MEDICAL Dr. I/iOnroe Sutter Memorial, A LABORATORY MANUAL AND TEXT- BOOK of EMBRYOLOGY CHAR AM PRENTISS, A.M., PH.D. PROFESSOR OF MICROSCOPIC ANATOMV IN THE NORTHWESTERN UNIVERSITY MEDICAL SCHOOL, CHICAGO WITH 368 ILLUSTRATIONS MANY OF THEM IN COLORS PHILADELPHIA AND LONDON W. B. SAUNDERS COMPANY 1915 Copyright, 1915 by W. B. Saunders Company PRINTED IN AMERICA PRESS OF W. B. SAUNDERS COMPANY PHILADILPHIA W6PI PREFACE THIS book represents an attempt to combine brief descriptions of the verte- brate embryos which are studied in the laboratory with an account of human embryology adapted especially to the medical student. Professor Charles Sedg- wick Minot, in his laboratory textbook of embryology, has called attention to the value of dissections in studying mammalian embryos and asserts that "dissection should be more extensively practised than is at present usual in embryological work " The writer has for several years experimented with methods of dissecting pig embryos, and his results form a part of this book. The value of pig embryos for laboratory study was first emphasized by Professor Minot, and the development of my dissecting methods was made possible through the reconstructions of his former students, Dr. F. T. Lewis and Dr. F. W. Thyng. The chapters on human organogenesis were partly based on Keibel and Mall's Human Embryology. We wish to acknowledge the courtesy of the pub- lishers of Kollmann's Handatlas, Marshall's Embryology, Lewis-Stohr's Histology and McMurrich's Development of the Human Body, by whom permission was granted us to use cuts and figures from these texts. We are also indebted to Professor J. C. Heisler for permission to use cuts from his Embryology, and to Dr. J. B. De Lee for several figures taken from his "Principles and Practice of Obstetrics." The original figures of chick, pig and human embryos are from preparations in the collection of the anatomical laboratory of the Northwestern University Medical School. My thanks are due to Dr. H. C. Tracy for the loan of valuable human material, and also to Mr. K. L. Vehe for several reconstruc- tions and drawings. C. W. PRENTISS. NORTHWESTERN UNIVERSITY MEDICAL SCHOOL, CHICAGO, ILL., January, 1915. CONTENTS INTRODUCTION 1 1 CHAPTER I. — THE GERM CELLS 17 The Ovum 17 Ovulation and Menstruation 20 The Spermatozoon 21 Mitosis and Amitosis 22 Maturation 24 Fertilization 30 The Chromosomes in Heredity 31 The Determination of Sex 31 CHAPTER II. — SEGMENTATION AND FORMATION OF THE GERM LAYERS 33 Segmentation in Amphioxus, Amphibian, Bird, and Reptile 33 Segmentation of the Rabbit's Ovum 36 Origin of the Ectoderm and Entoderm 37 Origin of the Mesoderm, Notochord and Neural Tube 42 Origin of the Mesoderm in Mammals 45 The Notochord 46 CHAPTER III. — THE STUDY OF CHICK EMBRYOS 48 Chick Embryo of Twenty-five Hours 48 Transverse Sections of Twenty-five-hour Chick Embryo 50 Origin of the Primitive Heart 52 Chick Embryo of Thirty-six Hours ( 18 segments) 54 Central Nervous System 54 Digestive Tube 55 Heart and Vessels 56 Mesodermal Segments 61 Ccelom 63 Mesenchyma 63 Derivatives of the Germ Layers 64 Chick Embryo of Fifty Hours (27 segments) 65 Nervous System 66 Digestive Organs 67 Blood System 67 Study of Transverse Sections 69 Amnion, Chorion 74 Yolk-sac and Allantois 75 CHAPTER IV. — THE FETAL MEMBRANES AND EARLY HUMAN EMBRYOS 77 Early Human Embryos 77 Fetal Membranes of Pig Embryos 77 Umbilical Cord 79 Fetal Membranes of Early Human Embryos 80 Chorion 81 Amnion 83 Allantois 84 Yolk-sac and Stalk 86 Anatomy of a 4. 2 mm. Human Embryo 88 Central Nervous System 89 Digestive Canal 89 Urogenital and Circulatory Organs 92 Age of Human Embryos 95 CHAPTER V. — THE STUDY OF PIG EMBRYOS 97 The Anatomy of a six mm. Pig Embryo 97 External Form 97 Internal Anatomy 99 The Study of Transverse Sections in 7 8 CONTENTS PAGE The Anatomy of a 10 mm. Pig Embryo 120 External Form 1 20 Central Nervous System and Viscera 122 Heart and Blood Vessels 128 The Study of Transverse Sections 132 CHAPTER VI. — METHODS OF DISSECTING PIG EMBRYOS: DEVELOPMENT OF THE FACE, PALATE, TONGUE, TEETH AND SALIVARY GLANDS 145 Directions for Dissecting Pig Embryos 145 Lateral Dissections of 6-35 mm. Embryos 146 Median Sagittal Dissections 148 Ventral Dissections 153 Development of the Face in Pig and Human Embryos 153 Development of the Hard Palate 155 Development of the Tongue 158 Development of the Salivary Glands 161 Development of the Teeth 162 CHAPTER VII. — ENTODERMAL CANAL AND ITS DERIVATIVES 168 The Pharyngeal and Cloacal Membranes 168 Pharyngeal Pouches and their Derivatives 168 The Thymus Gland 171 The Epithelial Bodies or Parathyreoids 172 The Thyreoid Gland 172 Larynx, Trachea and Lungs 173 Digestive Canal 177 Digestive Glands : Liver 183 Pancreas 186 Body Cavities, Diaphragm and Mesenteries 188 Primitive Coelom and Mesenteries 188 Septum Transversum 191 Pleuro-pericardial and Pleuro-peritoneal Membranes 192 Diaphragm and Pericardial Membrane 195 Omental Bursa, or Lesser Peritoneal Sac 197 The Mesenteries 200 CHAPTER VIII. — UROGENITAL SYSTEM 203 Pronephros 203 Mesonephros 205 Metanephros 207 Nephrogenic Tissue 210 Cloaca, Bladder, Urethra and Urogenital Sinus 213 Genital Glands and Ducts 216 Testis * . 218 Ovary 221 Union of Genital Glands and Mesonephric Tubules 225 Uterine Tubes, Uterus and Vagina 226 Ligaments of the Internal Genitalia 228 Descent of Testis and Ovary 230 External Genitalia 232 Male and Female Genitalia Homologized . 235 The Uterus during Menstruation and Pregnancy 238 The Decidual Membranes 239 Chorion Laeve and Frondosum 243 Decidua Vera 243 Decidua Capsularis 244 The Placenta 245 The Relation of Fetus to Placenta 249 CHAPTER LX. — VASCULAR SYSTEM 251 The Primitive Blood-vessels and Blood-cells 251 Erythrocytes 25 2 Leucocytes 253 Blood plates 254 The Development of the Heart Primitive Blood Vascular System Development and Transformation of the Aortic Arches 270 Vertebral and Basilar Arteries 272 Segmental Arteries 274 Umbilical and Iliac Arteries • 275 CONTENTS Arteries of the Extremities 275 Vitelline and Umbilical Veins: Vena Porta 276 Anterior Cardinal Veins: Superior Vena Cava 279 Posterior Cardinal Veins : Inferior Vena Cava 280 The Veins of the Extremities 283 The Fetal Circulation 284 The Lymphatic System 286 Lymph Glands 287 Haemolymph Glands and Spleen 288 CHAPTER X. — HISTOGENESIS 290 The Entodermal Derivatives 290 The Mesodermal Tissues 291 Sclerotomes and Mesenchyme 291 Supporting Tissues 292 Cartilage 294 Bone 295 Joints 298 "Muscle 298 The Ectodermal Derivatives . . 303 Epidermis 304 Hair 305 Sweat Glands 307 Mammary Glands 307 Nails 308 The Nervous Tissues 309 The Differentiation of the Neural Tube 310 Neurones of the Ventral Roots 313 Spinal Ganglia and their Neurones 313 The Neurone Theory . 315 The Supporting Tissue of the Nervous System 316 CHAPTER XL — MORPHOGENESIS or THE CENTRAL NERVOUS SYSTEM 319 The Spinal Cord 320 The Brain 325 The Differentiation of the Subdivisions of the Brain 330 Myelencephalon 330 Metencephalon 333 Cerebellum 334 Mesencephalon 335 Diencephalon 336 Hypophysis 337 Telencephalon 339 Chorioid Plexus of Lateral Ventricles 340 Cerebral Hemispheres 346 CHAPTER XII. — THE PERIPHERAL NERVOUS SYSTEM 351 The Spinal Nerves 351 Brachial and Lumbo-sacral Plexuses 353 Cerebral Nerves 355 Special Somatic Sensory Nerves . 355 Somatic Motor Nerves 358 Visceral Mixed Nerves . . -. 359 The Sympathetic Nervous System 364 Chromaffin Bodies: Suprarenal Gland 366 The Sense Organs 368 General Sensory Organs 368 Taste Buds 368 Olfactory Organ 369 Eye- • • - 373 Ear 381 INDEX 389 TEXT-BOOK OF EMBRYOLOGY INTRODUCTION The study of human embryology deals with the development of the individual from the origin of the germ-cells to the adult condition. To the medical student human embryology is of primary importance because it affords a comprehensive understanding of gross anatomy. It is on this account that only recently a prominent surgeon has recommended a thorough study of embryology as one of the foundation stones of surgical training. Embryology not only throws light on the normal anatomy of the adult, but it also explains the occurrence of many anomalies, and the origin of certain pathological changes in the tissues. From the theoretical side, embryology is the key with which we may unlock the secrets of heredity, of the determination of sex and, in part, of organic evolution. There is unfortunately a view current among graduates in medicine that the field of embryology has been fully reaped and gleaned of its harvest. On the contrary, much productive ground is as yet unworked, and. all well- preserved human embryos are of value to the investigator. An institute of embryology for the purpose of collecting, preserving and studying human embryos has re- cently been established by Professor F. P. Mall of the Johns Hopkins Medical School. Aborted embryos and those obtained by operation in case of either normal or ectopic pregnancies should always be saved and preserved by immersing them intact in 10 per cent, formalin or Zenker's fluid. The science of embryology is a comparatively new one, originating with the use of the compound microscope and developing with the improvement of micro- scopical technique. Chick embryos had been studied by Malpighi and Harvey previous to Leeuwenhoek's report of the discovery of the spermatozoon by Dr. Ham in 1677. At this period it was believed that the spermatozoa were both male and female and developed in the ovum of the mother; that the various parts of the adult body were preformed in the sperm-cell. Dalenpatius (1699) believed that he had observed a minute human form in the spermatozoon. Previous to this period, many animals were believed to be spontaneously generated from slime and decaying matter as asserted by Aristotle. The preformation theory was first combated by Wolff (1759) who saw that the early chick embryo was differentiated 12 INTRODUCTION from unformed living substance. This theory, known as cpigenesis, was proved correct when, in 1827, von Baer discovered the mammalian ovum and later demonstrated the germ-layers of the chick embryo. When, after the work of Schwann and Schleiden (1839), the cell was recognized as the structural unit of the organism, the ovum was regarded as a typical cell and, in 1843, Barry ob- served the fertilization of the rabbit's ovum by the spermatozoon. Hence- forth all multicellular organisms were believed to develop each from a single fertilized ovum, which by continued cell-division eventually gives rise to the adult body. In the case of vertebrates, the segmenting ovum differentiates first three primary germ-layers. The cells of these layers are modified in turn to form tissues, such as muscle and nerve, of which the various organs are composed, and the organs together constitute the organism, or adult body. Primitive Segments — Metamerism. — In studying vertebrate embryos we shall identify and constantly refer to the primitive segments or metameres. These segments are homologous to the serial divisions of an adult earth- worm's body, divisions which are identical in structure, each containing a ganglion of the nerve cord, a muscle segment, or myotome and pairs of blood- vessels and nerves. In vertebrate embryos the primitive segments are known as mesodermal segments, or somites. Each pair gives rise to a vertebra, to a pair of myotomes, or muscle segments, and to paired vessels; each pair of mesodermal segments is supplied by a pair of spinal nerves, consequently the adult verte- brate body is segmented like that of the earth-worm. As a worm grows by the formation of new segments at its tail-end, so the metameres of the vertebrate embryo begin to form in the head and are added tailwards. There is this dif- ference between the segments of the worm and the vertebrate embryo. The seg- mentation of the worm is complete, while that of the vertebrate is incomplete ventrally. GROWTH AND DIFFERENTIATION OF THE EMBRYO A multicellular embryo develops by the division of the fertilized ovum to form daughter cells. These are at first similar in structure and, if separated, any one of them may develop into a complete embryo, as has been proved by the experiments of Driesch on the ova of the sea-urchin. The further development of the embryo depends (i) upon the multiplication of its cells by division; (2) upon the growth in size of the individual cells; (3) upon changes in their form and structure. The first changes in the form and arrangement