5i*iyijiu,Uuiym ■} wm MEMCAL SCHOOL OEI^AIISY Digitized by the Internet Archive in 2007 with funding from IVIicrosoft Corporation http://www.archive.org/details/earlyembryologyoOOpattrich THE EARLY EMBRYOLOGY OF THE CHICK PATTEN THE EARLY EMBRYOLOGY OF THE CHICK BY BRADLEY M. PATTEN ASSISTANT PROFESSOR OF HISTOLOGY AND EMBRYOLOGY SCHOOL OF MEDICINE, WESTERN RESERVE UNIVERSITY WITH 55 ILLUSTRATIONS CONTAINING 182 FIGURES PHILADELPHIA P. BLAKISTON'S SON & CO 1012 WALNUT STREET Copyright, 1920, by P. Blakiston's Son & Co. • • • • • • • • • • • • • • • ••••» ; « • • • • • • , • ' -♦. .», .•• • ••: : TKK M^I>X.K X>HKBS YOKKL PA 151 PREFACE The fact that most courses in vertebrate embryology deal to a greater or lesser extent with the chick seems to warrant the treatment of its development in a book designed primarily for the beginning student. To a student beginning the study of embryology the very abundance of information available in the literature of the subject is confusing and discouraging. He is unable to cull the essentials and fit them together in their proper relationships and is Hkely to become hopelessly lost in a maze of details. This book was written in an effort to set forth for him in brief and simple form the early embryology of the chick. It does not purport to treat the subject from the com- parative view point, nor to be a reference work. If it helps the student to grasp the structure of the embryos, and the sequence and significance of the processes he encounters in his work on the chick, and thereby conserves the time of the instructor for inter- pretation of the broader principles of embryology it will have served the purpose for which it was written. In preparing the text, details have been largely omitted and controverted points avoided for the sake of clarity in outHning fundamental processes. While I would gladly have avoided the matters of cleavage and germ layer formation in birds, a brief description of them seemed necessary. Without some interpretation of the initial phases of development, the student has no logical basis for his study of the already considerably developed embryos with which his laboratory work begins. The treatment which it is desirable to accord to gametogenesis and maturation as processes leading toward fertilization would vary so greatly in extent and view point in different courses that it seemed inadvisable to attempt any general discussion of these phenomena. The account of development has not been carried beyond the first four days of incubation. In this period the body of the embryo is laid down and the organ systems are estabHshed. Courses in general embryology rarely carry work on the chick beyond this phase of development. More extensive courses in V VI PREFACE which a knowledge of mammalian embryology is the objective, ordinarily pass from the study of three or four day chicks to work on mammalian embryos. While the text has been kept brief, illustrations have been freely used in the belief that they convey ideas more readily and more accurately than can be done in writing. Direct labeling has been used in the figures to facilitate reference to them. Most of the drawings were made directly from prepara- tions in the laboratory of Histology and Embryology of Western Reserve University School of Medicine. However, figures from other authors, particularly Lillie and Duval, have been used extensively for comparisons and for schemes of presentation. Several figures have been reproduced directly or with only slight modifications. These are designated in the figure legends. I wish to acknowledge the assistance I received in the prepa- ration of material by Mrs. Mary V. Bayes, and in the drawing of the figures by Mrs. Bayes and Dr. Louis J. Karnosh. I am also indebted to my father. Prof. Wm. Patten of Dartmouth College for criticism of the figures, and to Dr. F. C. Waite of the School of Medicine, Western Reserve University for his helpful interest and cooperation in all phases of the preparation of the book and especially for his reading of the manuscript. Beadley M. Patten. Western Reserve University, School of Medicine. Cleveland, Ohio. CONTENTS Page Preface v CHAPTER I Introduction i CHAPTER II The Gametes and Fertilization 7 The ovarian ovum; maturation, ovulation, and fertilization; the formation of the accessory coverings of the ovum; the structure of the egg at the time of laying; incubation. CHAPTER III The Process of Segmentation 14 The effect of yolk on segmentation; the unsegmented blastodisc; the sequence and orientation of the cleavage di\dsions in birds. CHAPTER IV The Establishment of the Entoderm 20 The morula stage; the formation of the bias tula; the effect of yolk on gastrulation; gastrulation in birds. CHAPTER V The Formation of the Primitive Streak and the Establishment of THE Mesoderm 27 The location and appearance of the primitive streak; the origin of the primitive streak by concrescence of the blastopore; the formation of the mesoderm. CHAPTER VI From the Primitive Streak Stage to the Appearance of the Somites ss The primitive streak as a center of growth; the growth of the entoderm and the establishment of the primitive gut; the growth and differ- entiation of the mesoderm; the formation of the notochord; the forma- tion of the neural plate; the differentiation of the embryonal area. CHAPTER VII The Structure OF Twenty-four Hour CmCKS 44 The formation of the head; the formation of the neural groove; the regional divisions of the mesoderm; the coelom; the pericardial region; the area vasculosa. Vlll CONTENTS CHAPTER VIIl Page The Changes Between Twenty-four and Thirty-three Hours of Incubation 52 The closure of the neural tube; the diflferentiation of the brain region; the anterior neuropore; the sinus rhomboidalis; the fate of the primitive streak; the formation of additional somites; the lengthening of the fore-gut; the appearance of the heart and the omphalomesenteric veins; organization in the area vasculosa. CHAPTER IX The Structure op Chicks Between Thirty-three and Thirty-nine Hours of Incubation 59 The divisions of the brain and their neuromeric structure; the auditory pits; the formation of extra-embryonic blood vessels; the formation of the heart; the formation of intra-embryonic blood vessels. CHAPTER X The Changes Between Forty and Fifty Hours of Incubation 75 Flexion and torsion; the completion of the vitelline circulatory channels; the beginning of the circulation of blood. CHAPTER XI Extra-embryonic Membranes 80 The folding of! of the body of the embryo; the establishment of the yolk-sac and the delimitation of the embryonic gut; the amnion and the serosa; the allantois. CHAPTER XII The Structure of Chicks from Fifty to Fifty-five Hours of In- cubation 93 I. External Features. II. The Nervous System. Growth of the telencephalic region; the epiphysis; the in- fundibulum and Rathke's pocket; the optic vesicles; the lens; the posterior part of the brain and the cord region of the neural tube; the neural crests. HI. The Digestive Tract. The fore-gut; the stomodaeum; the pre-oral gut; the mid-gut; the hind-gut. IV. The Visceral Clefts and Visceral Arches. V. The Circulatory System. The heart; the aortic arches; the fusion of the dorsal aortse; the cardinal and omphalomesenteric vessels. VI. The Differentiation of the Somites. Vn. The Urinary System. CONTENTS IX CHAPTER XIII Page The Development of the Chick During the Third and Fourth Days OF Incubation 109 I. External Features. Torsion; flexion; the visceral arches and clefts; the oral region; the appendage buds; the allantois. II. The Nervous System. Summary of development prior to the third day; the formation of the telencephalic vesicles; the diencephalon; the mesen- cephalon; the metencephalon; the myelencephalon; the ganglia of the cranial nerves; the spinal cord; the spinal nerve roots. III. The Sense Organs. The eye; the ear; the olfactory organs. IV. The Digestive and Respiratory Systems. Summary of development prior to the third day; the establish- ment of the oral opening; the pharyngeal derivatives; the trachea; the lung-buds; the oesophagus and stomach; the liver; the pancreas; the mid-gut region; the cloaca; the procto- daeum and the cloacal membrane. V. The Circulatory System. The functional significance of the embryonic circulation; the vitelline circulation; the allantoic circulation; the intra-embry- onic circulation; the heart. VI. The Urinary System. The general relationships of pronephros, mesonephros, and metanephros; the pronephric tubules of the chick; the meso- nephric tubules. VII. The Coelom and Mesenteries. APPENDIX References for Collateral Reading 155 Index 161 CHAPTER I INTRODUCTION The only method of attaining a comprehensive understanding of embryological processes is through the study and comparison of development in various animals. Many phases of the development of any specific organism can be interpreted only through a knowledge of corresponding processes in other organisms. The beginning student, however, must acquire his knowledge of embryology through intensive study of one form at a time, depending at first on older workers in the field for interpretation of the phenomena encountered. Building on the f amiharity with fundamental processes of development thus acquired, he may later broaden his horizon by the comparative study of a variety of forms. The chick is one of the most satisfactory animals on which student laboratory work in embryology may be based. Chick embryos in a proper state of preservation and of the stages desired can be readily secured and prepared for study. Used as the only laboratory material in a brief course they afford a basis for understanding the early differentiation of the organ systems and the fundamental processes of body formation common to all groups of vertebrates. In more extended courses where several forms are taken up, the chick serves at once as a type for the development characteristic of the large-yolked eggs of birds and reptiles, and as an intermediate form bridging the gap between the simpler processes of development in fishes and amphibia on the one hand and the more complex processes in mammals on the other. In medical school courses where a knowledge of human embryology is the end in view the chick not only makes a good stepping stone to the understanding of mammalian embryology, but also provides material for the study of early developmental processes not readily demon- strable in mammalian material. This book on the development of the chick has been written 2 EARLY EMBRYOLOGY OF THE CHICK for those who are beginning the study of embryology and has accordingly been kept as brief and as uncomplicated as possible. Nevertheless it is assumed that the beginner in embryology will not be without a certain back-ground of zoological knowledge and training. He may reasonably be expected to be familiar with some of the aspects of evolution and heredity, with the recapitulation theory, the cell theory, the nature of the various types of tissues, and the more general phases of the morphology of vertebrates. Before laboratory work on the chick is begun in any course in embryology the nature of sexual reproduction, and the processes of gametogenesis, maturation, fertilization and cleavage, will have been taken up. It therefore seems unnecessary to include here any pre- liminary, general discussion of these phenomena. References for collateral reading on this and other phases of the subject are given in the appendix. Like other sciences embryology demands first of all accurate observation. It differs considerably, however, from such a science as adult anatomy where the objects studied are rela- tively constant and their component parts are not subject to rapid changes in their inter-relations. During development, structural conditions within the embryo are constantly chang- ing. Each phase of development presents a new complex of conditions and new problems. Solution of the problems presented in any given stage of development depends upon a knowledge of the stages which precede it. To comprehend the embryology of an organism one must, therefore, start at the beginning of its development and follow in their natural order the changes which occur. At the outset of his work the student must realize that proper sequence of study is essential and may not be disregard-ed. A knowledge of structural conditions in earlier stages than that at the moment under consideration, and an appreciation of the trend of the developmental processes by which conditions at one stage become transmuted into different conditions in the next, are direct and necessary factors in acquiring a real com- prehension of the subject. Without them the story of embryology becomes incoherent, a mere jumble of confused impressions. A knowledge of the phenomena of development is ordinarily INTRODUCTION 3 acquired by studying a series of embryos at various stages of advancement. Each stage should be studied not so much for itself, as for the evidence it affords of the progress of develop- ment. In the study of embryology it does not suffice to acquire merely a series of *' still pictures" of various structures, however accurate these pictures may be. The study demands a constant application of correlative reasoning and an appreciation of the mechanical factors involved in the relations of various structures within the embryo to each other, and in the relation of the embryo as a whole to its environment. In order to really comprehend the embryological significance of a structure one must know not only its relations within the embryo being studied at the time, but also the manner in which it has been derived and the nature of the changes by which it is progressing toward adult conditions. To get absolutely the whole story it is obvious that one would have to study a series of embryos with infinitely small intervals between them. Nevertheless the fundamental steps in the process may be grasped from a much less extensive series. The fewer the stages studied, however, the more careful must one be to keep in mind the continuity of the processes and to think out the changes by which one stage leads to the next. The outstanding idea to be kept in mind by the student begin- ning the study of embryology is that the development of an individual is a process and that this process is continuous. The conditions he sees in embryos of various stages are of importance chiefly because they serve as evidence of events in the process of development at various intervals in its continuity, as his- torical events are evidences of the progress of a nation. Just as historical events are led up to by preparatory occurrences and followed by results which in turn affect later events, so in em- bryology events in development are presaged by preliminary changes and when consummated affect in turn later steps in the process. In certain respects the laboratory study of embryological. material involves methods of work for which courses in general zoology do not entirely prepare the student. Some general suggestions as to methods of procedure are, therefore, not out of place. In dissecting gross material it is not unduly difficult to- 4 EARLY EMBRYOLOGY OF THE CHICK appreciate the complete relationships of a structure. The nature of embryological material, however, introduces new problems. Embryos of the age when the establishment of the various organ systems and processes of body formation are being initiated are too small to admit of successful dissection, but npt sufficiently small to permit of the satisfactory micro- scopical study of an entire embryo, except for its more general organization. To study embryos of this stage with any degree of thoroughness they must be cut into sections which are sufficiently thin to allow effective use of the microscope to ascertain cellular organization and detailed structural relation- ships. In preparing such material the entire embryo is cut into sections which are mounted on slides in the order in which they were cut. A sectional view of any region of the embryo is then available for study. While sections readily yield accurate information about local regions it is extremely difl&cult to construct a mental picture of any whole organism from a study of serial sections alone. For this reason it is necessary to work first on entire embryos which have been prepared by staining and clearing so they may be studied as transparent objects. From such preparations it is possible to map out the configuration of the body, and the location and extent of the more conspicuous internal organs. In this work the fact that embryos have three dimensions must be kept constantly in mind and the depth at which a structure lies must be determined as well as its apparent position in surface view. While conventionally entire chick embryos are represented in dorsal view, much additional information ma}^ be gained by following a study of the dorsal, with a study of the ventral aspect. Unless the preliminary study of entire embryos is carefully and thoroughly carried out the study of sections will yield only confusion. In studying a section of an embryo it is necessary first of all to determine its location. The plane of the section under consideration, and the region of the embryo through which it passes should be ascertained by comparing it with an entire embryo of the same age as that from which the section was cut. Only when the exact location of a section is known can the structures appearing in it be correlated with the organization of the embryo as a whole. Probably nothing in the study of INTRODUCTION 5 embryology causes students more difficulties than neglect to locate sections accurately with the consequent failure to ap- preciate the relationships of the structures seen in them. Too great emphasis cannot be laid on the vital importance of fitting the structures shown by sections properly into the general scheme of organization as it appears in whole-mounts. It must by no means be inferred that the possibilities of the whole- mounts have been exhausted by the preliminary study accorded them before taking up the work on sections. | Further and more careful study of entire embryos should constantly accompany the study of serial sections. Many details which in the initial observation of the whole-mount were inconspicuous or abstruse will become significant in the light of the more exact information yielded by the sections. In the discussion of structures and processes in embryology, it is necessary to use terms designating location and direction which are referable to the body of the embryo regardless of the position it occupies. The ordinary terms of location, which are primarily referred to the direction of the action of gravity, such as above, over, under etc. are not sufficiently accurate. In gross human anatomy, there still persist many terms that are referred to gravity, and are therefore, because of the erect posture of man, not applicable to comparative anatomy or to embryology. The most confusing of these are anterior and posterior as used in gross human anatomy to mean, respec- tively, pertaining to the belly and to the back. In comparative anatomy and in embryology, anterior has reference to the head region and posterior to the tail region. The use of these terms in embryology in the sense usual in gross human anatomy is likely to lead to confusion and is entirely avoided in this book. The terms anterior and posterior have been replaced to a large extent by their less confusing synonyms, cephalic and caudal. In addition to the adjectives of position, such as dorsal, ventral, cephalic, caudal, mesial, lateral, proximal, distal, corresponding adverbs of motion or direction are commonly used in embryology. These adverbs are formed by adding the suffix -ad to the root of the adjective, as dorsad meaning toward the back, cephalad meaning toward the head, etc. These must not be used as adjectives of position but should be ap- O EARLY EMBRYOLOGY OF THE CHICK 4 plied only to the progress of processes, or to the extension of structures toward the part indicated by the root of the adverb. Cultivation of the use of correct and definite terms of posi- tion and direction in dealing with embryological processes will greatly aid accurate thinking and clear understanding. CHAPTER II THE GAMETES AND FERTILIZATION The ovarian ovum; maturation, ovulation, and fertiliza- tion; THE FORMATION OF THE ACCESSORY COVERINGS OF THE ovum; the structure of the egg at the time of laying; incubation. The Ovarian Ovum. — The formation of the ovum, the phe- nomena of fertihzation, and the stages of development occurring prior to the laying of the egg have been more completely worked out in the pigeon than in the hen. The observations which have been carried out on the hen's egg indicate, as might be expected from the near relationship of the pigeon and the hen, that the processes in the two forms are closely comparable. The following account which is based chiefly on observations made on the pigeon's egg may, therefore, be taken to apply equally well in all essentials to the hen's egg. The part of the egg commonly known as the ''yolk" is a single cell, the female sex cell or ovum. Its great size as com- pared with other cells is