it also takes place naturally parallel to, and in a similar manner on each side of, the plane in which the primary and secondary axes lie, hence known as the streaming meridian. That end of this plane towards which the protoplasm moves in its ascent, the end, that is, marked by the primary vegetative pole, Ill. 1 GRAVITATION 85 is anterior (more correctly, anterodorsal), for it is here that the dorsal lip of the blastopore appears; the opposite end is posterior (more correctly, postero-ventral). The pigment moves with the cytoplasm; it is, however, unable to completely displace that yolk which remains at the upper surface in consequence of the greater viscosity of the superficial rind, and here a ‘white plate’ or ‘grey patch’ is formed. Similarly, at the lower surface the pigment is not necessarily wholly displaced by the descending yolk. There is one special case that may be noticed. When the inver- sion is complete (180°) the yolk flows radially and peripherally away from the upper pole while the cytoplasm ascends in the axis. Born’s observations make it perfectly clear that gravity re- arranges the contents of these inverted eggs, and so confers upon them a secondary structure like that of the normal, and symmetrical about a secondary axis which is likewise vertical. To this secondary axis the direction of elongation of the karyo- kinetic spindles, and consequently the cleavage planes, bears the same relation as in the normal egg; and there is certainly no more need to explain these directions by reference to gravity, to suppose, in fact, a causal connexion between the two, in the one case than in the other. The planes, indeed, may fall where they do simply because the mitotic figures elongate in the direc- tion of least resistance (Pfliger) or (O. Hertwig) in that of the greatest protoplasmic mass, or may be related, in some similar way, to the structure of the egg alone. The point can only be determined by an experiment in which the directive influence of gravity is eliminated. This experi- ment has been made by Roux. The eggs were fastened in small vessels, at distances of from one to eight centimetres from the centre, to a wheel rotating continually about a hori- zontal axis, but so slowly (one revolution in from one to two minutes) that the centrifugal force developed was insufficient to make the eggs turn with the white pole outwards, and therefore negligible. The direction of the force exerted by gravity upon them from moment to moment was thus not constant. Of the eggs some were free to rotate inside their jelly, others were fixed. To anticipate the objection that the plane of rotation, 86 EXTERNAL FACTORS IIl. x the plane of the wheel, is constant, a third set were packed loosely in test-tubes, and so able to roll over one another in all directions as they fell from one end of the tube to the other with each revolution. The first furrow appeared in all these eggs at the normal time and it was meridional, as in the normal egg; similarly the second was meridional, the third latitudinal ; but the egg-axes exhibited no definite relation either to the vertical or to the plane of the wheel. The eggs were allowed to continue their development on the apparatus, and gave rise to normal tadpoles. From this experiment Roux drew the conclusion that it is not gravity which determines the direction of the planes of cleavage, and that gravity is not an indispensable necessity for the normal development of the egg of the Frog. Incontrovertible though this conclusion appears to be on the evidence, it has nevertheless been disputed by certain embryo- logists, Schulze and Moszcowski, the controversy between whom and Roux upon the subject has now extended over many years. Schulze has urged (1) that eggs placed on such a machine do not develop normally, and (2) that the rotation of the eggs in their jelly exactly compensates for the rotation of the wheel. With regard to the latter point Roux has replied that on this supposition the egg-axes ought to be, at any moment, vertical, which is not the case. ‘To the first objection it is a sufficient answer that not only Roux, but subsequent investigators (Morgan, Kathariner and the present writer) have been able to produce normal tadpoles from such rotated eggs. It may be noticed here that Kathariner has repeated Roux’s experiment with a slight variation. The eggs were kept con- stantly rotating, not in one but in an indefinite number of planes by a stream of air-bubbles passing through a glass vessel filled with water. Development was normal. This result does not differ materially from that obtained by Roux with the test-tube eggs referred to above, which has indeed been also independently corroborated by Morgan. The criticisms of Moszcowski take a different form. This author urges that gravity always exercises an influence upon the egg in determining the bilateral symmetry of both egg and IIT. 1 GRAVITATION 87 embryo. The grey crescent which appears soon after fertiliza- tion and is regarded by Roux as a direct effect of this process, is supposed by Moszcowski to be produced by the action of gravity upon the egg-contents during the short interval before the perivitelline space is formed and the egg able to turn over, to be comparable, in fact, to the grey area or white plate described by Born in his forcibly inverted eggs. Every normal egg, therefore, has a ‘gravitation’ plane of symmetry which later on becomes, as in inverted eggs, the median plane of the embryo ; nor are the eggs on the rotatory apparatus exempt, for it is held that the work of gravity can be accomplished on them even in the few moments before they are placed on the machine. With regard to the latter point both Kathariner and Morgan have demonstrated that eggs kept in a state of perpetual rotation in all directions, from the very moment of insemination develop into perfectly normal, bilaterally symmetrical embryos, while Roux has replied to the first part of the criticism by pointing out that the grey area observed by Moszcowski was not the normal grey crescent produced by the entering sperma- tozoon, but the ‘white plate’ of Born due to the incomplete rearrangement of yolk and cytoplasm in an egg which had been quite unintentionally prevented from assuming its normal position. The grey crescent, indeed, Roux argues, could not possibly be due to gravity in a normal egg, for it does not appear until some time after the axis has become vertical. There seems, therefore, to be little room for doubt that Roux’s original contention, that gravity does not determine the sym- metry of the egg and embryo in the Frog, is correct, although it remains a result of considerable importance that this external factor may be made artificially to induce a bilaterality in the ege which is sufficiently strong to persist as the symmetry of the embryo. There is one other matter of interest in this connexion. It is obvious, and has been experimentally shown by O. Hertwig, that a centrifugal force can replace gravity. On a wheel rotated with sufficient velocity the eggs turn with their axes radial, their white poles outermost. If the velocity is great enough (145 revo- lutions a minute, radius from 24 to 32 cm.) the yolk is driven 88 EXTERNAL FACTORS ME 2 inside the egg towards the vegetative pole, and the distinction between it and the protoplasm accentuated. The segmentation of such eggs is meroblastic ; a cap of small cells is formed, a blasto- derm, resting upon an undivided, though nucleated, yolk, and these yolk-nuclei are large and irregular, resembling the giant nuclei of the large-yolked eggs of Elasmo- branchs and other forms (Fig. 46). An experimental confirmation is Fra. 46.—Segmentation of the thus afforded of Balfour’s hypo- Frog’s egg under the influence ‘ : of a centrifugal force (from Kor- thesis, put forward on comparative ca ee Heider, on "3 cs grounds, that it is on the varying wig). The egg consists of a blas- ; ee fodoah Ba undivided yolk quantity of yolk that differences (yolk-syncytium) : kh, blastocoel; in the segmentation of eggs pri- m, yolk-nuclei; d, yolk. marily depend. If removed from the centrifuge in time, such eggs may con- tinue to develop, though they frequently give rise to monstrosities (Spina bifida). LITERATURE G. Born. Ueber den Einfluss der Schwere auf das Froschei, Arch. mikr. Anat. xxiv, 1885. O. Hertwig. Welchen Einfluss tibt die Schwerkraft auf die Teilung der Zellen ? Jen. Zeitschr. xviii, 1885. O. HErtTwie. Ueber einige am befruchteten Froschei durch Centri- fugalkraft hervorgerufene Mechanomorphosen, S.-B. kinig. preuss. Akad. Wiss. Berlin, 1897. L. KATHARINER. Ueber die bedingte Unabhiingigkeit der Ent- wicklung des polar differenzirten Eies von der Schwerkraft, Arch. Ent. Mech. xii, 1901. L. KATHARINER. Weitere Versuche tiber die Selbstdifferenzirung des Froscheies, Arch. Ent. Mech. xiv, 1902. F. KEIBEL. Bemerkungen zu Roux’s Aufsatz ‘ Das Nichtnéthigsein der Schwerkraft fiir die Entwicklung des Froscheies’, Anat. Anz. xxi, 1902. T. H. MorG@AN. The dispensability of gravity in the development of the Toad’s egg, Anat. Anz. xxi, 1902. T. H. More@an. The dispensability of the constant action of gravity and of a centrifugal force in the development of the Toad’s egg, Anat. Anz. xxv, 1904. M. MoszcowskI. Ueber den Einfluss der Schwerkraft auf die Entste- hung und Erhaltung der bilateralen Symmetrie des Froscheies, Arch. mikr, Anat, 1x, 1902. Ill. 1 GRAVITATION 89 M. Moszcowski. Zur Analysis der Schwerkraftswirkung auf die Entwicklung des Froscheies, Arch. mikr. Anat. 1xi, 1903. E. Pritieer. Ueber den Einfluss der Schwerkraft auf die Teilung der Zellen, Pfliiger’s Arch. xxxi, xxxii, xxxiv, 1883. W. Roux. Ueber die Entwicklung des Froscheies bei Aufhebung der richtenden Wirkung der Schwere, Breslau drtz. Zeitschr., 1884; also Ges. Abh. 19. W. Roux. Bemerkung zu O. Schulze’s Arbeit iiber die Nothwendig- keit, etc., Arch. Ent. Mech. ix, 1900. W. Roux. Das Nichtnéthigsein der Schwerkraft fiir die Entwicklung des Froscheies, Arch. Ent. Mech. xiv, 1902. W. Roux. Ueber die Ursachen der Bestimmung der Hauptrichtungen des Embryo im Froschei, Anat. Anz. xxiii, 1903. O. ScuuuzE. Ueber die unbedingte Abhingigkeit normaler tierischer Gestaltung von der Wirkung der Schwerkraft, Verh. Anat. Ges. viii, 1894. O. ScouuzE. Ueber die Nothwendigkeit der freien Entwicklung des Embryo, Arch. mikr. Anat. lv, 1900. O. Scuutze. Ueber das erste Auftreten der bilateralen Symmetrie im Verlauf der Entwicklung, A7ch. mikr. Anat. lv, 1900. 2. MECHANICAL AGITATION The necessity of perpetual and violent agitation for the very numerous pelagic ova which are ordinarily exposed to the stress of wind and weather is well known to every zoologist who has attempted to rear such forms in an aquarium, and need not be further insisted on. There are also other eggs which require a small amount of movement. The Hen turns her eggs every day, and the opera- tion has to be artificially performed in an incubator. Its omission leads to serious consequences, for, as Dareste has shown, the allantois sticks to and ruptures the yolk-sac in unturned eggs, the ruptured yolk-sac cannot be withdrawn into the abdomen, and the Chick cannot hatch out. Death may ensue at an early stage. A violent agitation of the Hen’s egg, on the other hand, is equally fatal. Dareste subjected the unincubated eggs to violent shocks at the rate of 27 a second for varying periods (from 4+ hour to 1 hour). The percentage of monstrosities observed after three or four days of incubation was very high indeed, except when ? It seems probable that the principal value of the mechanical agita- tion to the larvae is to prevent the Diatoms and Algae, of which their food consists, from sinking to the bottom. 90 EXTERNAL FACTORS III. 2 the eggs were placed vertically with the blunt pole uppermost, the blastoderm therefore resting against the shell membrane. Marcacci has exposed the eggs, inside the incubator, to con- tinual rotation for 48 hours. The eggs were fastened to hori- zontal and vertical wheels rotating 40, 80, and 60 times a minute. At the last mentioned rate of revolution, the direction of rotation was reversed half-way through the experiment. Many of the eggs actually hatched out, but the chickens were feeble and liable to disease, and exhibited malformations of the muscles or skeleton. Others, however, died before hatching, in some cases at an early stage, and death seems to have been due to rupture of the vitelline membrane; this was always fatal. The vertical motion was, on the whole, more harmful than the horizontal, owing to the perpetual see-saw. It may be noted here that Féré has succeeded in producing retardation and abnormality of development in the Chick by means of short exposures to sound-vibrations. Mathews has shown that mechanical agitation — violent shaking in a test-tube—is sufficient to provoke development (artificial parthenogenesis) of the unfertilized eggs in Asferias, but not in Ardacia (see, however, below, p. 124). LITERATURE C. DArEste. Recherches sur la production des monstruosités par les secousses imprimées aux ceufs de poules, Comptes Rendus, xcvi, 1883. C. Dareste. Sur le réle physiologique du retournement des ceufs pendant l'incubation, Comptes Rendus, c, 1885. C. DAREsTE. Nouvelles recherches concernant l’influence des secousses sur le germe de l’ceuf de la poule pendant la période qui sépare la ponte de la mise en incubation, Comptes Rendus, ci, 1885. C. DAREsTE. Note sur l’évolution de l’embryon de la poule soumis pendant l’incubation & un mouvement de rotation continu, Comptes Rendus, exv, 1892. C. Fire. Note sur les différences des effets des vibrations mécaniques sur l’évolution de l’embryon de poulet suivant l’époque oi elles agissent, C. R. Soe. Biol. (10) i, 1894. C. Fir&. Note sur l’incubation de l’euf de poule dans la position verticale, C. R. Soc. Biol. (10) iv, 1897. A. Marcacci. Influence du mouvement sur le développement des cufs de poule, Arch. Ital. Biol. xi, 1888. A. P. MatHews. Artificial parthenogenesis produced by mechanical agitation, Amen. Journ. Phys. vi, 1901-2. 91 3. ELECTRICITY AND MAGNETISM An external agent, to which all eggs are inevitably exposed, is the natural magnetism of the earth. No evidence has, however, as yet been brought forward that this agent exercises any directive influence upon them, although their development may be distorted by excessive exposure to it. Thus Windle placed a number of Hens’ eggs between the poles of a large horse-shoe magnet. Over 50 % of these, when incubated, gave rise to abnormalities, the area vasculosa being affected in most cases. No relation could be detected between the position of the egg in the magnetic field and the kind of monstrosity produced. In the case of Trout ova similarly treated a very high death- rate was observed, but this was attributed by the experimenter to the action of the electric currents set up by the running water between the poles of the magnet. Weak electric currents had less effect. Silkworms’ eggs, however, suffered no harm. The effects of the electric current upon the eggs of Amphibia and Birds were tested by some of the older observers. Rusconi, Lombardini, and Fasola all found that the development of the Frog’s egg could be accelerated by weak currents. Lombardini produced monstrosities in the case of the Chick by this method. More modern experiments are due to Windle, Dareste, Rossi, and Roux. Windle observed a fairly high death-rate amongst Trout eggs exposed to the action of the current. Dareste has found a large percentage of monsters among embryos developed from Hens’ eggs subjected for from one to three minutes to the electric spark (12 cm. long from Bonnetty’s machine, 3-35 cm. long from a Rhumkorff coil). Development was, however, normal in the case of eggs placed for an hour in a Tesla’s solenoid traversed by a discharge of 500,000 periods a second. Rossi employed a continuous current passing through the eggs (of Salamandrina perspicillata) in the direction of the axis. Both yolk and pigment became aggregated at the animal pole, leading to the formation there of a grey raised area surrounded more or less 92 EXTERNAL FACTORS IIT. 3 completely by a furrow. When segmentation occurred the first two blastomeres were unequal and detached; the vegetative hemisphere was hardly segmented at all in later stages, the previous divisions having disappeared. The nuclei were affected in various ways, and the directions of the cleavage spindles altered. The capacity for resistance to these evil effects was noticed to increase as development advanced. The polar area produced in these experiments recalls the polar areas observed by Roux in Frog's’ eggs exposed to a horizontal eur- rent, at right angles, therefore, to the axis. Alternating currents of 50 and 100 volts were employed. The eggs were fertilized two or three hours before the commencement of the experiment. In from fifteen to thirty seconds after exposure two polar areas appeared in each egg. The polar areas were turned towards the electrodes. They were marked, dotted in various ways, and flecked with white extruded drops of yolk, and separated by furrows from a middle or ‘equatorial’ zone, the width of which varied directly with the distance of the egg from the electrode, inversely with the strength of the current and the duration of exposure. Unfertilized ova were found to react in the same way. So also eggs in which segmentation had begun, and in those cases where the furrow cut the equatorial zone obliquely, the two halves of the latter turned away from one another. The polar areas appear too in eggs which are exposed in the ‘morula’ stage, each cell having in addition a polar area of its own. The latter, however, do not appear in enfeebled eggs, but only the former. In the gastrula and later stages the reaction occurs, but less markedly. None of the eggs which have been exposed to the current develops any further. They stick to the jelly, and consequently lose their power of rotation. Similar results were obtained by the use of the continuous current (43 volts), but the anodic and the kathodic areas usually differed from one another in certain details. It is important to notice that neither in these experiments, nor in another in which the eggs were placed inside a glass III. 3 ELECTRICITY AND MAGNETISM 93 tube surrounded by a coil, could any definite relation be satis- factorily made out between the direction of the first furrow and that of the current. Indeed, though intrinsically interesting, the experiments throw no particular light upon the problem of development. Rather should they be classed with the investiga- tions of Verworn and others upon the behaviour of Protozoa in the electric current,investigationswhich promise to contribute to the understanding of the structure and movements of living substance. It may be noted here that Roux has _ himself produced these polar areas on such structures as the heart and gall-bladder of the Frog and other vertebrates. LITERATURE C. DAREsTE. Recherches sur |’influence de l’électricité sur l’évolution de l’embryon de la poule, Comptes Rendus, cxxi, 1895. U. Rossi. Sull’ azione dell’ elettricita nello sviluppo delle uova degli Anfibi, Arch. Ent. Mech. iv, 1897. W. Roux. Ueber die morphologische Polarisation von Hiern und Embryonen durch den electrischen Strom, sowie tiber die Wirkung des electrischen Stroms auf die Richtung der ersten Teilung des Kies, S.-B. kais. Akad. Wiss. Wien, ci, 1891, also Ges. Abh. 25. B. C. A. WINDLE. On certain early malformations of the embryo, Journ. Anat. and Phys. xxvii, 1892-3. B. C. A. Winnie. The effects of electricity and magnetism on development, Journ. Anat. and Phys. xxix, 1895. 4. LIGHT As Roux pointed out long ago in the case of the Frog, light exercises no directive influence upon the development of the ovum. Blanc, indeed, has attempted to prove that the direction of the embryonic axis in the egg of the Hen may be made to depend upon the direction of the incident light-rays, but the experiments are hardly conclusive. The method employed was to blacken the shell of the horizontally placed egg with the exception of one spot to right or left of the blastoderm. On this spot a beam of light was kept directed during incubation. — In some cases, but not in all, the embryonic axis was found to deviate from its normal position at right angles to the long axis of the shell. Further, the head of the embryo might be turned towards or away from the source of light, There was 94. EXTERNAL FACTORS Til. 4 no relation between the amplitude of the deviation and the length of the exposure. Nor are the processes of growth and differentiation necessarily affected in any way at all by the presence or absence of light, or by the kind of light to which the eggs are subjected. Thus Driesch, who has experimented with the eggs of Echinus, Planorbis, and Rana, maintains that neither red, yellow, green, blue, nor violet light has the slightest effect upon the eggs during the early stages of segmentation and gastrulation, in what he calls the organ-forming period of development; and Loeb has asserted that the development of the embryos of the fish Yundulus is as rapid in darkness as in the light, except that on the yolk-sac (not in the embryo) far fewer pigment-forming cells are produced. Yung, on the other hand, has brought forward evidence to show that in later stages, at any rate, the embryos of the Frog react differently to lights of various wave-lengths, some of which are harmful, others, apparently, beneficial. Yung obtained his colours from solutions of fuchsin (red), potassium bichromate (yellow), nickel nitrate (green), bleu de Lyon (blue), and viole de Parme (violet). The colours, it may be noticed, are not absolutely monochromatic. Freshly laid eggs of Rana temporaria were placed under the influence of these lights. After one month, samples of the tad- poles were measured, with the following result in millimetres :— TABLE IX Red. Yellow. Green. Blue. Violet. | White. Length 21.58 25-91 18-83 26-83 29-66 25-75 Breadth 4.83 5-58 4.16 5-75 6-83 5-25 The mortality in the green light was great. After two months the dimensions were as follows :— TABLE X Red. Yellow. Green. Blue. Violet. White. Length 26-25 31-83 All 33-50 41-30 31-00 Breadth 6-00 7-50 dead. 8-00 10-16 7:33 All the tadpoles in the red light eventually died. White and yellow light gave the greatest number of perfect frogs, but, as will be seen, those in the violet were larger. They Ul. 4 LIGHT 95 were, however, less differentiated, for they did not acquire their hind legs so soon as did those in the white light. It may be mentioned, however, that when the tadpoles reared under these conditions are replaced in ordinary light and starved, those from the violet exhibit a greater power of resistance. Experiments with Rana esculenta gave the same result. In this case the effect of darkness was also tried and found to be distinctly unfavourable. Thus after one month the lengths in darkness and white light were respectively 19-66 mm. and 23-10 mm., the breadths 4-66 mm. and 5-50 mm.; after two months the difference was intensified, the lengths being 21-50 mm. and 32-16 mm., the breadths 7-16 mm. and 7-66 mm. The death- rate in the dark was exceedingly high. . The eggs and embryos of the Trout were likewise found by Yung to be highly sensitive to green and red light, while the larvae reared in violet hatched out rather more quickly than those from yellow, blue, or white light. In an experiment on the eggs of Limnaca stagnalis, due to the same investigator, the effect is measured by the time required for the young to hatch out, as the following table shows :— TABLE XI Light. Time to hatching in days. Red ‘ : ‘ ; : ; 36 Yellow . a : : : é 25 Green . . The heart is formed, fhen death occurs. Blue ; ; : . F A 19 Violet . , 5 : z 7 White . ; 4 P ‘ ; 27 Dark ‘ : . c fe 33 Green light is evidently fatal; development is retarded in red light, less so in darkness; yellow has about the same effect as white light, while there is a considerable acceleration in blue and violet. The relative effect produced by the various lights is as in the preceding experiments. The results obtained by Vernon for Echinoid larvae are, however, not quite consonant with this, as may be seen in the table (Table XII), where the colours are arranged in the order of the effect they produce. It will be observed that yellow is 96 EXTERNAL FACTORS HT, 4 more harmful than red, while green exerts about the same effect as blue (copper sulphate). The author states, however, that in two other experiments the larvae were entirely killed off by the green light though developing perfectly in the white. He also adds that in violet light no development was possible owing to the swarms of bacteria. TABLE XII Percentage change of size. Semi-darkness ; ; é ; +25 Absolute darkness . ‘ : ‘ : —1.3* Blue (copper si ee i ; ‘ ; —4.5 Green : : , : : —4.8 Red : : , , —6-9 Blue (bleu de Lyon) : i , ‘ —7.4 Yellow . : , : ; - 8-9 * Almost within ee limits of experimental error. In the Pluteus Vernon found that both the oral and the aboral arm-length decreased in darkness, green and blue (bleu de Lyon) lights, while blue (copper sulphate), yellow, and red light exerted little influence on this magnitude. It only remains to be added here that Blanc and Féré have brought forward some not very satisfactory evidence to show that white light is favourable to the development of the Chick. Féré has also stated that red and orange lights are more harmful than white, while violet has about the same effect. The experiments are, however, vitiated by the fact that the eggs were not turned over. ~ LITERATURE L. Buanc. Note sur l’influence de la lumiére sur l'orientation de V’embryon dans l’euf de poule, C. R. Soc. Biol. (9) iv, 1892. L. Bane. Note sur les effets tératogéniques de la lumiére blanche sur l’ceuf de poule, C. R. Soc. Biol. (9) iv, 1892. H. Driescu. Entwicklungsmechanische Studien II, Zeitschr. wiss. Zool. liii, 1892. C. Fért. Note sur l'influence de la lumiére blanche et de la lumiére colorée sur l’incubation des ceufs de poule, C. R: Soc. Biol. (9) v, 1893. T. List. Ueber den Einfluss des Lichtes auf die Ablagerung von Pigment, Arch. Ent. Mech. viii, 1899. J. Lors. A contribution to the physiology of coloration in animals, Journ. Morph. viii, 1893. J. Lors. Ueber den Einfluss des Lichtes auf die Organbildung bei Thieren, Pfliiger’s Arch. \xiii, 1896. IIT. 4 LIGHT 97 H. M. Vernon. The effect of environment on the development of Echinoderm larvae: an experimental enquiry into the causes of variation, Phil. Trans, Roy. Soc. clxxxvi, 1895. E. Yuna. De Vinfluence des milieux physiques sur les étres vivants, Arch. Zool. Exp. et Gén. vii, 1878. EK. Yuna. De V'influence des lumieres colorées sur le développement des animaux, Mitt. Zool. Stat. Neapel, ii, 1881. 5. HEAT As is very well known, those activities by which every organism maintains its specific form can only be carried on within certain definite limits of temperature. So also a certain degree of heat is necessary for the due performance of the functions of growth and differentiation ; above or below certain limits—more or less definite for each organism, but varying in different organisms—development is unduly accelerated or re- tarded, or brought to a standstill, while its form is frequently distorted as well. To Oscar Hertwig we are indebted for a careful inquiry into the conditions of temperature under which the development of the Frog’s egg takes place. In the case of Rana fusca Hertwig has found the cardinal temperature-points to be as follows:—The normal is about 15°-16° C.; above this up to 20°-22° C. development is accele- rated without being otherwise altered ; this temperature is there- fore the optimum (Fig. 51). Above this point the form of develop- ment is altered, and at such a high temperature as 30°C. death follows very quickly. At low temperatures (6°-1° C.) there is con- siderable retardation, and at the zero-point a complete cessation of segmentation ; the eggs are often permanently injured. At the high temperatures referred to—from 23° C. upwards— it is the yolk-cells which are primarily affected. At from 29-6" - to 27-5° the yolk is unable to divide, though it is nucleated, and segmentation is confined to the animal hemisphere, and soon ceases even there (Fig. 47). At 26-5° the first furrow indeed passes through the yolk, but subsequent segmentation is mero- blastic, with the resulting formation of a cap of cells or blastoderm lying upon and separated by a segmentation cavity from the JENKINSON H 98 EXTERNAL FACTORS IL. 5 nucleated yolk. The eggs then die. At lower temperatures— 25°-23°—the yolk is also affected, and many eggs die in the ‘morula’ stage; such as do survive give rise to distortions or monstrosities (Figs. 48, 49). The injury to the yolk interferes a5 SY A B Fia. 47.—Meridional sections of eggs of Rana fusca developed (A) at 29.5° C., (B) 26-5° C. Five hours ten minutes after fertilization. k, nuclei; p, pigment. — Dp JEP Sen C722 Fia. 48.—Meridional sections of eggs of Rana fusca developed at 26-5° C. One day after fertilization. /, nuclei; &h, blastocoel ; 2, cells imbedded in unsegmented yolk. Fra. 49.—Abnormal embryos of Rana fusca, produced by heat. A, embryo two days old developed at a temperature of 24° C.; B, embryo three days old, reared at a temperature of about 25°C. br, brain; y.p, yolk-plug; t.b, tail-bud ; ¢, tail; s, sucker; g, gill. IIL. 5 HEAT 99 with the proper closure of the blastopore ; there is consequently a large, persistent yolk-plug surrounded by a thickened blasto- poric rim into which the separated halves of the medullary plate and notochord are differentiated (spina bifida) (Fig. 50). The Fie. 50.—Two transverse sections through the embyro shown in Fig. 49 a. A, passes through the blastopore and yolk-plug; B, through the anterior end. d, yolk-plug; m.p, medullary plate; ch, notochord; mk, mesoblast. front end of the archenteron is, however, normally developed if the temperature is not too high, and in this case the anterior portion of the nervous system and notochord are undivided; pos- teriorly, however, their right and left halves diverge round the blasto- pore, and are continued into the halves of the double tail when the latter is formed. Gill slits, proto- vertebrae, striated muscle-fibres, i t, and alae ee oe een the tail fin may all be differentiated. o¢ an ege of Rana fusca, de- The development of the organs veloped at a temperature of f the two sides of the body is fre- 72, Six hours fifty minutes 2 y after fertilization. kh, blasto- quently unequal. coel. At low temperatures segmentation and the closure of the blastopore take place very slowly, and at 0° cease altogether. The eggs are not, however, dead, but will resume their develop- ment when replaced under ordinary temperature conditions. They show abnormalities, however, due to injury of the yolk ; H 2 - 100 EXTERNAL FACTORS IIT. 5 Morgan has similarly found that the fertilized (not, however, the unfertilized) eggs of an American species (2. palustris) which have been subjected to a temperature of 1°C. and then allowed to develop under normal circumstances exhibit spina bifida and persistent yolk-plug. Schulze has also observed these abnormalities as the result of excessive cold. On one point, however, this author is not quite in agreement with O. Hertwig, for he states that eggs and embryos exposed to 0° in various stages do continue to develop, though of course very slowly. Thus, in the case of eggs exposed shortly after fertilization, the blastula stage was only reached in ten days, while a month elapsed before the blastopore was formed. Lillie and Knowltoh, however, state that in 2. virescens and Amblystoma tigrinum segmentation is totally inhibited at 0°. In another species of Frog (2. esculenta) which spawns much later in the year—in May and June—the cardinal points were found by Hertwig to be much higher, and the eggs endured a temperature of 33°C. without injury. They are, in fact, acclimatized to a higher temperature, and it is very interesting to notice that Davenport and Castle have succeeded in artificially acclimatizing the eggs of another Amphibian (Bufo lentiginosus) to a considerable degree of heat. Eggs were reared at 15°C. and 24°-25°C, After four weeks the heat rigor temperature was 40°C. for the former, 43-2° for the latter; and in another experiment the temperature was raised to 43-5° by allowing the ego's to develop at 33°-34° for seventeen days. A similar lowering of the minimum seems to have been observed by Lillie and Knowlton in the case of Amblystoma tigrinum, In this form, which spawns much earlier than Rana virescens, there is considerably less retardation of development at 4°. That temperature markedly influences the rate of development, or, as Hertwig puts it, that the quantity of developmental work performed per unit of time is a function of the temperature, is abundantly clear, and is well shown in the annexed diagrams (from Hertwig), in which the curves show the times taken to reach various stages at various temperatures (Figs. 52, 53). It will be seen that as the temperature sinks the rate of development, or rather of differentiation, decreases, but at an IIL. 5 HEAT 101 increasing rate, Lillie and Knowlton have made the same observation for the species investigated by them. pv i ve | 27 ns | E F / | | mS | est §. i als ine os AmTiT, ols Al UY als [IZ LIT S t & ah wl’ Fae Fg All sts It ri ivi a> ALI AIH a oh ys V | te} o @ | J / HALT] ae. Ae / ‘g : AA : Reams Bala eg 7110 9£ ra oe Vi f >a eA | ~~ y i == PLA A 7” oad BS ei gi arae i A be or eT L (a= —-Z —T 4 ad Z —— la | Temperature Centigrade SOP 5" S89 EF FT eS ROS eM ST ES? 4" Sa" 1° Fie. 52.—Curves showing the effect of temperature upon the rate of development of the Frog (Rana fusca). The abscissae give the tempera- ture in degrees Centigrade, the ordinates the days required to reach each of the stages Ito IX. I, gastrula; II, medullary plate; III, me- dullary folds closed, suckers; IV, tail-bud; V, tail and gills; VI, tail fin; VII, operculum beginning; VIII, operculum closing; IX, rudiments of hind legs. (After O. Hertwig, 1898.) The rate of growth, however, may increase at an increasing (or decrease at a decreasing) rate, as Lillie and Knowlton found 102 EXTERNAL FACTORS Ill. 5 for the tadpoles of Rana virescens and Bufo lentiginosus. The same authors state that at low temperatures (below 3° in the case of the Frog, below 6° in the case of the Toad) growth was altogether inhibited, while at 2° there was an actual shortening in length in the case of the Frog tadpole, due, it is suggested, to a diminution in the turgor of the cells. The cardinal points have also been determined for the Hen’s egg. According to Kaestner normal development occurs only D\& Fig. 53.—Effect of temperature upon the growth of the tadpole of the frog (Rana fusca). A, B, developed at a temperature of 14-5°-15° C.; A, two days old, circular blastopore (Stage I in Fig. 52); B, three days old (Stage II in Fig. 52); C, D, developed at a temperature of 20° C. ; C, three days old (Stage V in Fig. 52); D, four days old (Stage VI in Fig. 52). (From Minot, 1907, after O. Hertwig, 1898.) between 95° and 102° F. (35° and 39°C.). The maximum, the temperature above which the embryo dies, is 43°C,; the mini- mum, at which development stands still, 28°C. Edwards, how- ever, fixes the minimum or physiological zero at 20°-21°C., for, as the annexed diagram shows(Fig. 54),development may continue between 20° and 29°, though it is, of course, very much retarded. Edwards has further made the highly interesting observation IIL. 5 HEAT 103 DEVELOPMENT 60 50 30 20 30 Temecrarver 24 26 28 Fria. 54.—The index of development (percentage of normal develop- ment) for the egg of the Hen at temperatures varying from 20°C. to 30-75°C. (After Edwards, 1902.) AVERAGE DIAMETEROF soece BLASTODERM IN MM. t +++ 4H eeene 6.90 6.40 4444 aenee 5,90 5.40) 490 secs | 4.40 2i 92 23 24 25 RELATION OF TEMPERATURE TO GROWTH OF BLASTODERM _ Fie. 55.—Growth of the blastoderm of the Hen’s egg independently of the appearance of the primitive streak, at low temperatures. (After Edwards, 1902.) 104 EXTERNAL FACTORS TIT. 5 that at the low temperatures in question growth may occur without differentiation (Fig. 55). Thus in one series of experiments at 24.5° for six days the blastoderm increased in diameter from 4-4:mm. (the average diameter of the blastoderm in unincubated eggs) to 6-9 mm. The primitive streak was, however, not formed. A temporary exposure to low temperatures often inflicts a permanent injury on the egg and leads to malformations. Kaestner, by subjecting the eggs at many different stages to temperatures of 15°-25°, 10° and 5°, has discovered that the capacity of resistance decreases as development proceeds (though not with absolute regularity). Thus the maximum exposure to ‘21° consonant with subsequent normal development was 192 hours for embryos of six hours, 96 hours for embryos of one day, 72 hours for embryos of two to six days, 49 hours for embryos of eight days, and 24 hours for embryos of 20 days. At these low temperatures development is stated to be com- pletely arrested, though the heart never ceases beating, irregularly and convulsively. The cooling process may be repeated over and over again without altering the capacity for future growth and differentiation, or reducing or increasing the maximum capacity of endurance of cold. Malformations, as stated above, are of frequent occurrence, but only in those cases in which the embryo has been exposed in an early stage, during the first two or three days of incubation, and only after long exposures. The medullary folds may remain unformed anteriorly, the two halves of the heart may remain widely separate, the head amnion fold may be absent and abnormal gill slits be formed ; the heart and blood-vessels are often enormously distended, and haemorrhages are frequent. Kaestner attributes these mon- strosities not directly to the cold but to the pressure of the blastoderm against the shell, for in the cooled eggs, owing to some change in the specific gravity of the albumen or yolk, the latter rises up; if the egg is placed with the blunt end upper- most, so that the embryo is pressed against the shell-membrane, no monsters are produced. Mitrophanow is another observer who has utilized low tem- ~ peratures to cause malformations. High temperatures also give Ill. 5 HEAT 105 rise to abnormalities accompanied by acceleration (Féré, Mitro- phanow). The effects of extremes of heat and cold upon the ova and embryos of certain Invertebrates have been studied by Driesch, the brothers Hertwig, Vernon, Sala, and Greeley. Fie. 56.