of the cells give rise to three GROWTH AND DIFFERENTIATION OF THE EMBRYO 13 definite plates, or germ-layers, which are termed from their positions the ectoderm (outer skin), mesoderm (middle skin) and entoderm (inner skin). In function the ectoderm, as it covers the body, is primarily protective, and gives rise to the nervous system through which sensations are received from the outer world. The entoderm, on the other hand, lines the digestive canal and is from the first nutritive in function. The mesoderm, lying between the other two layers, naturally per- forms the functions of circulation, of muscular movement and of excretion; it gives rise also to the skeletal structures which support the body. While all three germ-layers form definite sheets of cells known as epithelia, the mesoderm takes also the form of a diffuse network of cells, the mesenchyma. The Anlage. — This German word is the term applied to the first ag- gregation of cells which will form any distinct part or organ of the embryo. The various anlages are differentiated from the germ-layers by a process of un- equal growth. At points where multiplication of the cells is more rapid than in the circular area surrounding them, outgrowths or ingrowths of the germ-layer will take place. The outgrowths or evaginations are illustrated by the development of the finger-like villi from the entoderm of the intestine ; ingrowths or invagina- tions by the formation of the glands at the bases of the villi. According to Minot, the development of evaginations and imaginations, due to unequal rapidity of growth, is the essential factor in moulding the organs, and hence the body of the embryo. Differentiation of Tissues. — The cells of the germ-layers which form organic anlages may be at first alike in structure. Thus the evagination which forms the anlage of the arm is composed of a single layer of like ectodermal cells, surrounding a central mass of diffuse mesenchyma (Fig. 131). Gradually the ectodermal cells multiply, change their form and structure and give rise to the layers of the epidermis. By more profound structural changes the mesenchymal cells also are transformed into the elements of connective tissue, tendon, cartilage, bone and muscle, aggregations of modified cells which are known as tissues. The development of modified tissue cells from the undifferentiated cells of the germ-layers is known as histogenesis. During histogenesis the struc- ture and form of each tissue cell are adapted to the performance of some special function or functions. Cells which have once taken on the structure and func- tions of a given tissue can not give rise to cells of any other type. In tissues like the epidermis, certain cells retain their primitive embryonic characters throughout life and, by continued cell-division, produce new layers of cells which are later cornified. In other tissues all of the cells are differentiated into the adult type and, during life, no new cells are formed. This takes place in the case of the nervous elements of the central nervous svstem. 14 INTRODUCTION Throughout life, tissue cells are undergoing retrogressive changes. In this way the cells of certain organs like the thymus gland and mesonephros degenerate and largely disappear. The cells of the hairs and the surface layer of the epider- mis become cornified and eventually are shed. Tissue cells may thus normally constantly be destroyed and replaced by new cells. The Law of Biogenesis. — Of great theoretical interest is the fact, con- stantly observed in studying embryos, that the individual in its develop- ment recapitulates the evolution of the race. This law of recapitulation was asserted by Meckel in 1881 and was termed by Haeckel the law of biogenesis. According to this law, the fertilized ovum is compared to a unicellular organism like the amoeba; the blastula embryo is supposed to represent an adult Volvox; the gastrula, a simple sponge; the segmented embryo a worm-like stage, and the embryo with gill-slits may be regarded as a fish-like stage. The blood of the human embryo in development passes through stages in which its corpuscles resemble in structure those of the fish and reptile ; the heart is at first tubular, like that of the fish; the kidney of the embryo is like that of the amphibian, as are also the genital ducts. Many other examples of this law may readily be observed. A more complete account of the general conceptions of embryology is given in Minot's "Laboratory Textbook of Embryology." Methods of Study. — Human embryos not being available for individual laboratory work, we employ instead the embryos of the lower animals which best illustrate certain points. Thus the ova of Ascaris, a parasitic round worm, are used to demonstrate the phenomena of mitosis; the larvae of echinoderms, or of worms, are frequently used to demonstrate the segmentation of the ovum and the development of the blastula and gastrula larvae; the chick embryo af- fords convenient material for the study of the early vertebrate embryo, of the formation of the germ-layers and of the embryonic membranes, while the struc- ture of a mammalian embryo, similar to that of the human embryo, is best ob- served in the embryos of the pig, .which are very readily obtained. An idea of the anatomy of the embryos is obtained first by examining the exterior of whole embryos and studying dissections and reconstructions of them. Finally, each embryo is studied in serial sections, the level of each section being determined by comparing it with figures of the whole embryo. Along with his study of the embryos in the laboratory, the student should do a certain amount of supplementary reading. Only the gist of human organo- genesis is contained in the following chapters. A very complete bibliography of the subject is given in Keibel and Mall's "Human Embryology," to which GROWTH AND DIFFERENTIATION OF THE EMBRYO 15 the student is referred. Below are given the titles of some of the more impor- tant works on vertebrate and human embryology, to which the student is referred and in some of which supplementary reading is required. TITLES FOR REFERENCE Duval, M. Atlas D'Embryologie. Masson, Paris. His, W. Anatomie menschlicher Embryonen. Vogel, Leipzig, 1885. Keibel, F. Normentafel zur Entwicklungsgeschichte der Wirbelthiere. Bd. I. Fischer. Jena, 1897. Keibel and Elze. Normentafel zur Entwicklungsgeschichte des Menschen, Jena, 1908. Keibel and Mall. Human Embryology. Lippincott, 1910-1912. Kollmann, J. Handatlas der Entwicklungsgeschichte des Menschen. Fischer, Jena, 1907. Lee, A. B. The Microtomist's Vade Mecum. Blakiston, Philadelphia. Lewis, F. T. Anatomy of a 12 mm. Pig Embryo. Amer. Jour. Anat., vol. 2. Minot, C. S. A Laboratory Textbook of Embryology. Thyng, F. W. The Anatomy of a 7.8 mm. Pig Embryo. Anat. Record, vol. 5. Wilson, E. B. The Cell in Development and Inheritance. Macmillan, New York. CHAPTER I THE GERM CELLS: MITOSIS, MATURATION AND FERTILIZATION THE GERM CELLS The human organism with its various tissues composed each of aggregations of similar cells is, like that of all other vertebrates, developed from the union of two germ cells, the ovum and spermatozoon. The Ovum. — The female germ cell or ovum is a typical animal cell pro- duced in the ovary [for structure of typical cell see histologic texts]. It is nearly spherical in form and possesses a nucleus with nucleolus, chromatin network, chromatin knots, and nuclear membrane (Fig. i). The cytoplasm of the ovum is distinctly granular, containing more or less numerous yolk granules and a minute centrosome. The nucleus is essential to the life, growth, and reproduction of the cell. The function of the nucleolus is unknown; the chromatin probably bears the hereditary qualities of the cell. The yolk granules, containing a fatty substance termed lecithin, furnish nutrition for the early development of the embryo. A relatively small amount of lecithin is found in the ova of mammals, the embryo developing within, and being nourished by, the uterine wall of the mother. It is much larger in amount in the ova of fishes, amphibia, reptiles, birds, and the primitive mammalia, the eggs of which are laid and develop outside of the body. The so-called yolk of the hen's egg (Fig. 2) is the ovum proper and its yellow color is due to the large amount of lecithin which it con- tains. The albumen, egg-membrane, and shell of the hen's egg are secondary envelopes of the ovum. The human ovum is of small size, measuring from 0.22 to 0.25 mm. in diam- eter (Fig. i A) . The cytoplasm is surrounded by a relatively thick radially striated membrane, the zona pellucida. The striated appearance of the zona pellucida is said to be due to fine canals which penetrate it and through which nutriment is carried to the ovum by smaller follicle cells during its growth within the ovary. The origin and growth of the ovum within the ovary are known as oogenesis, and will be described in Chapter VIII. We may state here that each growing ovum is at first surrounded by small nutritive cells known a.1*, follicle cells. These increase 2 17 l8 THE GERM CELLS: MITOSIS. MATURATION AND FERTILIZATION FIG. i A. Vitelline ;- membrane A f Zona. . pellucida. Nucleu ••?»• ,«g FIG. i B. ..Jfc,' ^r»K«wr '•w? FIG. i. — A, Human ovum examined fresh in the liquor folliculi (Waldeyer). The zona pellucida is seen as a thick, clear girdle surrounded by the cells of the corona radiata. The egg itself shows a central granular deutoplasmic area and a peripheral clear layer, and encloses the nucleus in which is seen the nucleolus; B, ovum of monkey. X 430. THE GERM CELLS FIG. 2.- — Diagrammatic longitudinal section of an un- incubated hen's egg (after Allen Thomson, in Heisler). (Somewhat altered) : b.l, germinal area; w.y, white yolk, which consists of a central flask-shaped mass, and a num- ber of concentric layers surrounding the yellow yolk (y.y.)', v.t, vitelline membrane; x, a somewhat fluid al- buminous layer which immediately envelops the yolk; w, albumen, composed of alternating layers of more and less fluid portions; ch.l, chalazae; a.ch, air-chamber at the blunt end of the egg — simply a space between the two layers of the shell-membrane; i.s.m, inner, s.m, outer layer of the shell-membrane; s, shell. FIG. 3. — Section of human ovary, including cortex; a, germinal epithel- ium of free surface; b, tunica albugi- nea; c, peripheral stroma containing immature Graafian follicles (d); e, well-advanced follicle from whose wall membrana granulosa has partially separated; /, cavity of liquor folli- culi; g, ovum surrounded by cell-mass constituting cumulus oophorus (Pier- sol). FIG. 4. — Section of well-developed Graafian follicle FIG. 5. — Ovary with mature Graafian from human embryo (von Herff); the enclosed ovum follicle about ready to burst (Ribemont- con tains two nuclei. Dessaignes). 20 THE GERM CELLS: MITOSIS, MATURATION AND FERTILIZATION in number during the growth of the ovum until several layers surround it (Fig. 3) . A cavity appearing between these cells becomes filled with fluid and thus forms a sac, the Graafian follicle, within which the ovum is eccentrically located. The cells of the Graafian follicle immediately surrounding the ovum form the corona radiata (Fig. i) when the ovum is set free. Ovulation and Menstruation. — When the ovum is ripe, the Graafian follicle is large and contains fluid, probably under pressure. The ripe follicles form bud-like projections at the surface of the ovary (Fig. 5) , and at these points Ovum Follicle cells T •"& ::--9'-»^*^Af9"iLfaf-»^St^. £*) 1 j4tf/#3liSra1lJ9££> Zona pellncida FIG. 6. — Immature follicle containing several ova. From the ovary of a young monkey. X 430. the ovarian wall has become very thin. It is probable that normally the bursting of the Graafian follicle and the discharge of the ovum are periodic and associated with the phenomena of menstruation. That ovulation or discharge of the ovum from the ovary may occur independent of the menstrual periods has been proven by the observations of Leopold. Also in young girls ovulation may precede the inception of menstruation and it may occur in women some time after the menopause. At birth, or shortly after, all of the ova are formed in the ovary of the female THE GERM CELLS 21 child. Hensen estimates that a normal human female may develop in each ovary 200 ripe ova. Most of the young ova, which may number 50,000, degenerate and never reach maturity. At ovulation but one ovum is normally ripened and dis- charged from the ovary. Several ova, however, may be produced in Such Head y GaJea Neck Body , end ring Main segment of Tail* -intend of Knob -Post end of Knob .Spiral fibers ^Sheath of axial thread a single follicle in rare cases, multiple follicles have been ob- served in human ovaries and are of frequent occurrence in the ovary of the monkey. Fig. 6 shows such a follicle containing five immature ova. The Spermatozoon. — The male cell or spermatozoon is a minute cell 0.05 mm. long, specialized for active movement. Because of their active movements, spermatozoa were, when first discovered, regarded as para- sites living in the seminal fluid. The sperm cell is composed of a flattened head, indistinct neck piece, and thread- like tail (Fig. 7). The head is about 5 micra in length. It appears oval in side view, pear-shaped in profile. When stained, the anterior two-thirds of the head may be seen to form a cap, and the sharp border of this cap is the perforatorium by means of which the spermatozoon penetrates the ovum. The head contains the nu- clear elements of the sperm cell. The neck is said to be disc-shaped and to contain the centrosomes as the anterior and posterior centro- some bodies. The tail is divided into a short connecting piece, a flagellum which forms about four-fifths of the length of the sperm cell and a short end-piece (Fig. 7). The connecting piece is marked off from the flagellum by the annulus. ~/Jxial thread -Capsule Terminal filament FIG. 7. — Diagram of a human spermato- zoon, highly magnified, in side view (Meves, Bonnet). 