due to the food material it contains. While the egg cell is still in the ovary, material which is later used by the embryo as food is deposited in its cytoplasm. This deposit which is known as deutoplasiii consists of a viscid fluid in which are suspended granules and globules of Various sizes. As the deutoplasm increases in amount the nucleus and the cyto- plasm are forced toward the surface so that eventually the deutoplasm comes to occupy nearly the entire cell. This abundance of deutoplasm accumulated in the ovum furnishes a readily assimilable food supply, which makes possible the extremely rapid development of the chick embryo. A section of the hen's ovary passing through a nearly mature ovum (Fig. i) shows the ovum and the tissues which surround it projecting from the ovary but connected to it by a constricted stalk of ovarian tissue. The protuberance containing the ovum is known as a follicle. The bulk of the ovum itself is made up of 7 8 EARLY EMBRYOLOGY OF THE CHICK the yolk. Except in the neighborhood of the nucleus the active cytoplasm is but a thin film enveloping the yolk. About the nucleus a considerable mass of cytoplasm is aggregated. The region of the ovum containing the nucleus and the bulk of the active cytoplasm is known as the animal pole because this subsequently becomes the site of greatest protoplasmic activity. The region opposite the animal pole is called the vegetative pole because while material for growth is drawn from this region it remains itself relatively inactive. young follicle connective tissue stalk of follicle germinal epithelium of ovary white yolk yellow yolk cellular (granular) zone of follicle theca folliculi Fig. I. — Diagram showing the structure of a bird ovum still in the ovary. {Modified from Lillie, after Patterson.) The section shows a follicle containing a nearly mature ovum, together with a small area of the adjacent overian tissue. Enclosing the ovum is a thin non-cellular membrane, the vitelline membrane, which is a secretory product of the cyto- plasm of the ovum. Outside the vitelHne membrane and very difficult to differentiate from it, is another secreted membrane the zona radiata, so called because of its delicate radial stria- tions. Immediately peripheral to the zona radiata is an invest- ment of small polygonal cells, the cellular or ** granular" zone of the follicle, which is in turn enclosed in a highly vascular coat of connective tissue, the theca folliculi. The nutriment for the growing ovum is supplied by the mother from the prod- GAMETES AND FERTILIZATION ucts of her digested food. It is brought in through the blood vessels of the theca, absorbed by the follicular cells and trans- ferred by them to the ovum. Within the ovum this material is elaborated into deutoplasm. Maturation, Ovulation and Fertilization. — When the full allotment of deutoplasm has accumulated in the ovum the nucleus undergoes its first maturation division. Maturation is a process occurring before fertilization, in which there is an equal mitotic division of the nucleus of the ovum but a markedly unequal division of the cytoplasm and its contents. y The result of this division is the formation of one very large cell containing the entire dower of deutoplasm and one very small cell containing practically no deutoplasm. This small cell is call- ed a polar body because it is budded off at the animal pole of the ovum. Since this unequal division of the ovum typically occurs twice we speak of the first and second maturation divisions and of the first and second polar bodies. In one of these maturation divisions the chromosomes do not split at the metaphase stage as happens in ordinary mitoses. Instead, half of the original number of chromosomes migrate bodily to each pole of the spindle, with the result that each daughter nucleus receives but half the number of chromosomes normal for the somatic cells of the species. Such a modified mitotic division is known as a reduction division. After the maturation divisions, one of which is a reduc- tion division, the nucleus of the ovum now ready for fertilization, is called the female pronucleus. Although maturation in the male sex cells differs in some respects from the maturation of the ovum, there also, a reduction division occurs. The result is that the nucleus of each matured cell contains but half the species number of chromo- somes. When in the process of fertilization the nucleus of the male cell unites with the female pronucleus the full species number of chromosomes is restored. At about the time of the first maturation division the follicle ruptures, and the liberated ovum passes into the oviduct. If Fig. 2.— S permatozoon of the pigeon. (After Ballo- witz.) lO EARLY EMBRYOLOGY OF THE CHICK insemination has taken place meanwhile, the spermatozoa (Fig. 2) make their way along the oviduct where for several days they may remain alive and capable of performing their function of fertilization. Penetration of the ovum by sperma- tozoa takes place in the region of the oviduct near the ovary, before the albumen and shell have been added to the ovum. Coincidently the second polar body is extruded. Although in birds normally several spermatozoa penetrate the ovum, only a single one unites with the female pronucleus. The fusion of the male and female pronuclei in fertilization initiates the develop- ment of the embryo and the cleavage divisions are begun while the ovum is passing through the oviduct toward the cloaca and receiving meanwhile its accessory coverings. The Formation of the Accessory Coverings of the Ovum. The albumen, the shell membrane, and the shell are non-cellular investments secreted about the ovum by the cells lining the oviduct. In the part of the oviduct adjacent to the ovary a mass of stringy albuminous material is produced. This ad- heres closely to the vitelline membrane and projects beyond it in two masses extending in either direction along the oviduct. Due to the spirally arranged folds in the walls of the oviduct, the egg as it moves toward the cloaca is rotated. This rotation twists the adherent albumen into the form of spiral strands pro- jecting at either end of the yolk, known as the chalazae (Fig. 3). Additional albumen, which has been secreted abundantly in advance of the ovum by the glandular lining of the oviduct, is caught in the chalazae and during the further descent of the ovum is wrapped about it in concentric layers. These lamellae of albumen may be easily demonstrated in an egg which has had the albumen coagulated by boiling. The albumen secreting region of the oviduct constitutes about one-half of its entire length. The shell membranes which consist of sheets of matted organic fibers are added farther along in the oviduct. The shell is secreted as the egg is passing through the shell gland portion of the oviduct. The entire passage of the ovum from the time of its discharge from the ovary to the time when it is ready for laying has been estimated to occupy about 22 hours. If the completely formed egg reaches the cloacal end of the oviduct during the middle of the day it is usually laid at once, GAMETES AND. FERTILIZATION II otherwise it is likely to be retained until the following day. This over night retention of the egg is one of the factors which accounts for the variability in the stage of development reached at the time of laying. The Structtire of the Egg at the Time of Laying. — The arrangement of structures in the egg at the time of laying is shown in Figure 3. Most of the gross relationships are already familiar because they appear so clearly in eggs which have been boiled. If a newly laid egg is allowed to float free in water until it comes to rest and is then opened by cutting nucleus of Pander _ blastoderm neck of latebra white yolk ^^55^..^ ^»^^^^v less dense albumen yeUow yolk^ 'vitelline membrane Pig. 3. — Diagram of the hen's egg in longitudinal section. (After Lillie.) The relations of the various parts of the egg at the time of laying are indicated schematically. away the part of the shell which lies uppermost, a circular whitish area will be seen to lie atop the yolk. In eggs which have been fertilized this area is somewhat different in appear- ance and noticeably larger than it is in unfertilized eggs. The differences are due to the development which has taken place in fertilized eggs during their passage through the oviduct. The aggregation of cells which in fertilized eggs lies in this area is known as the blastoderm. The structure of the blastoderm and the manner in which it grows will be taken up in the next chapter. Close examination of the yolk will show that it is not uniform throughout either in color or in texture. Two kinds of yolk 12 EARLY EMBRYOLOGY OF THE CHICK can be differentiated, white yolk, and yellow yolk. Aside from the difference in color visible to the unaided eye, microscopical examination will show that there are differences in the granules and globules of the two types of yolk, those in the white yolk being in general smaller and less uniform in appearance. The principal accumulation of white yolk lies in a central flask- shaped area, the latebra, which extends toward the blastoderm and flares out under it into a mass known as the nucleus of Pander. In addition to the latebra and the nucleus of Pander there are thin concentric layers of white yolk between which lie much thicker layers of yellow yolk. The concentric layers of white and yellow yolk are said to indicate the daily accumula- tion of deutoplasm during the final stages in the formation of the egg. The outermost yolk immediately under the vitelline membrane is always of the white variety. The albumen, except for the chalazae, is nearly homogeneous in appearance, but near the yolk it is somewhat more dense than it is peripherally. The chalazae serve to suspend the yolk in the albumen. The two layers of shell membrane lie in contact everywhere except at the large end of the egg where the inner and outer membranes are separated to forni an air chamber. This space is stated (Kaupp) to appear only after the egg has been laid and cooled from the body temperature of the hen (about io6°F.) to the ordinary temperatures. In eggs which have been kept for any length of time the air space increases in size due to evaporation of part of the water content of the egg. This fact is taken advantage of in the familiar method of testing the freshness of eggs by "floating them." The egg shell is composed largely of calcareous salts. These salts are derived from the food of the mother and if lime con- taining substances are not furnished in her diet the shell is defectively formed or even altogether wanting. The shell is porous allowing the embryo to carry on exchange of gases with the outside air by means of specialized vascular membranes arising in connection with the embryo but lying outside it, directly beneath the shell. Incubation. — When an egg has been laid, development ceases unless the temperature of the egg is kept nearly up to the body temperature of the mother. Cooling of the egg does not, how- GAMETES AND FERTILIZATION 13 ever, lesult in the death of the embryo. It may resume its development if it is brooded by the hen or artificially incubated even after the egg has been kept for many days at ordinary temperatures. The normal incubation temperature is that at which the egg is maintained by the body heat from the brood-hen. This is somewhat below the blood heat of the hen (io6°F.). When an egg is allowed to remain undisturbed the yolk rotates so that the developing embryo lies uppermost. Its position is then such that it gets the full benefit of the warmth of the mother. In incubating eggs artificially the incubators are usually regulated for a heat of ioo°-ioi°F. (37°-38°C.). At this temperature the chick is ready for hatching on the twenty-first day. Development will go on at considerably lower tempera- tures but its rate is retarded in proportion to the lowering of the temperature. Below about 21 degrees Centigrade develop- ment ceases altogether. In incubating eggs which have been cooled after laying for some particular stage of the embryo which it is desired to secure, three or four hours are ordinarily allowed for the egg to become warmed to the point at which development begins again. For example if an embryo of 24-hours incubation age is desired the egg should be allowed to remain in the incubator about 27 hours. Even with allowance made for the warming of the egg and with exact regulation of the temperature of the incubator, the stage of development attained in a given incubation time will vary widely in different eggs. The factor of individual variabiHty which must always be reckoned with in developmental proces- ses, undoubtedly accounts for some of the variation. The different time occupied by different eggs in traversing the ovi- duct, the over-night retention of eggs not ready for laying till toward sundown, and especially the varying time different eggs have been brooded before being removed from the nest, account for further variations. The designation of the age of chicks in hours of incubation is, therefore, not exact, but merely a con- venient approximation of the average condition reached in that incubation time. CHAPTER III THE PROCESS OF SEGMENTATION The effect of yolk on segmentation; the unsegmented blastodisc; the sequence and orientation of the cleavage division in birds. The Effect of Yolk on Segmentation. — Immediately after its fertilization the ovum enters upon a series of mitotic divisions which occur in close succession. This series of divisions constitutes the process of segmentation or cleavage. In birds segmentation takes place before the egg is laid, during the time it is traversing the oviduct. A mitotic division, whether it be a cleavage division of the ovum or the division of some other cell, is carried out by the active protoplasm of the cell. The food material stored in an egg cell as deutoplasm is non-living and inert. The deutoplasm has no part in mitosis except as its mass mechanically influences the activities of the protoplasm of the cell. It is obvious that any considerable amount of yolk will retard the division, or prevent the complete division, of the fertilized ovum. The amount and distribution of the yolk will therefore determine the type of segmentation. Figure 4 shows diagrammatically the manner in which the first cleavage division is carried out in three types of eggs having different relative amounts and different distributions of yolk and protoplasm. In the egg of Amphioxus the yolk is relatively meager in amount and fairly uniformly distributed throughout the cell. An ovum with such a yolk distribution is termed isolecithal (homolecithal). An isolecithal egg under- goes a type of cleavage which is essentially an unmodified mitosis. The yolk is not sufficient in amount, nor sufficiently localized to alter the usual mode of cell division. In Amphibia the ovum contains a considerable amount of yolk and the accumulation of the yolk at one pole has crowded the nucleus and active cytoplasm of the ovum toward the opposite pole. An egg in which the yolk is thus concentrated 14 PROCESS OF SEGMENTATION 15 at one pole is termed telolecithal. Cleavage in such an egg is initiated at the animal pole where the nucleus and most of the Q -g 3 -^ <^ > O rt to o O 43 iG z O Xi < a w ° 3 "s active cytoplasm are located. The division of the nucleus is a typical mitotic division. The division of the cytoplasm is effected rapidly at the animal pole of the egg where the active 1 6 EARLY EMBRYOLOGY OF THE CHICK cytoplasm is aggregated. When, however, the yolk mass is encountered, the process is greatly retarded. So slowly, in fact, is the division of the yolk accomplished, that succeeding cell divisions begin at the animal pole of the egg before the first cleavage is completed at the vegetative pole. The eggs of birds are also telolecithal, but the amount of yolk which they contain is both relatively and actually much greater than that in Amphibian eggs. Cleavage in bird's eggs begins as it does in the eggs of Amphibia, but the mass of the inert yolk material in them is so great that the yolk is not divided. The process of segmentation is limited to the small disc of protoplasm lying on the surface of the yolk at the animal pole, and is for this reason referred to as discoidal cleavage (Fig. 5). The fact that the whole egg is not divided is indicated by designating the process as partial (meroblastic) cleavage in distinction to the complete cleavage (holoblastic) seen in eggs containing less yolk. The cells formed in the process of segmentation are known as blastomeres whether they are com- pletely separated as results in holobastic cleavage or only partially separated as results in meroblastic cleavage. The Unsegmented Blastodisc. — In the egg of a bird which is about to undergo cleavage, the disc of active protoplasm at the animal pole (blastodisc) is a whitish, circular area about three millimeters in diameter. The central portion of the blastodisc is surrounded by a somewhat darker appearing marginal area known as the periblast. The protoplasm of the blastodisc, especially in the periblast region, blends into the underlying white yolk so that it is difficult to make out any line of demarca- tion between the two. It is in the central area of the blasto- disc that cleavage furrows first appear. Neither the nuclei resulting from the early cleavages nor the cleavage furrows invade the marginal periblast until very late in the process of segmentation. The Sequence and Orientation of the Cleavage Divisions in Birds. — The nature of the series of divisions in the meroblastic, discoidal cleavage characteristic of the eggs of birds is largely determined by the amount and distribution of the yolk. An- other determining factor is the tendency of mitotic spindles to develop so that the long axis of the spindle lies at right angles to the axis of least dimension of the mass of unmodified cyto- plasm. The cleavage furrow always arises at right angles to PROCESS OF SEGMENTATION 1 7 the long axis of the mitotic spindle. Figure 5 shows the succes- sion of the cleavage divisions in the egg of the pigeon. The diagrams represent surface views of the blastodisc and an area of the surrounding yolk, the shell and albumen having been removed. The observer is looking directly at the animal pole. Figure 5, ^, should be compared with Figure 4. The diagrams of Figure 4 are of sections cut in a plane which passes vertically through the blastodisc and which is at right angles to the plane of the first cleavage (Fig. 5, A, I-I). The first cleavage furrow cuts into the egg in a plane coinciding with the imaginary axis passing through the animal pole and the vegetative pole. The two daughter cells or blastomeres resulting from the first cleavage are not completely walled off but each remains unseparated from the underlying yolk (Fig. 4). In each of the two blastomeres resulting from the first cleav- age division, mitotic spindles initiating the second cleavage arise at right angles to the position which was occupied by the first cleavage spindle. This determines that the two second cleav- age furrows will be at right angles to the first. Since these two second cleavage furrows lie in the same plane and are apparently continuous they are usually considered together. They mark the position of the second cleavage plane which cuts the egg in the animal- vegetative axis but which lies at right angles to the first cleavage plane (Fig. 5, B, II-II). A very good way of getting a clear conception of the orientation of the ojeavage planesJs to cut them in an apple. Let the core of the apple represent the animal- vegetative axis of the egg. The first cleavage furrow can be represented by notching the apple lengthwise, that is as one ordinarily starts to split an apple into halves. The second cleavage furrow can be represented by cutting into the apple again in a plane passing through the axis of the core, but at right angles to the first cut, as one would start to quarter the apple. The third cleavage furrows are variable in number and in position. In the most typical cases each of the four blastomeres established by the first two cleavages divides again so that eight blastomeres are formed (Fig. 5, C). Frequently, however, the third cleavage appears at first in only two of the blastomeres, so that six cells result instead of eight. The fourth series of cleavages takes place in such a manner i8 EARLY EMBRYOLOGY OF THE CHICK Fig. 5. — Surface aspect of blastoderm at various stages of cleavage. {Based on Blount's photomicrographs of the pigeon's egg.) The blastodenn and the immediately surrounding yolk are viewed directly from the animal pole, the shell and albumen having been removed. The order in which the cleavage furrows have appeared is indicated on the diagrams by Roman numerals. A, first cleavage; B, second cleavage; C, third cleavage; D, fourth cleavage; £, fifth cleavage; F, early morula. PROCESS OF SEGMENTATION IQ that the central (apical) ends of the eight cells established by the third cleavage are cut off from their peripheral portions. The combined contour of the fourth cleavage furrows forms a small irregularly circular furrow the center of which is the point at which the first two cleavage planes intersect (Fig. 5, D). The central cells now appear completely separated in a surface view of the blastoderm, but sections show them still unseparated from the underlying yolk. After the fourth, the succession of cleavages becomes irregular. In surface view it is possible to make out cleavage furrows that divide off additional apical cells, and other, radial furrows that further divide the peripheral cells. Figure 5, E and F, show the increase in number of cells and their extension out over the surface of the yolk, resulting from the succession of cleavages. When the process of segmentation has progressed to the stage in which the succession of cleavages is irregular and the number of cells considerable, the term blastoderm is applied to the entire group of blastomeres formed by the cleavage of the blastodisc.