—The effect of heat upon the segmentation of the Echinoid egg. a,b, c,d, four successive stages in the segmentation of the same egg of Echinus ; e, f, two successive stages in the division of the same egg of Sphaerechinus. (After Driesch, 1893.) DOME Fie. 57.— Suppression of cell-, but not of nuclear, division by heat (Echinus). (After Driesch, 1893.) The first-named observed that by subjecting the fertilized ova of Sphaerechinus to a temperature of 30°-31° (the normal is 19°) de- velopment was accelerated and segmentation abnormal (Fig. 56). After the first furrow—though not after subsequent divisions— the blastomeres separated and sometimes remained apart, a fact which provided a means of watching the independent develop- ment of the first two blastomeres. After the four-celled stage the direction of division became irregular, one spindle being 106 EXTERNAL FACTORS Il. 5 perpendicular, instead of parallel, to the other three, or two perpendicular to two, or all irregular; in the next phase the formation of micromeres was partially or wholly suppressed. Nevertheless these abnormally segmented eggs produced perfectly normal Plutei. It is also possible for nuclear division to continue while cell- division is suppressed, as a result of exposure to high tempera- tures (Driesch) (Fig. 57). . Fre. 58.--The effect of heat upon the development of Sphaerechinus granularis. a, exogastrula; b, exogastrula, with tripartite gut; c, Pluteus, with tripartite gut; (d) Anenterion, with stomodaeum, but no gut. (After Driesch, 1895.) By exposing the blastulae to the same high temperature Driesch brought about a very interesting malformation, an Anen- terion (Fig. 58). The archenteron was formed and constricted into the normal three portions, but it was evaginated instead of invaginated. Later on it shrank up and disappeared ; the rest of the embryo, however, became a Pluteus, with a stomodaeum. Vernon finds the optimum temperature for Echinoid larvae IIL. 5 HEAT 107 to be from 17-5° to 215°C. Exposure to high or low tempera- tures after fertilization, either for longer or shorter intervals or continuously, produced a decrease in body-length of the Plutei. The arm-length, however, increased with increasing temperature. Vernon has also made the most interesting observation that the variability alters with the temperature. Eight-day larvae were measured, and the mean variability (Galton’s Q) of the body-length was found to be at 16° to 18°,. 22-2, at 18° to 20°, 26-3, at 20° to 22°, 24-8, and at 22° to 24°, 24.0. Thus the variability is greatest at the temperature most favourable for development, and conversely. It is also possible for the cell processes that occur during fertilization itself to be seriously affected by heat and cold, as the researches of O. and R. Hertwig have shown. Moderate exposure (twenty minutes) of the eggs of Strongylo- centrotus to a temperature of 31° C. so weakens the cytoplasm that many spermatozoa are enabled to enter. Hach sperm forms its own aster, and these combine with one another to form various irregular mitotic figures (triasters, tetrasters, and so on). The segmentation of such eggs is very irregular. With longer exposures the cones of entrance become feebly developed and the asters are not formed, while the numerous sperm-nuclei remain unaltered. Greater heat—over 40° C.— prevents the entrance of the spermatozoa altogether. This pathological polyspermy may also be produced by cold ; in this case also excessive exposure prevents the formation of the vitelline membrane, the cones of entrance, and the sperm-asters, while the spermatozoa remain in the peripheral layer of the egg. The effect of a low temperature on eggs which have already been normally fertilized is seen in the reduction of the astral rays and spindle fibres, though not of the spheres, and in the thickening and irregular aggregation of the chromosomes. At a normal temperature the achromatic figure reappears. Very similar phenomena have been described by Sala in Ascaris. This author kept the eggs (the females, that is, con- taining eggs in all stages of development) at low temperatures —from 3° to 8° C.—for from half an hour to five hours and longer. The effect of a short exposure to a very low tem- 108 EXTERNAL FACTORS IIT. 5 perature is not so harmful as a longer exposure to a less degree of cold. The processes of maturation and fertilization were both abnormal. Granules of chromatin took the place of the tetrads and were unequally distributed to the spindle-poles ; or, if the chromosomes (tetrads) had been normally formed before the commencement of the experiment, their division was irregular, in extreme cases all passing to one pole and into the first polar body. Again, the formation of the polar bodies might be suppressed altogether, or abnormal, the second only being formed, or both as one, or the first polar body might be as large as the egg itself. The achromatic figure was also deformed, the spindle being split at one or both poles (pseudo- triaster, pseudo-tetraster), and centrosomes appeared instead of the usual centrosomal granules. The cytoplasm became granular, the vitelline membrane was not formed, two or more eggs frequently fused together. Polyspermy, with consequent multi- plication of asters and centrosomes, was very noticeable, and, in fertilization stages, a separate pronucleus may be formed from each female chromosome, or fragment of a chromosome. Closely connected with the cytoplasmic effects brought about by these temperature changes is the phenomenon of artificial parthenogenesis, produced by Morgan and Greeley in Arbacia and Asterias by lowering the temperature of the sea-water to the freezing-point. Greeley has also shown that a lowering of the temperature, like the raising of the osmotic pressure, results in a withdrawal of water, the cause to which, as is well known, Loeb attributed the development of unfertilized ova in his experiments. . Greeley has shown that by the combination of a low tem- perature with a chemical reagent a higher percentage of swimming blastulae can be obtained. LITERATURE C. B. DAVENPORT and W. E. Caste. Studies in morphogenesis ; III. On the acclimatization of organisms to high temperatures, Arch. Ent. Mech. ii, 1896. H. Driescu. Entwicklungsmech. Stud. 1V: Experimentelle Verin- derung des Typus der Furchung und ihre Folgen (Wirkungen von Wirmezufuhr und Druck), Zeitschr. wiss. Zool. lv, 1893. IIL. 5 HEAT 109 H. Driescu. Entwicklungsmech. Stud. VII: Exogastrula und Anen- teria, Mitth. Zool. Stat. Neapel, xi, 1895. C. L. Epwarps. The physiological zero and the index of develop- ment for the egg of the domestic fowl, Gallus domesticus, Amer. Journ. Phys. vi, 1901-2. A. W. GREELEY. On the effect of variations in the temperature upon the process of artificial parthenogenesis, Biol. Bull. iv, 1903. O. Hertwie. Ueber den Einfluss verschiedener Temperaturen auf die Entwicklung der Froscheier, S.-B. kinig. preuss. Akad. Wiss. Berlin, 1896. O. Hertwic. Ueber den Einfluss der Temperatur auf die Entwicklung von Rana fusca und esculenta, Arch. mikr. Anat. li, 1898. 8. Kagstner. Ueber kiinstliche Kilteruhe von Hiihnereiern im Verlauf der Bebriitung, Arch. Anat. Phys. (Anat.), 1895. S. Karstner. Ueber die Unterbrechung der Bebriitung von Hiihner- eiern als Methode zur Erzeugung von Missbildungen, Verh. Anat. Gesellsch., 1896. F, R. Linnre and E. P, Knowxron. On the effect of temperature on the development of animals, Zool. Buil. i, 1898. L. Sava. Experimentelle Untersuchung iiber die Reifung und Be- fruchtung der Eier bei Ascaris megalocephala, Arch. mikr. Anat. xliv, 1895. O. ScHuuzE. Ueber die Einwirkung niederer Temperatur auf die Entwickelung des Frosches, I, Anat. Anz. x, 1895. : O. ScHutze. Ueber die Einwirkung niederer Temperatur auf die Entwickelung des Frosches, II, Anat. Anz. xvi, 1899. 6. ATMOSPHERIC PRESSURE. THE RESPIRATION OF THE EMBRYO The respiratory exchange, which is so characteristic a function of adult organisms, is a necessity for the embryo also, and in some cases can be detected in very early stages indeed. In the case of the Chick this need of oxygen is shown by the arrest or distortion of development, or the death of the embryo when the egg is placed in too confined a space, or when the shell is varnished, wholly or above only (Mitrophanow, Féré), though a coat of varnish on the lower side has no effect accord- ing to the latter author; or again, when the egg is placed in an atmosphere of hydrogen, or when the pressure of the ordinary atmosphere is reduced (Giacomini). Giacomini found that at a pressure of about 600 mm. the 110 EXTERNAL FACTORS IIT. 6 embryos were small and abnormal in respect of the medullary tube and amnion; the optic vesicles and cranial flexure were absent, and there were serious disturbances in the area vasculosa, where, though the blood islands were present, the capillaries were either not formed or failed to reach the embryo. No haemoglobin was produced, Embryos exposed at a later stage (four days) nearly all died in two days of asphyxia, the blood being dark red and haemorrhages numerous. That these effects were due not to the reduced pressure but to the want of oxygen was shown by the complete normality of embryos reared in an atmosphere of pure oxygen at the same pressure (except in certain characters always exhibited by such embryos ; see below). Similar methods may be employed to demonstrate the neces- sity of oxygen for the Frog’s egg, a necessity which is indeed patent to any one who has observed the inferior development, accompanied by spina bifida and open blastopore (Morgan) of the eggs in the middle of a mass of spawn. Thus, according to Rauber, development is retarded at a pressure of 3 atmosphere of ordinary air, and the mortality high, while at pressures of 4 or 4 atmosphere death very rapidly ensues. As a result of four days’ exposure to pure hydrogen or nitrogen (ordinary air from which the oxygen had been removed) Samassa observed retarded segmentation, and subse- quently irregularities in development of the type already referred to. Carbon dioxide produced irregular segmentation and death in twenty hours. Godlewski’s experiments are perhaps more thorough. The eggs subjected to ordinary air at a greatly reduced pressure (2 mm.), as well as those kept in thoroughly boiled water, segmented but little, and cell-division was confined to the animal hemisphere. In an atmosphere of pure oxygen at the same low pressure, however, development was, in many cases at least, neither retarded nor abnormal. Further experiments with pure oxygen, pure hydrogen, and an atmosphere composed of oxygen and carbon dioxide in equal parts, gave the same result, as the subjoined table shows (Table XIII). It is also clear that the absence of oxygen makes itself felt almost from the begin- ning, while pure oxygen accelerates development. III. 6 ATMOSPHERIC PRESSURE 111 TABLE XIII Oxygen and Hours. Oxygen. Hydrogen. Carbon Controls. Dioxide. 3 First furrow in|No furrow No furrow some 3% |All but one with | One - half Most with first first furrow with first furrow furrow / 4 |All but one 4 cells aoe a 8 All with 2 cells cells se 5 |Allwith4cells [Mostwith4| <4 Most with 2 cells ; cells ® a few with 4 17? | Blastomeres smaller | Blastomeres 5 Normal than in controls |smallerthan 2 1 in controls ° 22+ |Blastomeres very| Segmenta- a Normal small- tion ceases 47 | Blastopore closed White hemisphere visible 73 Medullary folds Blastopore closed This method has given similar results for the eggs of the fishes Ctenolabrus and Fundulus (Loeb). One or two points are, however, worthy of especial notice. The former develops at the surface of the sea, and is more sensitive to a lack of oxygen than the latter, the segmentation of whose egg will indeed continue for twenty-four hours in pure hydrogen, though an embryo is never formed. In Ctenolabrus, on the other hand, segmentation never advances further than the eight-celled stage, and the cell-boundaries already formed subsequently disappear, though they can be restored on removal to pure oxygen. In Fundulus the capacity for enduring a lack of oxygen decreases (or the need of oxygen increases) with the progress of development; the fatal exposure for a newly fertilized egg is four days, for a newly formed embryo thirty- two hours, for an embryo with the circulation established twenty-four hours, and for the newly hatched larva shorter still. Carbon dioxide is quickly fatal to both species, The lack of oxygen has also a noteworthy effect on the pig- ment cells which are found, especially round about the blood- vessels, on the yolk-sac of Fundulus. These pigment cells are of two kinds, black and red, and when the embryo is deprived of oxygen the former disappear, the latter diminish only a little. 112 EXTERNAL FACTORS IIT. 6 It has been noticed elsewhere that this pigment is less abundantly formed in darkness than in light, and Loeb has suggested that light may promote oxidation. The ova of Echinoids also require oxygen from the beginning of their development (Loeb), Without this element segmenta- tion is impossible, or, if segmentation has already begun before they are deprived of it, the blastomeres swell up and fuse, According to Lyon the eggs of Arbacia are only sensitive to a want of oxygen for from fifteen to twenty minutes after fertilization. Vernon has shown that water saturated with carbon dioxide and mixed in the proportion of 20 % or more with sea-water is fatal to the development of these forms. Exact quantitative determinations of the oxygen absorbed and the carbon dioxide excreted have been made by Godlewski for the Frog and by Pott and Preyer for the Chick. The results are shown in the tables annexed (Tables XIV, XV). TABLE XIV Showing the result of one experiment on the respiration of the Frog's egg (Godlewski). Amount in grammes per 24 hrs. owe cr and per 100 eggs of *| O absorbed. CO, excreted. 0-03908 1 ae 2 0-4502 0-0995 3 0-7033 0-2131 a 1-0539 0-4193 It thus appears to be the very general rule that the egg begins to respire at an early age. There is a case, however, Ascaris, in which not only can the egg endure an atmosphere of nitrogen or carbon dioxide or nitric oxide for prolonged periods and still develop, but is actually killed by pure oxygen (at 24 atmospheres) (Samassa). The adult worm, of course, is an endoparasite, and Bunge has shown that it can manage to produce carbon dioxide though denied access to free oxygen. The effect of pure oxygen has also been tried on various embryos. In such an atmosphere (at ordinary pressure) the development of the chick is normal, except that skin, allantois, limbs and amni- otic fluid are all very red with oxyhaemoglobin ; an excessive amount of carbon dioxide is produced. The amount of this gas IIT. 6 ATMOSPHERIC PRESSURE 1138 excreted by the undeveloped though incubated egg in pure oxygen is, however, less than in air. TABLE XV Showing the oxygen absorbed and carbon dioxide excreted by the Hen’s egg during incubation. (After Pott and Preyer, from Preyer, Spez. Phys.) Days of Amount in grammes per 24 hrs, of incubation. O absorbed. CO, excreted. Developed. | put incubated. | Developed. | jot Govaneted, 1 2 3 £ 5 -08 6 -10 -08 7 -09 10 -09 08 8 10 ‘11 15 9 . il 10 “11 ‘11 ‘11 17 1 a" ‘Ul 17 12 5 13 24 14 +24 33 14 15 35 15 40 15 40 36 16 -42 15 42 36 oe Y 3 09 15 53 39 18 65 15 +52 39 19 -67 54 20 -68 -16 DD -41 21 86 16 -68 43 * Pulmonary respiration begins. According to Samassa and Rauber the development of the Frog’s egg in pure oxygen is normal, but Godlewski states, as JENKINSON £ 114 EXTERNAL FACTORS III. 6 we have seen, that it is somewhat accelerated. At a pressure of 24 atmospheres, however, segmentation is arrested and death ensues (Samassa). When the tadpoles, newly hatched, are exposed to its influence, the hyoid becomes immensely thickened and the branchial chamber completely closed; the internal gills are weak (Rauber), and the same author states that ‘ gastrulae’ subjected to three atmospheres of ordinary air had their development temporarily arrested, while later embryos, in the stage of the medullary folds, became small and immobile in air at twice the atmospheric pressure. This result seems to be due to pressure, not to the oxygen. In the foregoing the general necessity of respiration for the life of the developing organism has alone been taken into con- sideration, but it should not be forgotten that oxygen may exert a stimulus on some part, the response to which results in a process of differentiation. Thus His has suggested that the growth of the blastoderm over the yolk is oxygenotropic, and Herbst that the migration of the blastoderm-forming cells to the surface in Arthropod ova, and the migration of spicule- forming cells in Echinoid larvae are cases of definite reaction to oxygenotactic stimuli. Loeb, we may note, has found that the regeneration of the head of Zubularia will only take place when the stem is supplied with fresh water, and the same author has suggested that the accumulation of the pigment cells round the blood-vessels on the yolk-sac of Fwadulus is also an oxygenotaxis. LITERATURE C. Fert. Note sur l’influence des enduits partiels sur l‘incubation de l’eeuf de poule, C. R. Soc. Biol. (10) i, 1894. | C. Giacomini. Influence de l’air rarifié sur le développement de l’ceuf de poule, Arch. Ital. Biol. xxii, 1895. EK. GoDLEWsKI. Die Einwirkung des Sauerstoffes auf die Entwicklung von Rana temporaria, Arch. Ent. Mech. xi, 1901. C. Hergst. Ueber die Bedeutung der Reizphysiologie fiir die causale Auffassung von Vorgiingen in der thierischen Ontogenese, Biol. Centralbl. xiv, xv, 1894, 1895. J. Lors. Ueber die relative Empfindlichkeit von Froschembryonen gegen Sauerstoffmangel und Wasserentziehung in verschiedenen Entwick- lungsstadien, Pfliiger’s Arch. lv, 1894. IIT. 6 ATMOSPHERIC PRESSURE 115 J, LoeB. Untersuchungen iiber die physiologischen Wirkungen des Sauerstoffmangels, Pfliiger’s Arch. 1xii, 1896. P. MirRoPpHANOW. Einfluss der veriinderten Respirationsbedingungen auf die erste Entwickelung des Hiihnerembryos, Arch. Ent. Mech. x, 1900. R. Pott. Versuche iiber die Respiration des Hiihner-Embryo in einer Sauerstoffatmosphiire, Pfliiger’s Arch. xxxi, 1883. R. Pott und W. PREYER. Ueber den Gaswechsel und die chemischen Veriinderungen des Hiihnereies wiihrend der Bebrutung, Pfliiger’s Arch. xxvii, 1882. W. PREYER. Spezielle Physiologie des Embryo, Leipzig, 1885. A. RAUBER. Ueber den Einfluss der Temperatur, des atmosphirischen Druckes und verschiedener Stoffe auf die Entwicklung thierischer Hier, S.-B. Naturf. Ges. Leipzig, x, 1883. H. Samassa. Ueber die féiusseren Entwicklungsbedingungen der Kier von Rana temporaria, Verh. Deutsch. Zool. Ges. vi, 1896. 7. OSMOTIC PRESSURE. THE ROLE OF WATER IN GROWTH That growth seems to depend in many cases on the absorption of water or a watery fluid—in the swelling of the Echinoderm blastula, for example, or the enlargement of the Mammalian blastocyst—has been noticed by several observers; in a few instances experimental proof has been given of the relation between the two. Although, as is very well known, the Hen’s egg loses weight daily throughout incubation by loss of water, this loss is due almost entirely to the slow evaporation of the albumen, and a humid atmosphere is necessary for development, as Pott and Preyer have found. Féré’s experiments with eggs incubated in desiccators demonstrated, during later stages, a slight re- tardation accompanied by abnormalities and a high death-rate ; in earlier stages, up to about the fourth day, there was on the contrary an acceleration of development. Davenport has shown for tadpoles of various Amphibia (Am- blystoma, Toads, Frogs) that increase in weight is very largely due to increase in weight of water. Known numbers of tad- poles, from which superficial water had first been carefully removed, were placed over sulphuric acid in a desiccator. Re- I 2 116 EXTERNAL FACTORS IIt. 7 peated weighings were made until a constant minimum was reached. The results are set forth in the accompanying table and figure (Table XVI, Fig. 59). It will be seen that the percentage of water rises very rapidly in the first fortnight, from 56% to 96%, then decreases slightly, afterwards becoming nearly constant. TABLE XVI Showing the rate of absorption of water by Tadpoles (after Davenport). Days after hatching. Percentage of water. 1 56 2 ; ; ‘ 59 5) . 4 r 77 7 : : 5 89 9 : ; : 93 14 ‘ ; ; 96 41 7 : : 90 84 : , : 88 100% DA en 603 | jos boy | 502) Days 20 30 40 SO 40 Jo $0 jo Fre. 59.—Curve showing change in percentage of water in Frog tad- poles from the first to the eighty-fourth day after hatching. Abscissae, days; ordinates, percentages. (After Davenport, from Korschelt and Heider.) A different, and a less satisfactory, method has been employed by Loeb, hypertonic solutions being used to prevent the absorp- tion of water. While the newly fertilized eggs of Fundulus IIT. 7 OSMOTIC PRESSURE 117 developed as normally in fresh water as in sea-water, only a blastoderm with occasionally a dwarf embryo was formed in a 5 % solution of sodium chloride in sea-water, and segmentation was arrested in the thirty-two-celled stage when the concentra- tion of the salt was raised to10%. Older eggs were, however, far less sensitive, and after three or four days the embryos could be placed directly in a 27-5% solution without arresting their development, though the heart beat more slowly and differentia- tion was less rapid. Fie. 60.—A and C, formation of ex-ovates in the egg of Arbacia by dilution of the sea-water; &, nucleus; m, egg-membrane; B and D, blastulae formed from A and C; B becomes constricted into two blas- tulae, each of which gives rise to a Pluteus; D produces a single Pluteus. (After Loeb, from Korschelt and Heider.) The eggs can nevertheless be acclimatized to the salt. Re- moved from the 10% solution after the thirty-two cells had been formed to ordinary sea-water for eighteen hours, they were capable, when once more replaced in the strong solution, of giving rise to embryos which lived for a considerable time. Similar experiments made on Arsacia showed that though eell-division is suppressed in the hypertonic solution (2 % sodium 118 EXTERNAL FACTORS ITL,7 chloride) nuclear division continues all the same, for when re- turned to sea-water the eggs divided at once into as many cells as had in the meantime been formed in the controls, a result confirmed by Morgan. That the normal egg is in a condition of osmotic equilibrium with the sea-water is further shown by its behaviour in sea- water diluted to twice its volume; in this experiment the ege 4g Fia. 61.—Variations in the segmentation of Echinus microtuberculatus produced by dilution of the sea-water. a, tetrahedral four-cell stage ; b, eight cells, three premature micromeres ; c, eight cells, two precocious micromeres; d, the same egg after the next division, the precocious micro- meres have divided unequally, two normal micromeres have been formed. (After Driesch, 1895.) (of Arbacia) absorbs water, swells and bursts its membrane and so produces a large ex-ovate which may develop independently of the rest of the ovum (Loeb) when replaced under ordinary conditions (Fig. 60). Driesch has produced irregularities of segmentation by the same means (Fig. 61). Although, therefore, it seems reasonable to suppose that in the cases just quoted the observed effects really are due to the IIT. 7 OSMOTIC PRESSURE 119 increased osmotic pressure of the medium and consequent with- drawal of water from, or prevention of imbibition of water by, the eggs, the weak point of the experiment, and of all such experiments, is our ignorance of the extent to which the ova or embryos are permeable to the substance employed, since the osmotic effect, or withdrawal of water, will obviously vary in- versely with the permeability. The neglect of this possibly disturbing factor has indeed led in some cases to quite un- warrantable conclusions. In 1895 O. Hertwig showed that certain abnormalities could be produced by growing the eggs of the Frog (2. /usea and \ df ot “ Fre. 62.—Three sodium-chloride embryos of Rana fusca. df, yolk- plug; hp, brain; ki, gills; s, margin of epidermic layer of ectoderm ; sch, tail; wr, lip of blastopore. (After O. Hertwig, from Korschelt and Heider.) esculenta) and of the Axolotl in a solution of common salt. In stronger solutions (1% to 0-8 %) segmentation was confined to the animal hemisphere, though nuclear division went on in the yolk, Weaker solutions (0-6%) allowed of further, but dis- torted, development; the yolk-cells were unable to move beneath the lip of the blastopore, so that the latter remained open with a persistent yolk-plug, and the medullary folds failed to close in the’ region of the brain, a condition recalling the abnormalities known in Human and Comparative Teratology as Hemicrania and Anencephaly (Figs. 62, 65). The exposed region of the brain underwent a grey degeneration with dis- 120 EXTERNAL FACTORS Ill. 7 integration of the epithelium. Other organs were, however, normally formed, the front end of the gut by invagination, the notochord and mesoderm, protovertebrae, heart, pronephros, audi- tory vesicles, optic vesicles, infundi- bulum, and liver, until the embryo died. The persistence of the yolk-plug has also been induced by Gurwitsch by means of halogen salts (sodium bromide and lithium chloride) and weak solu- tions of alkaloids (strychnine, caffein, nicotine) (Fig. 63), by C. B. Wilson in Rana, Chorophilus, and Amblystoma Fra. 63.—Meridional sec- by means of sodium chloride and tion through a lithium Ringer’s solution, and by Morgan with Perinat, Gee Sia ihinins “sed ae Haieation, witsch, from Korschelt and who has used isotonic solutions of cane- Heider.) : : sugar, sodium chloride, and a large number of other salts for the purpose, claims that in this case the results produced depend upon the osmotic pressure alone, and are therefore due to a withdrawal of water from the developing embryo. B Fie. 64.—Sections of Frogs’ eggs grown in solutions of, A, ammonium iodide (1-5%), and, B, urea (23%). In both cases segmentation is mero- blastic, although in a there are a few large divisions in the yolk. In B the multinucleate cell masses of the animal hemisphere protrude above the surface. The nuclei are large, lobed, and homogeneously. ace in both cases, (Ammonia is probably present in the solution of urea. Recent experiments made by the author do not, however, bear out this conclusion. In the first place, it is to be observed that isotonic solutions (isotonic with a 0-625 % NaCl solution) do not IIT. 7 OSMOTIC PRESSURE 121 produce the same, but markedly different effects. Some solutions arrest development at an early stage (during segmentation (Fig. 64), gastrulation, or the formation of the medullary folds) ; in others development proceeds but is distorted, the medullary folds remaining open in whole or in part, and the yolk-plug un- covered, or either of these malformations may occur without the other; in one case (dextrose) development is quite normal in form but very considerably retarded, while finally in urea de- velopment is normal both in form and rate (Figs. 66, 67). No legitimate deductions can be made from these experiments, how- Fie. 65.—Frog embryos grown in a -625% solution of sodium chloride. A and B after five days, c and D after six days. In all the yolk-plug is fully exposed. In A the medullary groove is wholly open, in B and ¢ it is closed behind, in D it is closed throughout. ever, until the permeabilities of the tissues to these solutions are ascertained. The tadpole requires water (Davenport), and the degree of shrinkage of the tadpoles in these solutions affords a means of determining the question ; it appears that they are per- fectly permeable to urea, more or less impermeable to cane-sugar, dextrose, and sodium chloride, the shrinkage being rather greater in the first than in the other two. On the assumption that the permeabilities of the embryo are the same as those of the tadpole, it follows that the greater effect produced on the former by sodium chloride than by cane-sugar, or, still more, than by dextrose, cannot be set down to the osmotic pressure of the solution alone, 122 EXTERNAL FACTORS III. 7 a result which is further corroborated by the constancy in the relative toxicities of the bases and the acids in the case of the Fie. 66.—Frog embryos grown in isotonic solutions of, A, sodium chloride (625%); B, cane-sugar (6-6%) ; c, dextrose (3-4%) ; and, D, urea (1-14%). In a the medullary folds are closed but the blastopore open ; in B the medullary groove is open but the blastopore closed; in c de- velopment is normal, but retarded ; in D development is normal, both in form and rate, though the embryos die soon after the stage shown in the figure. ~ Fie. 67.—A. Longitudinal section of a Frog embryo grown in a -45% solution of lithium chloride. The medullary groove is open, except in front and behind. The notochord is bent in several places and the gut roof much crumpled. B. Longitudinal section of a Frog embryo grown in a 6.6% solution of cane-sugar. The medullary groove is open, except in front, the cells in its floor degenerating. The gut roof is incomplete in part and there is an evident neurenteric canal. monobasic salts. The observed deformities are therefore to be attributed to some other—chemical or physical—property of a Ill. 7 OSMOTIC PRESSURE j 123 the solutions, though what this is is not known.) It may be added that in Gurwitsch’s experiments the concentrations of the alkaloids employed were certainly far below those which would be isotonic with a -625% solution of sodium chloride. It also follows that during the closure of the blastopore the Frog’s egg does not need to absorb water from the outside; it may, in fact, be exposed to a very considerable degree of desiccation at this period without interfering in the least with the closure of the blastopore or of the medullary folds, a result which is all the more surprising in that the newly hatched tadpole imbibes water at so rapid a rate. The experiments which have hitherto been considered relate to the need of water for normal development. There are, however, certain processes for which not the absorption, but, on the contrary, the abstraction, or at least the local abstraction, of water appears to be essential, the phenomena, namely, of fertilization. Cytologists have observed that the entrance cone and funnel, the mechanism by which the spermatozoon is swept into the interior of the egg, appear to be aggregations of a watery substance about the acrosome or apical body, and that the sperm sphere and aster are similarly due to the withdrawal of water by the centrosome in the middle-piece from the cyto- plasm; in other words, that the stimulus whereby the sperma- tozoon restores to the egg its lost power of cell-division is essentially a process of local dehydration. This inference is substantiated by the familiar experiments of Loeb, who has succeeded in rearing normal larvae from the unfertilized eggs of Echinoderms and certain worms by tem- porary immersion in certain solutions. In his earlier experiments he found that a mixture in equal parts of a 22 solution of magnesium chloride and sea-water produced more Plutei than any other solution tried, and hence believed the result to be specific and attributable to the magnesium ion. Later, however, this artificial parthenogenesis was successfully brought about by various isotonic solutions (chlorides of sodium, potassium and calcium, potassium bromide, nitrate and sulphate, cane-sugar ' In this view Stockard, as a result of experiments on Fundulus, concurs (Arch. Ent. Mech. xxiii, 1907, and Journ. Exp. Zool. iv, 1907). 124 EXTERNAL FACTORS III. 7 and others). The increased osmotic pressure was, therefore, considered to be the cause of the phenomenon, and it was suggested that in ordinary fertilization the spermatozoon intro- duces a substance which has a higher osmotic pressure than, and is therefore able to withdraw water from, the egg. Hunter has also shown that sea-water concentrated to 70% of its volume is sufficient to bring about the result. It must still be remembered that the permeabilities of the ova to the various solutions are not known; Sollmann, indeed, has proved the secondary swelling after the primary shrinkage of many eggs in hypertonic solutions, which must therefore enter and cause the dissociation of the cytoplasm. Further, Delage has, as a matter of fact, denied that the increased osmotic pressure is solely responsible for the results. The French zoologist succeeded in making the ova develop in solutions hypertonic to sea-water, but found that isotonic solutions of different chlorides or mixtures of chlorides did not all give the same percentage of larvae. He holds, therefore, that other factors are involved. Other methods, as noticed elsewhere, are low temperatures and mechanical agitation. Fischer has successfully demonstrated the phenomenon in the Chaetopods, Nereis and Amphitrite, Bullot in Ophelia, and Bataillon in Vertebrates (Rana fusca and Petromyzon planert); but in this last case segmentation did not continue for very long and the processes of nuclear division were highly irregular. An attempt made by Gies to incite development (of Echinoids) by means of extracts of spermatozoa was unsuccessful. Although in brilliancy of conception and completeness of execution Loeb’s experiments are certainly pre-eminent over those of any other investigator, it should not be forgotten that - about the same time Morgan had succeeded in inducing asters, and even the beginnings of segmentation, in the unfertilized ova of sea-urchins and some other forms by the use of salts and other substances, and that the way for all recent work was really paved by the original labours of O. and R. Hertwig, to be described in the next section. Loeb did not undertake an examination of the cytological changes, but Wilson has shown that ordinary nuclear division occurs with asters and centrosomes: a primary radiation III. 7 OSMOTIC PRESSURE 125 centring in the nucleus first appears; this then fades away, and a definite aster with a centrosome is formed just to one side of the nucleus; this divides to form the first amphiaster (cleavage-spindle). Asters also arise independently of the nucleus in the cytoplasm (cytasters); these contain centrosomes, and may divide, and the cytoplasm divide round them. The part played by the cytasters in development is, however, in- significant ; their activity soon comes to an end. The number of chromosomes is one-half the normal number. This latter statement is confirmed by Morgan, but denied by Delage, who asserts that, as in egg fragments enucleated and subsequently fertilized, the half number becomes doubled. LITERATURE EK. BATAILLON. | La pression osmotique et les grands problémes de la Biologie, Arch. Ent. Mech. xi, 1901. E. BATAILLON. Etudes expérimentales sur l’évolution des Am- phibiens, Arch. Ent. Mech. xii, 1901. C. B. Davenport. The rdle of water in growth, Proc. Boston Soc. Nat. Hist. xxviii, 1897-8. C. Fer&. Note sur l’influence de la déshydratation sur le développe- ment de l’embryon de poulet, C. R. Soc. Biol. (10) i, 1894. A.GuRwitscH. Ueber die formative Wirkung des veriinderten chemi- schen Mediums auf die embryonale Entwicklung, Arch. Ent. Mech. iii, 1896. O. Hertwie. Die Entwicklung des Froscheis unter dem Einfluss schwiicherer und stiirkerer Kochsalzlisungen, Arch. mikr. Anat. xliv, 1895. O. Hertwic. Die experimentelle Erzeugung thierischer Missbild- ungen, Festschr. Gegenbaur, Leipzig, 1896. J.W. JENKINSON. On the effect of certain solutions upon the develop- ment of the Frog’s egg, Arch. Ent. Mech. xxi, 1906. J. Lors. Investigations in physiological morphology, Journ. Morph. vii, 1892. J. Lors. Ueber die relative Empfindlichkeit von Fischembryonen gegen Sauerstoffmangel und Wasserentziehung in verschiedenen Ent- wicklungsstadien, Pfliiger’s Arch. lv, 1894. J. Lors. Ueber eine einfache Methode zwei oder mehr zusammen- gewachsener Embryonen aus einem Hi hervorzubringen, Pfliiger’s Arch. lv, 1894. LITERATURE ON ARTIFICIAL PARTHENOGENESIS EK. Batarnuon. Nouveaux essais de parthénogénése expérimentale chez les Vertébrés inférieurs, Arch. Ent. Mech. xviii, 1904. G. Butior. Artificial parthenogenesis and regular segmentation in an Annelid (Ophelia), Arch. Ent. Mech. xviii, 1904. - Y. DezacE. Etudes sur la mérogonie, Arch. Zool. Exp. et Gén. (3), vii, 1899. Y. Detace. Ktudes expérimentales sur la maturation cytoplasmique 126 EXTERNAL FACTORS III. 7 et sur la parthénogénése artificielle chez les EKchinodermes, Arch. Zool. Exp. et Gén. (8) ix, 1901. M. A. Fiscoer. Further experiments on artificial parthenogenesis in Annelids, Amer. Journ. Phys. vii, 1902. W. J. Gres. Do spermatozoa contain an enzyme having the power of causing the development of mature ova? Amer. Journ. Phys. vi, 1901-2. A.W. GREELEY. Artificial parthenogenesis in the star-fish produced by lowering the temperature, Amer. Journ. Phys. vi, 1901-2. i A. W. GREELEY. On the analogy between the effects of loss of water and lowering of temperature, Amer. Journ. Phys. vi, 1901-2. A.W. GREELEY. On the effect of variations in the temperature upon the process of artificial parthenogenesis, Biol. Bull, iv, 1903. R. Hertwia. . Ueber die Entwicklung des unbefruchteten Seeigel- eles, Festschr. Gegenbaur, Leipzig, 1896. S. J. Hunrer. On the production of artificial parthenogenesis in Arbacia by the use of sea-water concentrated by evaporation, Amer. Journ. Phys. vi, 1901-2. J. W. JENKINSON. Observations on the maturation and fertilization of the egg of the Axolotl, Quart. Journ. Micr. Sci. xlviii, 1904. K. Kostranecki. Ueber die Veriinderungen im Inneren des unter dem Einfluss von KCl-Gemischen kiinstlich-parthenogenetisch sich ent- wickelnden Eis von Mactra, Bull. Intern. Acad. Sci. Cracovie, 1904-5. J. Lorg. On the nature of the process of fertilization and the artificial production of normal larvae (Plutei) from the unfertilized eggs of the sea-urchin (two papers), Amer. Journ. Phys. iii, 1899-1900. J. Lozs. Further experiments on artificial parthenogenesis, and the nature of the process of fertilization, Amer. Journ. Phys. iv, 1900-1. J. Lozrs. Experiments on artificial parthenogenesis in Annelids (Chaetopterus), Amer. Journ. Phys. iv, 1900-1. A. P. MatHEWs. Artificial parthenogenesis produced by mechanical agitation, Amer. Journ. Phys. vi, 1901-2. T. H. Morgan. The fertilization of non-nucleated fragments of Echinoderm eggs, Arch. Ent. Mech. ii, 1895-6. T. H. Morean. The production of artificial astrospheres, Arch. Ent. Mech. iii, 1896. T. H. Morgan. The action of salt solutions on the unfertilized and fertilized eggs of Arbacia, Arch. Ent. Mech. viii, 1899. T. H. More@an. Further studies in the action of salt solutions and other agents on the eggs of Arbacia, Arch. Ent. Mech. x, 1900. E. B. Wiuson. A cytological study of artificial parthenogenesis in sea-urchin eggs, Arch. Ent. Mech. xii, 1901. 