22 THE GERM CELLS: MITOSIS, MATURATION AND FERTILIZATION It is traversed by the axial filament (filum principale), and surrounded (i) by the sheath common to the flagellum; (2) by a sheath containing a spiral filament; and (3) by a mitochondria sheath. The flagellum is composed of an axial filament surrounded by a cytoplasmic sheath and the end-piece is the naked continuation of the axial filament. The spermatozoa are motile, being propelled by the movements of the tail. They swim always against a current at the rate of about 25 micra per second, or i mm. every forty seconds. This is important, as the outwardly directed cur- rents induced by the ciliary action of the uterine tubes and uterus direct the sper- matozoa by the shortest route to the infundibulum. Keibel has found sperma- tozoa alive three days after the execution of the criminal from whom they were obtained. They have been found motile in the vagina twelve to seventeen days after coitus. They have been kept alive eight days outside the body by arti- ficial means. It is not known for how long a period they may be capable of fer- tilizing ova but, according to Keibel, this period would be certainly more than a week. MITOSIS AND AMTTOSIS Before the discharged ovum can be fertilized by the male germ cell, it must undergo a process of cell division and reduction of chromosomes known as matu- ration. As the student may not be familiar with the processes of cell division, a brief description may be necessary. (For details of mitosis see text-books of histology and E. B. Wilson's "The Cell".) Amitosis. — Cells may divide directly by the simple fission of their nuclei and cytoplasm. This process is called amitosis. Amitosis is said to occur only in moribund cells. It is the type of cell division found in the epithelium of the bladder. Mitosis. — In the reproduction of normally active cells, complicated changes take place in the nucleus. These changes give rise to thread-like structures, hence the process is termed mitosis (thread) in distinction to amitosis (no thread). Mitosis is divided for convenience into four phases (Fig. 8). Prophase (Fig. 8, I-III). — i. The centrosome divides and the two minute bodies resulting from the division move apart, ultimately occupying positions at opposite poles of the nucleus. 2. Astral rays appear in the cytoplasm about each centreole. They radiate from it and the threads of the central or achromatic spindle are formed between the two asters, thus constituting the amphiaster (Fig. 8, II). MITOSIS AND AMITOSIS III. 3. The nuclear membrane and nucleolus disappear, the nucleoplasm and cytoplasm becoming continuous. 4. During the above changes the chromatic network of the resting nucleus resolves itself into a skein or spireme, the thread of which soon breaks up into distinct, heavily-staining bodies, the chromosomes. A definite number of chromo- somes is always found in the cells of a given species. The chromosomes may be block- shaped, rod-shaped, or bent in the form of a U. 5. The chromosomes ar- range themselves in the equa- torial plane of the central spindle. If U-shaped the base of each U is directed toward a common center. The amphiaster and the chro- mosomes together constitute a mitotic figure and at the end of the prophase this is called a monaster. Metaphase. — The longi- tudinal splitting of the chro- mosomes into exactly similar halves constitutes the meta- phase (Fig. 8, IV, V). The aim of mitosis is thus accom- A. /' '••-. A plished, an accurate division Flc-'s'-Diagr^ Of the phases of m'i'tosis (Schafer). of the chromatin between the nuclei of the daughter cells. Anaphase. — At this stage the two groups of daughter chromosomes separate and move up along the central spindle fibers, each toward one of the two asters. Hence this is called the diaster stage (Fig. 8, VI). At this stage, the centrioles may each divide in preparation for the next division of the daughter cells. Telophase (Fig. 8, VII, VIII). — i. The daughter chromosomes resolve them- vir. . 24 THE GERM CELLS: MITOSIS, MATURATION AND FERTILIZATION selves into a reticulum and daughter nuclei are formed. 2. The cytoplasm di- vides in a plane perpendicular to the axis of the mitotic spindle. Two complete daughter cells have thus arisen from the mother cell. The complicated processes of mitosis, by which cell division is brought about normally, seem to serve the purpose of accurately dividing the chromatic sub- stance of the nucleus in such a way that the chromatin of each daughter cell may be the same qualitatively and quantitatively. This is important if we assume that the chromatic particles of the chromosomes bear the hereditary qualities of the cell. The number of chromosomes is constant in the sexual cells of a given species. The number for the human cell is in doubt. It has been given as 16, 24, and 32. According to Winiwarter's recent work, the number of chromosomes in each immature ovum or oocyte is 48, in each spermatogone 47. Wiemann (Amer. Jour. Anat., vol. 14, p. 461) finds the number of chromosomes in various human somatic cells varies from 34 to 38. In species of Ascaris megalocephala, a parasitic worm, but two or four chromosomes are found and in their cells the processes of mitosis are most easily observed. We have seen that reproduction in mammals is dependent upon the union of male and female germ cells. The union of two germinal nuclei (pronuclei) would necessarily double the number of chromosomes in the fertilized ovum and also the number of hereditary qualities which their particles are supposed to bear. This multiplication of hereditary qualities is prevented by the processes of matu- ration which take place in both the ovum and spermatozoon. MATURATION Maturation may be denned as a process of cell-division during which the number of chromosomes in the germ cells is reduced to one-half the number characteristic for the species. The spermatozoa take their origin in the germinal epithelium of the testis. Their development, or s per mato genesis, may be studied in the testis of the rat; their maturation stages in the testis tubes of Ascaris. Two types of cells may be recognized in the germinal epithelium of the seminiferous tubules, the sustentacu- lar cells (of Sertoli), and the male germ cells or s per mato gonia (Fig. 9). The spermatogonia divide, one daughter cell forming what is known as a primary spermatocyte. The other daughter cell persists as a spermatogone and, by con- tinued division during the sexual life of the individual, gives rise to other primary spermatocytes. The primary spermatocytes correspond to the ova before matu- ration. Each contains the number of chromosomes typical for the male of the species. The process of maturation consists in two cell divisions of the primary MATURATION FIG. 9. — Diagram showing cycle of phases in the spermatogenesis of the rat (Schafer, Brown). The numbered segments of the circle represent portions of different seminiferous tubules, a, spermato- gonia; a', sustentacular cells; b, spermatocytes actively dividing in 5; c, spermatids forming an irregular clump in i, 6, 7 and 8 and connected to sustentacular cell a' in 2, 3, 4 and 5. In 6, 7 and 8 advanced spermatozoa of one generation are seen between spermatids of the next generation, s', parts of sperma- tids which disappear when sperms are fully formed; s, seminal granules representing disintegration of s'; a", in i and 2 are atrophied sustentacular cells. C E, 3. 10. — Diagrams of the development of spermatozoa (after Meves in Lewis-Stohr) ; a.c., an- terior centrosome; a./., axial filament; c.p., connecting piece; ch.p., chief piece; g.c., galea capitis; n, nucleus; nk., neck; p., protoplasm; p.c., posterior centrosome. 26 THE GERM CELLS! MITOSIS, MATURATION AND FERTILIZATION spermatocytes, each producing first, two secondary spermatocytes, and these in turn four cells known as spermatids. During these cell divisions the number of chromosomes is reduced to half the original number, the spermatids possessing just half as many chromosomes as the spermatogonia. Each spermatid now be- K L FIG. ii. — Reduction of chromosomes in spermatogenesis in Ascaris megalocephala (bivalens) (Brauer, Wilson). A-G, successive stages in the division of the primary spermatocyte. The original reticulum undergoes a very early division of the chromatin granules which then form a doubly split spireme (B~). This becomes shorter (C) and then breaks in two to form two tetrads (Z>) in profile, (E, in end). F, G, H, first division to form two secondary spermatocytes, each receiving two dyads. 7, secondary spermatocyte. /, K, the same dividing. L, two resulting spermatids, each containing two single chromosomes. comes transformed into a mature spermatozoon (Fig. 10), the nucleus forming the larger part of the head, the centrosome dividing and lying in the neck or middle piece. The posterior centrosome is prolonged to form the axial filament, and the cytoplasm forms the sheaths of the middle piece and tail. MATURATION The way in which the number of chromosomes is reduced may be seen in the spermatogenesis of Ascaris (Fig. n). Four chromosomes are typical for Ascaris megalocephala bivalens and each resting primary spermatocyte contains this number. When the first maturation spindle appears only two chromosomes are formed, but each of these is double, so four are really present. Each represents the union of two chromosomes, shows a quadruple structure, and is termed a tetrad (Fig. 1 1 E, F) . At the metaphase (G) the two tetrads split each into two chromosomes which already show evidence of longitudinal fission and are termed dyads. . One pair of dyads goes to each of the daughter cells, or secondary sper- Spermatogonium Proliferation period Growth period Maturation period 1234 1234 FIG. 12. — Diagrams of maturation, spermatogenesis and oogenesis (Boveri). matocytes (Fig. n G, I). Before the formation of a nuclear membrane, the second maturation spindle appears at once, the two dyads split into four monads, and each daughter spermatid receives two single chromosomes, or one-half the number characteristic for the species. A diagram of maturation in the male As- caris is shown in Fig. 12 A. The first maturation division is reductional, each daughter nucleus receiving two complete chromosomes of the original four, whereas in the second maturation division as in ordinary mitosis, each daughter nucleus receives a half of each of the two chromosomes, these being split lengthwise. In the latter case the division is equational, each daughter nucleus receiving chro- mosomes bearing similar hereditary qualities. In many insects and some ver- 28 THE GERM CELLS: MITOSIS, MATURATION AND FERTILIZATION tebrates it has been shown that the number of chromosomes in the oogonia is even, the number in the spermatogonia odd, and that all the mature ova and half the spermatids contain an extra