^ In addition to the cleavages which are indicated on the sur- face, at about the 3 2 -cell stage sections show cleavage planes of an entirely different character. These cleavages appear below the surface and parallel to it. They establish a superficial layer of cells which are completely delimited. These superficial cells rest upon a layer of cells which are continuous on their deep faces with the yolk. Continued divisions of the same type eventually establish several strata of superficial cells. This process appears first in the central portion of the blastoderm. It progresses centrifugally as the blastoderm increases in size but does not extend to its extreme margin. The peripheral margin of the blastoderm remains a single cell in thickness and the cells there lie unseparated from the yolk. 1 While but a single spermatozoon takes part in fertilization other spermatoza become lodged in the cytoplasm of the blastodisc. The nuclei of these sperma- tozoa migrate to the peripheral part of the blastoderm where they are recog- nizable for some time as the so-called accessory sperm nuclei. Some of them appear to undergo divisions which are accompanied by slight indications of division in the adjacent cytoplasm. The short superficial grooves thus formed are termed accessory cleav^age furrows. No cells are formed by the accessory "cleavages." The sperm nuclei soon degenerate, the superficial furrows fade out, and usually as early as the 3 2 -cell stage all traces of the process have dis- appeared without, as far as is known, affecting in any way the development of the embryo. CHAPTER IV THE ESTABLISHMENT OF THE ENTODERM The morula stage; the formation of the blastula; the effect of yolk on gastrulation ; gastrulation in BIRDS. The Morula Stage. — It should by no means be inferred that cell division ceases with the cleavage divisions. The end of the segmentation stage is not marked by even a retardation in the succession of mitoses. Segmentation is regarded as ending when the progress of development ceases to be indicated merely by increases in the number of cells, and begins to involve locaHzed aggregation and differentiation of various groups of cells. Development progresses from phase to phase without abrupt change or interruption. The nomenclature and limitation of the various phases of development are largely arbitrary and the use of terms designating phases or stages of development should not be allowed to obscure the fact that the whole process is a continuous one. In eggs without a large amount of yolk, segmentation results in the formation of a rounded, closely packed mass of blasto- meres. This is known as a morula from its resemblance to the mulberry fruit which is in form much like the more familiar raspberry or blackberry. At the end of segmentation the chick embryo has arrived at a stage which corresponds with the morula stage of- forms with less yolk. It consists of a disc- shaped mass of cells several strata in thickness, the blastoderm, lying closely appUed to the yolk. In the center of the blasto- derm the cells are smaller and completely defined; at the per- iphery the cells are flattened, larger in surface extent, and are not walled off from the yolk beneath. The Formation of the Blastula. — The morula condition is of short duration. Almost as soon as it is established there begins a rearrangement of the cells presaging the formation of the blastula. A cavity is formed beneath the blastoderm by the 20 ESTABLISHMENT OF THE ENTODERM 21 detachment of its central cells from the underlying yolk while the peripheral cells remain attached. The space thus estab- lished between the blastoderm and the yolk is termed the seg- mentation cavity (blastocoele). The marginal area of the blastoderm in which the cells remain undetached from the yolk , and closely adherent to it, is called the zone of junction. With the establishment of the blastocoele the embryo is said to have progressed from the morula to the blastula stage. Figure 7, D, shows the conditions seen on sectioning the blastula of a bird. Only the blastoderm and the immediately underlying yolk are included in the diagram. At this mag- nification the complete yolk must be imagined as about three feet in diameter. The structure of the bird embryo in these stages may be brought in line with the morula and blastula stages of forms having little yolk if the full significance of the great yolk mass is appreciated. Instead of being free to aggre- gate first into a solid sphere of cells (morula) and then into a hollow sphere of cells (blastula), as takes place in forms with ^ little yolk, the blastomeres in the bird embryo are forced to grow on the surface of a large yolk sphere. Under such mechanical conditions the blastomeres are forced to be- come arranged in a disc-shaped mass on the surface of the yolk. If one imagines the yolk of the bird morula removed, and the disc of cells left free to assume the spherical shape dictated by surface tension its comparability with the morula in a form having little yolk becomes apparent. The process of blastulation also is modified by the presence of a large amount of yolk. There can be no simple hollow sphere formation by rearrangement of the cells if the great bulk of the morula is inert yolk. But the cells of the central region of the blastoderm are nevertheless separated from the yolk to form a small blastocoele. The yolk constitutes the floor of the blastocoele and at the same time by reason of its. great mass nearly obliterates it. If we imagine the yolk removed from the blastula and the edges of the blastoderm pulled together the chick blastula approaches the form of the blastula in embryos with little yolk. The Effect of Yolk on Gastnxlation. — The process of gastrula- -^ tion begins as soon as blastulation is accompHshed. Gastrula- tion as it occurs in birds is not difiicult to understand if one 22 EARLY EMBRYOLOGY OF THE CHICK grasps its fundamental similarity to the corresponding process in forms with scanty yolk. In Amphioxus, gastrulation is an inpocketing of the blastula (Fig. 6). A double layered cup is formed from a single layered hollow sphere much as one might GASTRULATION IN PORM WITH ISOLECITHAL EGG HAVING ALMOST NO YOLK— AMPHIOXU& GASTRULATION IN FORM WITH TELOLECITHAL EGG CONTAINING MODERATE AMOUNT OF YOLK— AMPHIBIA. GASTRULATION IN FORM WITH TELOLECITHAL EGG CONTAINING LARGE AMOUNT OF YOLK— BIRDS. Auu^^' .^•~S<^^^"^atic diagrams to show the effect of yolk on gastrulation. Abbreviations: blc, blastocoele; bid., blastoderm; blp., blastopore; ect., ectoderm; ent., entoderm; mit., cell undergoing mitosis; yk., yolk; vk.g., yolk granules: yk.p.. yolk plug. push in a hollow rubber ball with the thumb. The new cavity in the double walled cup is termed the gastrocoele. The open- inir from the outside into the gastrocoele is called the blastopore. ESTABLISHMENT OF THE ENTODERM 23 In gastrulation the single cell layer of the blastula is doubled upon itself to form two layers. The outer cell layer is known as the ectoderm and the inner layer as the entoderm. These layers differ from each other in their positional relationship to the embryo and to the surrounding environment. Each has different functional potentiaHties and each will in the course of development give rise to quite different types of structures and organs. It is the importance of their later history rather than any complexity or veiled significance about the way in which they arise that attaches such importance in embryology to the establishment of these two layers. In the gastrulation of Amphibian embryos (Fig. 6) the yolk forces the invagination of the blastoderm toward the animal pole, but the inpocketing takes place into the blastocoele and the interrelationships of ectoderm, entoderm, and gastrocoele are established in fundamentally the same way as in Amphioxus. Gastrulation in birds is greatly modified by the large amount of yolk present (Fig. 6). Infolding must be effected in a disc of cells resting like a cap on a large yolk sphere. The smallness of the blastocoele sharply restricts the space into which the invagination can grow. Instead of arising as a relatively large circular opening the blastopore appears as a crescentlc slit at the margin of the blastoderm. The crescentic blastopore may be regarded as a potejitially circular opening which has been flattened as it develops between the growing disc of cells and the unyielding yolk which underhes them. The invagi- nated pocket of entoderm which grows in from this compressed blastopore is also flattened, conforming to the restrictions of the shape and size of the blastocoele. Moreover the floor of the invagination is represented only by a few widely scattered cells lying upon the yolk. It is as if the lower layer in its in- growth was impeded and broken up by the yolk. The scattered cells representing the floor of the invagination soon disappear and the yolk itself comes to constitute the floor of the gastrocoele. Notwithstanding the great displacement of the blastopore and the gastrular invagination toward the animal pole ajid the restricted size and incomplete floor of the gastrocoele, the cell layers and the cavity established can be homologized with the corresponding features in forms where the course of develop- ment has not been so extensively modified by yolk. 24 EARLY EMBRYOLOGY OF THE CHICK A comparative review of the diagrams of Figure 6 will afford a general understanding of the infolding process of gastrulation. These diagrams aim to convey merely the scheme of the process. They are therefore simplified and emphasize the similarities of gastrulation in forms with widely varying amounts of yolk, rather than the details of the process in any one form. With this general groundwork we may now profitably return to the blastula stage and consider in somewhat more detail the process of gastrulation as it occurs in birds. Gastrulation in Birds. — We have already estabhshed the blastula as a disc of cells lying on the yolk but separated from it centrally by a flattened blastoccele or segmentation cavity. The peripheral part of the blastoderm where the marginal cells lie unseparated from the yolk has been termed the zone of junction (Fig. 7, Z^). This part of the blastoderm is also called the area opaca because in preparations made by removing the blastoderm from the yolk surface, yolk adheres to it and renders it more opaque. This opacity is especially apparent when a preparation is viewed under the microscope by transmitted light. The central area of the blastoderm, because it is sepa- rated from the yolk by the segmentation cavity, does not bring a mass of adherent yolk with it when the blastoderm is removed. It is for this reason translucent and is called the area pellucida. The area opaca later becomes differentiated so that three more or less distinct zones may be distinguished: (i) a peripheral zone known as the margin of overgrowth where rapid prolifera- tion has pushed the cells out over the yolk without their becom- ing adherent to it; (2) an intermediate zone known as the zone of junction in which the deep-lying cells do not have complete cell boundaries but constitute a syncytium blending without definite boundary into the superficial layer of white yolk and adhering to it by means of penetrating strands of cytoplasm; (3) an inner zone known as the germ wall made up of cells derived from the inner border of the zone of junction which have acquired definite boundaries and become more or less free from the yolk. The cells of the germ wall usually contain numerous small yolk granules which were enmeshed in their cytoplasm when they were, as cells of the zone of junction, unseparated from the yolk (Fig. 7, By E). The inner margin of the germ wall marks the transition from area opaca to area pellucida. ESTABLISHMENT OF THE ENTODERM The changes in the blastula which indicate the approach of gastrulation are, first, a thinning of the blastoderm at its caudal margin and, second, freeing of the blastoderm from the yolk in the same region (Fig. 7, Z)). The separation of the blasto- derm from the yolk is evidenced in surface views by a crescentic gap in the posterior quadrant of the zone of junction (Fig. y, A). 26 EARLY EMBRYOLOGY OF THE CHICK This region where the blastoderm is thin and free from the yolk marks the position of the blastopore. Gastrulation begins with the undertucking of the cells at the free margin of the blastoderm. Figure 7, B, is a diagrammatic surface view of the blastoderm represented as a transparent object. The position and the extent of the invaginated ento- derm seen through the overlying ectoderm are indicated by the cross hatched area. The appearance of the blastopore locates the caudal region of the future embryo and permits the definition of its longitudinal axis. This axis is indicated by the line b-b on Figure 7, B. A diagram of a section cut in the longitudinal axis and passing through the blastopore of an embryo of this stage is shown in Figure 7, E. The invaginated cells which constitute the entoderm form a layer extending cephalad from the thickened lip of the blastopore. The yolk forms the floor of the gastroccele. Figure 7, C, is a diagrammatic surface-view of a later stage in the same process. The extent of the entoderm is marked by cross-hatching as in the diagram of the previous stage. The undertucking of the cells at the blastopore has ceased by this time, and as indicated in Figure 7, C. by the black area, and in Figure 7, F, by the solid mass of cells seen in section, the blastopore has become closed. During the entire time that the process of gastrulation has been in progress there has been constant cell proliferation going on in the blastoderm as a whole. The growth of the blastoderm has been evidenced especially by increase in its surface extent which has resulted in a general spreading of its peripheral mar- gins over the yolk. This extension has taken place uniformly at all parts of the margin except in the posterior quadrant where the blastopore is located. Here the cells proliferated, instead of spreading out over the yolk have turned in at the lip of the blastopore to form the invaginated entoderm. This particular part of the margin of the blastoderm, having contributed the cells formed in its growth to the entoderm which grows back toward the center of the blastoderm, takes no part in the general peripheral expansion. As a result the blastopore region is, as it were, left behind and the rapidly extending margin of the blastoderm on either side sweeps around and encloses it. The blastopore at the time of its closure thus comes to lie within the recompleted circle of the germ wall (Fig. 7, C). CHAPTER V THE FORMATION OF THE PRIMITIVE STREAK AND THE ESTABLISHMENT OF THE MESODERM The location and appearance of the primitive streak; the origin of the primitive streak by- concrescence OF THE blastopore; THE FORMATION OF THE MESODERM. The Location and Appearance of the Primitive Streak. The stages of development described in the preceding chapters take place before the egg is laid. The first conspicuous struc- tural feature to make its appearance in the embryo after the laying of the egg is the primitive streak. In eggs that have been incubated about i6^hours the primitive streak is well developed cephalic end Hensen's node area pellucida area opaca primitive pit primitive groove primitive ridge Fig. 8. ■Dorsal view ( X 14) of entire chick embryo in the primitive streak stage (about 16 hours of incubation). as a linear groove flanked on either side by ridge-like thickenings, extending from the inner margin of the area opaca to approxi- mately the center of the blastoderm (Fig. 8). The primitive streak Hes in the longitudinal axis of the future embryo. The end adjacent to the area opaca is its posterior (caudal) end, the opposite extremity is its anterior (cephalic) end. The ce- 27 28 EARLY EMBRYOLOGY OF THE CHICK phalic end of the primitive groove is deepened and often some- what expanded to form a depression known as the primitive pit. Directly anterior to the primitive pit the right and left primitive folds merge with each other in the mid-line to form a small rounded elevation called Hensen's node. Hensen's node is of importance as a landmark rather than because it gives rise to any particular structure. As early as the beginning of gastrulation the shape of the blastoderm responds to local inequality in the rate of growth. One of the early manifestations of differential growth is the more rapid extension of the embryo cephalad than either laterad or caudad. This results in a definite elongation in the antero-posterior axis by the time the primitive streak is established (Fig. 8). The Origin of the Primitive Streak by Concrescence of the Blastopore. — The significance of the primitive streak has been the subject of much controversy. The divergences of opinion have been due chiefly to incomplete knowledge of the stages of development passed through prior to the laying of the egg. Our present knowledge of these early stages is, however, suffi- cient to furnish the basis of an interpretation of the primitive streak which is now widely accepted. This interpretation is outlined below without reference to other, opposed views. The primitive streak is to be regarded as a scar-like thicken- ing arising from the fusion of the edges of the anterior lip of the blastopore. To understand the origin of the longitudinally placed primitive streak from the marginally located, crescentic blastopore it is necessary to follow carefully the growth proc- esses taking place during the closure of the blastopore. We have already seen how the ingrowth of entoderm from the anterior lip of the blastopore, caused the blastopore to lag behind the other parts of the margin of the blastoderm in the process of radial extension over the yolk surface. During this process the blastopore is compressed from either side toward the mid-line by the rapidly extending margins of the blastoderm adjacent to it and is eventually encompassed by them (see Chap. IV and Fig. 7). Because of the insweeping, converging tendency of the growth which first causes the blastopore to be laterally compressed and finally causes its margins to grow together the process has been termed concrescence. FORMATION OF THE PRIMITIVE STREAK 29 A schematic interpretation of four steps in the concrescrnce of the margins of the blastopore is given in the diagrams of Figure 9. The blastoderm shown in surface- view plan in Figure 9, ^, is approximately at the same stage of gastrulation as that indicated in Figure 7, B. To avoid complicating the diagarm, the entoderm has not been shown in Figure 9. Num- bers have been placed along the lip of the blastopore to facilitate marginal notch Fig. 9. — Schematic diagrams to illustrate the concrescence theory of the origin of the primitive streak. {After Lillie.) For explanation see text. following the changes in position undergone by the points to which they are affixed. As the margins of the blastoderm adjacent to the blastopore grow, they tend to converge in the direction indicated by the arrows in Figure g, B. The anterior lip of the blastopore is folded on itself by this converging growth. The middle point of the lip, i, comes to lie within the margin of the blastoerdm, and points, 2, 2, which formerly lay laterally are 30 EARLY EMBRYOLOGY OF THE CHICK brought into apposition in the mid Hne. Figures C, and D, show how, by the continuation of the same converging growth, the edges of the blastopore are folded together into a line of fusion at right angles to the Original marginal position of the blastopore. At the completion of concrescence, the germ wall of the blastoderm has coalesced posterior to the blastopore leaving the line along which the blastopore lips have fused within the area pellucida. The non-committal term primitive streak was given to this structure before its origin by fusion of the lips of the blastopore was suspected. The Formation of the Mesodenn. — In its early condition the primitive streak is a scarcely recognizable thickening of the blastoderm marking the line of fusion of the hps of the blasto- pore. The well defined groove with thickened ridges on either side, seen in chicks of 15 to 1 6 hours incubation, is a later devel- opment. A new process, the formation of the mesoderm, is taking place at this region and the