8. THE CHEMICAL COMPOSITION OF THE MEDIUM By means of solutions of alkaloids and other substances the brothers Hertwig have been able to incite very remarkable cyto- logical changes in the eggs of sea-urchins (Strongylocentrotus). 4 itl iI. 8 CHEMICAL COMPOSITION 127 The effects of nicotine are perhaps the most striking (lig. 68, a-e), Various solutions—1% and less of a concentrated extract—were allowed to act upon the egg for different lengths of time (five to fifty minutes) before fertilization ; the ova were then replaced in sea-water and fertilized. The cytoplasm is so paralysed by the Fra. 68.—The effect of alkaloids and other poisons on the processes of fertilization and nuclear division in the egg of the sea-urchin, Strongylocentrotus lividus. (After R. and O. Hertwig, 1887.) a. The egg was exposed to nicotine (one drop in 200 c.c. of sea-water) for ten minutes, and then fertilized; drawn fifteen minutes later. b, c. The same for fifteen minutes; drawn after one and a half hours. d. The same for ten minutes; drawn after three hours, ten minutes. e. The same; drawn after three hours. Only part of the complex figure is shown ; the remainder lies in another plane. St, g, he. Exposed to a 0-05% solution of quinine for twenty minutes one and a half hours after fertilization ; drawn from one to two hours later. k, 1-5, male pronucleus, 6, female pronucleus. Exposed to chloral (0-5%) one minute after fertilization ; fixed after 150 minutes, l, m. Chloral 0-5% one minute after fertilization ; fixed after six hours. Male and female pronuclei reconstructed and metamorphosing, in m the ‘fan’ form with commencing division. n, 0. Placed in chloral 0-5% five minutes after fertilization ; preserved after ninety minutes. n. Female pronucleus (four-rayed rosette), and male pronucleus (three-rayed rosette). o. Fusion of pronuclei. p. The same, Female pronucleus in the pseudo-tetraster forms. 128 EXTERNAL FACTORS III. 8 poison that the normal vitelline membrane cannot be formed and consequently many spermatozoa enter. In such eggs segmenta- tion does not occur in the ordinary fashion by successive binary divisions, but many small cells are simultaneously formed. The resulting blastulae are abnormal, the segmentation cavity being filled with a solid granular mass (Stereoblastulae), and very few reach the Pluteus stage. The irregularities of segmentation are due to the complex mitotic figures and divisions which poly- spermy entails. One, two, three or more of the spermatozoa fuse with the female pronucleus; each has its own aster, which divides into two. Hence the most complex nuclear figures are formed. In the case where two sperm-nuclei unite with the egg- nucleus a tetraster is formed, that is four asters united by spindles in a square or rhombus, or a triaster with an odd aster united to one angle of the system. The chromosomes are grouped in the equators of the four, or three, united spindles, as the case may be, and the egg divides simultaneously into four, or three. The arrangement becomes still more involved when there are other sperms, whether these fuse with the female pronucleus or not. Each amphiaster is united by one pole to the tri-, tetra-, or polyaster developed round the combination nucleus, or to the poles of other amphiasters; in one case there were nineteen spindles in all, not, of course, all in one plane. Each centro- sphere receives half the chromosomes of the spindle attached to it, and each cell, when division occurs, contains one or more nuclei. Hydrochlorate of morphine will produce similar effects, but only with longer exposures—a 0-4 % solution for from two to five hours. Strychnine, however, is poisonous in very weak doses (-005 % to +25 %), and quite short exposures are sufficient to call forth marked results. Other solutions successfully tried were chloral hydrate (from 0-2 % to 0-5 % for from one to four and a half hours), cocaine (from 0-025 % to 1 % for five minutes), and sulphate of quinine (-05 % for ten minutes). In quinine (05 % for thirty minutes) and chloral (5%) the entrance cone was small and no asters were formed, from which the III. 8 CHEMICAL COMPOSITION 129 Hertwigs argue that the contractility of the cytoplasm is impaired in these solutions. Chloroform dissolved in sea-water has the very interesting property of stimulating — without the addition of spermatozoa—the formation and separation of the vitelline membrane. The male generative cells are also sensitive to the action of these alkaloids, but not necessarily in the same measure. They can resist, for example, the influence of a solution of nicotine, which is ten times as strong as one necessary to evoke pathological changes in the ova. Though chloral hydrate (0-5 %) and quinine (0-05 %) are both temporarily fatal to the motility of the spermatozoa, sea-water restores the capacity for fertilization. Strychnine (0-01 %) and morphine (0-5 %) are without effect. In the experiments just described the abnormalities seem to be directly due to the initial paralysis of the egg by the reagent and consequent polyspermy. Should, however, the egg have been first normally fertilized, the irregularities produced by the subsequent action of the poison are, though well marked, not of the same kind, for in this case the vitelline membrane has already been formed and only one spermatozoon has gained admittance. Chloral hydrate (Fig. 68, k-p) was employed for ten minutes and at varying intervals after insemination (one, one and a half, five and fifteen minutes). Exposure to the solution very shortly after insemination first retards the progress of the sperm-head and the formation of its aster, and when later on the chromosomes are formed they lie heaped together in the centre of an achromatic figure described as a pseudo-tri- or pseudo-tetraster. This consists of three or four conical groups of fibres, the bases resting on, and the fibres connected to, the chromosomes, the apices outwardly directed and sometimes with, sometimes without, asters; in any case, however, they are not united by spindles, as is the case in the complex figures observed in polyspermy. Isolated asters are also to be seen in the cytoplasm, and, which is perhaps more remarkable, the female chromosomes are themselves the centre of a unipolar (fan-shaped) or multipolar apparatus of the same kind. The reader will not fail to notice the similarity to the phenomena occurring in artificial parthenogenesis. JENKINSON K 130 EXTERNAL FACTORS 111. 8 Should the pronuclei unite—which is only possible before these pseudasters have been developed, if the eggs have been sub- jected to the action of the poison immediately (one minute) after fertilization—the conjugation nucleus itself becomes the focus of a similar system. In eggs poisoned after a longer interval (fifteen minutes) the male and female pseudasters may them- selves unite. The nucleus—or nuclei—divide irregularly, the chromosomes passing in unequal numbers to the poles of the figure. The several pseudasters and isolated asters, with which nuclei may possibly become secondarily associated, may be united by clear streaks of protoplasm, thus giving rise to a dendritic figure. Simultaneous and unequal division of the whole ovum follows. Should the spermaster have already been developed—fifteen minutes after insemination—it degenerates. The subsequent changes comprise the formation of multipolar figures and irregular cell-division. In later stages—when fertilization has been completed and segmentation is about to begin—the ova are almost or quite indifferent to nicotine, strychnine, and morphine; but chloral (0-5 %) destroys the asters which are already in existence and brings about a reconstitution of the combination nucleus with subsequent formation of a tetraster and quadruple division. In future mitoses, however, the spindles are bipolar. Cocaine and quinine (-05 %) (Fig. 68, #2) have the same effect. The importance of these experiments does not require to be emphasized. Not only do they throw a valuable light on the possible causes of those pathological mitoses that occur in . malignant growths, they also contribute very greatly to the understanding of the normal processes of fertilization and karyokinesis. Thus from the failure of the asters to appear in eggs treated with chloral before fertilization the brothers Hertwig argue that the contractility of the cytoplasm is diminished by this substance, and from the failure of the pronuclei to unite in eggs which have been immersed in the solution shortly after fertilization they suggest that it is the contractility of the ovum which normally brings about the union of the pronuclei. Since, III. 8 CHEMICAL COMPOSITION —. 131 however, both male and female nuclei are able to divide, this division must be normally incited, not by their union with one another, but by the separate action of the cytoplasm on each, a view which is fully borne out by the phenomena of artificial parthenogenesis and merogony (the development of fertilized enucleate egg fragments), whatever interpretation may eventuaily be put on the ‘ contractility ’ of the cytoplasm.! Another alkaloid which exerts an injurious influence on the ova of Echinoderms is atropine, the sulphate of which retards and dwarfs the development of Asterias and Arbacia (Mathews). Pilocarpine, on the contrary, has an accelerating effect, a result attributed by Mathews to its activity as an oxidizer, while atropine is regarded as a reducing agent, the property to which Loeb has also assigned the value of potassium cyanide in pro- longing the life of unfertilized ova. The eggs of sea-urchins, when once laid, are only capable of fertilization and develop- ment within a certain definite limit of time, after the expiration of which they degenerate and die; after twenty-four . hours, for example, they are only able, when fertilized, to reach the gastrula stage, and after thirty-two hours even fertilization is hardly possible. By treatment with an appropriate solution of potassium cyanide this limit may be considerably postponed. In the most successful series of experiments the ova were first placed in a solution of KCN 750 in sea-water, and then removed successively every twenty-four hours to nN nN 2500’ 3000’ lengths of time, then removed to pure sea-water and fertilized. As the table shows (Table X VII), segmentation was still possible after 168 hours’ sojourn in the solution, but the greatest number of Plutei was obtained after only 66 hours’ stay. It was also shown that better results could be obtained with artificial parthenogenesis if the ova were first kept in the eyanide solution. Loeb points out that in the higher animals n nu 1400’ 2000? In the last solution they were kept for various 1 Strictly speaking, only the division of the male chromosomes can be regarded as being stimulated by the egg cytoplasm. What exactly it is which excites the female nucleus to divide is not at all clear. K 2 132 EXTERNAL FACTORS III. 8 the effects of this substance are due to its inhibition of oxidation ; that this is the real cause of the prolongation of the life of the eggs is shown by the fact that when kept in an atmosphere of hydrogen for thirty-eight hours they were still capable of being fertilized and developing into swimming larvae. TABLE XVII Showing the effect of exposures of various length of Sea-urchin eggs to a solution of KCN (After Loeb.) 3000 Length of exposure in hours. Result. 66 80 % Plutei, vitelline membrane formed 90 30 % Plutei, no vitelline membrane formed 99} 20% Plutei,,, _,, : J 112 Less than 20 % Plutei 120 Gastrulae, but no Plutei 139 A few blastulae 140 Blastulae, not swimming 46h Eight-celled stage only Simultaneous lowering of the temperature to the freezing-point enhanced the value of the cyanide treatment. In later stages, however, immediately after fertilization and subsequently, the action of potassium cyanide is by no means beneficial ; at this time, as we know, oxygen is a necessity (see above, p. 112); and Lyon has shown that the moment at which the ova are particularly sensitive to both KCN and the lack of oxygen is the same, about fifteen minutes after insemination. Chemical agents are also able to incite irregularities of growth and abnormalities in later stages of development. In a long series of experiments Féré has shown that mon- strosities can be produced by exposing the Hen’s egg to the unfavourable influence of a large variety of substances. Vapours of ether, alcohol, essential oils, nicotine, mercury, and phosphorus, injections of alkaloids such as morphine, nicotine, strychnine, and others, of bacterial toxines (those of tubercle, diphtheria), of peptones, dextrose, glycerine, several alcohols, certain salts (K Br, IIL. 8 CHEMICAL COMPOSITION 133 KI, SrBr,), are all baneful, retarding and distorting the embryo to a greater or less extent. Ammonia, it may be noted, is fatal at once. It has already been shown (p. 123) that the malformations induced by sodium chloride in Amphibian embryos are to be set down to some other property than the osmotic pressure of the solution, and it is here only necessary to advert to saat Me een PANG So BeBe H/, Fre, 69.—Cane-sugar (6:6 %). Two stages in the formation of the noto- chord from the whole thickness of the roof of the archenteron in the Frog. Dextrose (3-4 %). Secondary degeneration of the gut roof and ventral part of notochord. some of the more interesting effects occurring in particular solutions. Although the more poisonous salts (e.g. Lil, CaCl,, SrBr,, and others) inhibit altogether the formation of the blastoporic fold, a cause which normally assists in its production—the proliferation of small cells in the roof of the segmentation cavity—may continue to operate, with the result that that roof is thickened and thrown into puckers and folds. 134 EXTERNAL FACTORS _ III. 8 Again, the notochord may be formed from the whole thickness of the archenteric roof (cane-sugar)recalling the mode of its develop- ment in Urodela and Petromyzon (Fig. 69); the solid medullary tube observed in potassium chloride and other salts reminds one of the rudiment of the nervous system in Teleostei and others, while the mode of closure of the medullary tube in, for example, some of the magnesium salts resembles that observed in Amphioxus ; the formation of notochordal tissue from the wall of the neural Fra. 70.—Formation of vacuolated notochordal tissue in the medullary tube of the Frog embryo under the influence of urea (1-6%). Underneath the notochord is the subnotochordal rod. tube and the roof of the archenteron (Fig. 70) in strong solutions of urea (1-17 % to 1-56 %) shows that the prospective potentialities of these organs are not yet fixed, while the development of an optic cup without a lens in urea, sodium chloride, and sodium bromide demonstrates that the formation of the former is inde- pendent of that of the latter of these two parts of the eye. The grey degeneration of the exposed part of the medullary plate (due.to the distribution of the pigment throughout the cell-body), the protrusion of cells (‘framboisia’ of Roux), and disintegration of the epithelium which is so characteristic in a Ill. 8 CHEMICAL COMPOSITION 135 many of these solutions (cane-sugar, NaCl, LiCl, MgCl,, MgSO,), have been noticed by many observers (Roux, Hertwig, Morgan, Bataillon). All the more violent solutions attack the yolk-granules. In some cases the effect produced appears to be specific ; thus in lithium salts the ectoderm is often pitted and wrinkled before any degeneration appears in the nervous system, and in ammonia salts, which are highly poisonous, the nuclei are much enlarged, lobed, highly chromatic, and homogeneous. The very similar appearance of the nuclei (Fig. 64) in those stronger solutions of urea which arrest development in an early stage suggests that the ammonia set free is the toxic agent in this case. In solution isotonic with -625 % NaCl urea permits of normal development up to a certain point, when the embryos die. In this connexion it is interesting to notice that Moore has found that sodium sulphate will act as an antidote to the poisonous effect of sodium chloride on tadpoles. Thus the average length of life of tadpoles in a Fs NaCl solution was four and a quarter days, but was prolonged to twenty-one days by adding from 4% to 8% of Na,SO,. The poisonousness of sodium chloride, sodium nitrate, calcium nitrate, and magnesium chloride to Fundulus embryos and the value of other salts as antidotes has been shown by Loeb, while Lillie has noted that sodium is fatal but magnesium and calcium beneficial to the ciliary movement of Arenico/a larvae, a result first obtained by Loeb for the Plutei of Hehinus; the muscular contractions of the larva, on the other hand, are inhibited wholly by magnesium, partly by calcium, while sodium is necessary for their con- tinuance. In an artificial solution which combines the three elements in the proper proportions normal development is possible. The nature of the part played by the ions—whether toxic or antitoxic—is, however, a very open question. Arguing from the fact that the evil effects of such salts as sodium chloride and nitrate may be counteracted by calcium and magnesium salts, Loeb has suggested that toxicity and anti- toxicity are functions of valency, and also of electrical charge, since it is further stated that toxicity increases with the valency 136 EXTERNAL FACTORS III. 8 of the anion, antitoxicity with that of the cation. Ions of the same valency are not, however, necessarily equally antitoxic (Loeb and Gies, Lillie, Mathews), and sodium sulphate, as we have seen, may act as an antidote to the chloride (Moore). Mathews has accordingly sought for the cause of toxicity in another physical property, the decomposition tension of the salt, and has certainly succeeded in showing that the poisonousness of solutions to the eggs of Fundulus varies inversely with the decomposition tension, and that a similar relation holds good in certain other cases. Lillie argues that a physiologically balanced solution is necessary, one in which the electrolytes are in a state of chemical equilibrium with the necessary ion-proteid compounds in the tissues. Solutions which only contain some of these substances, or solutions (for example, non-electrolytes) which contain none, are poisonous, because they permit of the outward diffusion of the needful ions. It must be pointed out, however, that this explanation will not fit the cases where the embryo develops perfectly well in fresh water (Fundulus) or in distilled water (the Frog), and that some other reason must be found for the poisonous effect of cane-sugar upon the latter. The whole question, however, is one which belongs more properly to the province of pharmacology. Poisonous although these salts are, the embryo can still be acclimatized to them. C. B. Wilson placed the unsegmented eggs of Amblystoma, Rana, and Chorophilus in a 0-05 % solution of sodium chloride; after twenty-four hours they were removed to 0-1 %, and then successively to stronger solutions by incre- ments of 0-1 % until 1-0 % was reached, a concentration which quickly causes death under ordinary circumstances. In this case, however, development was normal, and the larvae hatched out and lived for some time. The distortions of development which solutions of salts and other substances call forth in Amphibian embryos find a parallel in the malformations which Herbst has produced in Echinoderm larvae (Hehinus, Sphaerechinus) by similar means; as in the former case, the results were at first assigned to the increased osmotic pressure of the media. III. 8 CHEMICAL COMPOSITION 137 When potassium salts are added to the sea-water—for example, a 7 % solution in sea-water of a 3-7 % solution of KCl in tap-water—the egg gives rise to a Pluteus in which, though the gut is, as normally, tripartite, the skeleton is rudimentary and the arms suppressed (Fig. 71). Herbst suggests that the suppression of the arms is due to the absence of a stimulus normally exerted by the skeletal spicules. These abnormal forms may fuse together to form double monsters. Such ‘ potassium’ larvae are developed in sodium salts, but lithium has a more pronounced effect (Figs. 72,73). In this case Fie. 71.—Potassium larvae of Echinoids. a. Potassium larva of Sphaer- echinus (1860 c.c, sea-water + 140 c.c. 3-7% KNO,). There is no skeleton. The gut is tripartite, and the mouth surrounded by the ciliated ring. b, c. Potassium larvae of Echinus (20% of 3% KCl). Note the button- shaped apical tuft of cilia, and, in c, the secondarily evaginated archen- teron. (After Herbst, 1893.) the blastula becomes constricted into two portions, a thin-walled gastrula wall provided with long cilia, and a thick-walled archen- teron, which may be muscular and mobile, and is thickly covered with short cilia. The archenteron has, in fact, failed to in- vaginate, and the larva is an ‘ Exogastrula’. Occasionally there is an attempt at invagination at the end of the archenteric portion, and, after temporary exposure, the invaginated part may be divided into three, and a mouth formed. All the parts of the gut, however, remain in the same straight line. A middle section may be formed by further constriction of the archen- teron (Hehinus) or of the gastrula wall (Sphaerechinus). Double 138 EXTERNAL FACTORS Ill. 8 monsters sometimes arise by fusion of these larvae by their archentera. A skeleton is not usually developed; if present it is abnormal— in position, the spicules being placed near the animal pole and Fie. 72.—Lithium larvae of Sphaerechinus granular’s. a. Larva partially constricted into gastrula wall and archenteric portions, the former with long, the latter with short cilia (980 c.c. sea-water +20 c.c. 3-7% LiCl). b. Similar larva to the last, but a neck or connecting piece has been formed from the ectodermal portion. c,d. Progressive diminution of the ectodermal gastrula wall portion with increase in the quantity of Li. the arms of the Pluteus formed under their influence near the mouth instead of by the side of the anus, in the number of the spicules, and consequently the number of arms (three, four, or five, instead of two), and in the number of their radii (four or five, instead of three). Ill. 8 CHEMICAL COMPOSITION 139 The gastrula wall is often smaller than the archenteron, and, as the strength of the solution is increased, becomes still further reduced, until nothing of it is left but a small button at the Fie. 73.—a, Larva with three skeletal spicules, and a ‘ cell-rosette’ at the end of the archenteron. 6. Larva with skeleton—more than three spicules—and arms developed. The neck is invaginated into the ecto- dermal portion, the gut tripartite. c. Five-armed Pluteus with five skeletal rods. The gut is normally invaginated and tripartite. d. Larva of Echinus microtuberculatus. There’is a neck, and the gut is partly invaginated. In the blastocoel are aggregations of mesenchyme and pigment cells. (After Herbst, 1895.) animal pole, which only indicates its real character by the long cilia which it carries. Such larvae Herbst. terms ‘ Holoento- blastia’. This nearly complete suppression of the ectodermal 140 EXTERNAL FACTORS III.'8 region can, however, only be realized when the salt is allowed to act at a stage in the blastula when the differentiation into the two primary layers is already beginning. Should the embryos be removed before this stage is reached, after twenty- four hours’ exposure to the solution, only ‘ Exogastrulae’, not ‘ Holoentoblastia,’ can be obtained. Should, on the other hand, older blastulae, or gastrulae, or Plutei be placed in the solutions, they die without showing any signs of the characteristic abnormal development. From the fact that equimolecular solutions of monobasic lithium salts produced like effects (such solutions, it must be observed, are also chemically equivalent), Herbst con- cluded at first that the osmotic pressure was responsible for the abnormalities ; but the permanent after-effects of temporary im- mersion just referred to subsequently convinced him that the ova were permeable to the lithium ions to which he now attributes the specific nature of the monstrosity. He suggests further that they act upon the endoderm cells by increasing their absorptive activity and their power of cell-division, while at the same time they inhibit the functions of those mesenchyme cells which are devoted to the formation of the skeleton. As in other monstrosities, there is an alteration in the pro- spective potentialities of cells, elements which would normally be ectodermal becoming converted into endoderm, and additional mesenchyme cells being involved in the secretion of skeletal spicules. It is only by lithium salts that the typical ‘ Holoentoblastia ’ can be produced ; but Exogastrulae can be reared in others, in sodium butyrate, for example; in this solution a stomodaeum is formed, but is, like the archenteron, everted. Even lithium, however, is powerless to cause the ‘ holoentoblastic’ reduction of the gastrula wall in the larvae of Asterias, although exogastru- lation may, but need not, occur. A characteristic deformity is the absence of the pre-oral region, and the elevation of the mouth on a sort of hypostome. In Amphiowus and Ascidians it is impossible to obtain even exogastrulae by these methods. It is evident, therefore, that the specific morphological reaction depends not only on the nature of the substance employed, but also on the constitution of the reacting organism. IIT. 8 CHEMICAL COMPOSITION 141 Herbst has not omitted to point out the significance of these— and indeed of all—monstrosities for the theory of the origin of those larger, discontinuous variations kifown as ‘sports’, or, in more modern phraseology, ‘mutations’; and Vernon has been able to show statistically that the degree of continuous variation may also be altered by changes in the chemical environment. In all the foregoing experiments the effect is observed of the addition of some chemical substance to the medium in which the embryo is placed. We have now to consider a very remark- able series of investigations, for whose planning and execution we are indebted to the genius of Curt Herbst, investigations in which substances which are present in the normal environment of the larva are omitted, and an insight thus gained into the part they play, if any, in the normal development of the organism. Herbst has indeed succeeded in demonstrating in the most conclusive manner the necessity to the sea-urchin egg’ for the normal performance of this or that phase of develop- mental function of a large number of the elements present in sea-water. The sea-water at Naples, where Herbst. carried out his work, has the following composition :—- NaCl .. : ; ; ee KCl : , ; ; « OFX MgCl, . ‘ ; ; 4.) ee MgSO, . : ; : nH CaSO, . - - , Peo CaHPO, Ca,P,O, CaCO, Fe,CO, in small quantities. Si Br I It may be said at once that silicon, bromine, and iodine are unnecessary, and that, though earlier experiments led Herbst to believe that phosphorus and iron were essential, he has since assured himself that phosphorus is certainly, and iron probably, 142 EXTERNAL FACTORS IIL. 8 not. All the other elements, however, can only be omitted under penalty of retardation, abnormality, or death (Figs. 74 A and B). The method employed was a simple one. A series of artificial sea-waters was made up, from which, one by one, each of the elements was omitted, another being substituted in its place. Care was taken to make these artificial media approximately Fiq@. 74 A. Fie. 74.—The necessity of substances contained in sea-water for the normal development of the larvae of sea-urchins. a. Without OH. Ciliated stereoblastula of Sphaerechinus. b. KOH has been added. c. Normal blastula of Sphaerechinus. d. Blastula in a K-free medium. e. Reared in K-free and replaced in sea-water (Sphaer- echinus). f. Larva from a medium devoid of Mg (Sphaerechinus). g. Echinus Pluteus with tripartite gut, mouth and coelom sacs, but neither skeleton nor arms; reared without CaCO, or CaSO,. h. Normal Pluteus of Echinus. isotonic with sea-water, and so exclude a possibly disturbing factor, the alteration of the osmotic pressure. The rdle of each of these necessary elements—or ions—will be considered sepa- rately and in some detail. Sphaerechinus and Echinus were the forms principally employed. III. 8 CHEMICAL COMPOSITION 143 i. SO,. This is ordinarily provided by MgSO, and CaSO,; when the fertilized ova are placed in a solution in which MgCl, is sub- stituted for it (as, for example, in 3% NaCl+-07% KCl + 5% MgCl, + CaHPO, + CaCO,) then their development is retarded from the blastula stage onwards, the embryos are small and degenerate without reaching the Pluteus stage (Fig. 74 B). The gut is straight instead of bent, and not divided into the Fig. 74 B. a. Normal position of skeletal spicules in Sphaerechinus. 6, d. Ab- normal position and number after treatment with SO,-free medium, c. Larva of Echinus from a S-free solution. e. Pluteus of Sphaerechinus with three fenestrated skeletal arms, instead of two. Treated with a SO,-free medium and replaced in sea-water. jf. Normal Pluteus of Sphaerechinus. (After Herbst, 1897 and 1904.) usual three parts ; in Sphaerechinus no mouth is formed, the gut is evaginated (Exogastrula), The endoderm is very thick, the cells dark and dense. The sulphuric acid radicle (sulph-ion) is thus necessary for the proper development of the gut, and necessary from the very beginning, for in embryos which have been kept in SO,-free water up to the mesenchyme-blastula stage and then replaced in sea-water the alimentary tract is still abnormal. Deprived of SO,, in fact, the gut remains radially symmetrical, 144 EXTERNAL FACTORS III. 8 and the same must be said of the skeleton. Normally there are two tri-radiate spicules, one to the right, the other to the left of the gut and some little way from it. Without the needful sulphate the spicules become placed near the gut, and may with the growth of the latter be pushed towards the animal pole. The number of spicules may also be diminished or increased to one, three, or four, arranged in a circle round the gut. On timely removal to sea-water, however, a secondary bilateral symmetry may arise by two of these outgrowing the rest and stimulating the development of the typical arms of the Pluteus. It seems that a sulphate is present in the calcareous skeleton of the Pluteus, as there is in that of the adult urchin. A ciliated cireum-oral ring is formed, but is abnormal in its position, at right angles instead of parallel to the long axis of the body. The pigment which should be secreted by the secondary mesenchyme cells (separated off from the inner end of the archenteron) remains in abeyance, and the apical tuft of cilia is hypertrophied. Other processes, however—fertilization, segmentation, and ciliary motion—are independent of SO,. The development of eggs which are allowed to remain in ordinary sea-water until the blastula stage is no better, whence Herbst concludes that no SO, is taken up during segmentation. During the early stages of gastrulation, however, they appear to absorb a store of it for future needs, for gastrulae reared in sea-water develop further in the SO,-free solution than do those embryos which have been kept in it since fertilization. SO, is equally necessary for the continued life of the Pluteus and of the Bipinnaria larva of Asterias, and without it the rate of regeneration of the head of Zwbularia is retarded and the number of tentacles reduced, until eventually a completely tentacleless head is evolved. The necessary sulphate can be, to a certain extent, replaced by a thio-sulphate. The addition, for instance, of .35% Na,S,O, to the SO,-free solution renders it possible for the larvae to reach the Pluteus stage, though the arms are short, the skeleton small, and the pigment reduced. The larvae die. Selenium and tellurium are both poisonous in an early stage. III. 8 CHEMICAL COMPOSITION 145 ii. Cl. A solution was made up in which the sodium chloride was replaced by sodium formate, the magnesium chloride by magne- sium sulphate, the potassium chloride by potassium sulphate ; thus, NaCOOH 3-5 % + MgSO, -26% + MgSO, .4%+4K, SO, 12 %+CaSO, -1 %+ CaHPO, + CaCO,. The eggs did not segment, and even when KCl and MgCl, were used in their ordinary proportions, segmentation did not progress very far. Nor did the substitution of Na,SO, for NaCOOH give any better results. A considerable amount of chlorine appears therefore to be absolutely necessary for the earliest developmental processes, its function being, Herbst sug- gests, to transport certain necessary cations, the tissues being possibly more permeable to NaCl than to Na,SO,. Later stages —blastulae, gastrulae, Plutei—all die in the Cl-free mixture. Chlorine can be replaced in some measure by bromine. Plutei are formed, though with a distorted skeleton, Tubularia regene- rates its head and the eggs of the fish Labrax develop as well as in sea-water. Iodine is, however, poisonous; so also are chlorates. iii. Na. 2-96 % of MgCl, was added to a solution containing the usual amounts of KC], MgSO,, CaSO,, CaHPO,, and CaCO,. In this the ova indeed segmented, but abnormally, the blastomeres being of unequal size. Death followed; nor was the addition of a certain small amount (-84%) of NaCl sufficient to save them, though segmentation was normal and traces of an archenteron could be detected; with more NaCl (1-34 %) the gastrula stage was reached. The sodium which is thus necessary in the earliest period is also required later on; without it gastrulation is im- possible to eggs which have been reared in sea-water even as far as the mesenchyme-blastula stage. The part played by sodium is not clearly understood. It is known that it counteracts the evil effects of calcium and is necessary for the continuance of muscular contractions. Since calcium is necessary for the cohesion of cells (see below) Herbst opines that sodium may pull them apart; its action in that case is capillary. JENKINSON L 146 EXTERNAL FACTORS IIL. 8 Sodium cannot possibly be replaced by lithium, potassium, rubidium or caesium, all of which would at the necessary con- centrations inevitably be poisonous. iv. K. In the artificial solution employed the small quantity (-07 %) of potassium present in sea-water is simply omitted. Without it segmentation—except the first few phases—is impossible in Hehinus. Sphaerechinus, however, segments, but the blastocoel is reduced, the cells are opaque and not vacuolated, and the ova, though ciliated, are motionless and die (Fig. 74 A, d, e). Later stages are also sensitive to the want of potassium. Blastulae gastrulate, but are shrunken, with short archenteron, and in gastrulae the gut does not divide into three. Plutei, like all the others, die when deprived of it. In the K-free medium spermatozoa temporarily lose their motility, and such spermatozoa cannot effect fertilization. The fertilization, however, of eggs which have been kept without potassium is possible ; in fact, at this earliest stage, no potassium is absorbed, for eggs fertilized in sea-water develop no further in the K-free solution than do those fertilized and kept con- tinuously in it. The absence of potassium in segmentation leaves its effect upon later stages. Two days’ exposure is not too long to prevent normal development on removal to sea-water, but five days’ exposure causes abnormalities of the skeleton (asymmetrical with several triradiate spicules round the gut) and alimentary canal (no mouth). De Vries has shown the importance of potassium for the turgor of young plant-cells, and its function here is probably similar, to promote growth, as the subjoined table of measure- ments shows. Its absence also affects the rate of development (Table X VITT). Potassium can in a measure be replaced by rubidium and caesium. The use of lithium either has no effect or, in larger quantities, produces lithium larvae. Other forms to which the lack of potassium was found to be fatal were the ova of Asterias and Cotylorhiza, and the adult * III. 8 CHEMICAL COMPOSITION 147 Amphioxus. Potassium is also necessary for the contractions of muscles (umbrella and tentacles of Ol: /ia). TABLE XVIII Showing the effect of potassium upon the growth of Sea-urchin blastulae. (After Herbst.) Ratio (unit = ;4, mm.) of cross- diameter to long diameter. After Without K. With K. 18 hours . se po 24 hours . a at 45 hours . = a Showing the effect of potassium on the rate of development of Sea-urchin larvae. (After Herbst.) Artificial sea-water with _ After 008 % KCl. -016 % KCl. 024 % KCl. 36 hours Small gastrulae. Nearly Plutei. _Plutei. 