or accessory chromosome (see p. 32). Previous to fertilization, the ova undergo a similar process of maturation. Two cell divisions take place but with this difference, that the cleavage is un- equal and, instead of four cells of equal size resulting, there are formed one large ripe ovum or oocyte and three rudimentary or abortive ova known as polar bodies or polocytes. The number of chromosomes is reduced in the same manner as in the spermatocyte, so that the ripe ovum and each polar body contain one-half the number of chromosomes found in the immature ovum or primary oocyte. The female germ cells, from which new ova are produced by cell division, are called oogonia and their daughter cells after a period of growth within the ovary are the primary oocytes, comparable to the primary spermatocytes of the male. During maturation the ovum and first polocyte are termed secondary oocytes (comparable to secondary spermatocytes), the mature ovum and second polocyte, with the daughter cells of the first polocyte, are comparable to the spermatids (see diagram B, Fig. 12). Each spermatid, however, may form a mature sper- matozoon, but only one of the four daughter cells of the primary oocyte becomes a mature ovum. The three polocytes are abortive and degenerate eventually, though it has been shown that in the ova of some insects the polar body may be fertilized and segment several times like a normal ovum. The maturation of human ova has not been observed, but such a process probably takes place. The reduction of the chromosomes may be best observed in the ova of Ascaris and of insects. The mouse offers a favorable opportunity for studying the maturation of a mammalian egg as the ova are easily obtained. Their maturation stages have recently been studied by Mark and Long (Carnegie Inst. Publ. No. 142). Maturation of the Mouse Ovum. — The nucleus of the mature ovum is known as the female pronucleus. When the spermatozoon penetrates the mature ovum it loses its tail and its head becomes the male pronucleus. The aim and end of fertilization consists in the union of the chromatic elements contained in the male and female pronuclei and the initiation of cell division. In the mouse, the first polocyte is formed while the ovum is still in the Graafian follicle. In the forma- tion of the maturation spindle no astral rays and no typical centrosomes have been observed. The chromosomes are V-shaped. The first polar body is seg- mented from the ovum and lies beneath the zona pellucida as a spherical mass about 25 micra in diameter (Fig. 13). Both ovum and polar body (secondary oocytes) contain 10 or 12 chromosomes, or half the number normal for the mouse. MATURATION (According to Mark and Long, the chromosomes number 20.) The first matura- tion division is the reductional one and the chromosomes take the form of tetrads. After ovulation has taken place, the ovum lies in the ampulla of the uterine tube. If fertilization takes place, a second polocyte is cut off, the nucleus of the FIG. 13. — Maturation and fertilization of the ovum of the mouse. A, C-J, X 500; B X 750. (after Sobotta). A-C, entrance of the spermatozoon and formation of the second polar body. D-E, develop- ment of the pronuclei. F-J, successive stages in the first division of the fertilized ovum. ovum forming no membrane between the production of the first and second polar bodies (Fig. 13 A-D). The second maturation spindle and second polar body are smaller than the first. Immediately after the formation of the second polar body, the chromosomes resolve themselves into a reticulum and the female pro- nucleus is formed (Fig. 30 THE GERM CELLS: MITOSIS, MATURATION AND FERTILIZATION Fertilization of the Mouse Ovum. — Normally, a single spermatozoon enters the ovum six to ten hours after coitus. While the second polar body is forming, the spermatozoon penetrates the ovum and loses its tail. Its head is converted into the male pronucleus (Fig. 13 D). The pronuclei, male and female, approach each other and resolve themselves into a spireme stage, then into two groups of 1 2 chromosomes. A centrosome, possibly that of the male cell, appears between them, divides into two, and soon the first segmentation spindle is formed. The 1 2 male and 12 female chromosomes arrange themselves in the equatorial plane of the spindle, thus making the original number of 24 (Fig. 13 I). Fertilization is now complete and the ovum divides in the ordinary way. The fundamental results of the process of fertilization are (i) the union of the male and female chromosomes, (2) the initiation of cell division or cleavage of the ovum. These two factors are separate and independent phenomena. It has been shown by Boveri and others that fragments of sea-urchin's ova containing no part of the nucleus may be fertilized by spermatozoa, segment and develop into larvae. The female chromosomes are thus not essential to the process of segmentation. Loeb, on the other hand, has shown that the ova of invertebrates may be made to segment by chemical and mechanical means without the cooperation of the spermatozoon. It is well known that the ova of certain invertebrates develop normally with or without fertilization (parthenogenesis). These facts show that the union of the male and female pronuclei is not the means of initiating the development of the ova. In all vertebrates it is, nevertheless, the end and aim of fertilization. Lillie (Science, vols. 36 and 38, pp. 527-530 and 524-528) has recently shown that the cortex of sea-urchin's ova produces a substance which he terms fertilizin. This substance he regards as an amboceptor essential to fertilization with one side chain which agglutinates and attracts the spermatozoa, another side chain which activates the cytoplasm and initiates the segmen- tation of the ovum. Spermatozoa may enter the mammalian ovum at any point. If fertilization is delayed and too long a period elapses after ovulation, the ovum may be weak- ened and allow the entrance of several spermatozoa. This is known as poly- spermy. The fertilization of the human ovum has not been observed, but probably takes place in the uterine tube some hours after coitus. Ova may be fertilized and start developing before they enter the uterine tube. If they attach themselves to the peritoneum of the abdominal cavity, they give rise to abdominal pregnancies. If the ova develop within the uterine tube tubular pregnancies result. Normally, the embryo begins its development in the uterine tube, thence passes into the uterus and becomes embedded in the uterine mucosa. The time required for the passage of the ovum from the uterine tube to the uterus is unknown. It probably varies in different cases and may occupy a week or more. The ovum may in some cases be fertilized within the uterus. Fertilization is favored by the fact that the spermatozoa swim always against a current. As the cilia of the uterus and uterine tube beat downward and outward the sperms are directed upward and inward. They may reach the uterine tubes within two hours of a normal coitus. DETERMINATION OF SEX 31 *"^ Usually but one human ovum is produced and fertilized at coitus. The de- velopment of two or more embryos within the uterus may be due to the ripening and expulsion of an equal number of ova at ovulation, these being fertilized later. Identical twins are regarded as arising from the daughter cells of a fertilized ovum, these cells having separated, and each having developed like a normal ovum. The Significance of Mitosis, Maturation and Fertilization. — It is assumed by students of heredity that the chromatic particles of the nucleus bear the hereditary qualities of the cell. During the course of development these particles are probably distributed to the various cells in a definite way by the process of mitosis. The process of fertilization would double the number of hereditary qualities and they would be multiplied indefinitely were it not for maturation. At maturation not only is the number of chromosomes halved, but it is assumed also that the number of hereditary qualities is reduced by half. In the case of the ovum, this takes place at the expense of three potential ova, the polocytes, which degenerate, but is to the advantage of the single mature ovum which retains m^re than its share of cytoplasm and nutritive yolk. Mendel's Law of Heredity. — Experiments show that all hereditary characters fall into two opposing groups, which alternate with each other and are termed allelomorphs. As an example, we may take the hereditary tendencies for black and blue eyes. It is supposed that there are paired chromatic particles in the germ cells which bear these hereditary tendencies. Each pair may be composed of similar particles, both bearing black-eyed tendencies or both blue- eyed tendencies, or opposing particles may bear the one black, the other blue-eyed tendencies. It is assumed that at maturation these paired particles are separated, and that one only of each pair is retained in each germ cell, in order that new and favorable combinations may be formed at fertilization. In our example, either a blue-eyed or a black-eyed tendency bearing particle would be retained. At fertilization the segregated tendency-bearing particles of one sex may enter into new combinations with the allelomorphs of the other sex, combinations which may be favorable to the offspring. Three combinations may be possible. If the color of the eyes is taken as the hereditary character, (i) two "black" germ cells may unite; (2) two "blue" germ cells may unite; (3) a "black" germ cell may unite with a "blue" germ cell. The result- ing individual will be in (i) black-eyed; in (2) blue-eyed; in (3) either black-eyed or blue-eyed, according to whether one or the other tendency predominated. Were the black-eyed tendency in (3) predominant and the resulting individual black-eyed, there would still be blue-eyed bearing chromatin particles in his or her germ cells. In the next generation these recessive blue-eyed qualities may unite with similar qualities of another black-eyed individual. The offspring would be blue-eyed, though both the parents were black-eyed. DETERMINATION OF SEX The assumption that the chromosomes are the carriers of hereditary ten- dencies is borne out by the observations of cytologists on the germ cells of insects and some vertebrates. It has been shown that in some forms the nucleus of the spermatogonia contain 23 chromosomes, while those of the oogonia contain 24. When maturation and reduction of the chromosomes take place, half of the sper- matids contain 1 2 chromosomes, the other half only eleven, while all the oocytes THE GERM CELLS: MITOSIS, MATURATION AND FERTILIZATION and polocytes contain 12. There is thus one extra chromosome in each matur ovum and in each of half the spermatozoa. This chromosome is larger than the others in some insects, and is termed the accessory chromosome. McClung wa the first to assume that the accessory chromosome was a sex determinant. It ha since been shown by Wilson, Davis, and others that the accessory chromosome carries the female sexual characters. When the spermatozoan with 12 chromo- somes fertilizes an ovum, the resulting embryo is a female, its somatic nuclei containing 24 chromosomes. An ovum fertilized by a sperm cell containing only ii chromosomes (without the accessory chromosome) produces a male with so- matic nuclei containing only 23 chromosomes. Winiwarther (Arch. d. Biol. Bd. 27) has recently made similar observations on the human germ cells but they have yet to be confirmed by other investigators. It is probable, however, that sex is transmitted by the human chromosomes in much the same way as in insects. : CHAPTER II SEGMENTATION OF THE FERTILIZED OVUM AND ORIGIN OF THE GERM LAYERS SEGMENTATION The processes of segmentation, not having been observed in human ova, must be studied in other vertebrates. It is probable that the early development of all vertebrates is, in its essentials, the same. It is modified, however, by the presence in the ovum of large quantities of nutritive yolk. In many vertebrate ova the yolk collects at one end, the vegetal pole. Such ova are said to be telolecithal. Examples are the ova of Amphioxus, the frog and bird. When very little yolk is present, the ovum is said to be alecithal (no yolk). Examples are the ova of the higher mammals and man. The typical processes of cleavage may be studied most easily in the fertilized ova of invertebrates (Echinoderms, Annelids, and Mollusks). Among Chordates, the early processes in develop- ment are primitive in a fish-like form Amphioxus. The yolk modifies the development of the amphibian and bird's egg, while the early structure of the mammalian embryo can be explained only by assuming that the ova of the higher Mammalia at one time contained a considerable amount of yolk like the ovum of the bird and of the lower mammals. Amphioxus. — The ovum is telolecithal, but contains little yolk (Fig. 14). About one hour after fertilization it divides vertically into two nearly equal daugh- ter cells. The process is known as cell cleavage, or segmentation and takes place by mitosis. Within the same interval of time the daughter cells cleave in the same plane, forming four cells. Fifteen minutes later a third segmentation takes place in a horizontal plane. As the yolk is more abundant at the vegetal poles of the four cells the spindle lies nearer the animal pole. Consequently in the eight- celled stage the upper tier of four cells is smaller than the lower four. By suc- cessive cleavages, first in the vertical, then in the horizontal plane a 16- and 32- celled embryo is formed. The upper two tiers are now smaller and a cavity, the blastocoel, is enclosed by the cells. The embryo is called a morula (mulberry). In subsequent cleavages, as development proceeds, the size of the cells is di- minished while the cavity enlarges (Fig. 14). The embryo is now a blastula, 3 33 34 SEGMENTATION OF THE FERTILIZED OVUM nearly spherical in form and about four hours old. The cleavage of the Amphioxus ovum is thus complete and somewhat unequal. FIG. 14. — Segmentation of the egg of Amphioxus, X 220 (after Hatschek). i. The egg before the commencement of development; only one polar body, P.B, has been seen, but from analogy with other animals it is probable that there are really two present. 2. The ovum in the act of dividing, by a vertical cleft, into two equal blastomeres. 3 . Stage with four equal blastomeres. 4. Stage with eight blastomeres ; an upper tier of four slightly smaller ones and a lower tier of four slightly larger ones. 5. Stage with sixteen blastomeres in two tiers, each of eight. 6. Stage with thirty-two blastomeres, in four tiers, each of eight; the embryo is represented bisected to show the segmentation cavity or blastocoel, B. 7. Later stage: the blastomeres have increased in number by further division. 8. Blastula stage bisected to show the blastocoel, B. The Ovum of the Frog. — The ovum contains so much yolk that the nucleus and most of the cytoplasm lies at the upper or animal pole. The first cleavage spindle lies in this cyto- plasm. The first two cleavage planes are vertical and the four resulting cells are nearly equal SEGMENTATION (Fig. 15). The spindles for the third cleavage are located near the animal pole and the cleavage takes place in a horizontal plane. As a result, the upper four cells are much smaller than the lower four. The large yolk-laden cells divide more slowly than the upper small cells. At the blastula stage, the cavity is small, and the cells of the vegetal pole are each many times larger than those at the animal pole. The cleavage of the frog's ovum is thus complete but unequal. 4 5 FIG. 15. — Segmentation of the frog's ovum (Hatschek in Marshall). B, segmentation cavity; U, nucleus. Ova of Reptiles and Birds. — The ova of these vertebrates contain a large amount of yolk. There is very little pure cytoplasm except at the animal pole 36 SEGMENTATION OF THE FERTILIZED OVUM and here the nucleus is located (Fig. 2). When segmentation begins, the first cleavage plane is vertical but the yolk, being lifeless matter, does not cleave. The segmentation is thus incomplete or meroblastic. In the hen's ovum the cy- toplasm is divided by successive vertical furrows into a mosaic of cells which, as it increases in size, forms a cap-like structure upon the surface of the yolk. These cells are separated from the yolk beneath by horizontal cleavage furrows, and successive horizontal cleavages give rise to several layers of cells. The space between cells and yolk mass may be compared to the blastula cavity of Am- phioxus and the frog (Fig. 17). The cellular disc or cap is termed the germinal area or disc. The yolk mass which forms the floor of the blastula cavity and the greater part of the ovum may be compared to the large yolk-laden cells at the vegetal pole of the frog's blastula. The yolk mass never divides, but is gradu- ally used up in supplying nutriment to the embryo which is developed from the cells of the germinal area. Round the periphery of the germinal area new cells constantly form until they surround the yolk. The Ovum of the Rabbit. — The ovum of all the higher mammals, like that of man, is microscopic in size and nearly alecithal (no yolk). Its segmentation has been studied in several mammals but we shall take the rabbit's ovum as an example. The cleavage is complete and nearly equal (Fig. 16), a cluster of nearly equal cells being formed within the zona pellucida. This corresponds to the morula stage of Amphioxus. Next an inner mass of cells is formed which corre- sponds to the germinal area, or blastoderm, of the chick embryo (Fig. 16). The inner cell mass is overgrown by an outer layer which we term the troph-ectoderm because, in mammals, it supplies nutriment to the embryo from the uterine wall. Between the outer layer and the inner cell mass fluid next appears, separating them except at the animal pole. As the fluid increases in amount, a hollow vesicle results, its wall composed of the single-layered troph-ectoderm except where this is in contact with the inner cell mass. This stage is known as the germinal or Uastodermic vesicle. It is usually spherical or ovoid in form, as in the rabbit, and probably this is the form of the human ovum at this stage. In the rabbit it is of macroscopic size before it becomes embedded. Among Ungulates (hoofed animals) the vesicle is greatly elongated and attains a length of several centi- meters, as in the pig. If we compare the mammalian blastodermic vesicle with the blastula stages of Amphioxus, the frog and the bird, it will be seen that it is to be homologized with the bird's blastula, not with that of Amphioxus (Fig. 17). In each case there is an inner cell mass of the germinal area. The troph-ectoderm of the Outer cell. Outer cells. Polar bodies Inner cell Inner celh Inner cells. Outer cells. Inner cells Inner cells. Outer cells. FIG. 16. — Diagrams showing the segmentation of the mammalian ovum and the formation of the blasto- dermic vesicle (Allan Thomson, after van Beneden). THE FORMATION OF THE ECTODERM AND ENTODERM 37 mammal represents a precocious development of cells which, in the bird, later envelop the yolk. The cavity of the vesicle is to be compared, not with the blastula cavity of Amphioxus and the frog but with the yolk mass plus the rudi- mentary blastocoel of the bird's ovum. The mammalian ovum, although almost devoid of yolk, thus develops much like the yolk-laden ova of reptiles and birds. Its segmentation, however, is complete and the early stages in its development are abbreviated. Blastula cavity ( o I o D Yolk cavity MG. 17. — Diagrams showing the blastulae: A, of Amphioxus; B, of frog, and C of chick; D, blastodennic vesicle of mammal. In Primates, but one stage in segmentation has been observed. This, a four-celled ovum of Macacus nemestrinus figured by Selenka, shows the cells nearly equal and oval in form. This ovum was found in the oviduct of the monkey and shows that, in Primates and probably in man, segmentation as in other mammals takes place normally in the oviducts. THE FORMATION OF THE ECTODERM AND ENTODERM The blastula and early blastodermic vesicle show no differentiation into layers. Such differentiation takes place later in all vertebrate embryos and the three primary germ layers, ectoderm, entoderm and mesoderm, are formed. From these three layers all of the body tissues and organs are derived. Gastrulation. — In the case of Amphioxus and amphibia the entoderm is SEGMENTATION OF THE FERTILIZED OVUM formed by a process termed gastrulation. The larger cells at the vegetal the blastula either fold inward (invaginate) or are overgrown by the more dividing micromeres. Eventually the invaginating cells obliterate the blastula cavity and come into contact with the outer layer of cells (Fig. 18). The new cavity formed is the primitive gut, or archenteron. The mouth of this cavity is the blastopore. The outer layer of cells is the ectoderm, the inner, newly formed layer is the ento- derm. The entodermal cells are henceforth concerned in the nutrition and metabolism of the body. The embryo is now termed a Gastrula (little stomach). The Origin of the Entoderm in Reptilia, Birds and Mammals. — Here the entoderm arises in quite a differ- ent manner. Instead of a process of gastrulation by in- vagination of cells we have first a process of delamination. Cells are split off or delaminated from the under side of the germinal area, arrange themselves in a definite inner layer, and thus the yolk entoderm is formed. This layer is already apparent in a longitudinal section through the germinal area of a chick (Fig. 19). In mammals like the pole of rapidly FIG. 18. — Gastrulation of amphioxus (modified from Hatschek). A, Blastula; az, animal cells; m, vegetative cells; fh, cleavage-cavity. B, Beginning invagination of vegetative pole. C, Gastrula stage, the invagination of the vegetative cells being complete; ak, outer germ- layer; ik, inner germ-layer; ud, archenteron; u, blastopore (Heisler). 1 THE FORMATION OF THE ECTODERM AND ENTODERM rabbit, the entoderm is split from the under side of the germinal area and the cells soon grow around the inside of the blastodermic vesicle, and form an inner entodermal sac (Fig. 16). In Tarsius, a creature classed by Hubrecht Ectoderm of embryonic disc Ectoderm Yolk entoderm Blastopore Ectoderm ' Completion plate " Protentoderm Yolk entoderm Blastopore Peristomal mesoderm Completion plate " :$pN&MiiS - •••«•»•»» '' Completion plate " Yolk entoderm "Completion plate " FIG. 20. — From medial vertical sections through embryonic disk of lizard, showing five successive stages in gastrulation (Wenckeback, Bonnet). with the Primates, the entoderm cells, after splitting off, do not grow around the wall of the vesicle as in the rabbit, but soon form an entodermal sac separated by a space from the troph-ectoderm layer. Just how the vesicle is SEGMENTATION OF THE FERTILIZED OVUM formed is not known. It is attached only to the cells of the germinal area. Although this stage has not been observed in the human embryo it is probable from the structure of the youngest known human embryo that an entodermal vesicle is formed in much the same way as in Tarsius. Gastrulation in Reptiles and Birds. — After the formation of the primary or yolk entoderm in reptiles and birds, a process of invagination takes place which has been compared to gastrulation in Amphioxus and Amphibia. In the lizard after the FIG. 21. — Two germ-discs of hen's egg in the first hours of incubation (after Roller in Heisler) : df, area opaca; hf, area pellucida; s, crescent; sk, crescent-knob; es, embryonic shield; pr, primitive groove. B primary entoderm has developed by delamination a curved depression is formed at the posterior border of the germinal area. There is a true invagina- tion of cells at this point (Fig. 20). The cells grow cephalad and contain an invagination cavity. Later the floor of the cavity fuses with the yolk entoderm and disappears. The cells of the roof persist as the dorsal or notochordal plate. The crescentic depression, at which point invagination occurs, may be compared to the blastopore of Amphioxus. The invagination cavity is the gastrula cavity or archenteron and the dorsal plate represents the entodermal roof of the gastrula cavity. In the bird, invagination takes place at a crescentic groove (Fig. 21), but the ingrowing cells form a solid plate, at first without an invagination cavity. The result is the same, however, the formation of a notochordal plate which lies beneath the ectoderm. The crescentic groove is interpreted as representing the blastopore (Fig. 19). Formation of the Primitive Streak in Birds. — The germinal area increases in size by growth about its periphery, new cells constantly being formed here until eventually the germinal area surrounds the yolk. According to the