change in the configuration of the primitive streak is its outward manifestation. It will be recalled that the lip of the blastopore is in all forms a region of rapid cell proHferation. It is a region from which we can trace the addition of cells to the differentiated germ layers, but it is itself indifferent. Ectoderm and entoderm both merge into this indifferent area at the lip of the blastopore. It is impossible to fix, except arbitrarily, where ectoderm begins and entoderm ends. Later when the mesoderm appears, we can trace the origin of its cells directly or indirectly to the same area of indifferent, rapidly prohferating cells. It is therefore wholly in Hne with the embryology of other forms to find the mesoderm of the chick arising at the fused lips of the blastopore. The manner in which the mesoderm arises can be understood only by the study of sections or diagrams of sections. Figure 10, A, represents schematically the conditions which would be seen in a section cut in the hne h-h across the marginal notch of an embryo of the stage depicted in Figure 9, B. The mar- gins of the blastopore at the point where this section is located have been folded so they lie in close proximity to each other. A Httle later they would be fused as shown in Figure 10, B. At the region of fusion, that is to say at the primitive streak, the entoderm and ectoderm merge in a mass of rapidly dividing cells (Fig. 13, Z>). A section across the primitive streak at a FORMATION OF THE PRIMITIVE STREAK 31 somewhat later stage (Fig. 10, C) shows cells extending to either side of the undifferentiated cell mass, between the ecto- derm and the entoderm. These cells are the primordium of the third of the germ layers, the»mesoderm. The outgrowth of the mesoderm and the median depression in the primitive streak appear synchronously. This median depression in the primi- tive streak is the primitive groove. It is not unhkely that the formation of the primitive groove is due to cell rearrangement lips of blastopore A y^jj ^^-'"■^ I ^*^~ entodenn primitive g;ut yolk primitive gut primitive groove Fig. 10. — Diagrams showing schematically the relations of the germ layers during the formation of the primitive streak by concrescence of the margins of the blastopore. A, hypothetical section of blastoderm at the stage represented in Fig. 9, B. The plane of the section is indicated by the line h-h Fig. 9, B. B, hypothetical section of blastoderm at the stage represented in Fig. 9, D. The plane of the section is indicated by the line d-d. Fig. 9, D. C, schematic transverse section through the primitive streak at the stage represented in Fig. 8. in this region entailed by the rapid outgrowth of the cells con- stituting the mesoderm. (See arrows in Figure lo, C.) With the formation of the mesoderm the chick has estab- lished the three germ layers characteristic of all vertebrate embryos. The importance of these layers lies in the uniformity of their origin and history. From them the development of all the organ systems may be traced. The ectoderm gives rise to 32 EARLY EMBRYOLOGY OF THE CHICK the outer epithelial covering of the body and its derivatives (feathers, claws, skin glands, etc.) , the nervous system, and the sense organs. The entoderm gives rise to the epithelial lining of the digestive tube and of the respiratory organs and the epitheHum of their associated glands. The mesoderm becomes differentiated to form the fibrous and rigid connective tissues (except neuroglia) the muscle, the epithelial lining of the body cavities, the organs of the circulatory system, the* blood, the lymphatic organs and the major part of the urino-genital system of the adult. CHAPTER VI FROM THE PRIMITIVE STREAK STAGE TO THE APPEARANCE OF THE SOMITES The primitive streak as a center of growth; the growth of the entoderm and the establishment of the PRIMITIVE gut; the GROWTH AND DIFFERENTIATION OF THE MESODERM; THE FORMATION OF THE NOTOCHORD; THE FORMATION OF THE NEURAL PLATE; THE DIFFERENTIATION OF THE EMBRYONAL AREA. The Primitive Streak as a Center of Growth. — The impor- tance of the primitive streak embryologically, is due chiefly to the way it is involved in the estabHshment of the germ layers. Representing as it does the fused lips of the blastopore it marks the location of entoderm invagination. The mesoderm also arises at the primitive streak region. The general appearance and the location of the primitive streak are both well shown in embryos of i6 hours of incubation (Fig. 8). In embryos which have been incubated i8 hours (Fig. ii) the primitive streak is still the most conspicuous feature. Structurally it is little changed from the conditions seen in 1 6-hour chicks, but it appears to be somewhat more caudally located. In 21 to 2 2 -hour em- bryos (Fig. 14) the primitive streak lies still farther caudal in the blastoderm. Its change in position is relative rather than actual. The apparent change in the position of the primitive streak is due to the fact that growth is taking place more rapidly cephalic to it than caudal to it. This tendency is in evidence throughout the early growth of the embryo. The cephalic region is precocious in development. As development pro- gresses we shall find the primitive streak occupying a constantly more posterior position in the body and being more and more overshadowed by the greater growth of the structures lying cephalic to it. The structure of the primitive streak region is best shown by transverse sections. In the sections diagrammed in Figure 3 . 33 34 EARLY EMBRYOLOGY OF THE CHICK 13, a different conventional scheme of representation has been employed to indicate each of the germ layers. The ectoderm is vertically hatched, the cells of the mesoderm are represented by heavy angular dots when they are isolated or by solid black lines when they lie arranged in the form of compact layers, and the entoderm is represented by fine stippHng backed by a single line. This same conventional representation of the different germ layers is observed in all diagrams of sections in anterior border of mesoderm neural plate. embryonal area ■ area pellucida- area opaca -r: notochord -Hensen's node 'primitive streak caudal end Pig. II. — Dorsal view ( X 14) of entire chick embryo of 18 hours incubation. order to facilitate following the way in which the organ systems of the embryo are constructed from the germ layers. Details of cell structure are for the most part omitted with the expecta- tion that the student will acquire a knowledge of them in his own study of sections. The plane in which each of the sections diagrammed passes through the embryo is indicated by a line drawn on a small outline sketch of an embryo of corresponding stage. For interpretation these outline sketches should be compared with actual specimens or detailed drawings of entire embryos of the same stage of development. In embryos of the stage under consideration the relationship of the germ layers at the primitive streak still indicates their man- ner of derivation (Fig. 13, C and D). The ectoderm and the PRIMITIVE STREAK TO SOMITE FORMATION 35 entoderm are continuous with each other without any demarca- tion. The mesoderm arises from the primitive streak where ectoderm and entoderm merge and grows laterad on both sides of the primitive streak extending into the space between ectoderm and entoderm. The mass of cells in the floor of the primitive groove is to be regarded as constituting an undifferentiated area from which new cells are being proUferated rapidly and are emigrating to become components of one or another of the germ layers. To those who have studied the embryology of more primitive vertebrates, particularly the Amphibia, the fact that the lips of the blastopore constitute centers of growth from which cells are pushed forth to take part in the formation of the differenti- ated germ layers will already be famiHar. The fact that the blastopore of the chick has suffered a change in position due to concrescence, and has in the same process become closed by fusion of its Ups must not be allowed to obscure its homologies. In attempting to bring the relationships of the germ layers in the chick into Hne with the relationships of the germ layers in embryos having less yolk, it will be of great assistance to picture a chick lifted off the yolk and the lateral margins of the blasto- derm pulled together ventrally; or, the method of comparison may be reversed if one imagines the embryo of a form having less yolk, such as an amphibian, to be split open along the mid- ventral line and spread out on the surface of a sphere as a chick lies on the yolk. In Figure 13, D, a small region at the primitive streak has been drawn at higher magnification to show the characteristic cellular structure of the undifferentiated region in the floor of the primitive groove and of the various layers merging at this place. The cells of the ectoderm are much more closely packed together and more sharply delimited than those of the other germ layers. Where the ectoderm is thickened in the primitive ridge region, it is several cell layers thick (stratified). (Fig. 13, D.) In regions lateral to the primitive ridge it gradually be- comes thinner until it consists of but a single cell layer (Fig. 13, E). The rapid extension that the mesoderm is at this time undergoing is indicated by the loose arrangement and sprawling appearance of its cells. Their irregular cytoplasmic processes, make them look much Hke amoebae fixed during locomotion. 36 EARLY EMBRYOLOGY OF THE CHICK The cells of the entoderm are neither as closely packed nor as clearly defined as are the ectoderm cells. Nevertheless, in contrast to the condition of the mesoderm at this stage, the entoderm cells form a definite, unbroken layer. The Growth of the Entoderm and the Establishment of the Primitive Gut. — Sections of embryos of this stage show how the entoderm has spread out and become organized into a coherent layer of cells merging peripherally with the inner mar- gin of the germ wall and overlapping it to a certain extent (Fig. 13, C, E, F). The cavity between the yolk and the ento- derm which has been called the gastrocoele is now termed the primitive gut. The yolk floor of the primitive gut does not show in sections prepared by the usual methods. The reasons for this are to be found in the relations of the embryo to the yolk before it is removed for sectioning. In the entire central region of the blastoderm the yolk is separated from the ento- derm by the cavity of the primitive gut. When the embryo is removed from the yolk sphere the yolk floor of the primitive gut, not being adherent to the blastoderm, is left behind. In contrast the peripheral part of the blastoderm lies closely ap- pHed to the yolk. Some yolk adheres to this part of the blasto- derm when it is removed. This adherent yolk is shown in the section diagrams of Figure 13. Its presence clearly indicates why this region (area opaca) appears less translucent in surface views of entire embryos. In embryos of 18 hours the primitive gut is a cavity with a flat roof of entoderm and a floor of yolk. Peripherally it is bounded on all sides by the germ wall (Fig. 13, C, F). The merging of the cells of the entoderm with the yolk mass is shown in the small area of the germ wall drawn to a high mag- nification in Figure 13, £. In the germ wall cell boundaries are incomplete and very difiicult to distinguish but nuclei can be made out surrounded by more or less definite areas of cyto- plasm. This cytoplasm contains numerous yolk granules in various stages of absorption. It will be recalled that the nuclei of the germ wall arise by division from the nuclei of cells lying at the margins of the expanding blastoderm. They appear to be concerned in breaking up the yolk in advance of the ento- derm as it is spreading about the yolk sphere. About the twenty-second hour of incubation indications can PRIMITIVE STREAK TO SOMITE FORMATION 37 be seen of a local differentiation of that region of the primitive gut which underHes the anterior part of the embryo. By focus- ing through the ectoderm in the anterior region of a whole- mount of this age a pocket of entoderm can be seen (Fig. 14). This entodermal pocket is the first part of the gut to acquire a floor, other than the yolk floor, and is called from its anterior position the fore-gut. Consideration of the fore-gut except to note the location of its first appearance can advantageously be deferred because its origin and relationships are more readily appreciated from the study of somewhat older embryos. The Growth and Differentiation of the Mesoderm. — The mesoderm which arises from either side of the primitive streak spreads rapidly laterad and at the same time each lateral wing of the mesoderm swings cephalad. Figure 12 shows schematically the extension of the mesoderm during the latter part of the first day of incubation. The diagonal hatch- ing represents the mesoderm seen through the transparent ectoderm. The principal landmarks of the embryos are sketchily represented. It will be noticed that the manner in which the mesoderm spreads out leaves a mesoderm-free area in the anterior portion of the blastoderm. This region is known as the proamnion. The name might carry the inference that this area is the primor- dium of the amnion, a structure which first appears near this region somewhat later in development. Such is not the fact. The term proamnion was applied to this region before its true significance was understood. It is not the precourser of the amnion. In dorsal views of entire embryos the proamnion is readily located by reason of its lesser density. The proamnion is bounded anteriorly by the area opaca, posteriorly in the mid- line by the thickened anterior part of the embryo, and poste- riorly on either side by the anterior bordero f the mesoderm (Fig. 12). The importance of the proamnion lies chiefly in the indication it gives of the progress of mesoderm extension. The rapid growth that the mesoderm of the anterior region is under- going at this stage is clearly indicated by the diminution in area of the proamnion in embryos of 22 hours as compared with embryos of 18 hours (Fig. 12). Sections passing through the primitive streak of embryos of this stage show the pair of loosely aggregated masses of meso- 38 EARLY EMBRYOLOGY OF THE CHICK derm extending to either side between the ectoderm and ento- derm. As would be expected from the method of origin, little mesoderm appears in the mid-line except posterior to the primi- tive streak. Immediately to either side of the mid-line the mesoderm is markedly thicker than it is farther laterad (Fig. IS, B). In whole-mounts the positions of the regional thicken- ^.J^ primitive streak peUucida. CHICK OF ABOUT 14 HOURS. anterior horn of mesoderm ...^n'i^+^. ]2 CHICK OF ABOUT 18 HOURS, proamnion dorsal mesoderm primitive streak V^ CHICK OF ABOUT 22 HOURS. Pig. 12. — Schematic diagrams to show the extension of the mesoderm during the latter part of the first day of incubation. Some of the more prominent structural features of the embryos are drawn in lightly for orientation but the ectoderm is supposed to be nearly transparent allowing the mesoderm to show through. The areas into which the mesoderm has grown are indicated by diagonal hatching. ^^ ings of the mesoderm are evidenced by the greater opacity they impart to the embryo locally (Fig. 14). These thickened zones of the mesoderm are the primordia of the dorsal mesodermic plates. Because of the way in which they are later divided into PRIMITIVE STREAK TO SOMITE FORMATION 39 ■^^m^^^ prumtive gut nucleus cell in mitosis ^ ^^^ „f ^^j^ granules - entoderm indifferent cells I J High power thru primitive streak at region (a) on section C. "C^ High power thru edge of germ wall at region (b) on section C. Hensen'snode primitive neural plate | .primitive pit ridge 'j)rimitive groove ' extent of primitive gut and of area pellucida Pig. 13. — Sections of iS-hoxir chick. The location of each section is indicated by a line drawn on a small outline sketch of an entire embryo of corresponding age. The letters affixed to the lines indicating the location of the sections correspond with the letters designating the section diagrams. Each germ layer is represented by a different conventional scheme: ectoderm by vertical hatching; entoderm by fine stippling backed by a single line; and the cells of the mesoderm which at this stage do not form a coherent layer, by heavy angular dots. A, diagram of transverse section through notochord; B, diagram of transverse section through primitive pit; C, diagram of transverse section through primitive streak; D, drawing showing cellular structure in primitive streak region; E, drawing showing cellular structure at inner margin of germ wall; F, diagram of median longitudinal section passing through notochord and primitive streak. 40 EARLY EMBRYOLOGY OF THE CHICK metamerically arranged cell masses or somites they are fre- quently designated as the segmental zones of the mesoderm. The segmental zones are in early stages most clearly marked somewhat cephalic to Hensen's node, where the first somites will appear. As they extend caudad on either side of the primitive streak they gradually become less and less definite. The sheet-like layers of mesoderm which are characteristic of the mid-body region do not extend to the anterior part of the embryo. The mesoderm of the future head region is derived from mesoderm cells which invade the head from the more definitely organized layers of mesoderm lying posterior to it. The cephaHc mesoderm for this reason never shows the regional differentiations and the organization into definite layers which later appear in the mesoderm of the mid-body region. The Formation of theNotochord. — The notochord arises in the chick as a median out-growth from the rapidly proliferating, undifferentiated cells at the cephalic end of the primitive streak (Fig. is,F). The way in which the notochord grows cephalad from the anterior end of the primitive streak, just as in other vertebrate embryos it arises from the region of the anterior lip of the blastopore, is one of the points which confirms the identifica- tion of the primitive streak of the chick as the closed blastopore. Largely because of the way in which the notochord arises in Amphioxus, a primitive vertebrate of doubtful relationships, it has usually been considered of entodermal origin. In Amphibia and in birds it arises not from any definite germ layer but from the undifferentiated growth center about the blastopore which is giving rise to both entoderm and mesoderm. Even in Am- phioxus the notochord arises at the same time and in the same manner as the mesoderm. In its later differentiation the noto- chord resembles mesodermal derivatives more closely than entodermal. The common origin of notochord and mesoderm, and the unmistakably mesodermal characteristics of the fully developed notochord should be emphasized rather than the early association of the notochordal primordium with the entoderm and its doubtful origin therefrom. For these reasons the notochord is in this book treated as a mesodermal structure. In entire embryos of i8 to 22 hours (Figs. 11 and 14) the notochord can be seen in the mid-line extending cephalad from Hensen's node. Hensen's node is at once the posterior limit PRIMITIVE STREAK TO SOMITE FORMATION 41 of the notochord and the anterior end of the primitive streak. The notochord and the primitive streak together clearly mark the mid-line of the embryo and estabUsh definitely the longitu- dinal axis of the developing body. In sections (Fig. 13, ^, F) the notochord is not at this early stage sharply differentiated from the loosely arranged mesoderm cells adjacent to it. In later stages, however, the cells composing it become aggregated to form a characteristic rod-shaped structure, circular in cross section and with clearly defined boundaries (Fig. 52, C). The Formation of the Neural Plate. — In surface views of en- tire chicks of about 18 hours (Fig. 11) areas of greater density cephalic end ectoderm of head border of fore-gut margin of anterior intestinal portal notochord primitive streak area pellucida area opaca caudal end Fig. 14. — Dorsal view ( X 14) of entire chick embryo of about 21 hours incubation. may be made out on either side of the notochord. These areas extend somewhat anterior to the cephaUc end of the notochord where they appear to blend with each other in the mid-hne. Sections of this region (Fig. 13, A) show that the greater density seen in whole-mounts is due to thickening of the ecto- derm. Rapid cell proHferation has resulted in the ectoderm in the middle region becoming several cells in thickness. This 42 EARLY EMBRYOLOGY OF THE CHICK thickened area is known as the neural (medullary) plate. Laterally the thickened ectoderm of the neural plate blends without abrupt transition into the thinner ectoderm of the general blastodermic surface. Anteriorly the neural plate is more clearly marked than it is posteriorly. At the level of Hensen's node the neural plate diverges into two elongated areas of thickening one on either side of the primitive streak. In embryos of 21 or 22 hours (Fig. 14) the neural plate becomes longitudinally folded to estabHsh a trough known as the neural groove. The bottom of the neural groove lies in the mid-dorsal line. Flanking the neural groove on each side is a longitudinal ridge-like elevation involving the lateral por- tion of the neural plate. These two elevations which bound the neural groove laterally are known as the neural folds. The folding of the originally fiat neural plate to form a gutter, flanked on either side by parallel ridges, is an expression of the same extremely rapid cell proUferation which first manifested itself in the local thickening of the ectoderm to form the neural plate. The formation of the neural plate and its subsequent folding to form the neural groove are the first indications of the differentiation of the central nervous system. The Differentiation of the Embryonal Area.