60 hours No mouth; gut not Small Plutei. Fully formed Plutei. constricted. v. Mg. The fertilized ova were placed in a solution which did not include the 32% MgCl, and the -26% MgSO, present in sea- water. Segmentation proceeds normally, but the blastula is slightly smaller than when magnesium is present (the ratio of the diameters is 32). The skeleton, however, though bilaterally laid down, is retarded and deformed (magnesium is present in the skeleton of the sea-urchin and possibly in that of the Pluteus) and the gut is not properly differentiated, not tripartite and without a mouth (Fig. 74 A, /). When the MgSO, is replaced by an isotonic quantity of Na,SO, the results are the same. In the Mg-free solution cilia cease beating, development is retarded (Table XIX), and, though the spermatozoa retain their motility, the ova are so injured that fertilization is impossible unless they are restored to sea-water. The ova, in fact, seem to have a store of Mg which they lose in the Mg-free mixture. Fertilization can, however, take place without magnesium if the L 2 148 EXTERNAL FACTORS ITI. 8 eggs have been kept in sea-water, and such eggs develop, in Mg-free water, to precisely the same extent as those which have been fertilized in sea-water; at this period, therefore, the egg needs no external magnesium. The original store of this element apparently suffices also for the first steps in the formation of the gut; for the lack of it is felt equally in the later stages of its differentiation and in the first moments of its development; the alimentary canal is as abnormal in those larvae which have been kept without magnesium only up to the time when the mesen- chyme and archenteric plate arise as in those which remain in the solution throughout. TABLE XIX Showing the retardation of development of Sea-urchin larvae deprived of magnesium. (After Herbst.) Two solutions were employed, mixed in various proportions ; one (1) has no Mg; the other (2) contains Mg. Solution. Result after 24 hours. 1 Nearly motionless; archenteron formed. 14+10%o0f2 Larger. 1+20 %of2 Larger still. 1+30%o0f2 Larger; gut constricted; mouth formed. 1+40 % of 2 7% 7 is ~ 1+50 % of 2 i 5 9 5s 33 2 It is, in fact, only after the gastrula stage that magnesium is absorbed. Eggs, blastulae and young gastrulae, reared in sea- water develop far worse when placed in the Mg-free liquid than do gastrulae. ‘The formation of pigment and the contractility of muscles remain unaffected by the absence of Mg. In Mg-free media the blastomeres of dAsfertas fall apart and the adult medusae of Ode/ia degenerate. vi. Ca. The calcium salts present in normal sea-water are CaCO,, Ca,SO,, CaHPO,, and Ca,(PO,),. When the carbonate only is absent the blastulae are crumpled and opaque; a few gastrulate but are markedly abnormal, the ciliated ring being crumpled, the gut flattened on the oral side and the skeleton absent (Fig. 74 A,g). Should the skeleton have already been formed when the larvae are exposed it III. 8 CHEMICAL COMPOSITION 149 becomes dissolved. When the sulphate only is omitted (mag- nesium sulphate being present) development is still inferior to the normal, inferior even to development in the presence of CaSO, but in the absence of MgSO,; CaSO, may be replaced by CaCl,. It seems, therefore, that the sulphate is necessary as a caleium salt. : Fia. 75,—a-c. Separation of the blastomeres of Echinus microtubercu- latus in a medium containing NaCl, 3.07%, KCl, 0-08%, MgSo,, 0-66%, MgHPO, and FeCO,, but no Ca. Note the radially striate border, which is the altered uniting membrane. d. Blastula disintegrating in the same medium. (After Herbst, 1900.) If only the phosphate (Ca,(PO,),) is present the egg dies during segmentation, though, if the other phosphorus compound is substituted for it, the effect is the same as when the carbonate alone is omitted. Should, however, all calcium salts be removed the result is more serious still (Fig. 75). The blastomeres are unable to , 150 EXTERNAL FACTORS IIT. 8 cohere, and separate as fast as division takes place, swimming about independently for a time, and then dying. The same phenomenon is witnessed when later stages are placed in such a calcium-free mixture. On removal to sea-water division continues without separation, and should the egg membrane still be intact all the cells unite and a whole larva is formed. Even should the egg membrane be lost, a reunion of the cells is always possible so long as they remain in contact with one another. The separation is due to a change in the surface-tension of the cells; a visible change takes place, in fact, in the superficial layer which covers and unites the blastomeres; it becomes ill-defined and radially striated. The lack of calcium also affects the rate of development, and causes shrinkage, but leaves karyokinesis, ciliary motion, and pigment formation unaltered. Calcium is not replaceable by magnesium, strontium, or barium. vii. CO,. As has just been pointed out, calcium carbonate is necessary for the due formation of the skeleton, although a beginning may be made without it.! -Whether the crumpling of the larva, due to diminution of internal osmotic pressure, which is observed in the absence of calcium carbonate is attributable to the lack of CO, or the lack of the hydroxyl ion is difficult to determine, since, as Herbst points out, a carbonate necessarily introduces OH, while the latter can convert into carbonates the CO, of the atmosphere and of respiration. viii. OH. The alkalinity of the sea-water—reckoned by the number of free hydroxyl ions—is provided by the calcium carbonate and calcium hydrogenphosphate. By the omission of these a solution —neutral to litmus—may be obtained in which the ova give rise to thick-walled, opaque blastulae with granular contents, ciliated ? CaCO, is necessary for the formation of the skeleton of the larva of the sponge Sycandra setosa (O. Maas, S.-B. Ges. Morph. Phys. Miinchen, xx, 1905). In a medium devoid of all calcium salts the Amphiblastulae fall to pieces, II. 8 CHEMICAL COMPOSITION 151 but motionless, and doomed to eventual degeneration and death (Fig. 74 A,a,4). Very occasionally a gastrula with a short gut is formed. When the blastulae are immersed in the solution they give rise to small, opaque gastrulae. A certain degree of alkalinity is necessary for fertilization. The spermatozoa are less sensitive to a want of alkalinity, more sensitive to excessive alkalinity than the ova. By the addition of a small amount of sodium hydrate to the neutral medium development is accelerated, but an increase of the alkalinity of ordinary sea-water is unfavourable. Loeb, on the other hand, has found that the addition of from -006 % to 008 % sodium hydrate to sea-water accelerates the development of Arbacia. The formation of pigment and the vibration of the cilia are other processes which depend on the presence of the hydroxyl ion. Plutei die without it and their skeleton is dissolved. The function of the ion does not appear to be to neutralize any acids produced by the tissues, for these give a neutral reaction even in OH-free media. Since aeration improves the development of eggs in these media, and the more so if the air is deprived of its carbon dioxide, Herbst has concluded that one function of the OH ion is to neutralize the CO, and allow of the formation of the necessary carbonates. Another function is possibly, as Loeb suggested, the acceleration of processes of oxidation. The experiments which we have been considering are unique of their kind, and it is impossible to exaggerate their importance. For, whatever may be the ultimate explanation of the facts, there can be no doubt whatever that the most complete demonstration has been given of the absolute necessity of many of the elements ~ occurring in ordinary sea-water, its normal environment, for the proper growth and differentiation of the larva of the sea-urchin. Nor is this all. Some of the substances are necessary for one part or phase of development, some for another, some from the very beginning, others only later on. Thus potassium, mag- nesium, and a certain degree of alkalinity are essential for 152 EXTERNAL FACTORS III. 8 fertilization, chlorine and sodium for segmentation, calcium for the adequate cohesion of the blastomeres, potassium, calcium and the hydroxyl ion for securing the internal osmotic pressure neces- sary for growth, while without the sulph-ion and magnesium the due differentiation of the alimentary tract and the proper formation of the skeleton cannot occur; the secretion of pigment depends on the presence of some sulphate and alkalinity, the skeleton requires calcium carbonate, cilia will only beat in an alkaline medium containing potassium and magnesium, and muscles will only contract when potassium and calcium are there. The part played by each substance is therefore specific ; for some particular part of the morphogenetic process it is indis- pensable. Not, of course, independently of internal factors, but in co-operation with them, it does, in fact, determine the pro- duction of organic form ; and the relation between the embryo and the environment in which it develops is in this case, at any rate, of the closest and most intimate kind. LITERATURE C.B. DAVENPORT and H. V. NEAL. Studies in morphogenesis. V. On the acclimatization of organisms to poisonous chemical substances, Arch. _ Ent. Mech. ii, 1896. C. Fer&. A series of papers on the effect of various chemical bodies on the development of the Chick, C. R. Soc. Biol. (9) v, 1893-liii, 1901. C. W.GREENE. On the relation of the inorganic salts of blood to the automatic activity of a strip of ventricular muscle, Amer. Journ. Phys. ii, 1898-9. C. Hersst. Experimentelle Untersuchungen iiber den Einfluss der verinderten chemischen Zusammensetzung des umgebenden Mediums auf die Entwickelung der Tiere: I. Versuche an Seeigeleiern, Zeitsch. wiss. Zool. ly, 1898. C. Hergst. Experimentelle Untersuchungen iiber den Einfluss der verinderten chemischen Zusammensetzung des umgebenden Mediums auf die Entwickelung der Tiere: II. Weiteres iiber die morphologische Wirkung der Lithiumsalze und ihre theoretische Bedeutung, Mitt. Stat. Zool, Neapel, xi, 1895. C. Hersst. Experimentelle Untersuchungen iiber den Einfluss der verinderten chemischen Zusammensetzung des umgebenden Mediums auf die Entwickelung der Tiere: III-VI, Arch. Ent. Mech. ii, 1896. C. Hersst. Ueber die zur Entwickelung der Seeigellarven nothwendigen anorganischen Stoffe, ihre Rolle und ihre Vertretbarkeit: I. Die zur Entwicklung nothwendigen anorganischen Stoffe, Arch. Ent. Mech. v, 1897. IIT. 8 CHEMICAL COMPOSITION 153 C. Hersst. Ueber zwei Fehlerquellen beim Nachweis der Unentbehr- lichkeit vom Phosphor und Eisen fiir die Entwickelung der Seeigellarven, Arch. Ent. Mech. vii, 1898. C. Hergst. Ueber das Auseinandergehen von Furchungs- und Ge- webezellen in kalkfreiem Medium, Arch. Ent. Mech. ix, 1900. C. Hergst. Ueber die zur Entwickelung der Seeigellarven noth- wendigen anorganischen Stoffe, ihre Rolle und ihre Vertretbarkeit : Il. Die Vertretbarkeit der nothwendigen Stoffe durch andere iihnlicher chemischer Natur, Asch. Ent. Mech. xi, 1901. C. Herssr. Ueber die zur Entwickelung der Seeigellarven noth- wendigen anorganischen Stoffe, ihre Rolle und ihre Vertretbarkeit: III. Die Rolle der nothwendigen anorganischen Stoffe, Arch. Ent. Mech. xvii, 1904, O. and R. Hertwie. Ueber den Befruchtungs- und Teilungsvorgang des tierischen Kies unter dem Einfluss fiusserer Agentien, Jen. Zeitschr. xx, 1887, O,HEertwie. Experimentelle Studien am tierischen Hi vor, wihrend und nach der Befruchtung, Jen. Zeitschr. xxiv, 1890. W.H.Howe tt. On the relation of the blood to the automaticity and sequence of the heart-beat, Amer. Journ. Phys. ii, 1898-9. W.H.Howe.u. Ananalysis of the influence of the sodium, potassium, and calcium salts of the blood on the automatic contractions of heart- muscle, Amer. Journ. Phys. vi, 1901-2. R. IRVINE and G. Sims WoopHEAD. The secretion of carbonate of lime by animals, Proc. Roy. Soc. Edinburgh, xvi, 1889. R.S. Linyre. On differences in the effects of various salt-solutions on ciliary and on muscular movements in Arenicola larvac, Amer. Journ. Phys. v, 1901. R. 8. Liniie. On the effects of various solutions on ciliary and muscular movement in the larvae of Arenicola and Polygordius, Amer. Journ. Phys. vii, 1902. R. 8. Litu1e. The relation of ions to ciliary movements, Amer. Journ. Phys. x, 1903-4. D. J. Lineie. The action of certain ions on ventricular muscle, Amer. Journ. Phys. iv, 1900-1. F, 8. Lockr. On a supposed action of distilled water as such on certain animal organisms, Journ. Phys. xviii, 1895. J, Lores. Ueber den Kinfluss von Alkalien und Siiuren auf die embryonale Entwickelung und das Wachsthum, Avch. Ent. Mech. vii, 1898. J. Lors. On ion-proteid compounds and their réle in the mechanics of life-phenomena: I. The poisonous character of a pure NaCl solution, Amer. Journ. Phys. iii, 1899-1900. J. Lozrs, On the different effect of ions upon myogenic and neuro- genic rhythmical contractions and upon embryonic and muscular tissue, Amer. Journ. Phys. iii, 1899-1900. 154 EXTERNAL FACTORS III. 8 J. Logs. The toxic and anti-toxic effects of ions as a function of their valency and possibly their electrical charge, Amer. Journ. Phys. vi, 1901-2. J. Lozp and W. H. Lewis. On the prolongation of the life of the unfertilized eggs of sea-urchins by potassium cyanide, Amer. Journ. Phys. vi, 1901-2. J. LozB and W. J. Gres. Weitere Untersuchungen iiber die ent- giftenden Ionenwirkungen und die Rolle der Kationen bei diesen Vor- giingen, Pfliiger’s Arch. xciii, 1903. E. P. Lyon. The effects of potassium cyanide and of lack of oxygen upon the fertilized eggs and the embryos of the sea-urchin (Arbacia punctulata), Amer. Journ. Phys. vii, 1902. A. P. MatHews. The action of pilocarpine and atropine on the embryos of the star-fish and the sea-urchin, Amer. Journ. Phys. vi, 1901-2. A. P. Matuews. The relation between solution tension, atomic volume, and the physiological action of the elements, Amer. Journ. Phys. x, 1903-4. A. P. MatHews. The toxic and anti-toxic action of salts, Amer. Journ. Phys. xii, 1904-5. A. P. MarHEews. The nature of chemical and electrical stimulation : I. The physiological action of an ion depends upon its electrical state and its electrical stability, Amer. Journ. Phys. xi, 1904. II. The tension co-efficient of salts and the precipitation of colloids by electrolytes, Amer. Journ. Phys. xiv, 1905. S. S. MAXWELL and J. C. Hitu. Note upon the effect of calcium and of free oxygen upon rhythmic contraction, Amer. Journ. Phys. vii, 1902. 8S. S. Maxweu. The effect of salt-solutions on ciliary activity, Amer. Journ. Phys. xiii, 1905. H. McGuiean. The relation between the decomposition-tension of salts and their anti-fermentative properties, Amer. Journ. Phys. x, 1903-4. A. Moore. Further evidence of the poisonous effects of a pure NaCl solution, Amer. Journ. Phys. iv, 1900-1. A. Moore. The effect of ions on the contractions of the lymph hearts of the Frog, Amer. Journ. Phys. v, 1901. A. Moore. On the effects of solutions of various electrolytes and non-conductors upon rigor mortis and heat rigor, Amer. Journ. Phys. vii, 1902. A. Moore. On the power of Na,SO, to neutralize the ill effects of NaCl, Amer. Journ. Phys. vii, 1902. T. H. Morean. The action of salt-solutions on the unfertilized and fertilized eggs of Arbacia and of other animals, Arch. Ent. Mech. viii, 1899. T. H. Morgan. Further studies on the action of salt-solutions and of other agents on the eggs of Arbacia, Arch. Ent. Mech. x, 1900. T. H. More@an. The relation between normal and abnormal develop- ment of the embryo of the Frog as determined by the effect of lithium chloride in solution, Arch. Ent. Mech. xvi, 1903. III. 8 CHEMICAL COMPOSITION 155 H. Neruson. Further experiments on the antitoxic effects of ions, Amer. Journ. Phys. vii, 1902. S. RINGER and A, G. PHear. The influence of saline media on the tadpole, Journ. Phys. xvii, 1894-5. J. Rircure. The relation of chemical composition to germicidal action, Trans. Path. Soc. 1, 1899. W. Roux. Beitriige zur Entwicklungsmechanik des Embryo : I. Zur Orientierung iiber einige Probleme der embryonalen Entwicklung, Zeitschr. Biol. xxi, 1885. D. Rywoscu. Ueber die Bedeutung der Salze fiir das Leben des Organismus, Biol. Centralbl. xx, 1900. T. SoLLMANN. Structural changes of ova in anisotonic solutions and saponin, Amer. Journ. Phys. xii, 1904-5. C. B. Witson. Experiments on the early development of the Am- phibian embryo under the influence of Ringer and salt solutions, Arch. Ent. Mech. v, 1897. W. D. ZorrHout. The effects of potassium and calcium ions on striated muscle, Amer. Journ. Phys. vii, 1902. 9. SUMMARY In the numerous experiments which we have been considering the effect is observed upon the development of the embryo of certain alterations in the constitution of that embryo’s normal environment. Either some factor which is not usually present is added to the environment, or else some factor which is customarily found there is altered by increase or decrease, or removed altogether. In some cases development remains undisturbed by this treat- ment, in others it may be merely generally retarded or accelerated, in others again it may be altered not merely in rate but in form, with the production of an abnormality or monstrosity, and if its effect is too intolerable the death of the embryo may ensue. Throwing light as they do on the causes of the formation of natural monsters, such experiments are no doubt of the highest interest from a general teratological point of view. The mere possibility of the occurrence of such malformations is, however, itself a fact of the deepest morphological significance. A monster is an organism in which the development of some part or parts has either exceeded or fallen short of its normal limitations, and any such phenomenon points indubitably to a certain mutual 156 _ EXTERNAL FACTORS IIT. 9 independence of the parts in the growth and differentiation of the organism ; while some pursue their normal course, others deviate from it. It follows that when such deformations are due to changes in the external conditions the parts are not equally sensitive to the unusual influence to which they are exposed. Thus in the Frog embryos which exhibit persistent yolk-plugs and open brains when grown in solutions of various kinds, the yolk and the medullary folds are alone susceptible to the action of the poison, other parts are unaffected and continue their development as though under normal circumstances: or, again, a sea-urchin passes through the early stages of segmentation and gastrulation unchanged when placed in a sea-water from which magnesium has been removed, but the subsequent differentiation of the gut and the formation of the skeleton are abnormal ; magnesium is necessary for these, though not for the earlier processes. A means is thus afforded of watching the behaviour of one or more parts independently of others, as, for example, of the animal cells in the gastrulation of the Frog’s egg when the yolk-cells are injured, and the most valuable information con- tributed, often quite unexpectedly, to our understanding of the events of normal ontogeny. Quite apart from this such experiments have already con- tributed, and will probably contribute still more in the future, to the study of variation. Between conspicuous monstrosities and those milder abnormalities which are termed ‘sports’ or ‘mutations’ there is every intermediate gradation, just as there is, on the other hand, no sharply defined limit between these discontinuous and those far smaller continuous variations to which the term has been often exclusively applied. The embryo is particularly sensitive to a change in its environment and reacts to such change by a variation in its form of greater or less degree. And not only that ; as Vernon has shown, these changes can pro- duce also an alteration in the variability of the species; and so provide greater opportunities for the operation of natural selection. At the same time teratology is not the main inquiry with which the experimental embryologist is concerned. The pro- blem that confronts him is to determine the part played by each factor of the external environment in the processes of III. 9 SUMMARY 157 normal, specific growth and differentiation, and for the solution of this problem only those experiments, of course, are of avail in which such factors are either altered or removed. By this means, as we have seen, it has been shown that a certain constitution of the physical environment, fixed within certain limits, is needful for the embryo; to these conditions it is closely adapted ; those limits it can only transgress under pain of abnormality or death. Every factor, or nearly every factor, is necessary for this or that phase or part of the process, some for the whole. Light of a certain wave-length will accelerate development, light of another kind, or in some instances darkness, will retard it, or stop it altogether; a certain degree of heat is indispensable; oxygen is required for respiration, water for growth ; some eggs demand constant agitation, others comparative rest; fertilization, or segmentation, or gastrulation, or some one or other of the later phases of development may depend absolutely on the presence of some particular chemical element; remove the factor in question, whatever it may be, and that particular process will not occur, and the specific, typical end which is reached in normal development will not be attained. Nevertheless, the achievement of this end does not depend wholly upon extrinsic forces, for the ovum is no completely homogeneous ‘ isotropic’ substance in which the complex circumstances of its environ- ment conspire to produce heterogeneity and coherence. There is no evidence that any physical factor exerts a directive influence sufficient of itself to determine any part of the whole specific effect, although this may happen under extraordinary conditions, as when gravity impresses a bilateral symmetry upon the com- pulsorily upturned egg of the Frog, and so determines the median plane of the embryo. Intimately bound up though these external conditions are with the proper conduct of the whole series of events whereby the organism comes gradually to resemble the parents that gave it birth, they can only operate in conjunction with internal factors which must be sought for not only in the initial structure and constitution of the germ-cells, but in the mutual interactions of the developing parts. CHAPTER IV INTERNAL FACTORS 1. THE INITIAL STRUCTURE OF THE GERM AS A CAUSE OF DIFFERENTIATION § 1. THe Moprrn Form or tHE Prerormationist Doctrine. As has been already pointed out, it was in the hands of Roux that the principle of germinal localization first advanced by His assumed the rank and importance of a theory of development. On this ‘ Mosaik-Theorie’ of self-differentiation the precise relation observable between the several parts of the embryo and certain definite regions of the undeveloped germ is not merely customary or normal, but necessary and causal. The germ-cell is endowed with a preformed structure corresponding to the structure of the organism which is to arise from it; each part of this structure is predetermined for the formation of some particular member of the embryo, and out of it no other member can, under ordinary circumstances, be made. The causes of its development, regarded as a specific activity of the organism, as leading to the production of a form which is like that of the parents which gave it birth, lie wholly within this pre-existing structure, and of each part within each part. Although the influence of the environment and, in later stages, of the parts on one another is not entirely excluded, the factors on which the differentiation of the whole and of each part depends are essentially internal, and all that happens is that by a continued process of cell-division the parts are separated from one another and the structure thus made palpable and manifest. Cell-division in the embryo is therefore qualitatively unlike. The supposed proof, by Pfliiger’s experiment with forcibly inverted Frogs’ eggs, of the complete ‘isotropy’ or equivalence of all parts of the cytoplasm, compelled Roux to locate the 1V.1 INITIAL STRUCTURE OF THE GERM 159 self-differentiating substance, the Idioplasson, in the nucleus, a view adopted and elaborated by Weismann, while the facts of regeneration necessitated the assumption of a reserve Idioplasson endowed with the potentialities of the whole, which is not quali- tatively divided during development, but handed on intact to some or all of the tissues of the body. The hypothesis then assumes the existence in the nucleus of the fertilized ovum of as many separate units as there are separately inheritable characters in the embryo, arranged there according to a plan which conforms to the structural arrangement of the parts which they represent, the separation of these units by con- tinued ‘unlike’ nuclear division, and their distribution to the cytoplasm, where they determine the formation of the structures to which they are beforehand assigned. For Roux the theory was no mere speculation, but the result of observation and experiment. The natural occurrence of half or partial monsters, the correspondence of defects in the embryo to injuries to the egg, and the coincidence of the first furrow with the sagittal plane in a large percentage (80 %) of, unfortunately, only a small number of eggs, suggested the self- differentiation of predetermined parts, while the last-mentioned observation led directly to the experiment in which one of the first two blastomeres of the Frog’s egg was killed and a half- embryo produced from the survivor. By means of a hot needle Roux succeeded in at least partially destroying one of the first two blastomeres. The other con- tinued to segment, and passed through the ordinary stages ; first the Hemimorula, with animal and vegetative cells and a segmentation cavity (though the last was sometimes absent), then by continued cell-division the Hemiblastula, followed by the Hemigastrula and Hemiembryo. In all cases the living tissues formed an exact half—either a right half or a left half— of a normal embryo, and ended abruptly at the (sagittal) plane of separation of the living and the dead. The segmentation cavity, however, was observed to extend in some cases across this plane, while in others it was confined wholly within the living cells. In the Hemigastrula the greatest extent of the archenteron— 160 INTERNAL FACTORS © i he produced by the overgrowth of the blastoporic lip—was parallel to the plane of separation: the yolk-cells were pushed into the segmentation cavity. The Hemiembryo had half a medullary oS rt NY 0 6° 5) ° eo%og eo ° y Se, 9 ° So 029 0 99 foue e ° ° 9. oo, > LOND IOOS: S i mkVar > Fig. 76.—A and B. Meridional sections through Hemiblastulae of the Frog: kn, remains of nucleus; k’, large reticular nuclei; %, nuclei in the yolk; F, blastocoel; r, vacuoles. C. Hemigastrula lateralis. Oblique longitudinal section. D. Hemiembryo sinister, transverse section ; ss, median plane ; the right half of the egg*is already completely cellulated, post-generation of the germ-layers has begun; ch, notochord, of whole size; mw, medullary fold; u, gut; j, two yolk-cells that have remained embryonic. (From Korschelt and Heider, after Roux.) tube, mesoderm on one side, half a gut cavity—open towards the dead blastomere—but a whole notochord (attributed by Roux to premature regeneration). IV.1 INITIAL STRUCTURE OF THE GERM 161 Anterior half-embryos—occurring when the second furrow by an ‘anachronism’ appears first—were also observed, but posterior never. | : Quarter and three-quarter embryos were obtained by killing three or one of the first four cells, and Hemiblastulae superiores by killing the four yolk-cells in the eight-celled stage. The self-differentiation of the Idioplasson is thus demonstrated. Subsequently, however, the reserve Idioplasson comes into play, and the missing half is formed by a peculiar process, to which A Fie. 77.—A and B. Normal Frog embryos with medullary folds (m), open (A) and closed (B). C. Hemiembryo dexter with almost complete post-generation of the ectoderm; w, yolk-plug. D.'The same, older, but with less post-generation. £. Hemiembryo anterior (?) with beginning post-generation. (From Korschelt and Heider, after Roux.) Roux has given the name of ‘ post-generation’ to distinguish it from regeneration, in which lost parts are formed out. of already differentiated tissue. The injured blastomere first degenerates, is then reorganized, and finally post-generated. ; In the first of these phases the cytoplasm and yolk are seen to be vacuolated, while the injured but not completely killed nucleus has given rise to small and large nuclei of normal structure JENKINSON M 162 INTERNAL FACTORS IV.1 surrounded by masses of pigment and in addition to irregular chromatic masses —some pale, some deeply staining. Separating it from the uninjured half is.a thin layer of yolkless protoplasm. Reorganization is accomplished by any one of three methods. The first is nucleation followed by cellulation, that is to say, masses of cytoplasm arise round normal .nuclei, some of which are derivatives of the injured nucleus, while others have migrated across from the living blastomere. The second method involves an abundant immigration of whole cells from the living embryo which resuscitate or feed on the yolk and nuclei of the injured half, In the third method overgrowths take place at various points of the tissues of the living half-embryo, the cells dipping into and feeding on the yolk as they pass across. In post-generation, which only occurs after the first method of reorganization, each layer of the uninjured reforms the corre- sponding layer of the injured, the cells of the former exerting a directive stimulus upon the reorganized tissues of the latter, which thus pass through a process of ‘dependent’ differentiation. The ectoderm grows over from in front backwards and from below upwards, so that a structure superficially resembling a yolk-plug is formed at the hinder end; as it does so it becomes differentiated into the usual two layers. The medullary tube is formed in like manner. The mesoderm is formed from the ventral side and later divided into vertebral and lateral plates. The notochord was a whole from the beginning. The missing half of the gut is formed directly from the half-gut of the living embryo, not by any process of blastoporic invagination. The conclusions drawn by Roux from this experiment have - already been stated. Development is conceived of as a process governed essentially by a factor which is entirely internal, the preformed structure of the nucleus of the germ, Into the validity of this factor we have now to inquire by examining the evidence offered by the very numerous and similar experi- ments, performed on the eggs of animals of all kinds, which the ‘ Mosaik-Theorie’ has evoked. ‘We shall take these experiments in order, beginning with the form employed by Roux himself, the common Frog}. ’ For literature see following section. IV.1 INITIAL STRUCTURE OF THE GERM 163 § 2. AMPHIBIA. It will be remembered that the Frog’s egg possesses, when freshly laid, a symmetry which is radial about the axis, the line joining the centres (animal and vegetative poles) of the pigmented and unpigmented portions of the egg; the symmetry is marked internally by the arrangement of yolk and protoplasm, for the latter lies most abundantly in the smaller unpigmented, the former mainly in the larger pigmented portion. Since the yolk C Ree 5 Fie. 78.—Formation of the grey crescent in the Frog’s egg (R. tem- poraria), A,B from the side; c, D from the vegetative pole. In A, c there is no crescent, in B, D a part of the border of the pigmented area has become grey, is heavier than the protoplasm the white pole is turned down- wards, with the axis vertical, the egg being free to rotate inside its jelly membrane. Shortly after fertilization, however, the radial is replaced by a bilateral symmetry (in Rana fusca and it. temporaria), a grey crescent being formed on one side of the egg along the border of the pigmented area by the retreat of pig- ment into the interior (Fig. 78). The grey crescent eventually becomes white and added to the white area. In Rana esculenta M 2 164 INTERNAL FACTORS IV. it is stated that the egg-axis becomes inclined to the vertical. This would seem to be an error, due to a confusion between the original white area and the white area enlarged by the addition Fig, 79.—Diagrams of the closure of the blastopore in the egg of the common Frog (R. temporaria). In A-D the egg is viewed from the vegeta- tive pole, in E, F from below. The dorsal lip is at the top of the figures. In p the ventral lip has just been formed and the blastopore is circular. In E the rotation of the whole egg has begun, and in F is complete. of the grey crescent (compare Fig. 44 with Fig. 78). The ege is now bilaterally symmetrical about the plane which includes the egg-axis and the middle point of the crescent. IV.1 INITIAL STRUCTURE OF THE GERM 165 - Since the crescent is formed on the side opposite to the entry of the spermatozoon, the latter is considered by Roux to determine the symmetry-plane of the egg. The first furrow is ‘also stated to generally lie in this plane, and the dorsal lip of the blastopore, which marks, of course, the sagittal plane of the embryo, appears in the region of the grey crescent. The dorsal lip grows through 75°, starting 25° below the equator and passing beyond the vegetative pole; with the rotation of the whole egg and therefore also of its axis in the direction opposite to that through which the dorsal lip has travelled, the anterior - end of the embryo, whose dorsal side is now uppermost, comes to lie a little above and behind the animal pole, while its posterior end is marked by the now fast-closing blastopore (Fig. 79). According to Roux the first furrow and the sagittal plane coincide and the first two blastomeres are right and left, unless, by an anachronism, the second furrow, which separates anterior from posterior (this should be dcersal from ventral), occurs first. The third furrow, therefore; separates dorsal from ventral (cor- rectly speaking, anterior from posterior). In the small number of eggs examined by Roux the percentage of coincidences was found to be high (80%) and the deviations were attributed to experimental error. Oscar Hertwig has, however, stated—on the strength of a small number of observations on eggs compressed between hori- zontal glass plates—that the angle between the two planes may have any value; while Schulze and Kopsch think it probable that they coincide in the majority of cases. The recent exami- _ nation by the present author of a large number of cases has shown that the angle in question may indeed have any value from 0° to 90°, but that there is a decided tendency to small — values, that is to coincidence, as may be seen from the annexed frequency polygon (Fig. 80), although at the same time there is no correlation, and therefore no causal connexion, between the two planes. Between the plane of symmetry and the sagittal plane, however, the correlation is considerable, and the tendency of the two to coincide much greater (Fig. 81), a result in con- formity with the statements of other observers (Roux, Schulze, Morgan). Within certain limits, therefore, the right and left 166 : INTERNAL FACTORS ih Fas halves, the dorsal and ventral sides (since the dorsal lip always appears in the region of the grey crescent) and the anterior and posterior ends (since the anterior end is always a little above the H9 Sai 100 BA eet 60 Frequency. 20) -90 -80 -70 -60 -50 -40 -30 -20 -10 oO +10 +20 +30 +40 +50 +60 +70 +80 +90 Fie. 80.—Frequency polygon of the angle between the first furrow of the Frog’s egg and the sagittal plane of the embryo. n = 889, M = 2-12° +-914, o = 40-39° +-646. animal pole) are predetermined in the undivided egg. But the manner in which the material of the egg is cut up by the first two meridional divisions makes no difference to the result. In IV.: INITIAL STRUCTURE OF THE GERM 167 these two divisions the qualitatively distinct parts of the cyto- plasm’ may be separated from one another in any assignable manner, the nuclear material being distributed to these in any assignable order. The symmetry of segmentation has no relation 10 100 70 60 =a 50) Frequency, 40 30 ie -90 -80 -70 -60 50 40 -30 -20 -f0 Oo +10 +20 +30 +40 +50 +60 +70 +80 +90 Fig. 81.—Frequency polygon of the angle between the plane of symmetry of the Frog’s egg and the sagittal plane of the embryo. n = 509, M = 2-23+-889, o = 29-75 + -629. either to the symmetry of the embryo or to the symmetry of the undivided egg, for the first furrow tends to lie either in or at right angles to the plane of symmetry and is not correlated with it to any great extent4, The third furrow, however, being always 1 See, however, Appendix A. 168 INTERNAL FACTORS | IV.1 at right angles to the axis, invariably separates anterior from posterior. An observation of Morgan’s has some bearing on this question ; even when the first furrow divides the egg unequally a normal embryo results. Even when the first two blastomeres are equal the cells pro- duced by their subsequent division may migrate across the plane of separation (Kopsch). In Necturus there appears to be no relation between the first furrow and the sagittal plane (Eycleshymer). The hypothesis of qualitative nuclear division has been tested by O. Hertwig in another way. Under pressure Fie, 82.—The first four phases of segmentation of the Frog’s egg. Normally (the three upper figures) and under pressure between hori- zontal plates (the three lower figures) J, IJ, III, IV, the first, second, third, and fourth divisions. 