interpreta- tion of Duval and Hertwig, as the periphery of the germinal area extends itself, a middle point in the cranial lip of the crescentic groove remains fixed while the FIG. 22. — Diagram elucidating the forma- tion of the primitive groove (after Duval). The increasing size of the germ-disc in the course of the development is indicated by dot- ted circular lines. The heavy lines represent the crescentic groove and the primitive groove which arises from it by the fusion of the edges of the crescent (Heisler). THE FORMATION OF THE ECTODERM AND ENTODERM 41 edges of the lip on each side are carried caudad and brought together. Thus the crescent is transformed into a longitudinal slit, as in Fig. 26. The lips of the slit fuse, and the line of fusion is marked by the longi- tudinal primitive groove. This interpretation of the primitive groove and streak, shown in Fig. 22, is known as the con- crescence theory. According to the theory, the crescentic groove of reptiles and birds is homo- logous with the blastopore of Amphioxus. As it is trans- formed into the primitive streak and groove, these represent a modified blastopore. Accord- ing to this view, a large part of the entoderm of birds, rep- tiles, and mammals is formed by gastrulation, as in Amphi- oxus. Gastrulation in Mammals. — As in reptiles and birds so also in mammals, a process resembling gastrulation takes place but after the formation of the yolk entoderm. A primitive streak appears at the posterior border of the germinal FIG. 23. — The primitive streak of pig embryos (Kei- bel). A, embryo with primitive streak and primitive node; B, a later embryo in which the medullary groove is also present, cephalad in position. Post opening ofnotochord canal Primitive streak Ant. open iny of /Int. persisting portion of notochord canal notochorctal canal A/eurenteric canaJ FIG. 24. — Median longitudinal section through the blastoderm of a bat (Vespertilio murinus) (after Van Beneden). area (Fig. 23), with a crescentic opacity corresponding to the crescentic groove of birds. Longitudinal sections (Fig. 24) of the germinal area of the bat show the formation of a dorsal or notochordal plate which has replaced, and is fused later- ally with, the yolk entoderm. A blastopore or notochordal canal is present lead- 42 SEGMENTATION OF THE FERTILIZED OVUM ing from the dorsal surface of the germinal area into the space beneath the ento- derm, the archenteron. No gastrulation stage for the human embryo has yet been observed but the primitive streak may be recognized in later stages (Fig. 73 A). There is also evidence of an opening, the notochordal or neur enteric canal, leading from the exterior into the cavity of the primitive gut (archenteron). According to the view of Keibel and Hubrecht, the invagination of cells to form the noto- chordal plate in reptiles, birds and mammals is a secondary process not to be compared with formation of the entoderm by gastrulation, as in Amphioxus. The notochordal plate is not entodermal but ectodermal, and the primitive streak cannot be compared in its entirety to the blastopore of Amphioxus. THE ORIGIN OF THE MIDDLE GERM LAYER (Mesoderm), NOTOCHORD, AND NEURAL TUBE Amphioxus. — The dorsal plate of entoderm, which forms the roof of the archenteron, gives rise to paired lateral diverticula or ccelomic pouches (Fig. 25). These separate both from the plate of cells in the mid-dorsal line (which form the notochord}, and from the entoderm of the gut, and become the primary mesoderm. tnes. sonz. ect FIG. 25. — Origin of the mesoderm in Amphioxus (after Hatschek). n.g., neural groove; n.c., neural canal; ch., anlage of notochord; mes. som., mesodermal segment; ect., ectoderm; ent., entoderm; al., cavity of gut; coe., ccelom or body cavity. The mesodermal pouches grow ventrad and their cavities form the ccelom or body cavity. Their outer walls, with the ectoderm, form the body wall or somatopleure; their inner walls with the gut entoderm, form the intestinal wall (splanchnopleure) . In the meantime, a dorsal plate of cells cut off from the ec- toderm has formed the neural tube (anlage of central nervous system), and the notochordal plate has become a cord or cylinder of cells extending the length of the embryo (axial skeleton). In this simple fashion the ground plan of the THE ORIGIN OF THE MIDDLE GERM LAYER 43 Head process Neural groove A Primitive node Primitive groove Area opaca streak Blood island FIG. 26. — Dorsal surface view of a twenty-hour chick em- bryo showing primitive streak and extent of mesoderm (after Duval) . The lines A , B, and C indicate the levels of the cor- responding sections shown in Fig. 28. chordate body is developed. In Amphibia from the dor- sal plate of entoderm the mesodermal diverticula grow out as solid plates between ectoderm and en- toderm. Later, these plates split into two layers and the cavity so formed gives rise to the ccelom. Origin of the Meso- derm in Chick Embryos. — If we examine a chick em- bryo of twenty hours' in- cubation (Fig. 26), it will be seen that the primitive streak is formed as a linear opacity near the posterior border of the germinal area. Over a somewhat pear-shaped clear area the yolk has been dissolved away from the overlying entoderm. This area, from its appearance, is termed the area pellucida. It is surrounded by the darker and more granular area opaca. Whether or not the primitive streak represents the fused lips of the blastopore, it is certain that it' represents the point of origin for the middle germ layer. It also indicates the future longitudinal axis of the em- bryo. Proliferation of cells takes place here between ectoderm and entoderm and there grows out laterally and caud- ally between these layers a solid plate of mesoderm, as in amphibia. The shaded area in Fig. 26 shows the extent FIG. 27. — Surface view of a twenty-one- of the mesoderm. It extends at first hour chick embryo, in which the head-fold and -i, , , , ,, . ... ( . .' more rapidly caudad to the primitive first pair of primitive mesodermal segments J are present (after Duval). streak, at the cranial end of which Blood island 44 SEGMENTATION OF THE FERTILIZED OVUM appears a shaded thickening, the primitive knot or node (Hensen's). From this point it grows cranially, forming along the midline a thicker layer of tissue, the notochordal plate or head process (Fig. 26). At twenty-five hours (Fig. 31), the mesoderm forms lateral wings which extend cephalad beyond the limits of the area pellucida. The space between these wings is the proamniotic area. A transverse section through the primitive streak at twenty hours (see guide line C, Fig. 26) shows the three germ layers distinct Ectod Neural plate Mesodi •Notochorda.1 plate Cntoderm Ectoderm Primitive node En to derm Ectod, SfreaK . . -Entoderm Mesa derm FIG. 28. — Transverse sections through the embryonic area of a twenty-hour chick. A, through the head process; B, through the primitive node; C, through the primitive streak. X 165. laterally (Fig. 28 C). In the midline, a depression in the ectoderm is the primitive groove. In this region there is no line of demarcation between ectoderm and mesoderm. A transverse section through the primitive node (Fig. 28 B, guide line B, Fig. 26) shows in this region the marked proliferation of cells, which are growing cephalad to form the notochordal plate (head process) . A transverse section through the notochordal plate just beginning to form at this stage (Fig. 28 A, guide line A, Fig. 26) shows the thickening near the midline which will separate from the lateral mesoderm and form the notochord. THE ORIGIN OF THE MIDDLE GERM LAYER 45 After the notochordal plate becomes prominent at twenty hours the dif- ferentiation of the germinal area is rapid. A curved fold, involving the three layers of the germinal area, is formed cephalad to the notochordal process. This is the head-fold and is the anlage of the head of the embryo (Fig. 27). The ecto- derm has thickened on each side of the mid-dorsal line, forming the neural folds. The groove between these is the neural groove. The closure of this groove will form the neural tube, the anlage of the central nervous system. The notochord is now differentiated from the mesoderm and may be seen in the mid-dorsal line through the ectoderm. In the mesoderm lateral to the notochord and cephalad to the primitive node, transverse furrows have differentiated a pair of mesodermal segments. As development proceeds these increase in number, successive pairs being developed caudally. They will be described in detail later. To sum up, in the chick the mesoderm appears with the formation of the primitive streak. It originates from the primitive streak and node and spreads in all directions between the other germ layers as an undivided plate of cells. It grows cephalad in the midline as the notochordal process or plate from which the notochord is developed. As the mesoderm is derived from the entoderm in Amphioxus, its origin is generally regarded as entodermal in birds and mammals. This would certainly be the case if we interpret the notochordal process and entoderm as formed by a process of gastrulation. Keibel (in Keibel and Mall, vol. I), however, holds that the mesoderm and notochordal plate are derived from the ectoderm, and that any relation which they bear to the entoderm is of secondary origin. The Origin of the Mesoderm in Mammals. — As we have seen, the primitive streak is formed on the surface of the germinal area in mammalian embryos as in the chick. It has been described as due to a keel-like thickening of the ecto- derm, and the knob-like mass of cells at its cephalic end, the primitive node, is the first to appear. The mesoderm is formed precisely as in the chick, growing out in all directions from the primitive streak and node between the other two layers. Its extent in rabbit embryos is shown in Fig. 29 A and B. Cranial to the primitive node the notochord is differentiated in the midline, the meso- derm being divided into two wings. The mesoderm rapidly grows round the wall of the blastodermic vesicle until it finally surrounds it and the two wings fuse ventrally (Fig. 30 A and B). The single sheet of mesoderm soon splits into two, the cavity between being the codom or body cavity. The outer mesodermal layer (somatic), with the ectoderm, forms the somatopleure or body wall, the inner splanchnic layer, with the entoderm, forms the intestinal wall or splanchnopleure. The neural tube having in the meantime been formed from the neural folds of the 46 SEGMENTATION OF THE FERTILIZED OVUM ectoderm, we have the ground plan of the vertebrate body, the same in man as in Amphioxus. The origin of the mesoderm in the human embryo is unknown, but in Tarsius it has two sources, (i) The primary mesoderm derived by delamination from the FIG. 29. — Diagrams showing the extent of the mesoderm in rabbit embryos (Kolliker). In A the mesoderm is represented by the pear-shaped area at the caudal end of the embryonic area; in B by the circular area which surrounds the embryonic area. ectoderm at the caudal edge of the germinal area. This forms the extra-embryonic mesoderm and takes no part in forming the body of the embryo. (2) The secondary or intraembryonic mesoderm, which gives rise to body, tissues, takes Ectoderm Mesoderm Entoderm Archeriteron Fntodi rfrchenferon Splanchnic tnesoderm Coelotn FIG. 30. — Diagrams showing the origin of the germ layers of mammals as seen in transverse section (modified from Bryce). its origin from the primitive streak and node as in the chick and lower mammals. The origin of the mesoderm in human embryos is probably much the same as in Tarsius. The Notochord. — In mammals and in man the notochordal plate is described )RIGIN OF THE MIDDLE GEI as taking its origin directly from the entoderm. Keibel points out that this con- nection of the notochord is only secondary. The notochordal process grows cephalad from the primitive node and the tissue from which it is derived is of ectodermal origin, according to Keibel's view. In later stages, the notochord extends in the midline beneath the neural tube from the tail to a dorsal out-pocket- ing of the oval entoderm known as Seessel's pocket. It becomes enclosed in the centra of the vertebrae and in the base of the cranium, and eventually degenerates. In Amphioxus, it forms the only axial skeleton and it is persistent in the axial skeleton of fishes and Amphibia. In man, traces of it are found as pulpy