^Due to the thickening of the ectoderm to form the neural plate and also to the thickening of the dorsal zones of the mesoderm, the part of the blastoderm immediately surrounding the primitive streak and notochord has become noticeably more dense than that in the peripheral portion of the area pellucida. Because it is the region in which the embryo itself is developed this denser region is known as the embryonal area. Although the embry- onal area is at this early stage directly continuous with the peripheral part of the blastoderm without any definite Une of demarcation, they later become folded off from each other. The peripheral portion of the blastoderm is then spoken of as extra-embryonic because it gives rise to structures which are not built into the body of the embryo, although they play a vital part in its nutrition and protection during development. The anterior region of the embryonal area is thickened and protrudes above the general surface of the surrounding blasto- derm as a rounded elevation. This prominence marks the region in which the head of the embryo will develop (Fig. 14). PRIMITIVE STREAK TO SOMITE FORMATION 43 The crescentic fold which bounds it is termed the head fold and is the first definite boundary of the body of the embryo. Throughout the course of development we shall find the head region farther advanced in differentiation than other parts of the body. This is a repetition of race history in the develop- ment of the individual, for phylogenetically the head is the oldest and most highly differentiated region of the body. It is one of many manifestations of the law of recapitulation, in conformity with which the individual in its development rap- idly repeats the main steps in the development of the race to which it belongs. CHAPTER VII THE STRUCTURE OF TWENTY-FOUR HOUR CHICKS The formation of the head; the formation of the neural groove; the regional divisions of the mesoderm; the ccelom, the pericardial region; the area vasculosa. The Formation of the Head. — In embryos of 21 to 22 hours the anterior part of the embryonal area is thickened and ele- vated above the level of the surrounding blastoderm, with a well defined crescentic fold marking its anterior boundary. Between 21 and 24 hours this region has undergone rapid growth (Fig. 15). Its elevation above the blastoderm is much more marked and it has grown anteriorly so it overhangs the proamnion region. The crescentic fold which formerly marked its anterior boundary appears to have undercut the anterior part of the embryo and separated it from the blastoderm. The changes in relationships are due, however, not so much to a posterior movement of the fold as to the anterior growth of the embryo itself. This anterior region which projects, free from the blastoderm, may now properly be termed the head of the embryo. The space formed between the head and the blasto- derm is called the subcephalic pocket (Fig. 17, E). In the mid-Une the notochord can be seen through the over- lying ectoderm. It is larger posteriorly near its point of origin than it is anteriorly. Nevertheless it can be readily traced into the cephaUc region where it will be seen to terminate somewhat short of the anterior end of the head (Fig. 15). The Formation of the Neural Groove. — The neural plate in chicks of 18 hours was seen as a flat, thickened area of the ecto- derm. In embryos of 21 to 22 hours a longitudinal folding had involved it establishing the neural groove in the mid-dorsal line flanked on either side by the neural folds. At 24 hours of incubation the folding of the neural plate is much more clearly marked. In a dorsal view of the entire embryo (Fig. 15) the neural folds appear as a pair of dark bands. The folding which 44 STRUCTURE OF TWENTY-FOUR HOUR CHICKS 45 establishes the neural groove takes place first in the cephalic region of the embryo. At its cephalic end the neural groove is therefore deeper and the neural folds are correspondingly more prominent than they are caudally. The folding has not, at this stage, been carried much beyond the cephalic half of the embryo. Consequently as the neural folds are followed caudad they diverge slightly from each other, and become less and less distinct. ectoderm of head border of fore-gut subcephalic pocket Hensen's node .unsegmented Jy'^^'.'-'i'i^f^yC'^'^ mesoderm l^&piW^W^ ' V primitive i-<':-.A-iV=:s,..':35C*«- ■ -,treak border of mesoderm blood island area vasculosa Pig. 15. — Dorsal view ( X 14) of entire chick embryo having 4 pairs of meso- dermic somites (about 24 hours incubation). Study of transverse sections of an embryo of this stage affords a clearer interpretation of the conditions in neural groove for- mation than the study of entire embryos. A section passing through the head region (Fig. ly, A) shows the neural plate folded so it forms a nearly complete tube. Dorsally the mar- gins of the neural folds of either side have approached each other and lie almost in contact. The formation of the neural folds takes place first in about the center of the head region, and progresses thence cephalad and caudad. By following caudad the sections of a transverse series, the margins of the 46 EARLY EMBRYOLOGY OF THE CHICK neural folds will be seen less and less closely approximated to each other. The Establishment of the Fore-gut. — In the outgrowth of the head, the entoderm as well as the ectoderm has been involved. As a result the entoderm forms a pocket within the ectoderm, much like a small glove finger within a larger. This entodermic pocket, or fore-gut, is the first part of the digestive tract to ac- quire a definite cellular floor. That part of the gut caudal to the fore-gut where the yolk still constitutes the only floor, is termed the mid-gut. The opening from the mid-gut into the fore-gut is called the anterior intestinal portal (fovea cardiaca) . margin of anterior horn of mesoderm pourior margin of '^ subcephalic pocket margin of fore-gut -Js margin of Aiterior intestinal portal (entoderm) notochord 11 r : margin of area opaca ectoderm of head " — mesenchyme ■ „ border of mesoderm — — pericardial region of coelom ~ — thickened splanchnic mesoderm neural fold Pig. i6. — Ventral view ( X 37) of cephalic region of chick embryo having 5 pairs of somites (about 25-26 hours of incubation). The topography of the fore-gut region at this stage can be made out very well by studying the ventral aspect of entire embryos. The margin of the anterior intestinal portal appears as a well defined crescentic Une (Fig. i6). The lateral boun- daries of the fore-gut can be seen to join the caudally directed tips of the crescentic margin of the portal. Considerably cephalic to the intestinal portal an irregularly recurved hne can be made out. On either side it appears to merge with the ecto- derm of the head. This Hne marks the extent to which the head is free from the blastoderm. It is due to the fold at the bottom of the subcephalic pocket where the ectoderm of the under surface of the head is continuous with the ectoderm of the blastoderm. Comparison of Figure 16 with the sagittal section diagrammed in Figure 17, -E, will aid in making clear the rela- STRUCTURE OF TWENTY-FOUR HOUR CHICKS 47 tionships of fore-gut to the head. From the sagittal section it will also be apparent why the margins of the intestinal portal and of the subcephalic pocket appear as dark lines in the whole- mount. In viewing an entire embryo under the microscope by transmitted light one depends largely on differences in density for locating deep-lying structures. When a layer is folded so the light must pass through it edgewise, the fold stands out as a dark hne by reason of the greater thickness it presents. The Regional Divisions of the Mesoderm. — The first con- spicuous metamerically arranged structures to appear in the chick are the mesodermic somites. The somites arise by divi- sion of the mesoderm of the dorsal or segmental zone to form block-Hke cell masses. In the embryo shown in Figure 15 three pairs of somites are completely delimited and a fourth pair can be made out which is not as yet completely cut off from the dorsal mesoderm posterior to it. The regular addition of somites as embryos increase in age makes the number of somites the most reliable criterion of the stage of development. Chicks which have been incubated for a given number of hours show wide variation in the degree of development attained; chicks of a given number of somites vary but little among themselves. ' Cross sections passing through the rnid-body region show the formation of the somites and the beginning of other changes in the mesoderm (Fig. 17, C, cf. also Fig. 28, E). Following the mesoderm from the mid-line toward either side three regions or zones can be made out: (i) the dorsal mesoderm which at this level has been organized into somites, (2) the intermediate mesoderm, a thin plate of cells connecting the dorsal and lateral mesoderm and (3) the lateral mesoderm which is distinguished from the intermediate by being split into two layers with a space between them. The somites are compact cell masses lying immediately lateral to the neural folds The cells composing them have a fairly definite radial arrangement about a central cavity which is very minute or wanting altogether when the somites are first formed but which later becomes enlarged (Fig. 38). Cephalic and caudal to the region in which somites have been formed the dorsal mesoderm is differentiated from the rest of the mesoderm simply by its greater thickness and compactness. 48 EARLY EMBRYOLOGY OF THE CHICK In 24-hour embryos the intermediate mesoderm shows very little differentiation. In the chick it never becomes segmentally divided as does the dorsal mesoderm. The fact that it is potentially segmental in character is indicated, however, by the way in which it later gives rise to segmentally arranged proamnion region primitive ridge Fig. 17. — Diagrams of sections of 24-hour chick. The sections are located on an outline sketch of the entire embryo. The conventional representation of the germ layers is the same as that employed in Fig. 13 except that here where its cells have become aggregated to form definite layers the mesoderm is repre- sented by heavy solid black lines. nephric tubules. Because of the part it plays in the establish- ment of the excretory system the intermediate mesoderm is frequently called the nephrotomic plate. STRUCTURE OF TWENTY-FOUR HOUR CHICKS 49 In the chick the lateral mesoderm like the intermediate mesoderm, shows no segmental division. In 24-hour embryos (Fig. 17, C) it is clearly differentiated from the intermediate mesoderm by being split horizontally into two layers with a space between them. The layer of lateral mesoderm lying next to the ectoderm is termed the somatic mesoderm, the layer next to the entoderm is termed the splanchnic mesoderm, and the cavity between somatic and splanchnic mesoderm is the coelom. Because in development the somatic mesoderm and ectoderm are closely associated and undergo many foldings in common, it -is convenient to designate the two layers together by the single term somatopleure. Similarly the splanchnic mesoderm and the entoderm together are designated as the splanchnopleure. The Coelom. — The coelom, like the cell layers of the blasto- derm, extends over the yolk peripherally beyond the embryonal area (Fig. 17, C). Later in development foldings mark off the embryonic from the extra-embryonic portion of the germ layers. This same folding process divides the coelom into intra-em- bryonic and extra-embryonic regions. In the 24-hour chick, however, embryonic and extra-embryonic coelom have not been separated. It is evident from the manner in which the coelomic chambers arise in the lateral mesoderm that the coelom of the embryo con- sists of a pair of bilaterally symmetrical chambers. It is not until later in development that the right and left coelomic chambers become confluent ventrally to form an unpaired body cavity such as is found in adult vertebrates. The Pericardial Region. — In the region of the anterior intes- tinal portal the coelomic chambers on either side show very marked local enlargements. Later in development these dilated regions are extended mesiad and break through into each other ventral to the fore-gut to form the pericardial cavity. In their early condition these enlarged regions of the coelomic chambers are usually called amnio-cardiac vesicles. With their later fate in mind we may avoid multiplication of terms and speak of them from their first appearance as constituting the pericardial region of the coelom. The relationships of the pericardial region of the coelom in embryos of 24 hours can be most readily grasped from a study 50 EARLY EMBRYOLOGY OF THE CHICK of transverse sections. Figure 17, B, shows the great dilation of the coelom on either side of the anterior intestinal portal as compared with its condition farther, caudad (Fig. 17, C). Where the splanchnic mesoderm lies closely applied to the entoderm at the lateral margins of the portal it is noticeably thickened. It is from these areas of thickened splanchnic mesoderm that the paired primordia of the heart will later arise. In entire embryos of this age the thickened splanchnic mesoderm can be made out as a dark band lying close against the crescentic entodermal border of the anterior intestinal portal (Fig, 16). If the preparation is favorably stained the boundaries of the pericardial regions of the coelom can be traced (see Fig. 16). Following mesiad from the easily located thick- ened areas, the mesodermic borders can be seen to extend from either side parallel to the entodermic margins of the portal nearly to the mid-line. They then turn cephalad. When they encounter the ectodermal fold which constitutes the posterior boundary of the subcephalic pocket they swing laterad parallel with it and can be traced outside the embryonic region where they constitute the cephalic borders of the anterior horns of the mesoderm (see also Fig. 27, A). The portion of the coelom, the borders of which we have just located between the subcephalic pocket and the anterior in- testinal portal, is an important landmark from another stand- point than the part it is destined to play in the formation of the pericardial region. It is the most cephalic part of the coelom. There is no coelom in the head. In the head region the meso- derm is not aggregated into definite masses or coherent cell layers. The mesodermic structures of the head are derived from cells which migrate into the cephahc region from the meso- derm lying farther caudally. These migrating cells are termed mesenchymal cells in distinction to the more definitely aggre- gated cell layers of the mesoderm. By careful focusing on the whole-mount the mesenchyme of the head can be seen as an indefinite mass lying between the superficial ectoderm and the entoderm of the fore-gut. The distribution of the mesenchymal cells and the characteristic irregularity of shape correlated with their active amoeboid movement may be readily made out from sections (Fig. 17, ^4). STRUCTURE OF TWENTY-FOUR HOUR CHICKS 5 1 The Area Vascvilosa. — In a 24-hour chick the boundary be- tween area opaca and area pellucida has the same appearance and significance as in chicks of 18 to 20 hours. There is, how- ever, a very marked difference between the proximal portion of the area opaca adjacent to the area pellucida and the more distal portions of the area opaca. The proximal region is much darker and has a somewhat mottled appearance (Fig. 1 5) . The greater density of this region is due to its invasion by mesoderm which makes it thicker and therefore more opaque in transmitted light (Fig. 17, D). The boundary between the inner and outer zones of the area opaca is established by the extent to which the mesoderm has grown peripherally. The distal zone is called the area opaca vitellina because the yolk alone underlies it. The proximal zone into which mesoderm has grown is known as the area opaca vasculosa, because it is from the meso- derm in this region that the yolk-sac blood vessels arise. The mottled appearance of this region is due to the aggregation of mesoderm into cell clusters, or blood islands, which mark the initial step in the formation of blood vessels and blood corpus- cles. Later in development the formation of blood islands and vessels extends in toward the body of the embryo from its place of earhest appearance in the area opaca and involves the mesoderm of the area pellucida. The histological nature of the blood islands will be taken up in connection with later stages where their development is more advanced. CHAPTER VIII THE CHANGES BETWEEN TWENTY-FOUR AND THIRTY- THREE HOURS OF INCUBATION The closure of the neural tube; the differentiation of the brain region; the anterior neuropore; the sinus rhomboidalis; the fate of the primitive streak; the lengthening of the fore-gut; the appearance of the heart and omphalomesenteric veins; organ- IZATION IN THE AREA VASCULOSA. In dealing with developmental processes the selection of stages for detailed consideration is more or less arbitrary and largely determined by the phenomena one seeks to emphasize. There is no stage of development which does not show some- thing of interest. It is impossible in brief compass to take up at length more than a few stages. Nevertheless it is important not to lose the continuity of the processes involved. By calling attention to some of the more important intervening changes, this brief chapter aims to bridge the gap between the 24-hour stage and the 33-hour stages of the chick both of which are taken up in some detail. The Closure of the Neural Tube. — In comparison with 24- hour chicks, entire embryos of 27 to 28 hours of incubation (Fig. 18) show marked advances in the development of the cephalic region. The head has elongated rapidly and now pro- jects free from the blastoderm for a considerable distance, with a corresponding increase in the depth of the subcephalic pocket and in the length of the fore-gut. In 24-hour chicks the anterior part of the neural plate is already folded to form the neural groove. Although the neural folds are at that stage beginning to converge mid-dorsally the ^ groove nevertheless remains open throughout its length (Fig. ly. A, B, C). By 27 hours the neural folds in the cephalic region meet in the mid-dorsal line and their edges become fused. The fusion which occurs is really a double one. Careful following of Figures 26, A to £, will aid greatly in understanding 52 CHANGES BETWEEN 24 AND ^^ HOURS 53 the process. Each neural fold consists of a mesial component which is thickened neural plate ectoderm, and a lateral com- ponent which is unmodified superficial ectoderm (Fig. 26, A), When the neural folds meet in the mid-dorsal line (Fig. 26, -B, C) the mesial, neural plate components of the two folds fuse with each other, and the outer layers of unmodified ectoderm also become fused (Fig. 26, D). Thus in the same process the neural groove becomes closed to form the neural tube and the proamnion prosencephalon anterior / neuropore border of fore-gut subcephalic pocket mesenchyme omphalo- mesenteric vein blood island border ot ..... .^. -y -.» . inesodenn. ' v^^^v^'^5?iT-;> '*•. rhomboidalis Hensen's node .■.-^' ^'&f.