1-16, the cells produced by successive divi- sions, numbered as follows :— The egg divides into a 1 2 1 3 2 4 PS leita este | es PS Rae 2 1 5 3 7 2 6 4 8 Peotone ina cay 7: 15) 0.8) 16 2 1 BT The animal cells in the normal egg and the corresponding cells in the compressed egg are stippled. (After O. Hertwig’s account.) The eggs are affixed to glass plates by their jelly, fertilized, and allowed to rest until the axis has become vertical. Pressure is then applied by a second plate, in the direction of the axis, the plates being horizontal, or at right angles to the axis, the plates being vertical or oblique. In such compressed eggs the direction of elongation of the nuclear spindles is distorted, and rg IV.1 INITIAL STRUCTURE OF THE GERM 169 consequently the ‘qualitatively unlike’ nuclei compelled to assume an abnormal arrangement. Between horizontal plates the first two furrows are, as normally, meridional and vertical (Fig. 82); the third, however, is parallel to the first (instead of latitudinal), and the fourth latitudinal (instead of meridional), Between vertical plates the first is meridional and at right angles to the plates, the second latitudinal (horizontal) and near the animal pole, instead of meridional, the third furrows are parallel to the first, and the fourth meridional and at right angles to the first in the four upper blastomeres. Now if nuclear division is a qualitative process, if, further, the nuclei of the compressed egg are divided in the same way in successive mitoses as are the nuclei of the normal egg (that is to say if the first two nuclei are right and left, each of these then divided into dorsal and ventral, and each of these again into an ‘anterior and a posterior), then as a result of com- pression their distribution and arrangement must be altered (as the figures show), parts which should be anterior will be lateral or ventral, and vice versa, and a monster will be formed. Such eggs give rise to normal embryos. Nuclear division is there- fore not qualitative. From this conclusion there is only one escape; it may be argued that the orientation of the nuclei remains unaltered when the direction of the spindles is changed by the pressure, and that therefore the order of sequence of the cell-divisions may be varied ad libitum, may be as ‘ anachronistic’ as is pleased, without affecting in the least the mode of distribu- tion of the, qualitatively unlike, parts, a contention, surely, which would only be urged for the sake of supporting a thesis. O. Hertwig has also repeated Roux’s experiment, killing, or at least injuring, one of the first two blastomeres by a needle or by means of electricity. The uninjured half segments to form a mass of cells lying on the top of the dead blastomere, as a blastoderm lies on the yolk of a meroblastic egg, and separated from it by a segmentation cavity, although the latter may be wholly within the living cells. This is Roux’s Hemiblastula, but in Hertwig’s case the dead cell lies below, a point, as we shall see, of considerable importance. Later on a blastopore is formed, but always, according to Hertwig, within the bounds of 170 INTERNAL FACTORS IVs the living portion, not at the edge; the blastopore is usually symmetrical to the plane of separation of the two blastomeres. Beneath the lip of the blastopere an archenteron is formed, and notochord and mesoderm are differentiated. The closure of the blastopore is, however, prevented by the resistance offered by the dead yolk-mass, which lies ventrally and posteriorly, just as it is retarded by the yolk ina large-yolked Fish egg ; in fact, were this dead yolk removed, the living portion, Hertwig maintains, would develop normally. A nearly normal embryo may, in fact, be formed, but more usually there are considerable abnormalities. The mass of dead yolk, though partially enclosed by the grow- ing edge of the ectoderm, is sufficiently great to impede the ultimate closure of the blastopore, and sometimes of the medullary folds as well; the latter frequently diverge round the yolk-plug of dead and living tissue, the chorda being split as well; they may be symmetrical, or one side may be much less developed than the other, owing, it is asserted, to an asymmetry in the resistance offered by the dead cell, and in the more extreme cases this inequality may be so pronounced as to give rise to a condition which does not differ in any way from Roux’s Hemiembryo lateralis; there is one medullary fold, a notochord, mesoderm on one side, and a gut cavity in the yolk-mass; to the bare side of the yolk-cells is attached the dead blastomere, only partially covered by extensions of the ectoderm of the living half. Such cases are, however, very rare ; in the majority more, sometimes much more, than a half is produced from the living blastomere, for Hertwig denies that the missing parts are post-generated, as Roux maintains, though he admits the overgrowth and immigration of cells, as well as the persistence of living nuclei where the injury has been only partial; all these contribute to the formation of the embryo, which would be complete were its development not hindered by the presence of the inert mass of yolk. -The differences of interpretation put by Roux and Hertwig on the same phenomena appear to be radical. A reconciliation is, however, possible, for Morgan has observed that the whole or half development of the injured cell depends upon the position it takes up. If the original position—with the black pole IV.: INITIAL STRUCTURE OF THE GERM 171 uppermost—is retained, then a half-embryo is formed, as is indeed the case, according to Hertwig’s own evidence, when the egg is prevented from turning over by compression. In such half-embryos Morgan finds no traces of Roux’s post- generation, although a whole embryo may be eventually formed by regenerative processes, referred by the author to the retarded development of the living parts of the injured half. It should be noticed, however, that Roux’s statements are confirmed by Endres and Walter. Should, however, the white pole be uppermost, then a whole embryo of half-size results. In Hertwig’s expertments, of course, . the original position was usually not retained. Fie. 83.—Double embryo of Rana fusca, from an egg compressed in the direction of the axis and inverted in the two-cell stage. (After Schultze, from Korschelt and Heider.) A B Fie. 84.—Double monsters of Rana fusca, obtained by the same method. (After Schultze, from Korschelt and Heider.) This conclusion is still further strengthened by Schultze’s observations on the development of eggs inverted in the two- celled stage and kept so. Each blastomere develops independently and a double monster is produced (Figs. 83, 84). The result is not due to the pressure, for controls similarly compressed developed normally. The details of development of these eggs have been carefully worked out by Wetzel (Figs. 85, 86). In each inverted blasto- 172 INTERNAL FACTORS IV.1 mere the yolk sinks next to the plane of separation, while the protoplasm and pigment rises to the outer side. The two cells are now related as two whole eggs united by their vegetative poles, their axes in one and the same (horizontal) straight line; each has, in fact, acquired a totally new polarity of its own. When segmentation has been completed a groove appears, in the plane of separation, and gradually extends round the whole circumfer- ence; the ends of the groove are forked, but the branches of each fork unite as the groove grows round. The groove is, in fact, a blastoporic lip common to the two, the branches the individual lips, the material between them and finally covered over by them a common yolk-plug, the space between the two h Fie. 86.—Section through a double blastula of the Frog —— (Rana fusca). k, blastocoels. Fie. 85.—Double embryo ob- (After Wetzel, from Korschelt tained by the same method: h, and Heider.) heads; m, medullary grooves; c, line of union of the latter. (After Wetzel, from Korschelt and Heider.) a common archenteron extending into an archenteric space in each individual. Subsequently medullary folds and notochord are developed in each below the groove, that is on the vegetative or postero-dorsal side of each, but anteriorly each grows out free of the other, and in this region medulla, notochord, and gut are single, The result is, therefore, two embryos placed back to back, and united by a common yolk-plug. Experiments in which the four animal or the four vegetative cells of the eight-celled stage are killed are not very conclusive, as the development of the survivors does not go very far. IV.1 INITIAL STRUCTURE OF THE GERM 173 > Samassa has, however, shown that a short archenteron with dorsal lip, together with traces of notochord and mesoderm, may be formed from the four animal cells alone, the dead yolk-cells making a floor to the archenteric cavity and protruding as a large yolk-plug; and Morgan has obtained a similar result with the four vegetative cells alone. From all these investigations it seems reasonable to infer that each of the first two blastomeres of the Frog’s egg may under certain circumstances acquire the polarity of a whole, and be capable of giving rise to an embryo whose complete development is only prevented by the impediment offered by the presence of the other, whether living or dead. Were it possible to com- pletely separate the two blastomeres, we may surmise that each would become a perfect embryo: a surmise which is raised to a certainty by our knowledge of what happens in the newt. For in this form it is possible to separate the two cells by means of a noose of fine hair tied round the egg in the plane of the first furrow, as Herlitzka has shown, yg. 87._Egg of the newt and in this ease each half seg- (Triton cristatus), with two com- pletely normal embryos, obtained ments as a whole and develops },y tying a thread (sf) round the into a whole larva of rather more egg = the Gree Heike jelly than half-size (Fig. 87). Her- Xorchelt and Heider.) litzka has further investigated the dimensions of the organs in these embryos ; those of the me- dulla and notochord he finds are the same as in embryos developed from a whole egg, those of myotomes and gut a little less. The size of the nuclei and cells in the medulla and myotomes is the same as in the whole (3) embryo!; the number of nuclei in the medulla is the same, in the myotomes one-half. He concludes that some organs need a certain minimum number of cells for their differentiation, while the cells must attain to certain dimensions. 1 It will be convenient henceforward to designate the whole egg, or the embryo or larva developed from it, by the symbol }, each of the first two blastomeres and the embryo or larva, whether complete or incom- plete, formed from it by 3, and so on; thus a ? embryo means one which arises from three out of the first four blastomeres. 174 INTERNAL FACTORS Va The discovery of Herlitzka has been followed up by the researches of Spemann, who has employed the same method of constriction, but with variations of degree, direction, and time. These differences have given the most interesting results. The first furrow, according to this author, is usually at right angles to the sagittal plane and separates the material for the dorsal and ventral halves of the embryo; only rarely do sagittal plane and first furrow coincide. Both cases were, however, experimentally investigated. Fig. 88.—Three stages in the production of a double monster by strong median constriction of the Newt’s egg. (After Spemann, 1903.) a. Beginning of gastrulation; there is a separate*lip in each half. b. 7. and r. Med., Medullary folds of left and right embryos; *, point where the medullary grooves separate; Bl, blastopore. c. The double- headed larva. When the first furrow is in a horizontal plane, a slight con- striction in the two-celled stage separates the dorsal lip of the blastopore in one portion from the ventral lip in the other. Medullary folds are developed in the first half only, the second forms a sort of yolk-sac appendage which is later absorbed by the single normal embryo. With tighter, but still incomplete, constriction the dorsal half alone becomes an embryo ; it contains either the whole or only the dorsal portion of the blastopore. The ventral half contains either no portion of the blastopore or IV.1 INITIAL STRUCTURE OF THE GERM 175 only the ventral lip; it develops mesoderm but undergoes no further differentiation, and eventually drops off. Should the constriction be delayed until after the dorsal lip has appeared both halves may form an embryo, but the ventral embryo is usually imperfect. These differences are attributed by Spemann to differences in the time or place of constriction, When a transverse constriction is made after the appearance of the medullary plate the anterior half develops as a whole, with complete nervous system and optic vesicles; the posterior forms a medullary plate, but no folds, and dies. After the appearance of the medullary folds, however, the anterior and posterior halves produced by transverse constriction develop as halves, although a case is described where each had a = of auditory vesicles. When the sagittal plane coincides with the first furrow, con- striction in the two-celled stage gives rise to double-headed monsters ; if the constriction is slight the most anterior organs only are involved (duplicitas anterior), the cerebral hemispheres, epiphysis, hypophysis, and paraphysis being doubled; there may be two complete pairs of eyes, or the median eyes may be more or less fused (Diprosopus triophthalmus), Tighter constriction brings about a reduplication of the chorda, auditory vesicles, and fore limbs (Dicephalus tetrabrachius). Similar effects are obtained by constriction in the blastopore stage, but not later ; halves separated in the median plane when the medullary folds have arisen die as halves. We shall now briefly consider another experiment by which the independence of one another of the parts is demonstrated. Schaper removed the brain, eyes, and, probably, the auditory vesicles from newly hatched tadpoles of Rana esculenta. The wound healed up. The mouth moved to the anterior end and the suckers up the sides of the tadpoles, which lived for nearly a week, in the course of which they grew 2mm. They then showed signs of weakness and were preserved. It was found that the mouth had opened, that labial cartilages, pterygo- _ palatine bar, jaw-muscles, gill-bars, gills, heart and blood- vessels, trigeminus and vagus ganglia, oesophagus, pronephros and glomus, and dorsal muscles had all been differentiated. 176 INTERNAL FACTORS V8 There was, however, no operculum. ‘The anterior end was occupied -by a mass of mesenchyme. There was no sign of a regeneration of .any of the lost organs except the anterior end of the notochord. The nerve ganglia were normal, but the spinal cord underwent degeneration. In spite of this the creatures could execute spontaneous and reflex movements. What this experiment shows is, of course, that the organs of the trunk are not dependent for their development upon the presence of the brain; fresh researches would be necessary to determine how far in each case the parts are self-differentiating. In the second place the brain and eyes, when removed at this stage, cannot be remade by the tissues, but remain behind. So far, therefore, the body is at this stage an inequipotential system, as Driesch would call it. We know, however, that at an earlier stage the parts of the body are equipotential. We have, in fact, only another instance of that loss of totipotentiality, of that in- crease of independence and self-differentiation which takes place as development proceeds. Here, however, we are anticipating a conclusion which can only be completely stated after a discus- sion of the experiments performed on eggs of other types. LITERATURE H. EnprREs and H. E. Wauter. Anstichversuche an Hiern von Rana Susca, Ite? und Iter Teil, Arch. Ent. Mech. ii, 1896. A. C. EycLesHymer. Bilateral symmetry in the egg of Necturus, Anat. Anz. xxv, 1904. A. HERLITZKA. Contributo allo studio della capacita evolutiva dei due primi blastomeri nell’ uovo di tritone (Triton cristatus), Arch. Ent. Mech. ii, 1896. A. HERLITZKA. Sullo sviluppo di embrioni completi da blastomeri isolati di uova di tritone, Arch. Ent. Mech. iv, 1897. O. HErtTwig. Ueber den Werth der ersten Furchungszellen fiir die Organbildung des Embryo, Arch. mikr. Anat. xlii, 1893. F. Kopscu. Ueber das Verhiiltniss der embryonalen Axen zu den drei ersten Furchungsebenen beim Frosch, Internat. Monatschr. Anat. u. Phys. xvii, 1900. T. H. Morgan. Half-embryos and whole-embryos from one of the first two blastomeres of the Frog’s egg, Anat. Anz. x, 1895. T. H. More@an and E. ToreLue. The relation between normal and abnormal development (iv) as determined by Roux’s experiment of injuring the first-formed blastomeres of the Frog’s egg, Arch. Ent. Mech. xviii, 1904. IV.1 INITIAL STRUCTURE OF THE GERM 177 T. H. Monegan. The relation between normal and abnormal develop- ment of the embryo of the Frog, (v) as determined by the removal of the upper blastomeres of the Frog’s egg, Arch. Ent. Mech. xix, 1905. - W. Roux. Ueber die Zeit der Bestimmung der Hauptrichtungen des Froschembryo, Leipzig, 1883; also Ges. Abh. 16. W. Roux. Ueber die Bestimmung der Hauptrichtungen des Frosch- embryo im Ki und iiber die erste Theilung des Froscheies, Bresl. drtz. Zeitschr., 1885; also Ges. Abh. 20. W. Roux. Ueber die kiinstliche Hervorbringung halber Embryonen, Virchow’s Arch., 1888; also Ges. Abh. 22. W. Roux. Ueber das entwicklungsmechanische Vermégen jeder der beiden ersten Furchungszellen des Hies, Verh. Anat. Ges., 1892; also Ges. Abh. 26. W. Roux. Ueber Mosaikarbeit und neuere Entwicklungshypothesen, Anat. Hefte, I'* Abt., ii, 1892-3; also Ges. Abh. 27. W. Roux. Ueber die Specification der Furchungszellen und iiber die bei der Postgeneration und Regeneration anzunehmenden Vorginge, Biol. Centralbl. xiii, 1893; also Ges. Abh. 28. W. Roux. Ueber die ersten Theilungen des Froscheies und ihre Be- ziehungen zu der Organbildung des Embryo, Anat. Anz. viii, 1893; also Ges. Abh, 29. W. Roux. Die Methoden zur Hervorbringung halber Frosch-Embry- onen, und zum Nachweis der Beziehung der ersten Furchungsebenen des Froscheies zur Medianebene des Embryo, Anat. Anz. ix, 1894; also Ges. Abh, 31. W. Roux. Ueber die Ursachen der Bestimmung der Hauptrichtungen des Embryo im Froschei, Anat. Anz. xxiii, 1903. P. SamassA. Studien tiber den Einfluss des Dotters auf die Gastrula- tion und die Bildung der primiren Keimblitter der Wirbelthiere : II. Amphibien, Arch. Ent. Mech. ii, 1896. A. ScHaperR. Experimentelle Studien an Amphibienlarven, Arch. Ent. Mech. vi, 1898. O. Scnuutzze. Die kiinstliche Erzeugung von Doppelbildungen bei Froschlarven mit Hilfe abnormer Gravitationswirkung, Arch. Ent. Mech. i, 1895: O. ScHuLTzE. Ueber das erste Auftreten der bilateralen Symmetrie im Verlauf der Entwicklung, Arch. mikr. Anat. lv, 1900. H. Spemann. Entwickelungsphysiologische Studien am Triton-Hi, _I, II, III, Arch. Ent. Mech. xii, xv, xvi, 1901, 1903. G. WerzEL. Ueber die Bedeutung der cirkuliren Furche in der Ent- wicklung der Schultze’schen Doppelbildungen von Rana fusca, Arch. mikr. Anat. xlvi, 1895. JENKINSON N 178 INTERNAL FACTORS IV.1 § 3. Pisczs. Morgan has shown that in the fishes Crenolabrus, Serranus, and Fundulus there is no definite relation between the first (or second) furrow and the median plane of the embryo. In Fundulus when one of the first two blastomeres is removed the other gives rise to a perfect embryo of more than half-size. In segmentation the first furrow is in the plane of what would be the second furrow of the whole egg, the second in that of the third, and the third more or less in that of the fourth; but as the successive furrows are at right angles to one another in both cases it is permissible to assert that the segmentation of the half blastomere is total. Some of the protoplasm of the half that has been removed flows in underneath the other, is nucleated by it and added to its mesoderm. The size of the nuclei is the same in 4 and + embryos. Morgan also found it possible to remove two-thirds of the yolk, but not more, without interfering with the normal develop- ment of the whole egg. Injury to the germ-ring on one side of the dorsal lip resulted in a deficiency in the mesoderm of that side posteriorly ; anteriorly both sides were equally well formed. So Kastschenko has shown for Elasmobranchs and Kopsch for Teleostei and Elasmobranchs that injury to the lip of the blasto- pore will only interfere with the normal bilaterality of the embryo when effected quite close to the middle line. A series of experiments similar to the last has been carried out by Sumner on the eggs of various fish (principally Yundulus, also Hxocoetus, Salvelinus, and Batrachus). By means of needles inserted into various parts of the blasto- derm the exact mode in which the material of the latter is used in the formation of the embryo is first determined. It is shown that the material for the embryo is brought into position not by a conerescence of the lateral lips of the blastopore (sides of the germ-ring), but rather by an axial concentration of the cells originally situated in that ring at the posterior margin or dorsal lip, the cells so concentrated being continually pushed forwards in the middle line until the anterior end of the embryo comes to lie near the original centre of the blastoderm. IV.1 INITIAL STRUCTURE OF THE GERM = 179 Injuries to various parts of the blastoderm and its margin (the germ-ring) prove that it possesses a certain degree of ‘ isotropy ’. Thus the entire embryonic region of the posterior margin was destroyed by electrocautery, The adjacent edges closed up and - completed the germ-ring; at the posterior point of this a new embryonic shield was formed, in which ectoderm, endoderm, and notochord became differentiated. Or, again, a lateral piece of the germ-ring immediately adjacent to the embryo was removed without preventing the formation of a normal, bilaterally symmetrical embryo, although, as Morgan found, the mesoderm might be deficient posteriorly on the injured side. LITERATURE N. KAstscHEeNnKo. Zur Entwickelungsgeschichte des Selachierembryos, Anat. Anz. iii, 1888. F. Kopscu. Experimentelle Untersuchungen tiber die Keimhaut der Salmoniden, Verh. Anat. Gesell., 1896. F. Kopscou. LExperimentelle Untersuchungen am Primitivstreifen des Hiihnchens und an Scylliwm-Embryonen, Verh. Anat. Gesell., 1898. T. H. Morgan. Experimental Studies on Teleost eggs, Anat. Anz. viii, 1893. T. H. Morean. The formation of the fish-embryo, Journ. Morph. x, 1895. F. B. Sumner. A study ot early fish-development, experimental and morphological, Arch. Ent. Mech, xvii, 1904. § 4. AMPHIOXxUs. In this form the blastomeres may be separated by shaking. Their development has been observed by Wilson, whose account (r) Fig. 89. A. Normal 4 gastrula of Amphioxus. B. Gastrula from a } blas- tomere. C. Gastrula from a} blastomere. D. Gastrula of 4 normal size from an egg-fragment. (After Wilson, from Korschelt and Heider.) has since been confirmed by Morgan. The isolated blastomeres soon become rounded; the first division is always transverse to the long axis. N 2 180 INTERNAL FACTORS IV.1 A 4 blastomere segments like a whole ovum, except that the second division may be unequal, and gives rise to a normal blastula, gastrula, and embryo of half the normal size; 2 blasto- meres behave in the same way (Fig. 89). Incomplete separation of the cells after the first or second division leads to double embryos in the first case, double, triple, or quadruple embryos in the second. The double embryos may make any angle with one another and continue to live for some time (Fig. 90). A 2 blastomere segments like a whole egg (the second division is, however, always unequal) and gives rise to a normal blastula and gastrula, more rarely to an embryo of one-quarter size. ‘ Fie. 90. Double gastrulae of Amphioxus, from incompletely separated + blastomeres. «', u*®, separate blastopores; w, common blastopore. (After Wilson, from Korschelt and Heider.) A 2 blastomere will segment normally, or nearly so. A blas- tula is rarely formed, usually an open curved plate of cells, which are ciliated and swim about but do not gastrulate. Lastly #; blastomeres divide but produce only an irregular heap of cells. It is obvious that the potentiality of a blastomere to form a whole embryo diminishes with its germinal value, the ratio it bears to the whole ovum; but it is not so clear whether this diminished capacity is due to the lack of some necessary, specific, organ-forming substance or merely to the small size and lack of IV.1 INITIAL STRUCTURE OF THE GERM 181 undifferentiated material. Morgan has attempted to answer this question by counting the number of cells in the various organs of whole and partial larvae. It appears from his estimate that the number of cells in the archenteron, in the notochord — and in the nerve-cord of 4, $, and 4 larvae is constant, and that though the size of the whole body is less, the dimensions of the notochord and nerve-cord are about the same in all three. It would seem, therefore, that there is a minimum size and a minimum number of cells necessary for the formation of these organs. Whether the failure of the 3 blastomere to develop into a larva is, however, due to mere lack of material or the absence of some specific substance needful for the development of some particular organ is still undecided ; for the 4 and + cells contain substance from both the animal and vegetative portions of the egg, while the $ are composed of one or the other substance exclusively, the furrows of the third phase being equatorial. LITERATURE T. H. Morgan. The number of cells in larvae from isolated blasto- meres of Amphioxus, Arch. Ent. Mech. iii, 1896. E. B. WiLson. Amphioxus and the Mosaic theory of development, Journ. Morph. viii, 1893. § 5. CoxELENTERATA. Many years ago Metschnikoff observed a perfectly normal © separation of the blastomeres at a certain period in the develop- ment of certain medusae (Oceania). If when the cells reunite in a later stage the order of their rearrangement is not constant, the egg-substance must be in some measure isotropic.’ More recently the equipotentiality of the blastomeres of these forms has been experimentally demonstrated by Zoja (Fig. 91). The cells were separated by means of a needle. 4 and 4 blastomeres (in the case of Liriope, Geryonia, Mitrocoma, Clytia, and Laodice) and 4 and 5 blastomeres in the case of the two last mentioned will give rise by normal segmentation to blas- 1 It should not be forgotten that in 1869 Haeckel had cut the blastulae of Crystallodes (a Siphonophor) in pieces and obtained from the larger fragments normal larvae. The development of small pieces was re- tarded and abnormal (Zur Entwickelungsgeschichte der Siphonophoren, Utrecht, 1869). 182 INTERNAL FACTORS avcx tulae with closed blastocoel and these to normal Planula larvae. In Clytia a hydroid was eventually reared and in Liriope a medusa with four primary tentacles from 4 and 3 cells. It is to be noticed that, since the third division in these egg’s is equatorial, the 2 and #4, blastomeres must be either animal or vegetative. Zoja also finds that in Laodice and Clytia the number of cells in partial larvae is proportional to the germinal value. In Liriope the number of cells in the endoderm is equal to that in the whole (4) larva. Fie. 91. Partial development in Coelenterata. (After Zoja, 1896.) a-d. Laodice cruci- ata: a, blastomeres: b, # blastomeres; c¢, blastula from } blasto- mere; d,4 larva during formation of endo- derm. e-l. Liriope mu- cronata. e-i. Develop- ment of 3 blastomere : e, division of } blasto- mere into 7; f, 439, 353 h, end of endoderm formation in } em- bryo; 7, medusa reared from +4 blastomere. j-l. Development of + blastomere: j, 2; &, is; 4, embryo with endoderm. LITERATURE KE. METscHNIKOFF. Embryologische Studien au Medusen, Wien, 1886. R. Zosa. Sullo sviluppo dei blastomeri isolati dalle uova di aleune meduse, Arch. Ent, Mech. i, ii, 1895-6. IV.1 INITIAL STRUCTURE OF THE GERM 183 § 6. EcHINODERMATA. Easy to obtain in large numbers and eminently suitable for experiment, the eggs of the sea-urchins and starfishes have pro- vided the analytical embryologists with abundance of material; and it is in this group that the possibility of rearing a perfect larva from a single blastomere was first demonstrated by the classical researches of Driesch. : The experiments which have been carried out, mainly by Driesch, but also subsequently by others, are very numerous and fall into two principal classes. In the first the development of parts—whether isolated blasto- meres or groups of blastomeres, fragments of unsegmented eggs fertilized or unfertilized, or pieces of blastulae and gastrulae—has been observed, the rate of their development recorded, and the relation of the number of cells and dimensions of the partial larvae to their germinal value determined. By the second series of investigations the effect of alteration of the type of nuclear or cell-division and consequent displacement of the blastomeres upon the subsequent development has been discovered. Before discussing these experiments, however, a word must be said as to the normal behaviour of the whole egg. The structure, segmentation and formation of the primary germinal layers have been very completely studied by Boveri in the sea-urchin, Strong ylocentrotus lividus (Fig. 92). The axis of the ovarian egg is marked by the excentric position of the nucleus ; at the point on the surface nearest which it lies the polar bodies are formed, and this point is the animal pole. At this point there is a fine canal (micropyle) in the jelly which surrounds the egg. After the extrusion of the polar bodies a brown pigment which had previously been uniformly distributed through the cytoplasm becomes aggregated in a dense but quite superficial subequatorial zone, the upper somewhat ill-defined border of which may, however, extend into the animal hemisphere. The pigment-free region left about the vegetative pole occupies from go to #5 the volume of the whole ovum. The spermatozoon may, but need not, approach the egg by the micropyle. The fertiliza- tion spindle lies in a plane parallel to the equator of the egg (the vi Fig. 92.—Normal development of the sea-urchin, Strongylocentrotus lividus. (After Boveri, 1901.) - The animal pole is uppermost in all cases, and in the first two figures the jelly with the canal (micropyle) is shown. ; a, primary oocyte ; the pigment is uniformly peripheral. b, ovum after extrusion of polar bodies: the pigment now forms a subequatorial band. The nucleus is ex-axial. c, d, first division (meridional). e, 8 cells, the pigment almost wholly in the vegetative blastomeres. J, formation of mesomeres (animal cells) by meridional division: the vegetative cells have divided into macromeres and micromeres. g, blastula. h, mesenchyme blastula. i, j, k, invagination of the pigmented cells to form the archenteron of the gastrula. In j the primary mesenchyme is separated into two groups, in each of which, in &, a spicule has been secreted. In & the secondary, pigmented mesenchyme is being budded off from the inner end of the archenteron. IV.1 INITIAL STRUCTURE OF THE GERM 185 ‘karyokinetic plane’) and elongates at right angles to the sperm- path, which thus lies in the plane of the first, a meridional, furrow (a similar observation had been previously made by Wilson and Mathews on Yowxopneustes). The second furrow is likewise meridional and at right angles to the first, the third equatorial or a little nearer to one or other pole; the animal cells get only a very little of the pigment. In the next phase the animal cells divide meridionally and equally to form the eight mesomeres, while the vegetative cells divide latitudinally and very unequally into four large pigmented macromeres next the equator, and four small quite unpigmented micromeres grouped round the vegetative pole. In the blastula stage all the cells are of the same size, vacuo- lated and ciliated, and the polarity of the egg is only deter- minable by the position of the pigment zone. In the next stage the clear vegetative cells derived (presumably) from the micro- 2 | Kaela Fie. 93.—Echinus: segmentation under pressure. a, preparation for third division (radial); 6, preparation for fourth division (tangential); b’, after fourth division; c, another form of the 8-cell stage (third division parallel to first); d, the same after removal of the pressure. (After Driesch, 1893.) meres wander in to form the primary mesenchyme—from which the first triradiate spicules are developed—and this is followed by the invagination of the pigmented cells to form the archenteron. Secondary pigmented mesenchyme cells are budded off from the inner end of the latter. The character and sequence of the divisions in segmentation are the same in other Echinid eggs, but the polarity is not marked by pigment, nor indeed recognizable till the micromeres have been formed, and again when the mesenchyme cells wander into the blastocoel. By means of pressure—under a coverglass—Driesch succeeded (Echinus) in altering the direction of the spindles and consequently of the cell-divisions (Fig. 98). The first and second furrows were at 186 INTERNAL FACTORS IV.1 right angles to one another and to the coverglass, but whether meridional or not does not appear. The third furrows were again at right angles to the coverglass and at 45° to the first two; a flat plate of eight cells was thus formed. The next division resulted in the formation of sixteen equal cells still all lying in the same plane ; the formation of micromeres is thus suppressed. Such eggs gave rise to normal larvae. In the case just quoted the egg-membrane was intact; but similar results were obtained when it was broken. The second furrow was sometimes at right angles to, sometimes parallel to, the first, the third at right angles to both in the latter case, and the fourth Fig. 94.—The effect of heat upon the segmentation of the Echinoid egg. a,b, c, d, four successive stages in the segmentation of the same egg of Echinus; e, f, two successive stages in the division of the same egg of Sphaerechinus. (After Driesch, 1893.) parallel to the third ; but if the pressure was released the blasto- meres became rounded off and the sixteen-celled stage consisted of two plates of eight cells each; one or two micromeres were sometimes formed. The development of these eggs is also quite normal, These results have been confirmed by Ziegler. In this, as in the similar experiment of Hertwig on the Frog’s egg; the normal distribution and arrangement of the nuclei is interfered with without prejudice to the normality of subsequent development. Nuclear division cannot therefore, Driesch contends, be a qualitative process. Boveri has also shown in similar fashion that the order of segmentation may be disarranged and IV.1 INITIAL STRUCTURE OF THE GERM 187 the formation of the micromeres suppressed in Strongylocentro- ¢us and a normal larva still result. The relation, however, of the egg-axis, as determined by the pigment-ring, to the symmetry of the larva remains the same as in the undisturbed egg. Increased temperature, violent shaking, dilute sea-water, and calcium-free sea-water are all means by which Driesch has suc- ceeded in disarranging the blastomeres of Echinus in various ways (Fig. 94). Thus when the temperature was raised from 19° C. (the Fie. 95.—Variations in the segmentation of Echinus microtuberculatus produced by dilution of the sea-water. a, tetrahedral four-cell stage ; b, eight cells, three premature micromeres; c, eight cells, two precocious micromeres; d, the same egg after the next division, the precocious micromeres have divided unequally, two normal micromeres have been formed. (After Driesch, 1895.) ’ normal) to 31°C. the first two (not subsequent) blastomeres separated, though they sometimes reunited. The next division was normal, but with the next phase one or two blastomeres divided in a direction perpendicular to that of the others, or the direction of division was different in each. In the following stage the micromeres were wholly or partly suppressed. So also in dilute sea-water (20% fresh water) the third division was unequal 188 INTERNAL FACTORS IV ‘3 in two or more blastomeres, and in the following an excessive number of small cells was formed (Fig. 95). Ina less dilute mix- ture (16% fresh water) the blastulae divided to form each two Plutei. Again by shaking the eggs in the eight-celled stage the blastomeres were disarranged and made to lie almost in one plane ; micromeres were, however, formed as usual from the vegetative cells, but not, of course, in the normal position (Fig. 96). In spite of these very considerable alterations in the size and posi- tions of their constituent cells all these ova nevertheless gave rise to perfectly normal larvae. By the use of calcium-free sea-water either the mesomeres or the macromeres and micromeres were separated into two groups. In the latter case the larva had two guts, in the former a normal larva resulted, so that, as Driesch points out, the ectoderm at the Fig. 96.—Disarrangement of the blastomeres of Echinus by shaking, a, eight cells; b, sixteen cells ; notice that the micromeres are not close together. (After Driesch, 1896.) antiblastoporic (animal) pole must in this case have been formed from the macromeres. The conclusion to be drawn from these experiments seems obvious ; not only the nuclei, but the parts of the cytoplasm too are equivalent; within at any rate fairly wide limits their normal arrangement may be disturbed without affecting in the least the normality of subsequent development ; or, as Driesch phrased it, the destiny of a nucleus or blastomere is not deter- mined by its original situation in the preformed structure of the egg, rather it is a function of its position in the whole embryo to which that egg gives rise. Its character is decided not by its origin, but by its final position. Whether there really is a limit to the rearrangement is a question which must be reserved for future discussion. We are IV.r1 INITIAL STRUCTURE OF THE GERM 189 A a Fie. 97.—A and C, formation of ex-ovates in the egg of Arbacia by dilution of the sea-water; K, nucleus; m, egg-membrane. B and D, blastulae formed from A and C; B becomes constricted into two blas- tulae, each of which gives rise toa Pluteus; D produces a single Pluteus. (After Loeb, from Korschelt and Heider.) 