masses in the intervertebral discs. CHAPTER III THE STUDY OF CHICK EMBRYOS In the following descriptions we shall use the terms dorsad and ventrad to indicate "towards the back" or "towards the belly"; cephalad and cranially to denote "headwards," caudad to denote "tailwards," and later ad when the loca- tion is at the side. As there is no single word in English to express the primi- tive cellular germ of a structure, the German word anlage has been adopted by embryologists and will be used here. Chick embryos may be studied whole and most of the structures identified up to the end of the second day. The eggs should be opened in normal saline solution at 40° C. With scissors, cut around the germinal area, float the embryo off the yolk and remove the vitelline membrane. Then float the embryo dorsal side up on a glass slide, remove enough of the saline solution to straighten wrinkles, and carefully place over the embryo a circle of tissue paper with opening large enough to leave the germinal area exposed. Add a few drops of fixative (5 per cent, nitric acid gives good fixation) and float embryo into a covered dish. After fix- ing and hardening, stain in acid Haematoxylin (Conklin) or in acid Carmine. Extract sur- plus stain, clear, and mount on slide supporting cover-slip to prevent crushing the embryo. Acid Hsematoxylin gives the best results for embryos of the first two days. For a detailed ac- count of embryological technique see Lee's "Microtomist's Vade Mecum." EMBRYO OF SEVEN SEGMENTS (TWENTY-FIVE HOURS' INCUBATION) In this embryo (Fig. 31) there is a prominent network of blood-vessels and blood-cells in the caudal portion of the area opaca. In its cranial portion isolated groups of blood and blood-vessel forming cells are seen as blood-islands. To- gether, they constitute the angioblast from which arises the blood vascular system. The area pellucida has the form of the sole of a shoe with broad toe directed for- ward. The head-fold has become cylindrical and the head of the embryo is free for a short distance from the germinal area. The mesoderm extends on each side beyond the head leaving a median clear space, the proamniotic area. The en- toderm is carried forward in the head-fold as the fore-gut, from which later arise the pharynx, esophagus, stomach and a portion of the small intestine. The opening into the fore-gut faces caudad and is the fovea cardiaca. The way in which the entoderm is folded up from the germinal disc and forward into the head is seen well in a longitudinal section of an older embryo (Fig. 32). The 48 EMBRYO OF SEVEN SEGMENTS 49 tubular heart lies ventral to the fore-gut and cranial to the fovea cardiaca. In later stages it is bent to the right. Converging forward to the heart on each side of the fovea are the vitelline veins just making their appearance at this stage. The lips of the neural folds have met throughout the cranial two-thirds of the embryo but have not fused. The neural tube formed thus by the closing of the ectodermal folds is open at either end. Cephalad, the neural tube has begun to Pharyn* iefi vitelline meso derma! Segment 3 Area > pelludda I\lo1o chord Area opaca Primitive direaK Anterior neuropore Forebram Free portion of head Fbvea cardiaca L l\ ff/'ght v/fe///fie I vein Neural groove Segmental zone ~ Primitive node Blood island FIG. 31. — Dorsal view of a twenty-five-hour chick embryo with seven primitive segments. X 20. expand to form the brain vesicles. Of these only the fore-brain is prominent, and from it laterally the optic vesicles are budding out. The paraxial mesoderm is divided by transverse furrows into seven pairs of primitive segments. Caudally between the segments and the primitive streak there is undifferentiated meso- derm, but new pairs of segments will develop in this region. Looking through the open neural tube (rhomboidal sinus), one may see in the midline the chorda dor- salis extending from the primitive node cephalad until it is lost beneath the 4 THE STUDY OF CHICK EMBRYOS AN BF BM SP neural tube in the region of the primitive segments. The primitive streak is still prominent at the posterior end of the area pellucida, forming about one-fourth the length of the embryo. Transverse sections through the primitive streak and open neural groove show approximately the same conditions as in the twenty-hour embryo (Figs. 26 and 28). A Transverse Section through the Fifth Primitive Segment (Fig. 33) is characterized by the differentiation of the mesoderm, the approxi- mation of the neural folds and the presence of two vessels, the descending aortae on each side between the mesodermal segments and the entoderm. The neural folds are thick and the ectoderm is thickened over the embryo. The notochord is a sharply de- nned oval mass of cells. The mesodermal segments are somewhat triangular in outline and connected by the intermediate cell mass with the lateral meso- derm. This is partially divided by irregular flat- tened spaces into two layers, the dorsal of which is the somatic, the ventral the splanchnic layer of mesoderm. Later, the spaces unite on either side to form the ccelom or primitive body cavity. Transverse Section Caudal to the Fovea Cardiaca (Fig. 34). — The section is characterized (i) by the closing together of the neural folds to form the neural tube; (2) by the dorsad and laterad folding of the entoderm which, a few sections nearer the head end, forms the fore-gut or pharynx; (3) by the presence of the vitelline veins laterally be- tween the entoderm and mesothelium; (4) by the wide separation of the somatic and splanchnic mesoderm and the consequent increase in the size of the ccelom. In this region, it later sur- rounds the heart and forms the pleuro-pericardial cavity. The neural tube in this region forms the third brain vesicle or hind-brain. The neural folds have not yet fused. Mesodermal segments do not develop in so SP FIG. 32. — Median longitu- dinal section of a thirty-six-hour chick embryo (Marshall). AN, amnion fold; BF, fore-brain; BH, hind-brain; BM, mid-brain; CH, notochord; CP, pericardial cav- ity; GF, fore-gut; H, entoderm; NS, spinal cord; NT, neurenteric canal; PS, primitive streak; RV, ventricle of heart; SO, somato- pleure; SP, splanchnopleure; TA, allantois. EMBRYO OF SEVEN SEC this region, instead a diffuse network of mesoderm partly fills the space between ectoderm, entoderm and mesothelium. This is termed mesenehyma and will be described later. Neural plate Neural groove Ectoderm \ / .Mesodertnal segment •Somatic, mesoderm \ jg^ / jfs^ / £oe/on Splanchnic, mesoderm / \ ^\ Notochord Descending aorta ' ^ Entoderm FIG. 33. — Transverse section through the fifth pair of mesodermal segments of a twenty-five-hour chick embryo. X 90. Mured crest: Neural tube Ectoderm Descending aorta. Efftbderm of Forty ut Vltelline vein FIG. 34. — Transverse section caudal to the fovea cardiaca of a twenty-five-hour chick embryo. X 90. Neural tube Ectoderm Som. mesoderm *•$* ffl*1 Deseendintj aorta \lj; •Splanchnic mesoderm (Myocardium) Endolhehum of heart lube Entoderm Splanchnic mesoderm j/ FIG. 35. — Transverse section through the fovea cardiaca of a twenty-five-hour chick embryo. X 90. Transverse Section through the Fovea Cardiaca (Fig. 35).— This section is marked by a vertical layer of the entoderm at the point where it is folded into the head as the fore-gut. The entoderm is thickened laterally and forms a continuous mass of tissue between the vitelline veins. The splanchnic mesoderm 52 THE STUDY OF CHICK EMBRYOS is differentiated into a thick walled pouch on each side lateral to the endothelial layer of the veins. Transverse Section through the Heart (Fig. 36). — As we pass cephalad in the series of sections the vitelline veins open into the heart just in front of the fovea cardiaca. The entoderm in the head-fold now forms the crescentic pharynx or fore-gut separated by the heart and splanchnic mesothelium from the entoderm of the germinal disc. The descending aortae are larger, forming conspicuous spaces between the neural tube (hind-brain) and the pharynx. The heart, as will be seen, is formed by the union of two endothelial tubes, similar to those which form the walls of the vitelline veins in the preceding sections. The median walls of these tubes disappear at a slightly later stage to form a single tube. Thick- Ectoderm Somatic mesoc/er Notoehord Myocardium Entoderm 'eural tube Descending aorta Splanchnic mes. FIG. 36. — Transverse section through the heart of a twenty-five-hour chick embryo. X 90. ened layers of splanchnic mesoderm which, in the preceding section, invested the vitelline veins laterally, now form the mesothelial wall of the heart. In the median ventral line,, the layers of splanchnic mesoderm of each side have fused, separated from the splanchnic mesothelium of the germinal disc and thus the two pleuro-pericardial cavities are in communication. The mesothelial wall of the heart forms the myocardium and epicardium of the adult. Dorsally, the splanch- nic mesoderm is continuous with the somatic mesoderm and forms the dorsal mesocardium. Origin of Primitive Heart. — From the two sections just described, it is seen that the heart arises as a pair of endothelial tubes lying in the pockets of the splanchnic mesoderm. Later, the endothelial tubes fuse to form a single tube. The heart then consists of an endo- thelial tube within a thick-walled tube of mesoderm. The origin of the endothelial cells of the heart is not surely known. They may be split from the entoderm, or arise from the mesoderm. According to another view, the endothelium arises in the vascular area and grows into the body EMBRYO OF SEVEN SEGMENTS 53 ie embryo. The vascular system is primitively a paired system, the heart arising as a double tube with two veins entering and two arteries leaving it. Origin of the Blood-vessels and Blood. — We have seen that in the area opaca a network of blood-vessels and blood-islands are differentiated as the angioblast. This tissue gives rise to all of the primitive blood-vessels and blood-cells and probably is derived from the splanchnic mesoderm. The vessels arise first as reticular masses of cells, the so-called blood- islands. These cellular thickenings undergo differentiation into two cell types, the innermost becoming blood-cells, the outermost forming a flattened endothelial layer which encloses the blood-cells. All the primitive blood-vessels of the embryo are composed of an endothelial layer only. The endothelial cells continue to divide, forming vascular sprouts and in this way new vessels are produced. The first vessels arising in the vascular area of a chick embryo form a close network, some of the branches of which enlarge to form vascular trunks. One pair of such trunks, the vitelline veins, is differentiated opposite, and later connects with, the posterior end of the heart. Another pair, the vitelline arteries, are developed in connection with the aortae of the embryo. The vessels of the vascular area thus appear before those of the embryo have developed, probably arise from the splanchnic mesoderm, and, both arteries and veins, are com- posed of a simple endothelial wall. As the ccelom develops in the region of the vascular area of the embryo soon after the differentiation of the angioblast the anlages of the blood-vessels are formed only in the splanchnic layer. (For the development of the heart and blood-vessels see Chapter IX.) Ectoderm AfesencJy S-^c-i'SIr .. •'£?;•.'/ ,*??> ^s^W ^J/lfg^ ^k*^ ..titistStk ..JR* *^-<- *"»•_' Ectoderm of proamnion FlG. 37. — Transverse section through the pharyngeal membrane of a twenty-five-hour chick embryo. X 90. Transverse Section through the Pharyngeal Membrane (Fig. 37). — This section passes through the head-fold and shows the head free from the underlying germinal area. The ectoderm surrounds the head and near the mid-ventral line is bent dorsad, somewhat thickened, and in contact with the thick entoderm of the pharynx. The area of contact between ectoderm and pharyngeal entoderm forms the pharyngeal plate or membrane. Later, this membrane breaks through and thus the oral cavity arises. The expanded neural tube is closed in this region and forms the middle brain vesicle or mid-brain. The dorsal aortae appear as small vessels dorsal to the lateral folds of the pharynx. The germinal area in the region beneath the head is composed of ectoderm and entoderm only. This is the proamniotic area. Laterad may be seen the layers of the mesoderm. 