^"':: < :: ■ ■* extra-embryonic ^- vascular plexus Fig. 18. — Dorsal view ( X 14) of entire chick embryo having 8 pairs of somites (about 27-28 hours incubation). superficial ectoderm closes over the place formerly occupied by the open neural groove. Shortly after this double fusion the neural tube and the superficial ectoderm become somewhat separated from each other leaving no hint of their former con- tinuity (Fig. 26, E). The Differentiation of the Brain Region. — By 27 hours of incubation the anterior part of the neural tube is markedly enlarged as compared with the posterior part. Its thickened 54 EARLY EMBRYOLOGY OF THE CHICK walls and dilated lumen mark the region which will develop into the brain. The undilated posterior part of the neural tube gives rise to the spinal cord. Three divisions, the three primary brain vesicles, can be distinguished in the enlarged cephahc region of the neural tube (Fig. i8). Occupying most of the anterior-part of the head is a conspicuous dilation known from its position as the fore-brain or prosencephalon. Posterior to ectoderm of head prosencephalon optic vesicle ventral aortic root ventral aorta epi-myocardium endocardium line of endocardi al fusion margin of anterior intestinal porta! anterior horn of mesoderm anterior neuropore infundibulum cephalic mesenchyme extra-embryonic vascular plexus Fig. 19. — Ventral view ( X 45) of head and heart region of chick embryo of 9 somites (about 29-30 hours incubation). the prosencephalon and marked off from it by a constriction is the mid-brain or mesencephalon. Posterior to the mesenceph- alon with only a very slight constriction marking the boundary is the hind-brain or rhombencephalon. The rhombencephalon is continuous posteriorly with the cord region of the neural tube without any definite point of transition. In somewhat older embryos (Fig. 19) the lateral walls of the prosencephalon become out-pocketed to form a pair of rounded dilations known as the primary optic vesicles. When the CHANGES BETWEEN 24 AND 33 HOURS 55 optic vesicles are first formed there is no cgnstriction between them and the lateral walls of the prosencephalon, and the lumen of each optic vesicle communicates mesially with the lumen of the prosencephalon without any definite hne of demarcation. The relation of the notochord to the divisions of the brain is of importance in later developmental processes. The notochord extends anteriorly as far as a depression in the floor of the prosencephalon known as the infundibulum (Fig. 19). There- fore, the rhombencephalon, mesencephalon, and that part of the prosencephalon posterior to the infundibulum he immediately dorsal to the notochord (are epichordal) while the infundibular region and the parts of the prosencephalon cephalic to it project anterior to the notochord (are pre-chordal) . The Anterior Neuropore. — The closure of the neural folds takes place first near the anterior end of the neural groove and progresses thence both cephalad and caudad. At the extreme anterior end of the brain region closure is delayed. As a result the prosencephalon remains for sometime in communication with the outside through an opening called the anterior neuro- pore. The anterior neuropore is still open in chicks of 2 7 hours (Fig. 18). In embryos of 33 hours the neuropore appears much narrowed (Fig. 21). A little later it becomes closed but leaves for some time a scar-like fissure in* the anterior wall of the prosencephalon (Fig. 23). The anterior neuropore does not give rise to any definite brain structure. It is important simply as a landmark in brain topography. Long after it has disap- peared as a definite opening the scar left by its closure serves to mark the point originally most anterior in the developing brain. TheSinusRhomboidalis. — The rhombencephaUc region of the brain merges caudally without any definite line of demarcation into the region of the neural tube destined to become the spinal cord. The neural tube as far caudally as somite forma- tion has progressed is completely closed and of nearly uniform diameter. Caudal to the most posterior somites the neural groove is still open and the neural folds diverge to either side of Hensen's node (Fig. 18). In their later growth caudad the neural folds converge toward the mid-line and form the lateral boundaries of an unclosed region at the posterior extremity of the neural tube known because of its shape as the sinus rhom- 56 EARLY EMBRYOLOGY OF THE CHICK boidalis (Fig. 21). Hensen's node and the primitive pit lie in the floor of this as yet unclosed region of the neural groove and subsequently are enclosed within it when the neural folds here finally fuse to complete the neural tube. This process in the chick is homologous with the enclosure of the blastopore by the neural folds in lower vertebrates. In forms where the blastopore does not become closed until after it is surrounded by the neural folds, it for a time constitutes an opening from the neural canal into the primitive gut known as the neurenteric canal or posterior neuropore. In the chick the early closure of the blastopore precludes the estabHshment of an open neurenteric canal but the primitive pit represents its homologue. The Fate of the Primitive Streak. — In embryos of about 27 hours the primitive streak is relatively much shorter than in younger embryos (Cf. Figs. 8, 11, 14, 15, and 18). This is due partly to its being overshadowed by the rapid growth of structures lying cephalic to it, and partly to actual decrease in the length of the primitive streak itself. The cells in the primitive stieak region would appear to be contributed to surrounding structures. Whatever the exact fate of its cells may be, the primitive streak becomes less and less a conspicuous feature in the developing embryo. By the time the caudal end of the body is delimited, the primitive streak as a definitely organized structure has disappeared altogether (Cf. Figs. 18, 21, 29, 34). The Formation of Addifional Somites. — The division of the dorsal mesoderm to form somites begins to be apparent in embryos of about 22 hours. By the end of the first day three or four pairs of somites have been cut off (Fig. 15). As develop- ment progresses new somites are added caudal to those fiist formed. In embryos which have been incubated about 27 hours eight pairs of somites have been established (Fig. 18). It was formerly beHeved that some new somites were formed anterior to the first pair. The experiments of Patterson would seem to indicate quite definitely that the first pair of completely formed somites remains the most anterior and that all the new somites are added posterior to them. The experiments referred to were carried out on eggs which had been incubated up to the time of the formation of the first somite. With thorough CHANGES BETWEEN 24 AND 33 HOURS $7 aseptic precautions the eggs were opened and the first somite marked, in some cases by injury with an "electric needle" in other cases by the insertion of a minute glass pin. Following the operation the shell was closed by sealing over the opening a piece of egg shell of appropriate size. After being again in- cubated for varying lengths of time the eggs were reopened. In all cases the injured first somite was still the most anterior complete somite. All the new somites except the incomplete ''head somite" had appeared caudal to the first pair of somites formed. The Lengthening of the Fore-gut. — Comparison of the rela- tions of the crescentic margin of the anterior intestinal portal in embryos between 24 and 30 hours shows it occupying pro- gressively more caudal positions (Fig. 27). This change in the position of the anterior intestinal portal is the result of two distinct growth processes. The margins of either side of the portal are constantly converging toward the mid-Une where they become fused with each other. Their fusion lengthens the fore- gut by adding to its floor and thereby displaces the crescentic margin of the portal caudad. At the same time the struc- tures cephalic to the anterior intestinal portal are elongating rapidly so that the portal becomes more and more remote from the anterior end of the embryo with the further lengthening of the fore-gut. As a result of these two processes the space between the sub- cephalic pocket and the margin of the anterior intestinal portal is also elongated (Fig. 27). This is of importance in connection with the formation of the heart for it is into this enlarging space that the pericardial portions of the coelom extend and in it that the heart comes to Ue. The Appearance of the Heart and Omphalomesenteric Veins. Although the early steps in the formal ion of the heart take place in embryos of this range, detailed consideration of them has been deferred to be taken up in connection with later stages when conditions in the circulatory system as a whole are more advanced. In dorsal views of entire embryos the heait is largely con- cealed by the overlying rhombencephalon (Fig. 18) but it may readily be made out by viewing the embryo from the ventral surface (Fig. 19). At this stage the heart is a nearly straight 58 EARLY EMBRYOLOGY OF THE CHICK tubular structure lying in the mid-line ventral to the fore-gut. Its mid-region has noticeably thickened walls and is somewhat dilated. Anteriorly the heart is continuous with the large median vessel, the ventral aorta, posteriorly it is continuous with the paired omphalomesenteric veins. The fork formed by the union of the omphalomesenteric veins in the posterior part of the heart lies immediately cephalic to the crescentic margin of the anterior intestinal portal, the veins lying within the fold of entoderm which constitutes its margin. Organization in the Area Vasculosa. — The extra-embryonic vascular area at this stage is undergoing rapid enlargement and presents a netted appearance instead of being mottled as in the earlier embryos. The peripheral boundary of the area vasculosa is definitely marked by a dark band, the precursor of the sinus terminalis (marginal sinus) . Its netted appearance is due to the extension and anastomosing of blood islands. The formation of the network is a step in the organization of a plexus of blood vessels on the yolk surface which will later be the means of absorbing and transferring food material to the embryo. The afferent yolk-sac or vitelline circulation is estab- lished in the next few hours of incubation when this plexus of vessels developing on the yolk surface comes into communica- tion with the omphalomesenteric veins already developing within the embryo and extending laterad. The efferent vitelUne circulation is established somewhat later when the omphalo* mesenteric arteries arise from the aorta of the embryo and become connected with the yolk-sac plexus. (Cf. Figs. 15, 18, 21). CHAPTER IX THE STRUCTURE OF CHICKS BETWEEN THIRTY-THREE AND THIRTY-NINE HOURS OF INCUBATION The divisions of the brain and their neuromeric struc- ture; THE auditory PITS; THE FORMATION OF EXTRA-EM- BRYONIC BLOOD vessels; THE FORMATION OF THE HEART; THE FORMATION OF INTRA-EMBRYONIC BLOOD VESSELS. Chicks which have been incubated from S3 to 39 hours are in a favorable stage to show some of the fundamental steps in the foimation of the central nervous system, and of the circu- latoi;y system. In this chapter, therefore, attention has been concentrated on these two systems. During this period of incubation there are also changes in the fore-gut region and in the somites, and differentiation in the intermediate mesoderm which presages the formation of the urinary organs. Consideration of these structures has, however, been defeired until their development has progressed somewhat farther. The Divisions of the Brain and Their Neuromeric Structure. The metameric arrangement of structures which is so striking a feature in the body organization of all vertebrates, is masked in the head region of the adult by superimposed specializations. In the brain of young vertebrate embryos, however, the meta- merism is still indicated. Dissections of the neural plate of chicks at the end of the first day of incubation show a series of eleven enlargements marked off from each other by contric- tions (Fig. 20, A). Concerning the precise homologies of indi- vidual enlargements with specific neuromeres in other forms there is not complete agreement. The controversies center about the question of neuromeric fusions in the anterior part of the brain. For the beginning student the fact that meta- merism is present is to be emphasized rather than the contro- versies concerning the homologies of neuromeres. With the reservation that some of the anterior enlargements may repre- 59 6o EARLY EMBRYOLOGY OF THE CHICK sent fusions of more than one neuromere, the series of enlarge- ments seen in the brain region of the chick may be regarded as neuromeric. For convenience in designation the neuromeres are numbered beginning at the anterior end. anterior neuropore cut ectoderm neural groove Hft neural fold neuromeric enlargement line of fusion neural folds rhombencephalon prosencephalon mesencephalon metencephalon myelencephalon Pig. 20. — Diagrams to show the neuromeric enlargements in the brain region of the neural tube. (Based on figures by Hill.) A , lateral view of neural plate from dissection of chick of 4 somites (24 hours) ; B, dorsal view of brain dissected out of 7-somite (26 to 27-hour) embryo; C, dorsal view of brain trom lo-somite (30-hour) embryo; D, dorsal view of brain from 14-somite (36-hour) embryo. \\ ith the closure of the neural tube and the establishment of the three primary brain vesicles we can begin to trace the fate of STRUCTURE OF THIRTY-THREE HOUR CHICKS 6l the vaiious neuromeric enlargements in the formation of the brain regions. The three anterior neuromeres form the prosen- cephalon; neuromeres four and five are incorporated in the mesencephalon; and neuromeres six to eleven in the rhom- prosencephalon proamnion anterior neuropore optic vesicle omphalomesenteric vein lateral m sinus rhomboidalis primitive streak Pig. 21. — Dorsal view ( x 14) of an entire chick embryo of 12 somites (about 33 hours incubation). b^ncephalon (Fig. 20, B). Anteriorly the interneuromeric constrictions soon disappear except for two; namely, the one between the prosencephalon and mesencephalon, and the one 62 EARLY EMBRYOLOGY OF THE CHICK between the mesencephalon and rhombencephalon. The rhombencephalic neuromeres, however, remain clearly marked for a considerable period. By about 33 hours of incubation the optic vesicles are estab- lished as paired lateral outgrowths of the prosencephalon. They soon extend to occupy the full width of the head (Fig. 20, C and Fig. 21). The distal portion of each of the vesicles >-'5- ^fir ***»»»„,„ -v.^> prosencephalon cctodeim S' ^"'m,,^ ^ optic vesicle infundibulum -—^ ' :!'^' -^^m L^m ^^^^ mesencephalon *' ^^m^ ^^_^....r^-^ aortic arch ventral -''1 r^'HrTfWR f^f J . if He Vi m3 T j ,- — i*^ notochord ■> ■i^JR y i jMI i f / siSm { ^^Km Ki HeHI << '''^^n'ln^ / ^ ** r f region of ganglion V VWF Imv^''^1 }j metencephalon ^ 4" -^H^^P^ '/ t , myelencephalon ^- { SQ ^^^\ " ' irteriosus cephalic neural ere at — region of ganglion ^^T^n-i^Jv*?^ i VII VIII t^iUHB ;AnL#'«HSpid ■ ' ■■r 'AjPjBK-glW ■ ^ -, i 'WmU^ *^ V >• L^^^ S-fll^^ - I ^^S vl^^ I vein Pig. 22. — Dorsal view ( x 45) of head and heart region of a chick embryo of 17 somites (38-39 hours incubation). thus comes to lie closely approximated to the superficial ecto- derm, a relationship of importance in their later development. At first the cavities of the optic vesicles (opticoeles) are broadly confluent with the cavity of the prosencephalon (prosoccele) . Somewhat later constrictions appear which mark more defi- nitely the boundaries between the optic vesicles and the prosen- cephalon (Fig. 20, D and Fig. 22). There arises also at this stage a depression in the floor of the STRUCTURE OF THIRTY-THREE HOUR CHICKS 63 prosencephalon known because of its peculiar shape as the infundibulum (Figs. 23 and 24). The infundibular region is the site of important changes later in development. At this stage, conditions are not sufficiently a^dvanced to warrant more than calling attention to its origin from, and relations to, the prosencephalon, and to the anterior end of the notochord as shown in the figures referred to. prosencephalon nfundibulum bulbo-conus arteriosus cut epi-myocardium ventricular region . atrial region -anterior intestinal portal ventral aortic roots cut ectoderm dorsal aortae stnus venosus lateral mesoderm cut splanchnopleure Pig. 23. — Diagrammatic ventral view of dissection of a 35-hour chick embryo. {Modified from Prentiss.) The splanchnopleure of the yolk-sac cephalic to the anterior intestinal portal, the ectoderm of the ventral surface of the head, and the mesoderm of the pericardial region, have been removed to show the under- lying structures. Figure 24 should be referred to for the relations of the peri- cardial mesoderm. In chicks of about 38 hours indications of the impending division of the three primary vesicles to form the five regions characteristic of the adult brain are already beginning to ap- pear. In the establishment of the five-vesicle condition of the brain, the prosencephalon is subdivided to form the 64 EARLY EMBRYOLOGY OF THE CHICK STRUCTURE OF THIRTY-THREE HOUR CHICKS 65 telencephalon and diencephalon, the mesencephalon remains undivided, and the rhombencephalon divides to form the metencephalon and myelencephalon. The division of the prosencephalon into telencephalon and diencephalon is not completed until a much later stage of development, but the median enlargement at this stage ex- tending anterior to the level of the optic vesicles indicates where the telencephalon will be established (Fig. 20, D). The optic vesicles and that part of the prosencephalon lying between them go into the diencephalon. The mesencephalon, as stated above, undergoes no subdivi- sion. The original mesencephalic region of the three-vesicle brain gives rise to the mesencephalon of the adult. This region of the brain does not undergo any marked differentiation until relatively late in development. At this stage the division of the rhombencephalon is clearly marked (Fig. 20, D and Fig. 22). The two most anterior neuromeres of the original rhombencephalon form the meten- cephalon and the posterior four neuromeres are incorporated in the myelencephalon. The Auditory Pits. — As is the case with the central nervous system, the organs of special sense arise early in development. The appearance of the optic vesicles which later become the sensory part of the eyes has already been noted. The first indication of the formation of the sensory part of the ear becomes evident at about 35 hours of incubation. At this age a pair of thickenings termed the auditory placodes arise in the superficial ectoderm of the head. They are situated on the dorso-lateral surface opposite the most posterior inter-neuro- meric constriction of the myelencephalon. By 38 hours of incubation (Fig. 22) the auditory- placodes have become depressed below the general level of the ectoderm and form the walls of a pair of cavities, the auditory pits. When first formed the walls of the auditory pits are directly continuous with the superficial ectoderm, and their cavities are widely open to the outside. In later stages the openings into the pits become narrowed and finally closed so that the pits become vesicles lying between the superficial ectoderm and the myelen- cephalon. As yet they have no connection with the central nervous system. 