190 INTERNAL FACTORS IV.1 also indebted to Driesch for a long series of experiments on the behaviour of isolated blastomeres and egg-fragments. In the forms which we have hitherto considered—the Vertebrates and Coelentera—an isolated cell segments as a whole and gives rise to a total larva. In these Echinoderms the isolated blastomere also gives rise to a total larva, but its segmentation is partial ; only after segmentation has been completed does the open blastula close up and resume the polarity of a whole. Various methods have been employed for separating the blastomeres. Loeb showed that in dilute sea-water (50 %) the egg Fra. 98.—Development of the isolated 4 blastomere of Echinus microtu- berculatus. Two micromeres, two macromeres, four mesomeres: a, 4; b, 58; c, Hemiblastula: on the right is the remaining $ blastomere, dead. (After Driesch, 1892.) swells, bursts its membrane and protrudes an ex-ovate which may be large and develop independently (Fig. 97). Driesch has used heat, pressure, violent shaking with fragments of cover- glasses, and the calcium-free sea-water introduced by Herbst. The isolated 4 blastomere in Hehinus becomes rounded and seg- ments as a half, as though the other blastomere were still present. It forms four mesomeres, two macromeres, and two micromeres, IV.1 INITIAL STRUCTURE OF THE GERM 191 and by division of these a hemispherical curved plate, a half- blastula(Fig.98). When, as may happen, the other blastomere dies without being separated, it is embraced by the open side of the survivor. Only after the 32-celled stage is passed do the edges meet and close. A blastula of half-size is the result, which becomes later a gastrula and a Pluteus of perfectly normal form but of half the normal size. 2S © & PE Sen 99.—Segmentation of an isolated } blastomere of Echinus ; be t , ¢, zs, One micromere, one macromere, . two mesomeres, 4, § fy. (A fter Tio 1893.) 3 blastomeres behaved in the same way, segmenting as parts, developing ultimately into whole normal Plutei. Similarly 4 blasto- meres segmented partially (Fig. 99), with, in some cases, irregu-— larities : two micromeres, for instance, were formed in the second division. The # blastulae will gastrulate, but progress no further. Sa ° ya vee O Fie. 100.—a, Gastrula with mesenchyme cells and triradiate spicules reared from a z animal cell of Sphaerechinus. B, The same from a } cell of Echinus: the gut is tripartite. (After Driesch, 1900.) In the next stage—3 blastomeres—a difference becomes notice- able between the behaviour of the animal and vegetative cells. Both kinds of cells will gastrulate, secrete triradiate spicules and 192 INTERNAL FACTORS , | eae develop a normally tripartite gut (Fig.100). But this only occurs in a small proportion of cases, smaller for the animal than for the vegetative cells. Further, the former frequently give rise to blastulae provided with a row of long cilia (Fig. 101), while the latter are delicate, and many die. Boveri has pointed out that the ability of animal cells to gastrulate depends, in Strongylocentrotus, on whether or not they contain a portion of the pigment-zone, and this on the position of the third furrow. Garbowski, however, denies that the pigment is itself an organ-forming substance. For in the first place there is a variety of Strongylocentrotus lividus, in which the pigment remains diffuse and never becomes concentrated to form a band at all; and secondly, even though, when present, it is usually in the subequatorial posi- tion described by Boveri, this is not always so; it may be oblique to the egg-axis, or wholly in the Fic. 101.—Long-ciliated blastula from imal, or wholly in the a 3 animal cell of Echinus. (After vegetative hemisphere. eee: 1) It would appear, there- fore, either that it is ; merely associated with some other sub- stance which has up to the: present remained invisible, or that in the cases described by Garbowski animal cells, if they were pigmented, would gastrulate as readily as ordinary vegetative blastomeres, in which case the manner in which the pre-deter- mined material is cut up in segmentation would be a matter of indifference. The differences in the behaviour of the 4, cells are still more marked, The micromeres will only divide to form a heap of about ten cells; the mesomeres give rise to either long-ciliated _blastulae or imperfect gastrulae, with or without skeleton and 1V.1: INITIAL STRUCTURE OF THE GERM 193 mesenchyme, and with an undivided archenteron (Fig. 102); the macromeres, on the other hand, become gastrulae provided with mesenchyme and spicules (Fig. 103). Lastly, even +; blastomeres will occasionally gastrulate, pro- bably only if derived from a macromere. This gastrulation was Fic. 102.—Echinus. Larvae reared from mesomeres (animal cells) of the 16-celled stage. a and b, mesenchyme gastrulae, a@ has spicules ; ec, gastrula without mesenchyme; d, a long-ciliated blastula. (After Driesch, 1900.) not, however, observed directly, but the germinal value of the smallest gastrulae found was calculated by a method which will be described below. ~; cells will not reach the gastrula stage. 4 Fig. 103.—Two gastrulae of Echinus reared from ;4; macromeres; both have mesenchyme cells, one has triradiate spicules. (After Driesch, 1900.) The differences between the developmental capacities of animal and vegetative cells may be studied in another way. The four micromeres were removed by Driesch in the 16-celled stage ; the remaining twelve meso- and macromeres produced a normal Pluteus (an experiment also performed by Zoja on Strongylocen- trotus). The eight (or four in the previous stage) animal cells alone, the eight vegetative cells alone also formed, in some cases, a perfect larva. Segmentation was in all these cases partial. JENEINSON (6) 194 _ INTERNAL FACTORS IV. 1 From the fact that the four animal cells together may form a Pluteus while each individually fails to pass beyond the gastrula stage, Driesch has argued that this failure depends solely on lack of sufficient material, not on the want of any specific gut-forming substance, and has supported his contention by other evidence. By placing the eggs of Hchinus in dilute sea-water the third division was made unequal ; the cells were then isolated in calcium- free water. The large cells thus formed must contain either more of Boveri’s pigment-ring (or rather of the substance of the vegetative hemisphere) or else more of the animal hemisphere, probably the former, since a larger percentage gastrulate than is usual with animal blastomeres. According to Boveri, however, all ought to gastrulate if all contain the specific substance for the archenteron. Further, these large cells are able to form mesen- chyme even when the gut is lacking although they possess the middle region of the egg in any case and may lack the micromere area. Driesch also points out that a + blastomere and a 3 vegetative blastomere have both the same amount of Boveri’s pigment-ring and mesenchyme area, the latter, however, only half as big a gut and half as many mesenchyme cells as the former, and lastly that 4 and 54, animal blastomeres will not only gastrulate but form mesenchyme as well. The limitation of the potentialities of ectoderm and endoderm in later stages has been investigated also by Driesch—by observing the development of pieces or fragments of the blastulae and gastrulae. These pieces are obtained by cutting or shaking. Loeb has employed dilute sea-water to make the blastula swell, protrude through its membrane and become constricted into two. In Echinus, Sphaerechinus, and Asterias, any piece of a blastula when first cut out is crumpled, but soon becomes rounded, and swims about and eventually gastrulates. Monsters: exhibiting a certain degree of duplicity have been produced by Driesch by shaking the egg of Asterias in the two-celled stage (this produces apparently a partial separation of the blastomeres) and by placing the blastulae of Hehinus in diluted sea-water. In the latter case the gut is single, the skeleton double; in the former the gut is doubled, though the two may subsequently fuse, or even trebled, or may be IV.1 INITIAL STRUCTURE OF THE GERM 195 merely forked. The two guts may be similarly oriented or turned in opposite directions, and it is interesting to observe that Driesch, who believes the first furrow to coincide with the plane separating the two guts, accounts for the latter case by supposing that one blastomere had been rotated upon the other, so that their vegetative ends—the J/ocus of the gut-forming _substance—faced in opposite directions. Neither the ectoderm of the early gastrula alone nor the endoderm is capable of giving rise to a larva, though the former can develope a stomodaeum, a statement confirmed by Morgan. The vegetative half of a gastrula of Sphaerechinus will give rise to a normal small Pluteus whether the ectoderm only, or together with it the tip of the archenteron, has been removed (Fig. 104) ; the animal half will not. When the spicules, one or Bie” fy Ic Fra. 104.—The potentialities of the cells of embryonic organs; a, b. The vegetative and animal portions of a gastrula of Sphaerechinus granularis, cut equatorially in two; c. Pluteus reared from a fragment of a gastrula ; d. Normal tripartite pluteus gut. (After Driesch, 1896.) cA both, are removed the skeleton is one-sided and the Pluteus con- sequently one-armed. In Asterias the archenteron can form a new terminal vesicle and mesenchyme cells afresh when these have been removed (Fig. 105); but should the coelom sacs be cut off ata later stage, the (secondary) archenteron can form no more, though it divides into the usual three portions. Parallel to the behaviour of isolated blastomeres is the segmenta- tion and development of egg-fragments, obtained by shaking, cutting, or (Loeb) dilute sea-water. The fragments may be taken from already fertilized eggs, or from unfertilized: in the latter case each, whether nucleate or enucleate, must be subsequently fecundated. In segmentation there are a great many differences, which appear to depend on the nature of the fragment, that is to say, O02 / 196 INTERNAL FACTORS {V4 on the part of the egg from which it has been removed. In the case of the colourless egg of Lchinus it is only possible to ee Fig. 105.—The potentialities of the cells of embryonic organs; a. Normal Pluteus of Sphaerechinus ; b. Pluteus reared from a frag- ment ofa gastrula; c, e. Normal Bipinnaria of Asterias glacialis; d, f. Bipinnaria from the vegetative half of a gastrula; g. Larva of Asterias with typical tripartite gut, but no coelom, from the vegetative half of a gastrula removed after development of the coelom sacs. (After Driesch, 1896.) In another case what appears to guess at the nature of the piece by observing the mode of segmenta- tion. The first and second divisions may both be equal, as also the third and fourth ; in this case (Driesch supposes) a large fragment is in- volved. Again, the first, second, and third may be normal, but the fourth and fifth equal: no micromeres, therefore, are formed, and the fragment is probably derived from the animal hemisphere. Or while the first and second are equal, one of the four cells will divide un- equally; in the next division a large or small number of micromeres is formed, according, presumably, as a larger or smaller portion of the micromere region has been included in the fragment (Fig. 106). be a meridional half divides equally twice, and then two of the four equally (meso- meres), two unequally (macro- and micromeres) (Fig. 107), while a supposed vegetative half is segmented to form four large and four small cells. Thus the type of segmentation is determined by the initial 1V.1 INITIAL STRUCTURE OF THE GERM 197 structure, including the physical constitution, of the ovum alone, a conclusion in which Boveri concurs. All these fragments will give rise to normal larvae, provided they are not toosmall. The least egg-fragment that will gastru- late has the same germinal value as the least blastomere, namely, Fie. 106.—Segmentation of an egg-fragment of Echinus supposed to contain the whole of the micromere area of the egg, and some of the animal hemisphere. a. Hight cells, three equal pairs and one unequal pair ; b. sixteen cells, four of the eight having formed micromeres, the other four divided equally. (After Driesch, 1896.) =z (calculated from the volume of the smallest gastrulae found). Morgan, however, estimates the smallest egg-fragment capable of giving rise to a normal larva at from 75 to go, the least blasto- mere that will gastrulate only 2. a Fre. 107.—Segmentation of an egg-fragment (Echinus) supposed to be an exact meridional half. a. Eight cells, four being mesomeres, in macromeres and two micromeres; b. sixteen cells. (After Driesch, 896.) In partial blastulae, gastrulae, or larvae the number of cells is proportional to the germinal value ; and this is true not merely of the whole embryo or larva, but of each of its organs, the 198 INTERNAL FACTORS NZI ectoderm, the mesenchyme, the archenteron. This assertion of Driesch’s is corroborated by Morgan for the whole larvae, but not for the organs; according to him the number of cellsinvaginated to form the archenteron in a partial tends to approach that found in a whole larva, about fifty. Driesch has also determined the relation to their germinal value of the dimensions of partial blastulae, gastrulae, and Plutei and their organs (Figs. 108- Pra. 108.—Outlines of }, 3, }, 112). As the table (Table XX) ate eek LO echinus. shows, the surfaces are very nearly as the germinal values, the ratios of the radii of the partial and total larvae con- sequently greater, of the volumes less than these values, Fig. 109.—Early gastrulae of Eehinus: a. 4, b. 4, ¢. 4, d. (After Driesch, 1900.) IV.1 INITIAL STRUCTURE OF THE GERM 199 TABLE XX Relation of dimensions of partial blastulae to their germinal value. Germinal values. 1 1 1 | 1 I 2 ri 8 Radii 2 1.2 1 -75 Surfaces 4 2.25 1 -5625 Volumes . 8 3-375 1 4 Fie. 110.—Plutei of Echinus: a. 4, b. 4 shown. (After Driesch, , « 4. The skeleton is 1900.) From the proportionality of the number of cells to the germinal value it follows that the cells of the whole and of the partial blastulae are of the same size. If R, S, and V are the radius, surface, and volume of the whole : F ; 1 egg, and 7,, 8,, v, the corresponding magnitudes in a = larva, \ then s, = 5, whence 7,, = se and therefore v, = ee this formula the germinal value of a larva of unknown origin may be calculated. Finally, although segmentation takes place at the normal rate By 200 INTERNAL FACTORS IV.% in the isolated blastomeres, their subsequent development is pro- gressively retarded as the germinal value decreases (Table X XI). TABLE XXI Showing the rate of development in partial larvae. Germinal value. = Ft ec Day | t 2 t i 1 Gastrula Early Gastrula| Still earlier | Blastula | Gastrula | eer Pluteus Early Pluteus | Gastrula with | Blastula and Skeleton Gastrula 3 Pluteus Pluteus Karly Pluteus | Gastrula 4 Pluteus Pluteus Pluteus Gastrula | a ¢ 7) Fig. 111.—Sphaerechinus Plutei, showing the gut. «a 3, b.4,¢.}4. (After Driesch, 1900.) It is evident from these experiments that, in the Echinoderms, while the isolated parts of the egg, whether blastomeres or egg- fragments, are only able to segment as they would have done had they remained in connexion with the whole, they are never- IV.1 INITIAL STRUCTURE OF THE GERM 201 theless capable of total development ina very marked degree. This capacity is not, however, unlimited, but diminishes as develop- ment proceeds, and it appears highly probable, and is indeed often admitted by Driesch, that the chief cause of this restriction is the lack of some specific substance and not the deficiency of mere material, though this may be a subsidiary factor. The progressive loss of potentialities is exhibited also by the embryonic organs as they are formed later on. a eo SS Fig. 112.--Echinus, the tripartite gut of the complete Pluteus : a. },b.4,¢.4. (After Driesch, 1900.) Before concluding this section we must notice an experiment which is the converse of those considered above, namely, the production of one embryo from two eggs. An unsuccessful attempt was made by Morgan to obtain this result. This author only succeeded in showing that the eggs of Sphaerechinus, when deprived of their membranes by being shaken, tend to fuse together, and that from such pairs double monsters may be produced with two guts and two skeletons. One skeleton may, however, very largely predominate over the other, which indeed remains rudimentary, and the guts may coalesce. 202 INTERNAL FACTORS IV. 1 Driesch, however, who has employed the same method as Morgan and has corroborated his results, has been able to produce, from the fusion of two ova, a larva single in all its parts—mesenchyme, skeleton, and gut. The organs are in correct proportion but larger than the normal (Fig. 113). The number of mesenchyme cells is about twice the normal. From this experiment it may of course be concluded that the egg substance possesses a degree of isotropy, but not that that iso- tropy is absolute. Nothing, it should be remarked, is known of the way in which the eggs which fuse to form one embryo are oriented upon one another. Garbowski, however, has stated A B Fig. 113.— A. Normal gastrula of Sphaerechinus. B. Single gastrula formed by the fusion of two blastulae. (After Driesch, from Korschelt and Heider.) that fragments of different eggs of Hchinus (in segmentation stages) may be grafted on one another, and that the product of their union will give rise to a normal embryo, whatever the relative positions of the fragments. The fusion of blastulae to form giant Planulae has been noticed by Metschnikoff in the medusa, Mitrocoma annae, and in Ophryotrocha Korschelt has observed a similar fusion of distinct egos in the body cavity of the parent when the latter is herma- phrodite. Lastly, Sala and Zur Strassen have observed the fusion in twos, threes, or more of the eggs of Ascaris, a result experimentally produced by the first author by exposure to a low temperature, while Zur Strassen has found that such double eggs, provided they had united before fertilization and then been fertilized by a single sperm, will produce perfectly normal embryos. Ty.1 INITIAL STRUCTURE OF THE GERM 203 LITERATURE T. Boveri. Die Polaritit von Ovocyte, Ei und Larve des S/rongylo- centrotus lividus, Zool. Jahrb. (Anat.), xiv, 1901. T. Bovert. Ueber die Polaritiit des Seeigeleies, Verh. d. phys.-med. Ges. zu Wiireburg, xxxiv, 1902. H. Driescu. Entwicklungsmechanische Studien: I, ll, Zeitschi. wiss. Zool. liii, 1892. H. DriescH. Entwicklungsmechanische Studien: II-VI, Zeitschr-., wiss. Zool. lv, 1893. H. Drrescu. Entwicklungsmechanische Studien: VII-X, Mitt. Zool. Stat. Neapel, xi, 1895. H. Driescu. Zur Analysis der Potenzen embryonaler Organzellen, Arch. Ent. Mech. ii, 1896. H. Driescu. Betrachtungen iiber die Organisation des Eies und ihre Genese, Arch. Ent. Mech. iv, 1897. H. DriescH. Primiire und sekundiire Regulationen in der Entwick- lung der Echinodermen, Arch. Ent. Mech. iv, 1897. H. Drrescu. Die Verschmelzung der Individualitit bei Echiniden- keimen, Arch. Ent. Mech. x, 1900. H. Drrescu. Die isolirten Blastomeren des Echinidenkeimes, Arch. Ent. Mech. x, 1900. H. DrrescH. Neue Ergiinzungen zur Entwicklungsphysiologie des Echinidenkeimes, Arch. Ent. Mech. xiv, 1902. H. DriescH. Drei Aphorismen zur Entwicklungsphysiologie jiingster Stadien, Arch. Ent. Mech. xvii, 1904. H. Drrescu. Altes und Neues zur Entwicklungsphysiologie des jungen Asteridenkeimes, Avch. Ent. Mech. xx, 1906. M. T. GarBowski. Ueber Blastomerentransplantationen bei See- igeln, Bull. Internat. de lV Acad. des Sci. de Cracovie, 1904. M. T. GARBowskI. Ueber die Polaritit des Seeigeleies, Bull. Internat. dle l Acad. des Sci. de Cracovie, 1905. M.T.GARBowsKI. Ueber Blastomerentransplantationen bei Seeigeln, Bull. Internat. Ac. Sc. Cracovie, 1905. _K. Korscuetr. Ueber Kerntheilung, Eireifung und Befruchtung bei Ophryotrocha puerilis, Zeitschr. wiss. Zool. 1x, 1895. J. Logs. Ueber eine einfache Methode, zwei oder mehr zusammen- gewachsene Embryonen aus einem Ei hervorzubringen, Pfliiger’s Arch. lv, 1894. J. Lorn. Beitrige zur Entwicklungsmechanik der aus einem Ei entstehenden Doppelbildungen, Arch. Ent. Mech. i, 1895. KE. Merscunikorr. Embryologische Studien an Medusen, Wien, 1886. T. H. Morean. Experimental studies on Echinoderm eggs, Anat. Anz. ix, 1894. “T. H. Mor@an. Studies of the ‘ partial’ larvae of Sphaerechinus, Arch. Ent. Mech. ii, 1896. 204 INTERNAL FACTORS IV.1 T. H. Morgan. A study of a variation in cleavage, Arch. Ent. Mech. ii, 1896, T. H. Morgan. The formation of one embryo from two blastulae, Arch. Ent, Mech. ii, 1896. L. Sana. Experimentelle Untersuchungen iiber die Reifung und Befruchtung der Hier bei Ascaris megalocephala, Arch. mikr. Anat. xliv, 1895. f O. L. zur Strassen. Ueber die Riesenbildung bei Ascavis-Kiern, Arch. Ent. Mech, vii, 1898. H. E. ZreatER. Ueber Furchung unter Pressung, Verh. Anat. Geseil., 1894. R. Zosa. Sullo sviluppo dei blastomeri isolati dalle uova di alcune meduse (e di altri organismi), Arch. Ent. Mech. ii, 1896. : § 7. NEMERTINEA. In the behaviour of its isolated blastomeres the Nemertine egg resembles the Echinoderm very closely ; the cells segment as parts but ultimately give rise to wholes. The limit of this capacity is, however, sooner reached, for there is a sharper distinction between the potentialities of animal and vegetative blastomeres. Experiments have been made on Cerebratulus laeteus by Wilson, on Cerebratulus marginatus by Zeleny ; Zeleny and Yatsu have further investigated the development of fragments removed from various regions of the egg. The axis of the egg is marked by the excentricity of the nucleus and the point. of extrusion of the polar bodies. Maturation precedes fertilization. The first two furrows are meridional and divide the egg into four equal cells (A, B, C, and J). Subsequent cleavage is, however, on the spiral plan seen in Tur- bellarian, Annelidan, and holoblastic Molluscan eggs. In the Nemertine the egg-axis becomes the axis of the Pilidium larva, the animal being the aboral, the vegetative the oral, pole. In both the 4 and the 4 blastomeres cleavage is partial and an open blastula is formed, or sometimes a flat plate of cells merely, sometimes a closed sphere. The 4 later becomes a larva with an apical organ, mesenchyme and archenteron, but no lappets; the + larva has a solid archenteron but neither apical organ nor lappets. Development is slightly retarded; 2 blastomeres give rise to a IV.1 INITIAL STRUCTURE OF THE GERM 205 larva with mesenchyme and apical organ, but the archenteron is solid. After the next phase—eight cells—the upper quartette Fie. 114.—Cerebratulus. Larvae from upper quartettes of the 8-cell stage. .A. Larva from whole quartette. B, C. The same, but one cell was injured. D, E, F. Slightly older, from complete quartette. (After Zeleny, 1904.) (1 a1 d) produces a larva with an apical organ and mesenchyme but devoid of an archenteron ; the larva developed from the lower quartette (4—D) has an archenteron but no apical organ, while a Le A aoe Fie. 115.—Cerebratulus. Larvae from lower (vegetative) quartettes of 8-cell stage. Note absence of apical organ, presence of large, hollow or solid, archenteron. (After Zeleny, 1904.) meridional half, comprising two macromeres and two micromeres, develops both organs, though it is usually asymmetrical. In the 16-celled stage the isolated apical cells (1 a@ 1-1 d 1) will give rise to a ciliated blastula with mesenchyme and an apical 206 INTERNAL FACTORS TV. % organ, but without an archenteron, while from the remaining twelve is derived a ciliated embryo with a large solid archenteron, but destitute of an apical organ and lappets. Wilson asserts that the size of cells in partial is the same as in total larvae ; their number appears to be proportional to their germinal value. From fragments of blastulae normal, or nearly normal, dwarf Pilidia may arise, but the degree of development again depends on the origin of the fragment. While the lower third becomes Fig. 116.—Cerebratulus. Larvae from portions of the 8-cell stage. A, B. Larva from lateral 4-cell group. Note presence of both apical organ and archenteron. C. Similar, but younger. D. Larva from upper quartette plus two cells of lower quartette. Note that there are three apical organs, a large blastocoel, a small archenteron, and two in- penton of ectoderm at the sides of the archenteron. (After Zeleny, 4.) a ciliated embryo without an apical organ, a piece of the upper two-thirds developed (in one case) into a larva with a gut, with two apical organs, but no lappets (Zeleny). Wilson states that in Cerebratulus lacteus the archenteron is small in animal frag- ments, the apical organ frequently, though not apparently always, absent in pieces of the vegetative hemisphere. Animal fragments of the blastula have a greater total poten- tiality, a greater regulative capacity, than the animal blastomeres IV.1 INITIAL STRUCTURE OF THE GERM 207 of the eight-celled stage. This is attributed by Zeleny to the greater time and opportunity afforded them of regaining the polarity of the whole. The behaviour of egg-fragments depends not merely on the part of the egg from which they are taken but also on the time at which they are removed. Pieces taken from the ovum, no matter in what way, before the disappearance of the germinal vesicle segment as wholes and give rise to perfect Pilidia. Only in a small percentage (15 %) of cases is their development defective, the lappets being absent, or the gut abnormal, or the apical organ absent or multiplied (Yatsu). The proportion of perfect Pilidia obtained from fragments removed a little later, at the time of formation of the first polar spindle, is, however, less, only 52 Z. It is during the conjugation of the pronuclei that the definite localization of specific substances seems to begin, for now the fate of the fragment de- pends upon the direction in which the cut ismade, Eggsfrom which theanimal ‘ , Fie. 117.— Cerebratulus. portion—about one-third of the whole Diagram of egg with one —has been taken away become perfect polar body, and basal pro- Pilidia; when, however, by removal of haar a eon tet , > ions of horizontal (#H), the vegetative third, or by an oblique vertical (V), and oblique cut, the fragment does not contain ‘Fras (See AT. the whole of the lower two-thirds, the lappets are defective and the apical organ sometimes absent as well. If, therefore, there is present at this stage a special material for this organ it cannot as yet be located in its ultimate position. The cleavage of these fragments is always partial (Zeleny). After the first furrow the material for the apical organ has still not reached the animal pole: the animal region may be removed from both blastomeres without inhibiting its formation. Perfect Pilidia may’also be produced from eggs in the two-celled stage when part of one blastomere has been obliquely cut away. Should, however, the blastomeres be cut apart before their complete 208 INTERNAL FACTORS IV.i separation, each develops into a Pilidium with one lappet but no apical organ; it seems, therefore, that the material for this struc- ture is at this stage placed in the bridge of plasma connecting the two cells, and is fatally injured by the operation. Taken together these experiments make it, to say the least, highly probable that there are in the Nemertine egg definite substances connected necessarily, that is causally, with the forma- tion of certain organs. - These specific organ-forming substances may be said to be preformed, but it is equally clear that they are not prelocalized, but only reach their ultimate destination in the course of develop- ment, LITERATURE EK. B. Witson. Experiments on cleavage and localization in the Nemertine-egg, Arch. Ent. Mech. xvi, 1903. N. Yatsu. Experiments on the development of egg-fragments in Cerebratulus, Biol. Bull. vi, 1904. C. ZELENY. Experiments on the localization of developmental factors in the Nemertine egg, Journ. Exp, Zool. i, 1904. § 8. CreNOPHORA. With the Ctenophora we come to a group of animals in which the development of a total larva from a single blastomere is no longer possible, though the missing parts may eventually be regenerated. The egg consists of a central yolk-mass surrounded by a super- ficial layer of granular protoplasm. After fertilization this protoplasm passes wholly to one side—the animal hemisphere—and the egg divides meridionally into equal parts. The protoplasm then spreads itself over the outer surface of each cell, but returns to the animal side prior to the second cleavage, which is again meridional and at right angles to the first. Once more the protoplasm is distributed over the whole outer surface of each blastomere, only to be again concentrated before the third division, an unequal one, but nearly meridional. There are now four large cells lying together in a square and four smaller cells lying above them in two groups of two each, one at each end of the plane of IV.1 INITIAL STRUCTURE OF THE GERM 209 the second furrow. This becomes the transverse, while the first furrow marks the sagittal plane of the future organism. Each ‘of these cells contains beth granular protoplasm and yolk, but at the succeeding division nearly all of the former material passes into the eight small cells, which are nipped off and lie in an oval ring on the vegetative side of the egg! (Fig. 118). These small cells or micromeres will give rise to the ectoderm, the large cells or macromeres to the endoderm and mesoderm, the mesoderm _ Fre. 118.—Normal segmentation of the Ctenophore egg. «a, Stomach or sagittal plane ; bb, funnel or transverse plane. C, #, from micromere pole; D, F, from the side. (After Ziegler, from Korschelt and Heider.) being separated off later on at the vegetative pole, which becomes the oral pole of the embryo. In normal development, therefore, the embryonic axes are marked out at quite an early stage. Chun was the first to isolate the 4 and 2 blastomeres of 1 Ziegler’s description is that in this (the fourth) division the furrow first appears near the animal pole, but yolk then streams from the larger vegetative into the smaller animal portion, until the latter becomes the macromere, the former the micromere. Division is then completed and the micromeres are at the vegetative pole. This explains the divergences in the accounts of earlier observers (Agassiz, Kowalewsky, Chun, Fol, and Metschnikoff). JENKINSON P 210 INTERNAL FACTORS IVs Eucharis by shaking. These gave rise to half larvae, provided with four costae, four meridional canals communicating with two _ subsagittal and two subtransverse canals in the ordinary way, one subgastric canal, one tentacle, but a whole funnel and a whole stomach, the latter formed by an oblique invagination ; the side turned towards the missing blastomere was covered over by ectoderm. Later on the missing half was regenerated, first the subgastric vessel, then the meridional canals, over these the costae, and finally a tentacle. Fig. 119.—Development of isolated blastomeres in Ctenophora. a, D- Segmentation of 3 blastomere of Bete ovata: a, two large, two small cells; b, each gives off a micromere ; c. Larva from a } blastomere with four costae ; d, e. Segmentation of } blastomere; /. The resulting larva with two costae; g. Larva with six costae from ~ blastomeres. (After Driesch and Morgan, 1896.) h. Isolated 38; blastomeres of Bere ovata; j. The resulting larva with four costae, one sense-organ, and one stomodaeum ; 7. Isulated 35; blastomeres; /. The resulting larva with three costae, one sense-organ, and one stomodaeum. (After Fischel, 1898.) Such half larvae are found in the tow-net after a storm and may become (Bolima) sexually mature. These experiments have been repeated and confirmed on Berde by Driesch and Morgan. The authors add that the segmentation of these isolated blastomeres is partial. The 3 larva has two costae only and a large and a small canal, the former passing to the costae, the latter to the opposite side and representing apparently IV.1 INITIAL STRUCTURE OF THE GERM 211 the other half of the funnel (Fig. 119 a-g). Fischel has carried the analysis of the potentialities of the blastomeres a step further (Fig. 119 4-z). Though cleavage is partial, as described by Driesch and Morgan, there are slight irregularities in the position of the blastomeres. A } blastomere produces one costa only, 8; blastomeres (four macro- and four micromeres) four costae, 42 (five macro- and five micromeres) five, =, +2, and 34, when separated in the same way, respectively three, six, and two costae. Similar results were obtained by meridional division of the egg in later stages, Fischel confirms the statements of the other Fig. 120.—a. Berve ovata: egg in the 16-cell stage. The micromeres disarranged by pressure; b. The resulting larva with two sense-organs, and four costae radiating from each: one stomodaeum. (After Fischel, 1898.) c, Normal segmentation of the egg of Berde from which a small portion of the vegetative hemisphere has been removed; d. Larva pro- duced from the same, with eight costae, four endodermal canals, and one (central) stomodaeum. (After Driesch and Morgan, 1896.) investigators as to the structure of the half larvae, In 2 larvae, however, not three, but four, canals are formed, and the stom- odaeum is vertical. The sense-organ is not found in embryos developed from small fragments, Removal of the macromeres left the number of costae unaffected, but displacement of the micromeres by pressure led to the formation of an abnormal larva with two sense-organs and eight costae arranged in two groups of four each round each sense-organ (Fig. 120 a, 4), or, if the micromeres were separated into two lots, a group of four round one organ, a group of three round the other, and some scattered combs. With greater pressure three sense-organs were produced and the PZ 212 INTERNAL FACTORS IV.1— costae were very irregular. In these eggs there was, however, only one stomodaenm, and only the normal number of canals. It appears, therefore, that the individual costae are definitely related to the meridional divisions of the segmented egg, and further that the material which is concerned in their formation becomes located in the micromeres of the 16-celled stage. Driesch and Morgan have also shown that if the vegetative hemisphere be taken away from the undivided egg, the animal portion, though it segments normally, is able to produce neither costae nor stomodaeum; it has, however, endodermal canals. Loss of a small portion of the vegetative hemisphere, however, does not interfere with subsequent normal development (Fig. 120, @). On the other hand an egg which has been deprived of a lateral portion divides, by three unequal divisions, into eight cells, all of which form micromeres. Nevertheless, the larva has only four, five, or six costae, and some of these rudimentary, only three or four canals, and the stomodaeum, as in half larvae, is oblique. As Driesch points out, the whole of the nucleus is here, and the embryo is still defective. Differentiation thus depends on the cytoplasm, but is quite independent of segmentation, which may be perfectly normal without determining the formation of a normal embryo. Driesch further argues that the observed defects are due to lack of sufficient material merely, not to lack of any preformed specific substance. Whether such substances are already present in the unsegmented as they undoubtedly appear to be in the segmented egg, it is perhaps impossible, on the evidence, to decide, but it may be observed that the develop- ment of perfect larvae from those eggs from which a portion of the vegetative hemisphere has been detached does not necessarily support the view advanced by Driesch. LITERATURE C.Cuun. Die Dissogonie, eine neue Form der geschlechtlichen Zeugung: 7. Zur Entwicklungsmechanik der Ctenophoren, Festschr. Leuckart, 1892. H. DriescH and T. H. Morgan. Zur Analysis der ersten Entwick- lungsstadien des Ctenophoreneies, Arch. Ent. Mech. ii, 1896. H. Fiscuen. Experimentelle Untersuchungen am Ctenophorenei, 1, Arch. Ent. Mech, vi, 1898; II, HI, IV, ibid. vii, 1898. H. E. ZIEGLER. Expesimentello Studien tiber die Zelltheilung: Die Furchungszellen von Berde ovata, Arch. Ent. Mech. vii, 1898. IV.1 INITIAL STRUCTURE OF THE GERM 213 § 9. CHarropopa AnD Mottusca. In the Chaetopoda and Mollusca (except the Cephalopods) it is possible to trace back almost every organ of the body to some parti- cular cell, or group of cells, of the segmenting ovum, to describe, in fact, its lineage in terms of individual cells. Specific materials for the formation of the various parts seem to be sundered from one another by the process of cleavage, which thus presents the characters of a ‘Mosaic’ work in an exceptional degree. ‘To | what extent such a preformed structure does in reality exist experiment alone can decide, and the answer given by experiment, so far, at least, as the nucleus is concerned, is in the negative. By means of pressure Wilson succeeded in preventing in Nerezs the normal formation of the first quartette of micromeres ; instead, the egg divided into a flat plate of eight equal cells. The pressure was then removed. All eight cells formed micromeres, and later on eight instead of the usual four macromeres were found in the Trochophore larva. Four of these necessarily contained nuclei which would normally be placed in cells of the prototroch, and yet the larva was normal. In this case, then, the causes of differentiation cannot lie in the nucleus; they must be sought for, if anywhere, in the cytoplasm. In the eggs of these forms segmentation is holoblastic and of the spiral type, with the cleavages alternately dexiotropic and laeotropic, and it is easy not only to determine the lineage of each organ, but to compare the origin and the destiny of indivi- dual cells in a large series of forms. The first, second, and third quartettes of micromeres are usually destined for ectoderm formation, the second quartette cell in the D quadrant, 2 d, being the first somatoblast from which the ectodermal structures of the trunk are derived. The ciliated ring or prototroch is formed, in most cases, from certain derivatives of the first quartette, namely la2—1d 2, the primary trochoblasts, each of which divides into a group of four; but it may be reinforced by secondary trochoblasts from the same or the second quartette. The second and third quartettes may provide larval mesoderm or mesenchyme. The trunk mesoderm is usually derived from 4.7. The remaining 214 INTERNAL FACTORS LV. 3 cells of this quartette (4a—4c) become with the residual macro- meres (A—D) the endoderm (or mesendoderm). A peculiar structure seen in some forms (J/yanassa, Dentalium, AX ‘de Fra. 121.