54 THE STUDY OF CHICK EMBRYOS Transverse Section through the Fore-brain and Optic Vesicle (Fig. 38). — The neural tube is open here and constitutes the first brain vesicle or fore-brain. The opening is the anterior neuropore. The ectoderm is composed of two or three layers of nuclei and is continuous with the much thicker wall of the fore-brain. The lateral expansions of the fore-brain are the optic vesicles, which eventually give rise to the retina of the eye. The two ectodermal layers are in contact Ectoderm Optic vesicle Neuropore Neural tube Proamnion FIG. 38. — Transverse section through the fore-brain and optic vesicles of a twenty-five-hour chick. X 90 with each other except in the mid-ventral region, where the mesenchyma is beginning to penetrate between and separate them. The proamnion consists of a layer of ectoderm and of entoderm. CHICK EMBRYO OF EIGHTEEN PRIMITIVE SEGMENTS (THIRTY-SIX HOURS) The long axis of this embryo is nearly straight (Fig. 39), the area pellucida is dumb-bell shaped and the vascular network is well differentiated throughout the area opaca. The tubular heart is bent to the right, and opposite its posterior end the vascular network converges and becomes continuous with the trunks of the vitelline veins. Connections have also been formed between the descending aortse and the vascular area, but as yet the vitelline arteries have not appeared as distinct trunks. The proamniotic area is reduced to a small region in front of the head, which latter is now larger and more prominent. In the posterior third of the vascular area blood-islands are still prominent. Central Nervous System and Sense Organs. — The neural tube is closed save at the caudal end where the open neural folds form the rhomboidal sinus. In the head the neural tube is differentiated into the three brain vesicles marked off from each other by constrictions. The fore-brain (prosencephalon) is charac- terized by the outgrowing optic vesicles. The mid-brain (mesencephalon) is undifferentiated. The hind-brain (rhombencephalon) is elongated and gradually merges caudally with the spinal cord. It shows a number of secondary constric- tions, the neuromeres. The ectoderm is thickened laterally over the optic ves- CHICK EMBRYO OF EIGHTEEN PRIMITIVE SEGMENTS icles to form the lens placode of the eye (Fig. 41). The optic vesicle is flattened at this point and will soon invaginate to produce the inner, nervous layer of the retina. In the hind-brain region, dorso-laterally the ectoderm is thickened and invaginated as the auditory placode (Fig. 43). This placode later forms the otocyst or otic -vesicle from which is differentiated the epithelium of the internal ear (membranous labyrinth). Forsbram Mid-brain Hind-bram VilellineVein Notochord Proammon Optic vesicle Free portion of Head Heart Neural lube Rhorrthoidal sinus Primitive tfreaK FIG. 39. — View of the dorsal surface of a thirty-six-hour chick embryo. X 20. Digestive Tube. — The entoderm is still flattened out over the surface of the yolk caudal to the fovea cardiaca. In Fig. 40 the greater part of the entoderm is cut away. The flattened fore-gut, folded inward at the fovea, shows indications of three lateral diverticula, the pharyngeal pouches. Cephalad the pharynx is closed ventrally by the pharyngeal membrane. THE STUDY OF CHICK EMBRYOS Heart and Blood-vessels. — As seen in the dorsal view of the embryo, the heart tube is bent to the right. Viewed from the ventral side, the bend is to the left (right of embryo) (Fig. 40). After receiving the vitelline veins cephalad to the fovea cardiaca the double-walled tube of the heart dilates and bends ventrad Optic vesicle Paired ventral aorta Ventral aorta Bulbus cordis Ventricle Splanchnic mesoderm Fovea cardiaca R. descending aorta ' » »j Vascular plexus — *tg 9** , Splanchnic mesoderm Notochord Mes. segment Segmental zone Fore-brain Phar. pouch I Descending aorta Phar. pouch II Left vitelline vein Entoderm Section medullary tube Somatopleure Descending aorta Medullary tube Splanchnic mesoderm Capillary plexus Somatopleure Neural groove FIG. 40. — Ventral reconstruction of a thirty-six-hour chick embryo. The entoderm has been removed save about and caudal to the fovea cardiaca. X 38. and to the right (left of Fig. 43). It then is flexed dorsad and to the median line, and narrows to form the ventral aorta. The aorta lies ventrad to the pharynx and divides at the boundary line between the mid- and hind-brain into two ventral aorta. These diverge and course dorsad around the pharynx. Before CHICK EMBRYO OF EIGHTEEN PRIMITIVE SEGMENTS 57 reaching the optic vesicles they bend caudad, and as the paired descending aortae may be traced to a point opposite the last primitive segments. In the region of the fovea cardiaca they lie close together and have fused to form a single vessel, the dorsal aorta. They soon separate and opposite the last primitive segments they are connected by numerous capillaries with the vascular network. In this region at a later stage the trunks of the paired mtelline arteries will be differen- tiated. The heart beats at this stage, the blood flows from the vascular area by way of the vitelline veins to the heart, thence by the aortae and vitelline arteries back again. This constitutes the mtelline circulation and through it the embryo receives nutriment from the yolk for its future development. Farebram 5S?** ' Spknchno, — pleure FIG. 41. — Transverse section through the fore-brain of a thirty-six-hour chick embryo. X 75. In studying transverse sections of the embryo the student should not only identify the structures seen, but should locate the level of each section by compar- ing with Figs. 39 and 40, and trace the organs from section to section in the series. Transverse Section through the Fore-brain and Optic Vesicles. (Fig. 41). — The optic stalks connect the optic vesicles laterally with the ventral portion of the fore- brain. Dorsally the section passes through the mid-brain. We have alluded to the thickening of the lens placode. Note that there is now a considerable amount of mesenchyma between the ectoderm and the neural tube. In the germinal area the layers of mesoderm are present. Transverse Section through the Pharyngeal Membrane and Mid-brain (Fig. 42). — In the mid- ventral line the thickened ectoderm bends up into contact with the entoderm of the rounded pharynx. At this point the oral opening will break through. On either side of the pharynx a pair of large vessels are seen; the ventral pair are the ventral aorta. Two sections cephalad their cavities open into those of the dorsal pair, the descending aorta. The section is thus just caudad to the point where the ventral aortae bend dorsad and caudad to form the descending aortae. The section passes through the caudal end of the mesencephalon which is here thick walled with an oval cavity. Note the large amount of undifferentiated 58 THE STUDY OF CHICK EMBRYOS mesenchyma in the section. The structure of the germinal area is complicated by the presence of collapsed blood-vessels. Transverse Section through the Hind-brain and Auditory Placodes (Fig. 43). — Besides the auditory placodes already described as the anlages of the internal ear, this section is characterized (i) by the large hind-brain, somewhat flattened dorsad; (2) by the broad dorso-ventrally flattened pharynx, above which on each side lie the dorsal aorta; (3) by the Ectoderm. Mesencfyma Neural tube NotochorcL Foreyuf Pharyngeal membrane Splanchnopleure FIG. 42. — Transverse section through the pharyngeal membrane of a thirty-six-hour chick embryo. X75- Ectoderm Notochord Descending Qorta Fkncardial cavity Somafi'c. tne. Neural tube /4nt. Cardinal Vein Auditory placode Foregut Ectoderm Endol helium ofhearf Entoderm Endotheli'um of ventral aorta. Myocardium FIG. 43. — Transverse section through the hind-brain and auditory placodes of a thirty-six-hour chick embryo. X 75. presence of the ventral aorta and bulbar portion of the heart. The descending aortas are located on each side dorsal to the pharynx. The ventral aorta is suspended dorsally by the mesoderm, which here forms the dorsal mesocardium. The bulbus of the heart lies to the left in the figure (right of embryo) and a few sections caudad in the series is continuous with the ventral aorta (see Fig. 40). Between the somatic and splanchnic mesoderm is the large pericardial cavity. It surrounds the heart in this section. CHICK EMBRYO OF EIGHTEEN PRIMITIVE SEGMENTS Transverse Section through the Caudal End of the Heart (Fig. 44.) — The section passes through the hind-brain. The descending aortas are separated only by a thin septum which is ruptured in this section. The mesothelial wall of the heart is continuous with the somatic mesoderm. On the right side of the section there is apparent fusion between the myocardium of the heart and the somatic mesoderm. Lateral to the aortae are the anterior cardinal Ectoder. Mes. seqmenf 'eural tube ForequT Ant. cardinal Vein c I L • i Coelo( opianchmc. mesoaer/n Entoderm Vitelllne Vein / I '^v x Splanchnic fnesoderm Heart , FIG. 44. — Transverse section through the caudal end of the heart of a thirty-six-hour chick embryo. X75- FIG. 45. — Transverse section through the fovea cardiaca of a thirty-six-hour chick embryo. Ant. card, -vein, anterior cardinal vein; L. vit. vein, left vitelline vein; Mes. segment, mesodermal segment; Spl. mesoderm, splanchnic mesoderm. X go. veins. A pair of primitive mesodermal segments may be seen in this section lateral to the hind- brain. It may be noted here that the primitive segments were not present in the sections of the head previously studied. Transverse Section through the Fovea Cardiaca (Fig. 45). — The descending aorta now form a single vessel, the dorsal aorta, the medium septum having disappeared. The section passes through the entoderm at the point where it is folded dorsad and cephalad into the 6o THE STUDY OF CHICK EMBRYOS head as the fore-gut. The cavity is the fovea cardiaca and two sections caudad it communicates with the flattened space between the entoderm and the yolk. On each side of the fore-gut are the large vitelline veins, sectioned obliquely. As the splanchnic mesoderm overlies these veins dorsad, it is pressed by them on each side against the somatic mesoderm and the cavity of the ccelom is thus interrupted. Transverse Section Caudal to the Fovea Cardiaca (Fig. 46). — This section re- sembles the preceding save that the primitive gut is without a ventral wall. The vitelline vein on the left is still large. Neural lube fNeural Cavt'fy Ectoderm focHord \Entoderm Jplanchnopleun Open FIG. 46. — Transverse section caudal to the fovea cardiaca of a thirty-six-hour chick embryo. X 90. Mesonephrii. clucf Splanchnic . mesoderm Coelom Descending aorta /VotOchord Encode. FIG. 47. — Transverse section through the fourteenth pair of mesodermal segments of a thirty-six-hour chick embryo. X 90. Section through the Fourteenth Pair of Primitive Segments (Fig. 47). — The body of the embryo is now flattened on the surface of the yolk. The dorsal aortae have sepa- rated and occupy the depressions lateral to the primitive segments. The section is characterized by the differentiated mesoderm which forms the primitive segments, nephrotomes, somatic and splanchnic mesoderm, structures soon to be described. Transverse Section through the Rhomboidal Sinus (Fig. 48). — The neural groove is open, the notochord is oval in form. The ectoderm is characterized by the columnar form of its cells. At the point where the ectoderm joins the neural fold a crest of cells projects ventrally on either side. These projecting cells form the neural crests, and from them the spinal ganglia are formed. The mesodermal plates have split laterally into layers, but the ccelomic cavities are mere slits. Between the splanchnic mesoderm and the entoderm blood-vessels may be seen. CHICK EMBRYO OF EIGHTEEN PRIMITIVE SEGMENTS 6l Transverse Section through the Primitive (Hensen's) Node or Knot (Fig. 49). — The section shows the three germ layers bound together at the "knot" or node into a mass of undifferentiated tissue. The mesoderm is split laterally into the somatic and splanchnic layers. Transverse Section through the Primitive Streak (Fig. 50). — In the mid-dorsal line is the primitive groove. The germ layers may be seen taking their origin from the undiffer- Ecfod, Neural tube Neural Crest Sey menial zone «Somaf/c mesoderm ."••.-*-.-•- ,,:, '« - * i£jr