66 EARLY EMBRYOLOGY OF THE CHICK The Formation of Extra-embryonic Blood Vessels. — In dealing with the circulation of the chick we must recognize at the outset two distinct circulatory arcs of which the heart is the common center. One complete circulatory arc is estab- lished entirely within the body of the embryo. A second arc is established which has a rich plexus of terminal vessels located in the extra-embryonic membranes enveloping the yolk. These are the vitelline vessels. The vitelline vessels communicate with the heart over main vessels which traverse the embryonic body. The chief distribution of the vitelline circulation is, however, extra-embryonic. Later in development there arises a third circulatory arc involving another set of extra-embryonic vessels in the allantois, but with that we have no concern until we take up later stages. Neither the intra-embryonic, nor the vitelline circulatory channels have as yet been completed but the heart and many of the main vessels have made their appearance. The formation of extra-embryonic blood vessels is presaged by the appearance of blood islands in the vascular area of chicks toward the end of the first day of incubation (see Chapter Vll). Figure 25 shows the differentiation of blood islands to form primitive blood corpuscles and blood vessels. At their first appearance the blood islands are irregular clusters of meso- derm cells lying in intimate contact with the yolk-sac entoderm (Fig. 25, A). When the lateral mesoderm becomes split forming the somatic and splanchnic layers with the coelom between, the blood islands lie in the splanchnic mesoderm ad- jacent to the entoderm. In embryos of 3 to 5 somites fluid filled spaces begin to appear in the blood islands with the result that in each blood island the peripheral cells are separated from the central ones (Fig. 25, -B). As the fluid accumulates and the spaces expand the peripheral cells become flattened and^ushed outward, but they remain adherent to each other and com- pletely enclose the central cells. At this stage the single layer of peripheral cells may be regarded as constituting the endo- thelial wall of a primitive blood channel (Fig. 25, C). Exten- sion and anastomosis of neighboring blood islands which have undergone similar differentiation results in the establishment of a network of communicating vessels. Meanwhile the cells enclosed in the primitive blood channels have become separated STRUCTURE OF THIRTY-THREE HOUR CHICKS 67 from each other and rounded. They soon come to contain haemoglobin and constitute the primitive blood corpuscles. The fluid accumulated in the blood islands serves as a vehicle in which the corpuscles are suspended and conveyed along the vessels. yolk ectoderm central cells of blood island peripheral cell of blood island ectoderm blood cells entoderm cell somatic mesoderm coelom endothelial cell lumen yolk Fig. 25. — Drawings to show the cellular organization of blood islands at three stages in their differentiation. The location of the areas drawn with reference to the body of the embryo and other structtires of the blastoderm can be ascertained by reference to Fig. 17, D. A, from blastoderm of 18-hour chick; B, from blastoderm of 24-hour chick;. C, from blastoderm of 33-hour chick. The differentiation of the blood islands in the manner de- scribed begins first in the peripheral part of the area vasculosa and from there extends toward the body of the embryo. By 33 hours of incubation the extra-embryonic vascular plexus has extended inward and made connection with the omphalomesen- teric veins which, originating within the body of the embryo 68 EARLY EMBRYOLOGY OF THE CHICK have grown outward. Thus are established the afferent vitel- line channels (Fig. 21). The efferent vitelline channels have not yet appeared and there is no circulation of the blood corpuscles which are being formed in the area vasculosa. Th^ intra-embryonic blood vessels remain empty until the extra-embryonic circuit is com- pleted. The embryo meanwhile draws its nutrition from the yolk by direct absorption. The Formation of the Heart. — The structural relations of the heart and the way in which it is derived from the mesoderm can be grasped only by the careful study of sections through the heart region in several stages of development (Fig. 26). The fact that the heart, itself an unpaired structure, arises from paired primordia which at first lie widely separated on either side of the mid-line, is likely to be troublesome unless its significance is understood at the outset. The paired condition of the heart at the time of its origin is due to the fa.ct that the early embryo lies open ventrally, spread out on the yolk sur- face. The rudiments of all ventral structures which appear at an early age are thus at first separated, and lie on either side of the mid-line. As the embryo develops, a series of foldings undercut it and separate it from the yolk. This folding off process at the same time establishes the ventral wall of the gut and the ventral body wall of the embryo by bringing together in the mid-line the structures formerly spread out to right and left. The primordia of the heart arise in connection with layers which are destined to form ventral parts of the embryo, but at a time when these layers are still spread out on the yolk. As the embryo is com- pleted ventrally the paired primordia of the heart are brought together in the mid-line and become fused (Fig. 27). The first indication of heart formation is to be seen in trans- verse sections passing through a 2S-hour chick immediately caudal to the anterior intestinal portal. Where the splanchno- pleure of either side bends toward the mid-line along the lateral margin of the intestinal portal there is a marked regional thick- ening in the splanchnic mesoderm of either side (Figs. 26, A and 27, yl). This pair of thickenings indicates where there has been rapid cell proliferation preliminary to the differentiation of the heart. Loosely associated cells can already be seen STRUCTURE OF THIRTY-THREE HOUR CHICKS 69 somewhat detached from the mesial face of the mesoderm layer. These cells soon become organized to form the endocardial primordia. In a chick of about 26 hours, sections through a corresponding region show distinct dfferentiation of the endocardial and epi- myocardial primordia (Fig. 26, B). The endocardial primordia are a pair of delicate tubular structures, a single cell in thick- ness, lying between the entoderm and mesoderm. They arise from the cells seen separating from the adjacent thickened meso- derm in the 25-hour chick. As their name indicates they are destined to give rise to the endothelial lining of the heart. By far the greater part of each of the original mesodermic thicken- ings becomes applied to the lateral aspects of the endocardial tubes as the epi-myocardial primordium which is destined to give rise to the external coat of the heart (epicardium) and to the heavy muscular layers of the heart (myocardium). In chicks of 27 hours the lateral margins of the anterior intes- tinal portal have been undergoing concrescence lengthening the fore-gut caudally and involving the heart region. In this process the former lateral margins of the portal swing in to meet each other and fuse in the mid-line, and the endocardial tubes of the right and left side are brought toward each other beneath the newly completed floor of the fore-gut (Figs. 26, C and 27, B). In the 28-hour chick the endocardial primordia are approximated to each other (Figs. 26, D and 27, C) and by 29 hours they fuse in their mid-region to form a single tube (Figs. 26, E and 27, D). At the same time the epi-myocardial areas of the mesoderm are brought together first ventrally (Fig. 26, D) and then dor- sally to the endocardium (Fig. 26, E). Where the splanchnic mesoderm of the opposite sides of the body comes together dor- sal and ventral to the heart it forms double layered supporting membranes called respectively the dorsal mesocardium and the ventral mesocardium. j The ventral mesocardium is a transitory structure, disappearing almost as soon as it is formed (Fig. 26, E). The dorsal mesocardium, although the greater part of it disappears in the next few hours of incubation, persists in em- bryos of the stage under consideration, suspending the heart in the pericardial region of the coelom. Conditions reached in the heart region at 33 hours of incubation are shown in section 70 EARLY EMBRYOLOGY OF THE CHICK in Figure 28, C. The heart here is enlarged and displaced somewhat to the right of the mid-line but its fundamental neural plate ectoderm donal meaoderm ■uperficial ectoderm neural groove j — somatopleure splanchnopleure splanchnic mesoderm gut itrnncdiaoely caudal to anterior intestinal portal neural groove epi-myocard I somatopleure \- splanchnopleure myocardium endocardium gut immediately caudal to anterior intestinal portal dorsal mesoderm fore-gut epi-myocardium endocardium line of fusion of lateral margins of anterior intestinal portal dorsal mesoderm fore-gut dorsal mesocardium ventral mesocardium |— somatopleure }- splanchnopleure epi-myocardium dorsal mesoderm notochord dorsal mesocardium endocardium } — splanchnopleure epi-myocardium Fig. 26. — Diagrams of transverse sections through the pericardial region of chicks at various stages to show the formation of the heart. For location of the sections consult Fig. 27. A, at 25 hours; B, at 26 hours; C, at 27 hours; D, at 28 hours; E, at 29 hours. relations are otherwise the same as in a 29-hour embryo (Fig. 26, E). STRUCTURE OF THIRTY-THREE HOUR CHICKS 71 The gross shape of the heart and its positional relations to other structures are best seen in entire embryos. The fusion of the paired cardiac primordia establishes the heart as a nearly straight tubular structure. It lies at the level of the rhombencephalon in the mid-line, ventral to the fore-gut (Fig. 19). By ^^ hours of incubation the mid-region of the A. pericardial _ _ _ region of coelom ^ epi-myocardium margin of • anterior intestinal - — - - - y' portal 1*.:\ e ventral aortic root pericardial ^-•region of coelom -^.^ epi-myocardium endocardium omphalomesenteric vein Fig. 27, — Ventral-view diagrams to show the origin and subsequent fusion of the paired primordia of the heart. The lines A, C, D, and E indicate the planes of the sections diagrammed in Fig. 26, A, C, D, E, respectively. A, chick of 25 hours; B, chick of 27 hours; C, chick of 28 hours; D, chick of 29 hours. heart is considerably dilated and bent to the right (Fig. 21). At 38 hours the heart k bent so far to the right that it extends beyond the lateral body margin of the embryo (Fig. 22). This bending process is correlated with the rupture of the dorsal mesocardium at the mid-r-egion of the heart. The breaking 72 EARLY EMBRYOLOGY OF THE CHICK through of the dorsal and ventral mesocardia is of interest aside from the fact that it leaves the heart free to undergo changes in shape. It makes the right and left ccelomic cham- bers confluent, the pericardial region thus being the first part of the coelom to acquire the unpaired condition characteristic of the adult. Although there are as yet no sharply bounded subdivisions of the heart, it is convenient to distinguish four regions which later become clearly marked off from each other (Fig. 23). The most caudal part of the heart where the omphalomesenteric veins join is the sinus venosus; the caudal part of the region of the heait which is dilated and bent to the right will become the atrium; the cephalic part of the heart bend is the ventricular region; and the region where the ventricle swings into the mid- line and becomes narrowed is known as the bulbo-conus ar- teriosus. Approximately at the stage of development indicated in Figure 23 irregular twitchings occur in the heart walls, but regular pulsations are not established until about the 44th hour of incubation. The Formation of the Intra-embryonic Blood Vessels. — Co- incident with the establishment of the heart, blood vessels have arisen within the body of the embryo. Concerning the exact nature of the process of blood vessel formation there is some disagreement. The weight of evidence seems to indicate that the early vessels are formed from mesodermal cells which lie in the path of their development. They grow by organi- zation of cells in situ as a drain might be built from bricks already deposited along its projected course. In later stages it seems probable that vessels extend by the formation of bud- like outgrowths from their walls, as well as by organization of cells in- their path of development. When first formed, the blood vessel walls are but a single cell in thickness. There is no structural differentiation between arteries and veins until a considerably later period. Recognition of the vessels depends wholly, therefore, on determining their course and relationships. The large vessels connecting with the heart are the first of the intra-embryonic channels established. From the bulbo- conus arteriosus the paired ventral aortic roots extend cephalad ventral to the fore-gut (Fig. 23). At the cephalic end of the STRUCTURE OF THIRTY-THREE HOUR CHICKS 73 fore-gut the ventral aortic roots turning dorsad curve around it, and then extend caudad, dorsal to the gut, as the paired dorsal aortae (Figs. 23, 24 and Fig. 28, B). Few conspicuous ectoderm of blastoderm extra-embryonic coelom mesoderm 1 lateral plate splanchnic mesoderm J of mesoderm Fig. 28. — Diagrams of sections of 33-hour chick. The location of each section is indicated on a small outline sketch of the entire embryo.t branches arise from the aortae at this stage but as development "progresses branches extend to the various parts of the embryo and the aortae become the main efferent conducting vessels of 74 EARLY EMBRYOLOGY OF THE CHICK the embryonic circulation. Both the ventral aortic roots and the omphalomesenteric veins are direct continuations of the paired endocardial primordia of the heart. The epi-myocardial coat is formed about the original endothelial tubes only where they are fused in the region destined to become the heart. The development of the heart at this stage is an epitome of its phylogenetic origin. The local investment of the endocardial tubes by the epi-myocardium, as seen in the formation of the chick heart, is a recapitulation of the evolutionary origin of the heart by the local addition of a heavy muscular coat about the walls of a blood vessel. During early embryonic life the cardinal veins are the main afferent vessels of the intra-embryonic circulation. The main cardinal trunks are paired vessels symmetrically placed on either side of the mid-line. There are two pairs, the anterior cardinals which return the blood to the heart from the cephalic region of the embryo, and the posterior cardinals which return the blood from the caudal region. The anterior and posterior cardinal veins of the same side of the body become confluent dorsal to the level of the heart. The vessels formed by the junction of the anterior and posterior cardinals are the ducts of Cuvier or common cardinal veins. The right and left ducts of Cuvier turn ventrad, one on either side of the fore-gut, and enter the sinus-venosus along with the right and left omphalomesen- teric veins, respectively (Fig. 24). In chicks of 33 hours the anterior cardinal veins can usually be made out in sections (Fig. 28, B, C). By 38 hours the an- terior cardinals and the ducts of Cuvier are readily recognized. The posterior cardinals appear somewhat later than the an- terior cardinals but are ordinarily discernible in the region of the duct of Cuvier by 33 to 35 hours and well established by 38 hours. For the sake of simplicity and clearness the cardinal veins have been represented in Figure 24 larger and more regularly formed than they are in actual specimens. Like all the other blood vessels of the embryo they arise as irregular anastomosing endothelial tubes, only gradually taking on the regularity of shape characteristic of fully formed vessels. CHAPTER X THE CHANGES BETWEEN FORTY AND FIFTY HOURS OF INCUBATION Flexion and torsion; the completion of the vitelline circulatory channels; the beginning of the circu- lation of blood. Flexion and Torsion.— Until 36 or 37 hours of incubation the longitudinal axis of the chick is straight except for slight for- tuitous variations. Beginning at about 38 hours, processes are initiated which eventually change the entire configuration of the embryo and its positional relations to the yolk. These proc- esses involve positional changes of two distinct types, flexion and torsion. As applied to an embryo, flexion means the bend- ing of the body about a transverse axis, as one might bend the head forward at the neck, or the trunk forward at the hips. Torsion means the twisting of the body, as one might turn the head and shoulders in looking backwards without changing the position of the feet. In chick embryos the first flexion of the originally straight body-axis takes place in the head region. Because of its loca- tion it is known as the cranial flexure. The axis of bending in the development of the cranial flexure is a transverse axis pas- sing through the mid-brain at the level of the anterior end of the notochord. The direction of the flexion is such that the fore-brain becomes bent ventrally toward the yolk. The proc- ess is carried out as if the brain were being bent about the anterior end of the notochord. Until the cranial flexure is well established it is inconspicuous in dorsal views of whole-mounts but even in its initial stages it appears plainly in lateral views (Fig. 24). To appreciate the correlation between the processes of flexion and torsion it is only necessary to bear in mind the relation of a chick of this stage to the yolk. As long as the chick lies with its ventral surface closely applied to the yolk, the yolk consti- tutes a bar to flexion. Before extensive flexion can be carried 76 EARLY EMBRYOLOGY OF THE CHICK out the chick must twist around on its side, i.e., undergo tor- sion, as a man lying face down turns on his side in order to flex his body. Torsion begins in the cephalic region of the embryo and pro- gresses caudad. The first indications of torsion appear almost as soon as the cranial flexure begins and the two processes then progress synchronously. In the chick, torsion is normally car- ried out toward a definite side. The cephahc region of the mesencephalon metencephalon myelencephalon auditory pit sinus resion somite, lateral mesoderm- lateral body fold unsegmented dorsal mesoderm prosencephalon optic vesicle margin of head fold of amnion bulbo-conus arteriosus ventricular region atrial region omphalomesenteric vein extra-embryonic vascular plexus oipphalo mesenteric artery neural tube primitive plate Fig. 29. — Dorsal view ( X 14) of entire chick embryo having 19 pairs of somites (about 43 hours incubation). Due to torsion the cephalic region appears in dextro-dorsal view. embryo is twisted in such a manner that the left side comes to lie next to the yolk and the right side away from the yolk. The progress of torsion caudad is gradual and the posterior part of the embryo remains prone on the yolk for a considerable time after torsion has been completed in the head region. Fig- ure 22 shows the head of an embryo of about 38 hours in which the cranial flexure and torsion are just becoming evident. In chicks of about 43 hours (Fig. 29) the further progress of both flexion and torsion is well marked. The processes of flexion and torsion thus initiated continue CHANGES BETWEEN 40 AND 50 HOURS 77 until the original orientation of the chick on the yolk is com- pletely changed. As the body of the embryo becomes turned on its side the yolk no longer impedes the progress of flexion. Following the accomplishment of torsion in the cephaUc region, the cranial flexure becomes rapidly greater until the head is practically doubled on itself (Fig. 34). As development pro- ceeds, torsion progresses caudad involving more and more of the body of the embryo. Finally the entire embryo comes to lie with its left side on the yolk. Concomitant with the progress of torsion, flexion also appears farther caudally, affecting in turn the cervical, dorsal, and caudal regions. The series of flexions which accompany torsion bend the head and tail of the embryo ventrally so that its spinal axis becomes C-shaped (Fig. 40). The flexions which bend the embryo on itself so the head and tail lie close together are characteristic of all amniote embryos. The torsion which in the chick accompanies flexion is correlated with the fact that it develops on the surface of a large yolk. The Completion of the Vitelline Circulatory Channels. — In chicks of $$ to 36 hours the omphalomesenteric veins have been established as postero-lateral extensions of the same endocardial tubes which are involved in the formation of the heart. As the omphalomesenteric veins are extending laterad, the vessels developing in the vitelline plexus are extending and converging toward the embryo. Eventually the vitelline vessels attain communication with the heart by becoming confluent with the omphalomesenteric veins. This establishes the afferent chan- nels of the vitelline circulation. The vessels destined to carry blood from the embryo to the vitelline plexus develop in embryos of about 40 hours (Fig. 29). Like the afferent vitelline channels, the efferent channels have a dual origin. The proximal portions of the efferent channels arise within the embryo as branches of the dorsal aortae, and extend peripherally. The distal portions of the channels arise in the extra-embryonic vascular area and extend toward the embryo. The efferent vitelHne vessels are estabhshed when these two sets of channels become confluent. In its early stages the connection is through a network of small channels rather than definite vessels, the aortae breaking up posteriorly into , small channels some of which communicate laterally with the 78 EARLY EMBRYOLOGY OF THE CHICK extra-embryonic plexus. Later some of these channels become confluent, others disappear, and gradually definite main vessels, the omphalomesenteric arteries, are estabHshed. For some time after their formation, the omphalomesenteric arteries are Hkely to retain traces of their origin from a plexus of small channels and arise from the aorta by several roots (Fig. 35). The Beginning of the Circulation of Blood. — At about 44 hours of incubation, coincident with the completion of the vitelline vessels, the heart begins regular contraction, and the blood which has been formed in the extra-embryonic vascular area is for the first time pumped through the vessels of the embryo. In tracing the course of either the embryonic or the vitelline circulation the heart is the logical starting point. From the heart the blood of the extra-embryonic vitelline circu- lation passes through the ventral aortae, along the dorsal aortae, and out through the omphalomesenteric arteries to the plexus of vessels on the yolk. In the small vessels which ramify in the membranes envelop- ing the yolk the blood absorbs food material. In young embryos, before the allantoic circulation has appeared, the vitelHne circulation is involved also in the oxygenation of the blood. The great surface exposure presented by the multitude of small vessels on the yolk makes it possible for the blood to take up oxygen which penetrates the porous shell and the albumen. After acquiring food material and oxygen the blood is collected by the sinus terminalis and the vitelline veins. The vitelline veins converge toward the embryo from all parts of the vascular area and empty into the omphalomesenteric veins which return the blood to the heart (Fig. 48) . The blood of the intra-embryonic circulation, leaving the heart enters the ventral aortae, thence passes into the dorsal aortae, and is distributed through branches from the dorsal aortae to the body of the embryo. It is returned from the cephalic part of the body by the anterior cardinals, and from the caudal part of the body by the posterior cardinals. The anterior and posterior cardinals discharge together through the ducts of Cuvier into the sinus region of the heart (Fig. 24). In the heart, the blood of the extra-embryonic circulation and of the intra-embryonic circulation is mixed. The mixed CHANGES BETWEEN 40 AND 50 HOURS 79 blood in the heart is not as rich in oxygen and food material as that which comes to the heart from the vitelhne circulation nor as low in food and oxygen content as that returned to the heart from the intra-embryonic circulation where these ma- terials are drawn upon by the growing tissues of the embryo. Nevertheless it carries a sufficient proportion of food and oxygen so that as it is distributed to the body of the embryo it serves to supply the growing tissues. CHAPTER XI EXTRA-EMBRYONIC MEMBRANES The folding off of the body of the embryo; the establish- ment OF THE YOLK-SAC AND THE DELIMITATION OF THE EMBRYONIC GUT; THE AMNION AND THE SEROSA; THE ALLANTOIS. The Folding off of the Body of the Embryo. — In bird embryos the somatopleure and splanchnopleure extend over the yolk peripherally, beyond the region where the body of the embryo is being formed. Distal to the body of the embryo the layers are termed extra-embryonic. At first the body of the chick has no definite boundaries and consequently embryonic and extra- embryonic layers are directly continuous without there being any definite boundary at which we may say one ends and the other begins. As the body of the embryo takes form, a series of folds develop about it, undercut it, and finally nearly separate it from the yolk. The folds which thus definitely estabHsh the boundaries between intra-embryonic and extra-embryonic regions are known as the limiting body folds or simply the body folds. The first of the body folds to appear is the fold which marks the boundary of the head. By the end of the first day of incu- bation the head has grown anteriorly and the fold originally bounding it appears to have undercut and separated it anteriorly from the blastoderm (Figs. 15 and 17, E). The cephalic limit- ing fold at this stage is crescentic, concave caudally. As this fold continues to progress caudad, its posterior extremities become continuous with folds which develop along either side of the embryo. Because of the fact that these folds bound the body of the embryo laterally, they are known as the lateral bod}' folds (lateral hmiting sulci). The lateral body folds, at first shallow (Fig. 28, D) become deeper, undercutting the body of the embryo from either side and further separating it from the yolk (Fig. 36, £ and Fig. 30). 