--Normal cleavage of Ilyanassa, A, B. Formation of polar- lobe or yolk-lobe (d?); C. ‘Trefoil’ stage; D, FE, F. The polar-lobe passes into CD; G. The second polar-lobe protruded in H; it passes into D. (After Crampton, from Korschelt and Heider.) Chaetopterus, Myzostoma) is the yolk-lobe, or polar lobe, as it should properly be called. This is a mass of cytoplasm protruded at the IV.t INITIAL STRUCTURE OF THE GERM 215 vegetative pole after fertilization. It becomes attached entirely to one of the first two blastomeres, namely to CD, into which it is withdrawn ; prior to the next division it is again protruded, attached to D only, and then once more withdrawn: at the time of the next division it is protruded again, for the last time (Figs. 121, 122). Crampton has isolated the blastomeres of J/yanassa and watched their development (Figs. 123-6). 4, 2, + (A, B, and C), 4, 2 c3 Fig. 122.—Normal cleavage of Ilyanassa. (After Crampton, 1896.) a, 8-celled stage from above; b, 16-celled stage from above ; c, 24-celled stage, ? from above; d, optical section, slightly oblique, of a later embryo, from below. Division of mesoblast pole-cell (d+). (a macromere and a micromere), 3, 4 (a micromere), 355, 7 and 75 all continue to segment as though the missing blastomeres were present, except for certain small irregularities in the positions of the cells; the A, B, and C blastomeres, separately or together, bud off the usual four generations of micromeres, but D only the first three ; the division of the micromeres continues in nearly normal fashion, The 4 micromere divides twiceonly. Alldie, however, 216 INTERNAL FACTORS ENS without giving rise to larvae. A } macromere, on the other hand, or a 3}; or later blastomere, will not segment at all. By lowering the temperature the cleavage may be made to resemble that of whole ova. ©) CL oe Fig. 123.—Ilyanassa: cleavage of smaller } blastomere (AB). a, Un- divided; b, 2 embryo, from side ; c, 4 embryo, from above; d, ;% embryo, from above: the dot indicates the former centre of the egg; e, }embryo, from side; 7, growth of ectoderm over the macromeres; g, formation of the micromeres of the fouith generation, from below; h, ciliated partial embryo of 46 hours. (After Crampton.) The yolk-lobe influences the character of segmentation, for if it be removed CD divides like AB. More important than this is 1V.1 INITIAL STRUCTURE OF THE GERM 217 the relation of the yolk-lobe to the mesoderm. When the former is cut off the latter is notformed at all. There are four generations of micromeres and four equal macromeres, and the larvae, though ciliated and able to swim, never reach the veliger stage (Fig. 127). i - a b c ad é ft Jat I i Fie. 124.—TIlyanassa: cleavage of CD. a, Formation of yolk-lobe (yl) ; 6, ‘trefoil’; c, } embryo from above; d, early 4 embryo, from above: secondary yolk-lobe (yl) ; e, resting 4 embryo from above; f, 34; embryo from above; g, passage to 8; stage, # from above; h, 43 embryo, from side ; i, division of mesoblast pole-cell (d*), from below. (After Crampton.) The potentialities of the blastomeres are thus strictly limited both in segmentation and differentiation. This result is amply confirmed by Wilson's researches on Dentalium and Patella, 218 INTERNAL FACTORS 1¥e4 In the latter genus the development of the isolated blastomeres, especially of those resulting from later divisions, has been minutely followed. Each separate first quartette cell (1 a—l d) forms (Fig. 128) a closed ectoblastic structure with an apical organ at Fie. 125.—Ilyanassa: cleavage of blastomere (A). a, 2, from side; b, jz, from side; c, passage to ;'5, first form, from side; d, resting 4‘, second form, from side; e, passage to 4, from side; /, sg, from side. (After Crampton.) d Fie. 126.—Ilyanassa: a-e, cleavage of } blastomeres; a, 2; stage, from one micromere ; b, 3's stage, from two micromeres; c, 35; stage, from three micromeres. d-f, cleavage of } blastomeres; d, 32 embryo, from below; e, origin of fourth quartette, from below; f, embryo of 48 hours. (After Crampton ) IV.1 INITIAL STRUCTURE OF THE GERM 219 cD ae, a b Fie. 127.—Ilyanassa: cleavage of the egg after removal of the yolk- lobe. a, 3; b, 43. ¢, first quartette formed; d, second quartette formed, first quartette divided ; a-d, from above; e,from below: fourth quartette formed ; f, ciliated embryo, from side. (After Crampton.) a h ec Fie. 128.—-Patella: development of isolated } micromeres. a, } ~ micromere; b, ¢, first division; d, 3% stage, with two trochoblasts (stippled), one rosette-cell, and one primary cross-cell; e, larva of 24 hours, from the side, showing trochoblasts below, apical cells above. (After Wilson.) 220 INTERNAL FACTORS IV.1 one end, and four cells (derived from a primary trochoblast) provided with long cilia at the other. When the primary trocho- blasts (1a 2—1 d 2) are isolated each divides into four ciliated cells, the cilia arranged as normally ina single row, but no further (Fig. 129a-e). If the daughter-cells of these trochoblasts (1 42.1 —1d 2.1 and 1 a2. 2—1 4d 2. 2) are separated they divide once only (Fig. 129,/), while their daughter-cells do not divide at all (Fig. 1299,). The sister-cells to these (1 « 1—1 d 1) behave in the same way, forming a closed larva with an apical organ at OBOE h i j h Fie. 129.—Patella. Development of isolated primary and secondary trochoblasts. a, Primary trochoblast; b, c¢, first division; d, second division ; e, after 24 hours; f, pair of primary trochoblasts, the products of division of either 1%! or 1”, after isolation; g, hk, single primary trochoblasts, either 17!*, 1°4, 1°71, or 1?”; 7, a pair of secondary trocho- blasts, the products of 1%; j, k, single secondary trochoblasts. (After Wilson.) one end and secondary trochoblasts at the other, while the isolated apical cells and secondary trochoblasts divide as often as they would in the whole embryo and put out their cilia (Fig. 129 7-4, Fig. 180 ¢-e). Finally, the whole first quartette becomes an ecto- blastic larva with an apical organ at one end and a prototroch at the other, but with no archenteron (Fig. 1807, 7). The cells of the second quartette (2 a—2 d) likewise form each a hollow ectoblastic vesicle containing larval mesenchyme, ciliated secondary trocho- blasts, and a few (preanal) ciliated cells (Fig. 131). IV.1 INITIAL STRUCTURE OF THE GERM 221 The cells of the third and fourth quartettes do not develop at all. A 4 macromere (1 A—1 D), however, segments partially, to form a micromere of the second quartette, from which secondary e vA g Fre. 130.—Patella. Development of isolated cells of first quartette and of the whole quartette. a, Isolated 1'; b, its first division ; ¢, its second division ; d, after 12 hours; e, after 22 hours, showing apical cells and secondary trochoblasts ; f, first division of isolated first quartette; _ g, second division. (After Wilson.) a b c Fie. 131.— Patella. Isolated cells of second quartette. a-c, Cleavage ; d, after 24 hours, showing secondary trochoblasts and ? preanal cells. (After Wilson.) trochoblasts are later developed, and one of the third. Subsequently an archenteron is invaginated (Fig. 132). So the #4 macromere (2 A—2 D) forms a micromere (3 a—3 @) and gives rise to a larva with entomesoblast (D) or eatoblast only (A—C). 222 INTERNAL FACTORS IV." Isolated 4 (A—D) or 4 (AB or CD) larvae have an apical organ and a prototroch complete or semicircular; they may remain open or close up ; sometimes they gastrulate. It is evident that each cell has exactly the same potentialities for division and differentiation when isolated as when in con- nexion with its fellows. Only in the shifting of the cells and closing up of the whole cell-mass is there anything which approaches to total development. E> Qe | Be Fig. 132.—Patella. Development of isolated } basal (macromere). a, First; 6b, second division; c, d, 56- and 64-cell stage. In c¢ the position of the 2-group is normal, not in d; e, after 24 hours, showing two secondary trochoblasts (products of 2!) and two feebly ciliated cells (preanal cells ?). (After Wilson.) The dependence of this specification of the blastomeres upon definite substances preformed—though not prelocalized—in the unsegmented ovum is demonstrated by Wilson’s experiments on Dentalium. The newly laid egg of Dentalium contains a brick- red yolk-mass surrounded by a thin hyaline ectoplasm ; the latter is thickened a little to form an animal polar area and very con- siderably to form another large area at the vegetative pole. The nucleus is in the centre (Fig. 1383). When the sperm enters the nucleus breaks down, and its substance becomes confluent with both 4 1V.1 INITIAL STRUCTURE OF THE GERM 223 areas. This continuit y is soon lost and the egg becomes divided into three regions, a clear layer spread over the animal hemisphere, below this the yolk-mass with the fertilization spindle, and a large, y h t Fie. 133.—Dentalium. Normal cleavage. The red pigmented part is represented by stippling. «a, Freshly laid egg, polar view; b, the same 20 minutes later, after shedding the membrane; c, the same from the side; d, one hour after fertilization, showing polar bodies; e, first division and protrusion of first polar lobe; f, ‘trefoil’ stage; g, the polar Jobe has passed into CD; h, protrusion of second polar lobe (72) ; ?, second cleavage. (After Wilson.) nearly yolk-free area at the vegetative pole, which is now pro- truded as the polar lobe. This becomes attached, as described above for //yanassa, to the D blastomere. The first quartette of 224 INTERNAL FACTORS IV.1 micromeres is formed from the clear animal area; after this division the polar lobe of D moves up to meet the upper white area, and so it comes about that a large part of this lobe passes into the second micromere, 2 7, which is the first somatoblast. In the other quadrants the second micromeres are formed from the upper white area alone. All the cells of the third quartette and (it is not known certainly whether this is the mesoblast) 4 d in the fourth are quite white. The larva is spindle-shaped with Fig. 134.—Dentalium. Cleavage after removal of the first polar lobe ; a, first division: the isolated polar lobe is seen below; b, 4-cell stage, from animal pole; c, 8-cell stage, from animal pole; d, forma- tion of second quartette, from vegetative pole; e, same stage as last, open type; Jf, same stage, from animal pole. (After Wilson.) a broad, triple prototroch, There is an apical organ of long cilia and a tuft of sensory hairs at the opposite end (Fig. 135 a). When the polar lobe is removed in the ‘ trefoil ’ (2-celled) stage, segmentation continues, but no polar lobe is formed in any stage, the D cells are not larger than those in the other quadrants, the embryo has no lower white area, the segmentation cavity may be open below, and the larvae are deficient and die (Figs. 134, 1354). Post-trochal region and apical organ are both absent and IV.1 INITIAL STRUCTURE OF THE GERM 225 these missing parts are never regenerated ; the pretrochal region is, however, increased and the three rows of cilia in the proto- troch are usually developed. : The polar lobe is thus clearly connected with the formation of the trunk and the preoral sense-organ. The segmentation of the isolated blastomeres is partial, as in the types already examined, but their subsequent behaviour depends on the presence or absence of the polar lobe. AB larvae or A, B, or C larvae closely resemble the deficient trochophore just described ; CD or D larvae are normal, though the structures derived from the polar lobe are out of proportion to the rest a b Fie. 135.—Dentalium. a, Normal trochophore of 24 hours ; }, larva of 24 hours after removal of the first polar lobe. (After Wilson, 1904.) (Fig. 136 a-d,g). The failure of the egg to develop normally is not due to insufficiency of material, for the volumes of CD and D are less than that of a whole but lobeless egg. The micromeres 1a, 14, 1¢, when isolated, become actively swimming, ectoblastic embryos provided with a prototroch, but with no apical organ, gut, or post-trochal region; the other micromere, 1d, however, has an apical organ, though it is still without the hinder region and incapable of gastrulation (Fig. 136¢,/). It appears, therefore, that the material for the apical organ is now placed in this cell, and this is proved by experiment ; for if the polar lobe is cut away during the second cleavage, or JENKINSON Q 226 INTERNAL FACTORS TVEz - from the isolated CD blastomere, then the larva has an apical organ, but no post-trochal region. Hence between the first and second divisions the stuff which determines the formation of this organ must migrate out of the polar lobe into the animal hemi- sphere of the D cell. In the unsegmented egg the specific material for both apical organ and post-trochal region is situated in the vegetative Fria. 136.—Dentalium. Larvae from isolated blastomeres. a, b, Twin larvae from the isolated CD (a) and AB (b) halves of the same egg, 24 hours; c, d, twin larvae from the isolated D (c) and C (d) quadrants of the same egg, 24 hours; e, f, twin larvae from the isolated posterior micromere 1 d (e) and the 1 c micromere (jf) of the same egg, 24 hours; g, t larva from one of the A, B, or C quadrants, 72 hours. (After Wilson.) hemisphere. The animal half of the egg, whether removed after fertilization, or before and then fertilized, segments like an egg from which the polar lobe has been removed (Fig. 137 4), and develops neither apical organ nor post-trochal region (rarely an apical»organ if the fragment is large). The vegetative hemi- sphere, when cut off before the entry of the sperm and afterwards IV.1 INITIAL STRUCTURE OF THE GERM 227 fertilized, segments as a whole with the polar lobe in correct proportion and gives rise to a dwarf larva with all its parts complete (Fig. 137 a, c). Many, however, die. A meridional half-egg is able to develop normally, or nearly so. The behaviour of the enucleate vegetative fragment of the fertilized egg is interesting. Though deprived of nucleus and centrosome it forms its polar lobe synchronously with the divisions of the animal portion ; three times is the lobe protruded and three times withdrawn: the fourth time it is not taken back ; this is the moment when the first somatoblast is given off. In this enucleate fragment the polar lobe is not in proportion but of the same size as in the whole egg. Even the isolated polar lobe, or pieces of it, exhibits periodic movements, amoeboid, or of elongation, or protruding a smaller lobe. es 52) © B LT a b c Fie. 137.—Dentalium. Development of egg-fragments, the plane of section being indicated in the small figures. «a, b, Twins, after horizontal or oblique section near animal pole; ¢, trochophore developed from a fragment resembling that shown in a. (After Wilson.) The existence in the egg of preformed substances specified for the production of particular organs is thus incontrovertibly established, and as long as an egg-fragment or blastomere contains all these it will give rise to an embryo which is, in form, perfect, though lack of enough material (or the shock of the experiment) may bring its life to an untimely end. At the same time these substances, though preformed, are not necessarily prelocalized ; their original is not the same as their ultimate situation, and in their redistribution we have to deal with a process which is, as Wilson remarks, truly epigenetic. Before bringing this section to a conclusion reference must be made to another case in which it seems probable that definite Q2 228 INTERNAL FACTORS IV.1 organ-forming substances exist, though experimental proof is at present lacking. In the immature egg of the aberrant parasitic Polychaet Myzostoma there is, according to Driesch, at the vegetative pole a mass of greenish cytoplasm, the surface of which is convex towards the reddish brown mass of the rest of the egg. When the polar bodies are formed the red substance retreats to the animal pole, leaving a clear equatorial zone. Before segmentation a polar lobe is formed—as in IJ/yanassa Fig. 138.—Development of the egg of Myzostoma. The green sub- stance on the vegetative side is shaded finely ; this passes into the polar lobe (e), and into the D quadrant (i). The red substance of the animal hemisphere is coarsely dotted. (After Driesch.) Fic. 139.—Development of the egg of Myzostoma. Formation of the cells 1d (d' in c) and 2 d or X (d*? ine). The latter cell takes a portion of the substance of the polar lobe. (After Driesch.) and Dentalium—containing the green and part of the clear substance: it passes first to CD andthen to D. When the latter divides it is again protruded and withdrawn. The cell 1d, like la, 14, 1c, contains the reddish protoplasm. The second micro- mere in the D quadrant —2 d—takes part of the green substance, and Driesch suggests that the rest of the latter would eventually pass into the second somatoblast (4d = M) (Figs. 188, 189). _- IV.1 INITIAL STRUCTURE OF THE GERM 229 LITERATURE H. E. Crampton. Experimental studies on Gasteropod development, Arch. Ent. Mech. iii, 1896. H. Driescu. Betrachtungen tiber die Organisation des Kies und ihre Genese, Arch. Ent. Mech. iv, 1897. EK. B. Witson. Cleavage and mosaic work. Appendix to Crampton’s paper on Ilyanassa, Arch. Ent. Mech. iii, 1896. EK. B. Witson. Experimental studies on germinal localization. I. The germ regions in the egg of Dentalium, Journ, Exp. Zool. i, 1904, KE. B, Wriuson. Experimental studies on germinal localization. II. Experiments on the cleavage mosaic in Patella and Dentalium, Journ. Exp. Zool. i, 1904. § 10. Asorpra. As has already been shown, there is in the Vertebrata and Amphioxus no constant relation between the first furrow and the sagittal plane of the embryo, although the symmetry of the embryo does stand in a very definite relation to that of the un- segmented ovum. Further, experiment has proved that the isolated blastomeres, up to a ceitain point at any rate, are capable of giving rise to normal total larvae. It is, perhaps, all the more remarkable that in another group of the Chordata, the Ascidians, the symmetry of the embryo is not only predetermined in the symmetry of the egg but also by the planes of segmenta- tion, and that the potentialities of isolated blastomeres remain strictly what they would have been in the whole ovum. The earliest experiments are due to Chabry (Figs. 140, 141). According to this author the first furrow in the egg of Ascidiella aspersa is meridional and coincides with the sagittal plane; the second, likewise meridional, coincides with the transverse plane ; the third, equatorial, separates oral from aboral regions, and is therefore horizontal. | tite Isolated 4 blastomeres or 2 right or left blastomeres give rise to imperfect larvae with only one fixing papilla, only one atrial invagination and one pigment-spot, the eye; the last is absent in the 2 left larva, 2 anterior larvae have one fixing papilla, one sense-spot, a gut, a. cerebral vesicle and a posterior notochord ; 230 INTERNAL FACTORS EV.2 in } posterior larvae the chorda and brain are absent, though the three germ-layers are all present. In the segmentation of the isolated blastomeres the direction of division is altered, in that a furrow (the fourth) which in the whole egg is meridional and at an angle of 45° to the first two becomes parallel to the plane of separation, and so resembles the third furrow of the whole egg. A right or left 4 blastomere or 2, anterior or posterior, right Ss Fig. 140.—A. Egg of Ascidiella aspersa in the two-cell stage. One 4 blastomere has been killed (@). B.The survivor (D) divides to form, in C, a small normal gastrula. (After Chabry, from Korschelt and Heider.) Fie. 141.—Larva of Ascidiella aspersa produced from 7 blastomeres. F, sucker; At, atrium; No, notochord; En, endoderm. (After Chabry, from Korschelt and Heider.) or left, blastomeres consist after this division of two tiers of four cells each ; the cleavage of these cells resembles, therefore, that of a whole egg. It should be noticed that the blastomeres cannot be actually separated ; it is only possible to kill one, by a needle, and note the development of the survivor. The more recent researches of Conklin on Cynthia have con- firmed and extended Chabry’s results. The immature egg of Cynthia (Fig. 142) comprises a central grey yolk surrounded by a peripheral layer of yellow pigmented IV.1 INITIAL STRUCTURE OF THE GERM 231 Fie. 142.—Normal development of the egg of Cynthia partita. Matura- tion and fertilization. (After Conklin.) A. Unfertilized egg before the fading of the germinal vesicle (clear), showing central mass of grey yolk (lightly dotted), peripheral layer of yellow protoplasm (thickly dotted), test-cells and chorion. B. After the entrance of the spermatozoon the yellow protoplasm has streamed to the vegetative pole (v); in it, excentrically, is the sperm nucleus (sp.n). The clear protoplasm derived from the germinal vesicle partly forms a layer over the yellow protoplasm, in part remains at the animal pole (a). The grey yolk occupies the animal part of the egg. c. The yellow and clear protoplasmic substances have both streamed to what will be the posterior side (P). A, anterior, v, ventral (animal pole), D, dorsal (vegetative pole). D. View of the same egg from behind. The two pronuclei are seen side by side in the clear area. R, right; L, left. 232 INTERNAL FACTORS IV. 1 protoplasm ; near one pole (the animal), and so determining the egg-axis, is the large germinal vesicle. Prior to the extrusion of the polar bodies the latter breaks down and provides a third substance, a clear cytoplasm. On the entrance of the spermato- zoon near the vegetative pole a remarkable change takes place in the arrangement of these materials. The yellow protoplasm flows down to the vegetative pole and arranges itself symmetrically to the axis; above it isa zone of clear protoplasm derived from the germinal vesicle ; these two areas together occupy about one- third of the egg. The remaining, the animal two-thirds, are occupied by the grey yolk with only a small quantity of clear cytoplasm at the animal pole in which, after the formation of the polar bodies, the female pronucleus is embedded. The egg is still radially symmetrical, but by further shiftings of these substances it becomes bilateral. The spermatozoon moves in the clear protoplasm to the equator on one side, the posterior side as it will be, and the yellow proto- plasm follows suit; the first is now disposed in a broad equatorial band encircling half the circumference of the egg, the second forms a wide crescentic mass below it and reaches nearly to the vegetative pole. The female pronucleus, with its clear area, now joins the male. The egg is now bilaterally symmetrical about a plane which includes the axis, and passes through the middle of the clear and yellow substances and between the two closely apposed pro- nuclei. It seems doubtful whether the movement of the clear and yellow material to the posterior side is immediately due to the agency of the sperm, for the latter does not always take the shortest route to the equator; it may enter on what becomes ultimately the anterior side ; and further, when two sperms enter they both travel to the same spot, appearing to be passively carried along by the independent streaming of the egg cyto- plasm. The two pronuclei next move into the axis of the egg in the animal hemisphere, together with their clear protoplasm. The grey yolk is thus largely displaced and shifted into the vegeta- tive region. The egg, therefore, now consists of an animal IV.1 INITIAL STRUCTURE OF THE GERM 233 hemisphere composed almost entirely of the clear substance, and a vegetative herhisphere, the anterior portion of which includes | grey yolk and nothing else, the posterior portion the yellow protoplasm together with a little yolk near the vegetative pole. The cleavage furrows separate these substances from one another in a perfectly definite way (Fig. 143). The first is A Ce Fie. 143.—Cleavage of the egg of Cynthia partita. (After Conklin.) A. 8 cells, from the left side. _B. 8 cells, from above. The yellow crescent (thickly dotted) is limited to the posterior dorsal cells: the animal cells are clear, the grey yolk (sparsely dotted) is found in the dorsal cells, both anterior and posterior. c. 16 cells, from the dorsal side. D. 74 cells, dorsal view, showing the division of the 4 neural plate- cells (n.p), and 4 chorda cells (ch.). There are 10 endoderm cells (e), 6 muscle-cells (m.s.), 8 posterior (p.m.ch.) and 2 anterior (a.m.ch.) mesenchyme cells, 234 INTERNAL FACTORS WA Fie. 144.—Cynthia partita. Successive stages in the development of the same right half embryo, the left blastomere having been injured in IV.1 INITIAL STRUCTURE OF THE GERM 235 meridional and sagittal, the second meridional and transverse, the third equatorial and horizontal. Since the animal pole will be ventral, the vegetative dorsal, the right and left sides of the future embryo are simultaneously determined. The four ventral animal cells consist almost entirely of clear protoplasm, the two anterior dorsal entirely of grey yolk, the two posterior dorsal almost exclusively of the yellow substance. The next furrows are, roughly, meridional and at angles of 45° to the first two; they emphasize the bilateral symmetry, for in the anterior half they meet the first furrow above, the second below, and conversely in the posterior half. The direction of the divisions in the next phase may be most easily gathered from the figures. In the vegetative hemisphere four anterior chorda-neural cells are separated off; the six posterior cells include all the yellow substance and will give rise to the posterior mesenchyme and the muscles of the tail; six endoderm yolk-cells remain, four anterior, two posterior. The next division sees the separation of the chorda from the neural cells in front, of an outside ring of muscle-cells from an inside ring of mesenchyme cells behind, and of an anterior mesenchyme cell on each side from the anterior endoderm. A few anterior animal cells become associated with the neural cells in the formation of the nervous system; the remainder of this hemisphere is ectodermal. , In gastrulation the endoderm cells first sink inside, followed by the chorda in front and the anterior and posterior mesenchyme cells and the muscle-cells at the sides and behind, Overgrowth is greatest at the anterior dorsal lip, the blastopore thus becoming posterior. In normal development, therefore, the axes of the embryo are the 2-cell stage. The yellow protoplasm is indicated by coarse stippling. (After Conklin.) a, 8-cell stage, posterior view. v, ventral; D, dorsal. b, 16-cell stage, anterior view. Normally, A 5.1 and @ 5.3 lie in front of A 5.2 and a 5.4. c, 16-cell stage, posterior view. In normal eggs the cells B 5.2 and b 5.4 lie behind B 5.1 and 65.3. d, 80-cell stage, dorsal view. e, f, 34-cell stage; e, posterior, f, dorsal view. In normal eggs the cell B 6.4 lies laterally to B 6.3, and the cell B 6.1 lies between B 6.3 and A6.1. g, 46 cells: posterior view. h, 48 cells: dorsal view. The cells B 7.1 and B 7.2 should lie next the middle line. 236 INTERNAL FACTORS EV 2 predetermined by the arrangement of certain substances in the egg, and segmentation exhibits the character of a ‘ Mosaic’ work, segregating these specific materials into the rudiments of definite organs. The causal connexion between these materials and the organs to which they are beforehand assigned is demonstrated by experiment. Fie. 145.— Cynthia partita. Half and three-quarter embryos. (After Conklin.) a, right half gastrula, dorsal view. The neural plate, chorda, and mesoderm cells are present only on the right side and in their normal position and numbers. }b, left half of young tadpole, dorsal view. The notochord is normal except for size and number of cells: the muscle and mesenchyme cells are present only on one side: the neural plate (7.p.) is abnormal in form but not in position. c, right half of young tadpole, dorsal view: slightly younger than b. m’ch., mesenchyme, ms., muscle-cells. d, left anterior three-quarter embryo, dorsal view.. The anterior half is entirely normal with anterior mesen- chyme (m’ch.) on both sides, Posterior mesenchyme and muscle-cells upon the left side only. IV.1 INITIAL STRUCTURE OF THE GERM 237 The segmentation of a 4 or of 2 right or left blastomeres is partial exceptfor the alteration in the direction of the furrows of the fourth phase already described by Chabry. The blastomeres divide as though the other half of the egg had not been killed (Figs. 144, 146, 147). Eventually a half gastrula is formed with exactly half thenormal number of neural, chorda, endoderm, mesen- chyme and muscle-cells ; the gastrula cavity is open on the side of the dead blastomere. Many are, however, abnormal in that the endodermand muscle- and mesenchyme cells do not invaginate (exo- gastrulae) or show invaginations of the ectoderm (pseudogastrulae), The half gastrula may develop into a half larva, with the notochord, muscles, and mesenchyme lying on one side of the medullary folds. The only signs of the regeneration of the missing half are the overgrowth of the ectoderm on the naked side, and the passing over of a few muscle-cells ; the full number of the latter is, however, never found on both sides (Fig. 145 a-c), The 3 larva is especially interesting, particularly when a pos- terior blastomere has been killed, for here the anterior end is complete with anterior mesenchyme on both sides ; the posterior mesenchyme and muscles are, however, missing on either the right or left (Fig.145d). The chorda and neural plate are stated to be smaller than thenormal in these larvae,andthereis nosense-vesicle. An embryo developed from the two anterior blastomeres of the 4-celled stage alone possesses the full number of neural, chorda, anterior endoderm and anterior mesenchyme cells; its hind end is covered by ectoderm but shows no trace of muscles or posterior endoderm or mesenchyme (Fig. 146 «¢, @). The two posterior blastomeres produce an embryo devoid of neural plate and notochord, of anterior mesenchyme and endo- derm. By overgrowth of the ectoderm a sort of solid gastrula is formed with a central mass of endoderm flanked by muscle- cells and posterior mesenchyme. No tail appears (Fig. 147). 1, 4, and {4 blastomeres also segment and develop partially. An anterior + has half the proper number of neural, notochordal, anterior endoderm and mesenchyme cells, but the neural plate does not become folded and the chorda cells protrude irregularly behind. A posterior quarter may gastrulate in the way described for the posterior half, but exogastrulae and pseudogastrulae occur. 238 INTERNAL FACTORS IV.1 Conklin has also divided the gastrula into anterior and posterior halves ; neither can regenerate the missing parts, though the cut surface of each becomes covered by ectoderm. Driesch, however, states that in another form, Phallusia mammiliata, anterior and posterior half gastrulae will, if Fia. 146.—Cynthia partita. Anterior half and three-quarter embryos. (After Conklin.) a, b, anterior ventral three-quarter embryo, the posterior dorsal cells (B 4.1, B 4.1) containing all the yellow pigment having been killed in the 8-cell stage. «a, ventral view; b, dorsal view. c, d, anterior half embryo: c¢, dorsal view, showing the neural plate (stippled); d, deeper focus showing two rows of chorda cells (the nuclei are shaded), and ectoderm and endoderm. separated before complete closure of the blastopore, each develop into a larva lacking only the otolith and the eye. In a later stage of gastrulation this becomes impossible, although the IV.1 INITIAL STRUCTURE OF THE GERM 239 posterior portion of a young Ascidian will regenerate a new head with pharynx and eyes; the anterior half dies, Driesch has also maintained that the isolated blastomeres (2, 3, 4) of the same species give rise ultimately to total gastrulae and these to larvae. The larvae are, however, unable to hatch out and are defective—with rudimentary eyes, otolith and suckers—or devoid of these organs altogether. Fie. 147.—Cynthia partita. Posterior half embryos. (After Conklin.) a, 32-cell stage, dorsal view. Cleavage is quite normal. b, 76-cell stage, dorsal view. Muscle-cells (B 8.8, B 8.7 and B 7.8) and posterior mesenchyme cells (B 8.6, B 8.5, B 7.7, and B 6.3) and posterior endoderm cells (B 7.1, B 7,2) are seen. ¢, later stage, deep focus, showing ventral endoderm (v end.) with a mesenchyme (m’ch.) on each side. d, same stage as last, showing muscle-cells (ms.). LITERATURE L. CHABRY. Contribution & l’embryologie normale et tératologique des Ascidies simples, Journ. de l’ Anat. et de la Phys, xxiii, 1887. E. G. Conxury. The organization and cell-lineage of the Ascidian egg, Journ, Acad. Nat. Sci. Philadelphia, xiii, 1905. 240 INTERNAL FACTORS DVS E. G. ConKLIN. Mosaic development in Ascidian eggs, Journ. Exp. Zool. ii, 1905. H. Drrescu. Von der Entwicklung einzelner Ascidienblastomeren, Arch, Ent. Mech, i, 1895. H. Driescu. Ueber Aenderungen der Regulationsfihigkeiten im Verlauf der Entwicklung bei Ascidien, Arch. Ent. Mech. xvii, 1904. § 11, GenzeraL ConsIDERATIONS AND CONCLUSIONS TO BE DrAwN FROM THE ForEGOING EXPERIMENTS. From the foregoing experiments certain general conclusions may now be drawn. (1) First, thedivision of the nucleus during segmentation at least is not the qualitative process imagined by Roux and Weismann. The pressure experiments of Hertwig, Driesch, and Wilson have clearly demonstrated that the nuclei of the segmenting ovum may be disarranged without prejudice to the subsequent normal development of the embryo; and this result is amply confirmed by the normal behaviour of eggs (of sea-urchins) in which the mutual positions of the blastomeres, and therefore also of the nuclei, are altered, as well as by the very numerous cases in which it has been shown to be possible to rear a perfect embryo or larva from an isolated blastomere or blastomeres. It is evident that during segmentation at least the nuclei are equipotential, and the hypothesis of self-differentiation in the form originally propounded by Roux can no longer be upheld. It has, in fact, been now abandoned by its author. (2) But though in this direction its labours have ended negatively, modern experimental research is yet able to point toa positive achievement of the greatest value and significance. For the same series of investigations has shown that the cytoplasm of the undeveloped ovum is not the homogeneous and isotropic substance which the experiments of Pfliiger led Roux to consider it, but heterogeneous, containing various specific organ-forming stuffs which are definitely and necessarily connected with the production of certain parts or organs of the developing animal. If the polar lobe of the mollusc //yanassa be removed the larva has no mesoderm ; if the polar lobe of Dentalium be taken away the larva has no trunk and no apical organ; the egg of a Ctenophor IV.1 INITIAL STRUCTURE OF THE GERM 241 is capable of segmenting when deprived of its vegetative hemi- sphere, but the larva to which it gives rise has no combs and no sense-organ. The egg of the Ascidian Cynthia exhibits in its cytoplasm various substances, easily distinguishable by their colour ; in segmentation these are distributed to the various blasto- meres in a perfectly definite way. Remove one or more of these substances and the embryo will lack the organ or organs normally produced from them. Again, a Nemertine egg from which, at a certain stage, the ‘vegetative portion has been cut away, gives - rise to a Pilidium without lappets and without a sense-organ. In these cases, then, at least, the existence of specific organ- forming stuffs in the cytoplasm of the egg has been proved ; the removal of the stuffs inevitably entails the absence of the corresponding organ later on. But though it may therefore be justly said that such stuffs are preformed, it is not invariably true that they are prelocalized, present, that is to say, ab initio in what will be their eventual situation. For example, the stuff which determines the formation of an apical organ in the larva of Dentalium is originally in the polar lobe of the vegetative hemi- sphere : but between the first and the second divisions it migrates into the animal portion of the egg ; and the same may be said of the formation of the sense-organ in Cerebratulus and the Ctenophora. It seems highly probable, though it cannot be said to have been demonstrated, that such specific organ-forming substances are of universal occurrence. The behaviour of the isolated blastomeres of the eggs of different forms, as well as of the blastomeres of the same form at different stages, is markedly different. The 4 or + blastomere of an Ascidian egg can never produce more than 4 or 2 larva; the isolated blastomeres of Patel/a produce just so much, dividing just so many times, as they would have produced had they remained in connexion with their fellows; a radial group of blastomeres (one or more 4 blastomeres, or two or four or more ;3; blastomeres) of a Ctenophore ege gives rise to a larva with one, two or more costae and meridional canals, as the case may be. On the other hand, the isolated 4 or 4 blastomeres of a Nemertine ovum will produce a complete larva, while, after the next division, the totipotentiality is lost; for an isolated JENKINSON R 242 INTERNAL FACTORS | IV.1 animal cell gives rise to a larva without an archenteron, an isolated vegetative cell to one in which the apical organ is lacking. So in Amphiowus 4 and % blastomeres develop into whole embryos, but 3 blastomeres, eiatiee of animal or vegetative origin, will not Sannin In other cases, however, the potentia- lities do not become restricted so soon. Either the four animal or the four vegetative cells of a Frog’s egg will give rise to an embryo in which the blastoporic lip and archenteron are de- veloped, while in Echinoderms the least blastomere that will gastrulate is 4. It is not, nevertheless, every cell of the 32-celled stage that is capable of developing an archenteron, but only those, as Driesch has conceded, of vegetative origin. But whatever differences there may be in the potentialities of the cells of different ova, or of the same ova at different stages or in different regions, it is clear that sooner or later the capacity of a part to become a whole is lost; and it seems only reason- able to conclude that this loss of totipotentiality is due to the loss of one or more necessary cytoplasmic substances, to the lack of specific material, and not merely of material. In the egg of Amphioxus, which is telolecithal, there is an obvious difference, in the amount of yolk and size of the granules, between the animal and vegetative hemispheres ; a difference in the capacities of these two regions has been experimentally demonstrated in the Nemertine egg; and, although it may often appear homo- geneous, the ovum of the sea-urchin, in all probability, possesses that ‘stratification’ of potentialities, that is of organ-forming stuffs, at right angles to its axis, on which Boveri has so strongly insisted, for Driesch has admitted that vegetative cells ‘gastrulate’ more readily than animal cells ; it would appear, then, that a gut- forming substance is present,and a mesenchyme-forming substance too, but that the concentration of these substances steadily diminishes from the vegetative to the animal pole. Radially about the axis the distribution of these substances spel be Seance to be uniform, since meridional fractions of the egg, 4, 4, 2 or 2 or 3 blastomeres, are invariably totipotential. The same explanation may be applied to other cases. Hence, speaking generally, the limitation of the potentialities of the parts will depend simply on the original distribution of the IV.1 INITIAL STRUCTURE OF THE GERM 243 substances in the unsegmented egg. Should they be already segregated (wholly or in part) in distinct regions, as in Ascidians, Molluses, and Ctenophors, then even the first blastomeres will to that extent be unable, when isolated, to give rise to more than they would have done in the uninjured egg; should they be uniformly distributed about the axis, but unequally along the axis (Nemertines, Echinoderms, Amphiowus), then a limitation of potentialities will appear when the third division separates the animal from the vegetative cells'; should the arrangement be Fre. 148.—Diagrammatic sections through the unsegmented egg of Loligo pealii. (From Korschelt and Heider, after Watase.) A is in the transverse, B in the sagittal plane of the future embryo. In B the anterior side (vo.) is more convex than the posterior (h.). d, dorsal (animal pole); v., ventral (vegetative pole) ; ., left; 7, right. The protoplasm is black, the yolk shaded. uniform about the egg-centre, then animal and vegetative blastomeres will be alike totipotential (Coelenterata). At the same time it would appear to be true that the capacity of total development may be lost for mere lack of material, for Driesch found that while all four (or all eight in the next stage) animal cells of the sea-urchin egg might give rise to a Pluteus larva, the isolated cells would not. The isolated cells may, how- } We may notice that O. Maas (Arch. Ent. Mech. xxii, 1906) has found that the isolated anterior flagellated and posterior granular cells of the Amphiblastula larva of Sycon are not equipotential. The latter can fix and give rise to a young sponge, the former cannot. ; <3 R 2 244 INTERNAL FACTORS IV. ever, gastrulate ; when once the gastrula stage has been reached, therefore, further development may be simply a matter of sufficiency of substance. So again the octants of the Ctenophore egg are alike, but each can produce one costa, and one only. There are then certain preformed organ-producing substances, and the arrangement which they either possess before, or acquire B ane Yq: ‘oe ae! Fia. 149.—Three segmentation stages in the blastoderm of Sepia offici- nalis; the segmentation is of the bilateral type. 7, left; 7, right; J-V, first to fifth cleavages. The top sides of the figures are anterior. (After Vialleton, from Korschelt and Heider.) during, segmentation determines the fixed relation which can be observed in almost all cases between the structure of the egg and the axes of the embryo. Thus in Amphibia the head of the embryo is formed at or near the animal pole, and the plane of egg symmetry becomes the sagittal plane. In Mollusca (not Cephalopoda) and Annelida the apical sense-organ is developed at the animal pole, while the D quadrant is posterior. A blasto- IV.1 INITIAL STRUCTURE OF THE GERM 245 pore is in very numerous cases formed at the vegetative pole, the bilateral symmetry of the eggs of Cephalopoda (Figs. 148, 149), Insecta, some Crustacea, and Ascidia becomes that of the embryo; in Ascaris the first four cells lie in one plane, which becomes the median plane, the germ-cell is posterior and the animal pole dorsal; and so on. (3) The instances in which the segregation of the organ- forming substances into the blastomeres takes place at once are especially interesting, as they appear to fulfil exactly the require- ments of the ‘ Mosaik-theorie ’, if that doctrine be transferred from the nucleus to the cytoplasm, for here, undoubtedly, segmentation is a qualitative process, a sundering of potentialities. It must be pointed out, however, that even in these cases the factors which determine segmentation and those which determine differentia- tion may be as distinct as they are elsewhere. In the Frog there is a much greater correlation between the symmetry of the egg and the median plane of the embryo than between the former and the plane of the first furrow. . 4 or 4 blastomere of a Nemertine or a sea-urchin segments as a part, and yet it eventually develops into a whole larva: and the same is true of egg-fragments. All the isolated blastomeres of a Molluse segment as parts, yet in Dentalium, that one which contains all the necessary stuffs, CD or D, can give rise to a complete embryo, while its fellows cannot. The converse case is to be found in Ctenophors; here the egg which has been deprived of its vegetative portion segments as a whole and yet produces an embryo devoid of costae and sense-organs. There are also certain forms (Amphiorus and Veitebrates, Coelenterata) where the isolated cell segments as a whole, and this appears to depend on the rapidity with which the part can re- arrange its material or resume the polarity of the whole (Wetzel). It is further to be noticed that the capacity of a part for total segmentation, like its power of total differentiation, may differ in the same egg at different times and under different conditions ; the fragment of an immature egg of Cerebratulus divides like a whole egg, an isolated blastomere like a part; again, in //yanassa the cleavage of a part may be made total by lowering the temperature. When, further, it is remembered how closely similar the 246 INTERNAL FACTORS IV. 1 geometrical pattern of segmentation may be in the eggs of different animals, whether of the same or of different groups (spiral segmentation of the eggs of Molluscs, Chaetopods and Turbellaria, similar segmentation of the eggs of, for example, some Echinoderms, Polyzoa, and Vertebrates), without neces- sarily involving a similarity in the fate of cells which are identical in origin, it will, I believe, be conceded that the factors which are responsible for cleavage and those which determine differentiation are distinct, though the two may, as in the so- called ‘Mosaik’ segmentations, coincide: and where, as in Ascidians, the Ctenophora, the Cephalopoda, this coincidence is complete, the symmetry of the egg, the symmetry of segmenta- tion, and the symmetry of the embryo are one. The former factors must be sought for in the arrangement of yolk and protoplasm, possibly in the relative strengths of the centrosomes, in surface-tensions, in the pressure exerted by egg- membranes and so forth; the latter in definite cytoplasmic organ- forming stuffs. (4) Like the parts of the egg the parts of the elementary organs of the embryo are at first equipotential, but exhibit a gradual limitation of potentialities as their development proceeds. As Minot puts it, there is a ‘ genetic restriction’ of capacities. Thus the archenteron of Astertas can form a new terminal vesicle when this has been removed, but only before ‘the outgrowth of the coelom sacs. The anterior half of the newt embryo will develop into a whole embryo when cut off before the medullary folds have arisen, but not after. When one vitelline vein of a chick is destroyed its fellow will form a whole, not a half heart; and Sumner’s experiments quoted above have shown that the margin of the Teleostean blastoderm is isotropic. (5) There still remains to be noticed a point of great impor- tance. It certainly has not been demonstrated, and it cannot be pretended that there are in the developed egg as many specific substances as there are separately inheritable qualities in the body. On the contrary, the evidence of experiment speaks against such a supposition; for, as Driesch has urged, were every separately inheritable quality to be represented in the germ by a distinct morphological unit, the equipotentiality of IV.r INITIAL STRUCTURE OF THE GERM 247 the isolated parts would be inconceivable. Although the forma- tion of the larval and elementary organs (germ-layers) may de- pend upon such stuffs, yet within each such elementary organ the parts are at first and remain for a time equipotential. Differentiation is progressive. The origin of the primary differentiations has been found; it remains for the experi- ments of the future to discover how fresh distinctions arise until the organization of the whole is complete. Suggestions as to the importance, in this respect, of certain other internal factors have been made, notably of the structure of the nucleus, and the reactions of the parts on one another. These sugges- tions, for they are hardly more than that at present, we shall have to discuss. First, however, we must briefly consider the part played by the spermatozoon in determining the structure of the egg and the symmetry of the embryo. § 12. It is of the greatest interest to observe that the definitive distribution of these specific substances in the unsegmented egg, and so the determination of the embryonic axes, may be brought about, partly at least, by the spermatozoon. In the Frog (Rana fusca, R. temporaria, and R. palustris, pro- bably also 2. esculenta) the egg soon after fertilization becomes bilaterally symmetrical. As first Roux and later Schulze have shown, this is due to the appearance on one side of the border of the pigmented area of a grey crescent, caused by the retreat of the pigment into the interior. The grey crescent subsequently becomes white and added to the white area. The side on which it appears is opposite to that on which the spermatozoon had entered, and later becomes the dorsal side of the embryo; there is also a considerable correlation between the plane of symmetry thus bestowed upon the egg and the future sagittal plane, since the latter lies in a large majority of cases in or close to the middle point of the crescent. This relation between the point of entry of the sperm and the egg- and embryonic symmetry has also been experimentally demonstrated by Roux, who, by applying the sperm to any arbitrarily selected meridian of the egg (by means of a fine cannulus, a brush, or a silk thread), was able to show that 248 INTERNAL FACTORS IV. 5 fertilization can take place from any meridian, and that the point of entry so selected becomes the ventral side later on; in other words, the fertilization meridian becomes the sagittal plane. This, as we have seen, is true, or approximately true. Roux, however, believed that the plane of the first furrow also coincided with the other two, in fact that it was the first furrow which determined the sagittal plane, and he brought forward evidence to show that the first furrow, if it did not pass through the point of entry of the sperm, at least either included, or was parallel to, the inner end of the crooked path of the spermatozoon within the egg, the so-called ‘ copulation’ path (Fig. 150). As we now know there is very little correlation between the first furrow and the sagittal plane. Nevertheless Roux’s work remains of Fia. 150.—Roux’s diagrams to show the relation of the sperm-path (Pig.) to the first furrow in the Frog’s egg. In A the furrow includes the sperm-path, in B it is parallel to it, in c it is parallel to the inner portion of the path (copulation path), in p it includes only the very last portion of the copulation path. (From Korschelt and Heider, after Roux.) the greatest significance, for it seems extremely likely that, while it is the actual point of entrance of the sperm and the first, radially directed, part (‘penetration path’) of its track within the egg, which determines the position of the grey crescent, and so of the sagittal plane, it is the second part which determines the direction of the first furrow, since the centro- some will divide at right angles to this ‘copulation’ path, or line of junction of the two pronuclei; the axis of the fertilization spindle is of course given by the line of separation of the two centrosomes, its equator by the plane including the two pro- nuclei and the egg-axis, and this spindle-equator is the plane of the first furrow.? In the egg of the sea-urchin Zoxopneustes the definitive egg- axis would appear to be fixed not by the original position of the egg- 1 See, however, Appendix A. IV.1 INITIAL STRUCTURE OF THE GERM 249 nucleus at all, but by the position of the excentric segmentation nucleus. Both male and female pronuclei move through the egg for a longer or shorter distance till they meet, and the combined nucleus then takes up its definitive excentric position (Fig. 151). The plane of the first furrow is given by the egg-axis and the point of entrance, approximately, of the spermatozoon, which may thus be said to determine the symmetry of segmentation, and so of the embryo, since this plane becomes, it is said (Selenka, Fie. 151.—Diagrams from successive camera drawings of the living eggs of Toxopneustes, to show the determination of symmetry during fertilization. The original position of the egg nucleus is shown by the position of the dotted circle marked 9; its path by the succession of such circles. E, the entrance-point of the spermatozoon, and cone of entrance. The male pronucleus is rendered in black, its path marked by the line uniting the black dots. M is the meeting-point of the pronuclei. The cleavage nucleus, ©, is shaded; its successive positions are indicated. The axis of the fertilization spindle is shown by the line with a small circle at each end passing through the cleavage nucleus; the arrow passing through this is the definitive egg-axis, its point the animal pole. The cleavage nucleus is slightly excentric, and nearer the animal than the vegetative pole. F is the meridian of the first furrow. A and B are opposed in polarity, similar in other respects. c and D differ in polarity, and otherwise as well. (After Wilson and Mathews.) ° 250 INTERNAL FACTORS EVE Garbowski, Driesch), the median plane of the Pluteus. In this case there is practically no distinction between ‘ penetration’ and ‘copulation’ parts of the sperm-track, and, the centrosome divid- ing perpendicularly to this path, the point of entrance naturally lies in, or nearly in, the first furrow. The other alternative, as Wilson has pointed out, is to suppose that the definitive polarity of the egg existed before fertilization, but exerted no influence upon the egg-nucleus, although able subsequently to compel the fertilization nucleus to take up a position in its axis.' There are other Echinoderms, however (Asterias, Strongylocentrotus), in which the original and the definitive egg-axes coincide. Another very interesting case is Cynthia (Fig. 142). In the immature egg of this Ascidian the cytoplasm is concentrically arranged, since the yellow pigmented protoplasm forms a peri- pheral investment for the central grey yolk: the egg-axis is de- termined only by the position of the germinal vesicle. Upon the entrance of the spermatozoon the yellow substance together with the clear plasma of the germinal vesicle, which has in the mean- time broken down, streams towards the vegetative pole and becomes radially arrayed about the axis. By a further streaming movement these two substances are carried up with the male pronucleus— or pronuclei in cases of dispermy—to the equator, so marking the future posterior side of the now bilateral egg. These changes are evidently an effect of fertilization: it cannot, however, be asserted that the point of entrance of the sperm determines the posterior end, since it may enter on the opposite side and get carried across. There are a few other instances in which an alteration of the egg-structure has been noticed to follow on fertilization. In Ctenophora the peripheral layer of granular cytoplasm becomes aggregated at the animal pole (Agassiz) ; in Polyclads (Lang) and Cirripedes (Groom) the egg becomes telolecithal ; and so in Physa 1 In Diplogaster longicauda also it appears to be the point of union of the two pronuclei which determines the definitive egg-axis and orientation of the embryo. The polar bodies are always extruded and the female pronucleus formed at that end of the ellipsoid egg which is turned towards the ovary, while the sperm enters at the opposite end. The pronuclei may meet and unite at either end; but it is at the end where their union takes place that the smaller of the first two blasto- meres is found and, later, the hinder end of the embryo (H. E. Ziegler, Zeitschr. wiss, Zool. 1x, 1890). IV.1 INITIAL STRUCTURE OF THE GERM 251 (Kostanecki and Wierzejski): the ‘polar rings’ appear in Allolobophora, Clepsine, and Rhynchelmis (K. Foot, Whitman, Vejdovsky), while in Teleostei the periblastic hyaline layer becomes concentrated as the blastodise at the animal pole. The case of Dentalium has been already referred to (p. 223, Fig. 133). Although the cases in which the réle played by the spermato- zoon in determining egg and embryonic structure are, as has been observed, not very numerous, yet it is fully to be expected that renewed investigation will show that some such rearrangement of the materials of the egg is in many, perhaps in all, instances one of the first results of fertilization. LITERATURE A. AGAssiz. Embryology of the Ctenophorae, Mem. Amer. Acad. Arts and Sciences, N.S. x, 1873. E. G. Conkuin. The organization and cell-lineage of the Ascidian egg, Journ. Ac. Nat. Sci. Philadelphia, xiii, 1905. K. Foor. Preliminary note on the maturation and fertilization of the egg of Allolobophora foetida, Journ. Morph. ix, 1894. T. T. Groom. On the early development of Cirripedia, Phil. Trans. Roy. Soc. elxxxv, B. 1894. K. von KosTaANEcKI and A. WIERZEJSKI. Ueber das Verhalten der sogen. achromatischen Substanzen im befruchteten Ei (Physa fontinalis), Arch. mikr. Anat. xlvii, 1896. A. Lane. Fauna und Flora des Golfes von Neapel: XI. Die Poly- claden, Leipzig, 1884. W. Roux. Die Bestimmung der Medianebene des Froschembryo durch die Copulationsrichtung des Eikernes und des Spermakernes, Arch. mikr, Anat. xxix, 1887; also, Ges. Abh. 21, Leipzig, 1885. F. VEspovskKY. Entwickelungsgeschichtliche Untersuchungen, Prag, 1888-92. C.O. WHITMAN. The embryology of Clepsine, Quart. Journ. Micr. Sci. xviii, 1878. KE. B. Writson and A. P. MatrHews. Maturation, fertilization and polarity in the Echinoderm egg, Journ. Morph. x, 1895. § 13. Wuar Part bors THE NUCLEUS PLAY IN DIFFERENTIATION ?! We have already criticized and rejected the hypothesis that the division of the nucleus during segmentation is a qualitative process. It may still be urged, however, that the nucleus is not insignificant in differentiation. In support of this contention the following arguments have been put forward. 1. It is urged that the nucleus is essential for the life of the cell, 1 See also Appendix B. 252 INTERNAL FACTORS IV.1 and not only for its metabolism (Figs. 152, 1538) but for the production of form (Fig. 154). 2. It is obvious that the germ-cells are the vehicles whereby the inheritable characters of the species are transmitted from one generation to the next; they are the material basis of in- heritance. The germ-cells of the two sexes are, however, as unlike as possible in every character except their nuclei, in which, as the study of maturation has abundantly shown, they are exactly alike. In fertilization, the union of the two germ-cells, two distinct processes are involved. ‘The first is the mutual stimulation whereby the lost power of cell-division is restored. This is a process independent of the nuclei (in Metazoa). Theother is, however, the union of the nuclei (or their chromosomes) ; and since the offspring are held to inherit equally from the two parents, it has been supposed that with the union of the nuclei, the only. parts of the cells that are Fig. 152.—Egg-cell of Dytiscus marginalis in its follicle with two nurse cells. From the nurse cells nutritive material passes into the egg-cell, towards which the nucleus exactly alike, the paternal and maternal contributions are intermingled, and that therefore it is in the chromatin of the nuclei that the vehicle of transmission of hereditary qualities must be looked for. sends out pointed pseudo- podia. (After Korschelt, 3, Thirdly, it has been pointed out Heider, eset A (originally by Roux) that karyokinesis appears to be a mechanism expressly adapted to the simultaneous division of a large number of qualita- tively different units. From the existence of karyokinesis it is argued that the nucleus is not homogeneous, and that in de- velopment the various units of which it is composed are directly concerned in the process of differentiation. The doctrine of the individuality of the chromosomes has been brought forward in support of this belief, as well as the constancy of their number. 4. The diminution of the chromosomes. Boveri has observed in Ascaris, in the nuclei of purely somatic cells, a peculiar process IV.1 INITIAL STRUCTURE OF THE GERM 253 to which he has given the name of the diminution of the chromatin. The middle parts of the chromosomes in somatic cells become divided transversely into small granules, the ends remain rod- shaped. The granules of the middle parts are alone divided and pass to the daughter nuclei, while the ends are cast out into the cytoplasm and there degenerate, The chromosomes of the germ-cells (or of the parent-cells of the germ-cells) do not undergo this change, but remain intact (Fig. 155). It might be argued that in such a case as this the deter- mination of certain characters —somatic—is brought about by the expulsion of the chro- matin into the cytoplasm. 5. A great deal of stress has been laid on the impor- tance of the experiment, due to Boveri, in which the enucleate fragment of the egg of one species of sea-urchin gives rise, when fertilized with the sperm of another species, to a larva # 2 : : d which exhibits the charactors ,, "10,1584, origin of « root:has of the male parent alone. placed at the point of origin. B, the 6. Boveri has brought for "ung im fuewbita pga. rot ba ward evidence to show that the growing point. (After Haberlandt, the abnormal development of fom Korschelt and Heider.) _ Echimoderm eggs which follows on polyspermy is in reality due to the irregular distribution of the chromosomes to the daughter- cells. Hence it is argued that the chromosomes are qualita- tively different. These are the reasons which are, or have been, brought forward in support of the belief that the nucleus is not insignifi- cant in differentiation. We may now discuss them in order, * ee GB ms 254 INTERNAL FACTORS > Twa premising only one thing, namely, that both maternal and paternal nuclei are certainly not necessary for the production of a new individual, however beneficial it may be for the elements of two parents to be commingled in the body of one offspring. In artificial parthenogenesis an egg is stimulated to development by a solution, and produces a normal larva; this creature possesses Guster of maternal origin. The Be converse case is seen in the phenomenon of ‘ merogony ’. An enucleate egg-fragment, fertilized with sperm of the same species, gives rise to a new organism, provided only with paternal nuclei. The older view that the union of the pronuclei was the essential act in fertiliza- tion can no longer be held, for, certainly, both nuclei are not necessary. The most, therefore, that can be said will be that a complete set of the chromosomes (or smaller chromatin units) of the species Fra. 154,—On the left the protozoon . Stentor, cut into three pieces, each con- capmeanaeas & for differentiation. taining a piece of the nucleus. On Whether this is so or not we the right, each piece has regenerated : ae the missing parts and become a com- must now a ae = plete Stentor. (After Gruber, from 1. It will of course be al- Korschelt and Heider.) lowed that the nucleus is neces- sary for the life of the cell and to a certain extent for the assump- tion of form. The numerous and familiar experiments on Protozoa have sufficiently established this point.!' A nucleate piece will live and regenerate lost parts, an enucleate piece will not (Fig. 154). 2. It will also be admitted that the maturation processes in the germ-cells of the two sexes are extraordinarily alike and end in the reception by each germ-cell of one-half the normal 1 See especially M. Verworn, Die physiologische Bedeutung des Zellkerns, Pfliiger’s Arch. li, 1892 ; in this paper an account of the work of Gruber and others will also be found. IV. INITIAL STRUCTURE OF THE GERM 255 Fie. 155.—The process of chromatin diminution as seen in the somatic cells of Ascaris megalocephala. (After Boveri, 1899.) 1. Mitosis in the 2-celled stage. In the first somatic cell (S, or AB), the primary ectoderm, the chromatin undergoes diminution, not in the germ-cell (P,). 1a chromosomes being ‘diminished’. 2. 4-cell stage, T-shape. In A and B the discarded masses of chroma- tin are seen. S, (H M St), second somatoblast or endomesostomodaeal rudiment. 3. 4cell stage, lozenge-shape. In A and B the next mitosis is begin- ning, in P, and £ M St the nuclei are in the resting stage. A is anterior, A and B are dorsal, all four cells lie in one plane, the sagittal plane of the embryo. 4. In EM St the chromatin is being diminished. Division of P, into P, and S, (C), the secondary ectoderm. a, b, primary ectoderm of right, a, 8, primary ectoderm of left side. 5. The endoderm (E,, E,) has now been separated from mesoderm and stomodaeum. P,; has just divided into P, and S,(d), tertiary ectoderm. 6. Diminution of chromatin in S,(D). The four endoderm cells (£) beginning to be invaginated: on each side two mesoderm cells (M) in which granular chromosomes may be seen, and two stomodaeal cells (St). Ventral view. 256 INTERNAL FACTORS FV.’ number of chromosomes of precisely the same shape and size. In fertilization the two sets of chromosomes—each.in half the normal number—are united. It is of course difficult to believe that this extraordinary resemblance of the nuclei, while all the other characters of the cells are unlike, is without significance. 3. With regard to the third point a distinction must be made between two hypotheses. The first is the individuality of the chromatin, the second the individuality of the chromosomes. For the first, as we shall see, independent evidence exists, and mitosis is certainly a mechanism admirably adapted to the simultaneous division (or separation of already divided halves) of a large number of qualitatively distinct bodies. But the second hypothesis in no way follows from the first, for the grouping of the unlike units may obviously be different at each successive division without in the least impairing their individuality. There is, indeed, evidence for the persistent in- dividuality of the chromosomes in only one or two cases. Boveri has noticed, in the segmenting egg of Ascaris, a constancy in the position of the pockets of the nuclear membranes which lodge the ends of the chromosomes, and a similarity in the arrangement of the chromosomes in the nucleus in successive nuclear divisions. Sutton again has observed that in Brachy- stola each chromosome forms a separate reticulum, situate in a separate pocket of the nucleus. There are other cases in which each chromosome forms its own vesicle (Echinoderm eggs). These instances are, however, few and far between. There are so many nuclei in which nothing can be observed of the chromosomes in the resting stage, often there is nothing but a fine granular mass, and a study of the early and end stages of mitosis seems to show that the chromosomes are precipitated from solution in the nuclear sap and redissolved when the division is over. In such a solution the chromatin elements would retain their individuality, their qualitative differences, just as each one of a number of crystals retains its distinctive properties in a mixed solution and exhibits them when recrystallization occurs. In such a sense, and such only, is it possible to speak in general of the individuality of the chromatin. 4, With regard to the diminution of the chromosomes in IV.1 INITIAL STRUCTURE OF THE GERM 257 Ascaris, Boveri has pointed out that the extruded outer ends of the chromosomes are very irregularly distributed to the daughter- cells, whereas they should surely, if they are determinants, be equally divided between the two. More than that, however, Boveri has found evidence which shows that not only does the diminished chromatin not determine the characters assumed by the cytoplasm, but that on the contrary it is the cytoplasm which decides which chromosomes shall be diminished, and which remain intact. - Boveri has observed certain cases of dispermy in Ascaris, which are followed by simultaneous division of the ovum into four, consequent, presumably, on a quadripolar mitosis which is due, in turn, to the presence of an extra pair of centrosomes. The total number of chromosomes present in such eggs is 3 x, where x is the reduced number, since each spermatozoon intro- duces x. This number becomes doubled by division, and the number is then 6z or 12, since 2 in Ascaris megalocephala »v. bivalens = 2. These twelve chromosomes have to be distributed over the four cells into which the ovum divides: their distri- bution is irregular. The next division is described. as being tangential in two of the cells, at angles of 45° to the first divisions in the other two: it leads to the formation of two groups of four cells each, in each of which the cells are arranged in the T shape characteristic of the normal four-celled stage. The normal four-celled stage is reached by (1) an equatorial division, (2) followed by a meridional division in the animal cell (A B), and a latitudinal division in the vegetative cell, which separates a cell S,, which like 4 and B is somatic, from a cell P, at the vegetative pole. The chromosomes in P, are intact, in A, B, and 8, they are diminished (Fig. 155). Boveri argues that if the diminution of the chromosomes were an intrinsic property of the chromatin, there should always be 6 (3 2) chromosomes intact and the rest diminished, just as there are always 4 (2) intact in the normal egg, no matter how the chromosomes had become distributed over the first four cells and their descendants. This, however, is not the case. Whole chromosomes are found only in those cells—one in each group of four—which correspond in position to the P, cell of the JENKINSON Ss 258 INTERNAL FACTORS IV.1 normal egg; the chromosomes in the other cells are all diminished, and no cell contains chromosomes of both kinds. Further, the number of whole chromosomes may be 5 in one cell and 3 in the other, or 4 and 3, or 8 and 8, or 4 and 2, or 3 and 2. Boveri concludes, it seems with perfect justice, that it is the (vegetative) cytoplasm which has determined that the chro- mosomes it contains shall remain whole, while, contrariwise, the animal cytoplasm of the other cells brings about the process of diminution. In Ascaris lumbricoides there is a diminution of the chromosomes very similar to that seen in 4. megalocephala, and, to judge from ————_———_ Fre. 156.—Pluteus of Sphaerechinus granularis from in front and from the side. (After Boveri, 1896.) certain abnormalities similar to those just mentioned, brought about by the same means (Bonnevie). In Dytiscus there is some- thing like a diminution of the chromatin in the nurse-cells, while the oocyte retains all the chromatic material intact (Giardina). The distinction seen here may, however, only be that observed in all ova between the nutritive and reproductive functions of the nucleus (trophochromatin and idiochromatin). 5. Boveri’s hybridization experiment, on which rests the assertion that the nucleus alone conveys the inheritable characters of the species, was as follows. The Plutei of Sphaerechinus granularis and of Echinus micro- tuberculatus were found (at Naples) to be markedly different. IV.1 INITIAL STRUCTURE OF THE GERM 259 The larva of the former (Fig. 156,) is short and squat, the oral lappet not divided into lobes, and the skeleton provided with a fenestrated anal arm—produced by three long parallel bars united by numerous cross-bars—an apical branch to the oral arm, and, at the apex, a square ‘frame’ formed by the union of twigs from the last mentioned and from the apical arms. The larva of Hehinus, on the other hand, is long and lank, the oral lappet is deeply cleft, and in the skeleton the anal arm is not fenestrated ; there is no apical branch of the oral arm, and the ex- Fie. 157.—Pluteus of Echinus microtuberculatus from in front and from the side. (After Boveri, 1896.) tremity of the apical arm is thickened and club-shaped (Fig. 157). Boveri first fertilized the ova of Sphaerechinus with the sperm of Lchinus, and so produced a hybrid larva whose characters were intermediate between those of the two parents in the following respects—in form, being shorter and broader than that of Hehinus, longer and narrower than that of Sphaerechinus, in having the oral lappet slightly divided, and in the skeleton, the extremity of the apical arm being swollen and branched, the anal arms being: double but not fenestrated, and the oral arms being (sometimes) provided with an apical branch (Fig. 158). 8 2 260 — INTERNAL FACTORS LV A mass culture of egg-fragments of Sphaerechinus—containing presumably whole eggs, and nucleate and enucleate fragments— was then made, and this was fertilized with the sperm of Lehinus. Amongst the larvae developed in such a culture Boveri found a small number (twelve) of dwarf individuals which possessed the paternal (Lchinus) characters alone (Fig. 159 4), and he suggested that these had come from enucleate fragments of eggs. He was further strength2ned in this opinion by the fact that the nuclei were small, which he attributed to their containing only one-half the normal number of chromosomes. This conclusion has been adversely criticized by Seeliger. Seeliger, working at Trieste and subsequently at Naples, has N 1896.) pointed out that the differences between the Plutei of these two sea-urchins are not as great as Boveri asserted them to be—the extremity of the apical arm in Eehinus is, for instance, not always club-shaped—and that therefore it cannot be so confidently asserted that the hybrid is intermediate. Secondly, he asserts that in an ordinary hybrid culture (whole eggs of Sphaerechinus fertilized by sperm of Hchinus) there are to be found together with variable larvae of more or less an intermediate type, individuals with purely paternal characters, though the pure Sphaerechinus type never occurs, and that the nuclei are variable in size. This contention has been upheld by Morgan, and, after a good deal of controversy, has finally been admitted by Boveri himself. No conclusion can therefore be drawn from the original experiment. IV.r INITIAL STRUCTURE OF THE GERM 261 An explanation of the discrepancies in the results of the different observers has been offered by Vernon. Vernon has found that Strongylocentrotus lividus (which has a Pluteus almost exactly like that of Hehinus) is at a minimum of sexual maturity in the summer months, but that from that time onwards the power of the male to transmit its characters to the hybrid larva, when crossed with Sphaerechinus 2 , increases, and that it is possible to obtain a culture consisting en- tirely of larvae of the paternal type, in respect, that is to say, of the skeleton. Vernon was inclined to look upon this difference in maturity as a seasonal varia- tion, but Doncaster has since attributed it to the effect of temperature alone. The transmission of the characters of the & skeleton may therefore be the function of the nucleus, though, as just pointed out, there is no stringent proof of this. There are other larval characters, however, which, as Driesch has justly urged, must depend on the cytoplasm of the ovum, the colour, for example, if the egg is pigmented, and the size of the larva, so far as this depends on the size of the egg. From a series of hybridization experiments between ~ ry Fie. 159.—a, Fully Echinus, Sphaerechinus, Strongylocentrotus, and