80 EXTRA-EMBRYONIC MEMBRANES 8 1 During the third day a fold appears bounding the posterior region of the embryo (Fig. 31, C). This caudal fold undercuts the tail of the embryo forming a sub caudal pocket just as the sub-cephaHc fold undercuts the head. The combined effect of the development of the sub-cephahc, lateral body, and the sub- caudal folds is to constrict off the embryo more and more from the yolk (Figs. 30 and 32). These folds which establish the contour of the embryo indicate at the same time the boundary between the tissues which are built into the body of the embryo, and the so-called extra-embryonic tissues which serve temporary purposes during development but are not incorporated in the structure of the adult body. The Establishment of the Yolk-sac and the Delimitation of the Embryonic Gut. — The extra-embryonic membranes of the . chick are four in number, the yolk-sac, the amnion, the serosa and the allantois. The yolk-sac is the first of these to make its appearance. The splanchnopleure of the chick instead of forming a closed gut, as happens in forms with little yolk, grows over the yolk surface. The primitive gut has a cellular wall dorsally only, while the yolk acts as a temporary floor (Fig. 31, ^). The extra-embryonic extension of the splanchno- pleure eventually forms a sac-like investment for the yolk (Figs. 30 and 32). Concomitant with the spreading of the extra-embryonic splanchnopleure about the yolk, the intra-embryonic splanchno- pleure is undergoing a series of changes which result in the establishment of a completely walled gut in the body of the embryo. The interrelations of the various steps in the forma- tion of the gut and of the yolk-sac make it necessary to repeat some points and anticipate other points concerning the forma- tion of the gut, in order that their relation to yolk-sac formation may not be overlooked. It will be recalled that the first part of the primitive gut to acquire a cellular floor is its cephalic region. The same folding process by which the head is separated from the blastoderm involves the entoderm of the gut. The part of the primitive gut which acquires a floor as the sub-cephalic fold progresses caudad is termed the fore-gut (Fig. 31, B). During the third day of incubation the caudal fold undercuts the posterior end of the embryo. The splanchnopleure of the gut is involved 82 EARLY EMBRYOLOGY OF THE CHICK embryo lateral amniotic fold lateral body fold ex -embryonic coelom ectoderm ^ mesoderm J mes") pleure splanch- hopleure yolk amniotic cavity lateral amniotic fold amnion / somatopleure) serosa Tsomatopleure) embryo allantois ( splanchnopleure ) yolk stalk yolk-sac /splanch- nopleure \ albumen vitelline membrane Fig. 30. EXTRA-EMBRYONIC MEMBRANES 83 allantoic cavity allantois amnion amniotic extra-embryonic coelom somatopleure yolk-sac / splanchno- pleure ) albumen allantoic cavity allantois serosa shell sero-ammotic cavity yolk-sac albumen vitelline membrane belly stalk Fig. 30. — Schematic diagrams to show the extra-embryonic membranes of the chick. {After Duval.) The diagrams represent longitudinal sections through the entire egg. The body of the embryo, being oriented approximately at right angles to the long-axis of the egg, is cut transversely. A, embryo of about two days incubation; B, embryo of about three days incubation; C, embryo of about five days incubation; D, embryo of about fourteen days incubation. •84 EARLY EMBRYOLOGY OF THE CHICK in the progress of the sub-caudal fold so that a hind-gut is established in a manner analogous to the formation of the fore- gut (Fig. 31, C). The part of the gut which still remains open to the yolk is known as the mid-gut. As the embryo is con- stricted off from the yolk by the progress of the sub-cephalic and sub-caudal folds, the fore-gut and hind-gut are increased in extent at the expense of the mid-gut. The mid-gut is finally diminished until it opens ventrally by a small aperture which flares out, like an inverted funnel, into the yolk-sac (Fig. 31, Z)). This opening is the yolk duct and its wall constitutes the yolk stalk. The walls of the yolk-sac are still continuous with the walls of the gut along the constricted yolk-stalk thus formed, but the boundary between the intra-embryonic splanchnopleure of the gut and the extra-embryonic splanchnopleure of the yolk-sac can now be established definitely at the yolk-stalk. As the neck of the yolk-sac is constricted the omphalomesen- teric arteries and omphalomesenteric veins, caught in the same series of foldings, are brought together and traverse the yolk- stalk side by side. The vascular network in the splanchno- pleure of the yolk-sac which in young chicks was seen spreading over the yolk eventually nearly encompasses it. The embryo's store of food material thus comes to be suspended from the gut of the mid-body region in a sac provided with a circulatory arc of its own, the vitelline arc. Apparently no yolk passes directly through the yolk-duct into the intestine. Absorption of the yolk is effected by the epithelium of the yolk-sac and the food material is transferred to the embryo by the vitelline circula- tion. In older embryos (Fig. 30, C and D) the epithelium of the yolk-sac undergoes a series of foldings which greatly increase its surface area and thereby the amount of absorption it can accomplish. During development the albumen loses water, becomes more viscid , and rapidly decreases in bulk. The growth of the allantois, an extra-embryonic structure which we have yet to consider, forces the albumen toward the distal end of the yolk- sac (Fig. 30, D). The manner in which the albumen is encom- passed between the yolk-sac and folds of the allantois and serosa belong to later stages of development than those with which we are concerned. Suffice it to say that the albumen EXTEA-EMBRYONIC MEMBRANES 85 ectoderm of neural plate ectoderm of blastoderm primitive pit primitive streak yolk lii-^v^r^ w&mmm^^^'^^mi^ii'^f^:^ ? '^ fore-gut neural tube ectoderm of head subcephalic pocket splanchnopleure; of yolk-sac open neural groove primitive pit B pericardial region ' anterior intestinal of coelom portal fore-gut subcephalic pocket extra-embryonic coelom •» ■d*'do*<'o''. antenor posterior intestinal portal intestinal portal hind-gur subcaudal pocket )- amnion — extra-embryonic ■3^:5;;^ coelom splanchnopleure of yolk-sac post-anal gut proctodaeum mid-gut splanchnopleure of yolk-sac allantoic bud yolk- stalk Fig. 31. — Schematic longitudinal-section diagrams of the chick showing: four stages in the formation of the gut tract. The embryos are represented as unaffected by torsion. A, chick toward the end of the first day of incubation; no regional differentia- tion of primitive gut is as yet apparent. B, toward the end of the second day^ fore-gut established. C, chick of about three days; fore-gut, mid-gut and hind- gut established. D, chick of about four days; fore-gut and hind-gut increased in length at expense of mid-gut; yolk-stalk formed. 86 EARLY EMBRYOLOGY OF THE CHICK like the yolk, is surrounded by extra-embryonic membranes by which it is absorbed and transferred over the extra-embryonic circulation to the embryo. Toward the end of the period of incubation, usually on the 19th day, the remains of the yolk-sac are enclosed within the body walls of the embryo. After its inclusion in the embryo both the wall and the remaining contents of the yolk-sac rapidly disappear, their absorption being practically completed in the first six days after hatching. The Amnion and the Serosa. — The amnion and the serosa are so closely associated in their origin that they must be con- sidered together. Both are derived from the extra-embryonic somatopleure. The amnion encloses the embryo as a saccular investment and the cavity thus formed between the amnion and the embryo becomes filled with a watery fluid. Suspended in this amniotic fluid, the embryo is free to change its shape and position, and external pressure upon it is equalized. Mus- cle fibers develop in the amnion, which by their contraction gently agitate the amniotic fluid. The movement thus im- parted to the embryo apparently aids in keeping it free and preventing adhesions and resultant malformations. The first indication of amnion formation appears in chicks of about 30 hours incubation. The head of the embryo sinks into the yolk somewhat, and at the same time the extra-embry- onic somatopleure anterior to the head is thrown into a fold, the head fold of the amnion (Fig. 32,-4). In dorsal aspect the margin of this fold is crescentic in shape with its concavity directed toward the head of the embryo. The head fold of the amnion must not be confused with the sub-cephalic fold which arises earlier in development and undercuts the head. As the embryo increases in length its head grows anteriorly into the amniotic fold. Growth in the somatopleure itself tends to extend the amniotic fold caudad over the head of the embryo (Fig. 32, B). By continuation of these two growth processes the head soon comes to lie in a double walled pocket of extra-embryonic somatopleure which covers the head like a cap (Fig. 29). The free edge of the amniotic pocket retains its original crescentic shape as, in its progress caudad, it covers more and more of the embryo. EXTRA-EMBRYONIC MEMBRANES 87 The caudally-directed limbs of the head fold of the amnion are continued posteriorly along either side of the embryo as the lateral amniotic folds. The lateral folds of the amnion grow dorso-mesiad, eventually meeting in the mid-line dorsal to the embryo (Fig. 30, A-C). During the third day, the tail-fold of the amnion develops about the caudal region of the embryo. Its manner of de- velopment is similar to that of the head fold of the amnion but its direction of growth is reversed, its concavity being directed anteriorly and its progression being cephalad (Fig. 32, B, C). Continued growth of the head, lateral, and tail folds of the amnion results in their meeting above the embryo. At the point where the folds meet, they become fused in a scar-like thickening termed the amniotic raphe (sero-amniotic raphe). (Fig. 32, C). The way in which the somatopleure has been folded about the embryo leaves the amniotic cavity completely lined by ectoderm which is continuous with the superficial ectoderm of the embryo at the region where the yolk-stalk enters the body (Fig. 30, D). All the amniotic folds involve doubling the somatopleure on itself. Only the inner layer of the somatopleuric fold is in- volved in the formation of the amniotic cavity. The outer layer of somatopleure becomes the serosa (Fig. 30, B). The cavity between serosa and amnion (sero-amniotic cavity) is part of the extra-embryonic coelom. The continuity of the extra- embryonic coelom with the intra-embryonic ccelom is most apparent in early stages (Fig. 30, A and B). They remain, however, in open communication in the yolk-stalk region until relatively late in development. The rapid peripheral growth of the somatopleure carries the serosa about the yolk-sac, which it eventually envelops. The albumen-sac also is surrounded by folds of serosa, and the allantois after its establishment develops within the serosa, between it and the amnion. Thus the serosa eventually encompasses the embryo itself and all the other extra-embryonic membranes. The relationships of the serosa and allantois and the functional significance of the serosa will be taken up after the allantois has been considered. ss EARLY EMBRYOLOGY OF THE CHICK w EXTRA-EMBRYONIC MEMBRANES 89 o Ci" u Xi 73 6 ^ 'd ^ (U H > 6 •^ CO Lill Fig. ibry t 0) o* w C^" § C ^-^ ^ aj +3 13 d M IH X» u :i S *s ^ 0) -IJ ^ tH +3 -S .1 1 •<-> 1 § •S s 2 ^ .0 s 'ti 'u Ox! >. Oi >» ^ -s-S 6 § e cq 0) 1 ^ 4> +a B ^ g 13 fj ^ ^ u Ul iw 5 g M J3 B na a £ xi ^ 'c3 2 ^1 y « a, o.lj ^1 "g g go EARLY EMBRYOLOGY OF THE CHICK The Allantois. — The allantois differs from the amnion and serosa in that it arises primarily within the body of the embryo. Its proximal portion is intra-embryonic throughout develop- ment. Its distal portion, however, is carried outside the con- fines of the intra-embryonic coelom and becomes associated with the other extra-embryonic membranes. Like the other extra- embryonic membranes the distal portion of the allantois functions only during the incubation period and is not incorpor- ated into the structure of the adult body. The allantois first appears late in the third day of incubation. It rises as a diverticulum from the ventral wall of the hind-gut and its walls are, therefore, splanchnopleure. Its relationships to structures within the embryo will be better understood when chicks of three and four days incubation have been studied, but its general location can be appreciated from the schematic diagrams of Figures 32 and 33. During the fourth day of development the allantois pushes out of the body of the embryo into the extra-embryonic coelom. Its proximal portion Hes parallel to the yolk-stalk and just caudal to it. When the distal portion of the allantois has grown clear of the embryo it becomes enlarged (Fig. 32, C). Its narrow proximal portion is known as the allantoic stalk, the enlarged distal portion as the allantoic vesicle. Fluid accumulating in the allantois distends it so the appearance of its terminal portion in entire embryos is somewhat balloon-like (Fig. 40). The allantoic vesicle enlarges very rapidly from the fourth to the tenth day of incubation. Extending into the sero- amniotic cavity it becomes flattened and finally encompasses the embryo and the yolk-sac (Fig. 30, C, D). In this process the mesodermic layer of the allantois becomes fused with the adjacent mesodermic layer of the serosa. There is thus formed a double layer of mesoderm, the serosal component of which is somatic mesoderm and the allantoic component of which is splanchnic mesoderm. In this double layer of mesoderm an extremely rich vascular network develops which is connected with the embryonic circulation by the allantoic arteries and veins. It is through this circulation that the allantois carries on its primary function of oxygenating the blood of the embryo and relieving it of carbon dioxide. This is made possible by the EXTRA-EMBRYONIC ME MBR ANE S 91 position occupied by the allantois, close beneath the porous shell (Fig. 30). In addition to its primary respiratory function the allantois serves as a reservoir for the secretions coming from neural tube notochord amniotic cavity sero-amniotic cavity hind- gut proctodaeum ' f« neural tube - notochord - mesenchyme :^'~*-^"'~ -"*^« ^V- mid-gut entoderm splanchnic mesoderm • yolk stalk If"'- If #if If 11 If [1 j^M'^%< 1 wt- V \ic yolk-sac-p^p^^ ^" J.^'^'^v;^^:^*,^ ^ ^ Mf , a'^ «* jr\ •'. -f ' d^'6'- '*■'''■ ," " ' 0" u j^^;jjj^:^£,^^m ^ . ,0 -0: amniotic cavity cloaca . cloaca 1 membrane post-anal gut sero-amniotK cavity proctodaeum Pig. 33. — Schematic longitudinal-section diagrams of the caudal half of the embryo to show the formation of the allantois. A, chick of about three days incubation; B, chick of about four lays in- cubation. the developing excretory organs and also takes part in the ab- sorption of the albumen. The fusion of the allantoic mesoderm and blood vessels 92 EARLY EMBRYOLOGY OF THE CHICK with the serosa is of particular interest because of its homology with the establishment of the chorion in the higher mammals.^ The chorion of mammalian embryos arises by the fusion of allantoic vessels and mesoderm with the inner wall of the serosa, and constitutes the embryos's organ of attachment to the uterine wall. In mammalian embryos the allantoic, or umbiUcal circulation as it is usually called in mammals, serves more than a respiratory function. In the absence of any appreciable amount of yolk, the mammalian embryo derives its nutrition through the allantoic circulation from the uterine blood of the mother. Thus the mammalian allantoic circula- tion carries out the functions which in the chick are divided between the vitelhne and the allantoic circulations. * By reason of this homology the serosa of the chick is sometimes called chorion. It seems less likely to lead to confusion if the use of the term chorion is re- stricted to mammalian forms, especially as the serosa alone is the homologue of only part of the mammalian chorion. In some books the term outer or false amnion will be found used to designate the structure called serosa in this book. The term false amnion is not, however, in general use in this country. CHAPTER XII THE STRUCTURE OF CHICKS FROM FIFTY TO FIFTY- FIVE HOURS OF INCUBATION I. External Features. II. The Nervous System. Growth of the telencephahc region; the epiphysis; the infundibulum and Rathke's pocket; the optic vesicles; the lens; the posterior part of the brain and the cord region of the neural tube; the neural crest. III. The Digestive Tract. The fore-gut; the stomodaeum; the pre-oral gut; the mid-gut; the hind-gut. IV. The Visceral Clefts and Visceral Arches. V. The Circulatory System. The heart; the aortic arches; the fusion of the dorsal aortae; the cardinal and omphalomesenteric vessels. VI. The Differentiation of the Somites. VII. The Urinary System. I. External Features In chicks which have been incubated from 50 to 55 hours (Fig. 34) the entire head region has been freed from the yolk by the progress caudad of the sub-cephalic fold. Torsion has involved the whole anterior half of the embryo and is completed in the cephalic region, so that the head now lies left side down on the yolk. The posterior half of the embryo is still in its original position, ventral surface prone on the yolk. At the extreme posterior end, the beginning of the caudal fold marks off. the tail region of the embryo from the extra-embryonic mem- branes. The head fold of the amnion has progressed caudad, together with the lateral amniotic folds impocketing the em- bryo nearly to the level of the omphalomesenteric arteries. The cranial flexure, which was seen beginning in chicks of about 38 hours, has increased rapidly until at this stage the brain is bent nearly double on; itself. The axis of the bending 93 94 EARLY EMBRYOLOGY OF THE CHICK being in the mid-brain region, the mesencephalon comes to be the most anteriorly located part of the head and the prosencephalon and myelencephalon lie opposite each other, ventral surface to ventral surface (Fig. 34). The original an- terior end of the prosencephalon is thus brought in close proximity to the heart, and the optic vesicles and the auditory vesicles are brought opposite each other at nearly the same antero-posterior level. mesencephalon metencephaMc region dorsal aortic root myelencephahc region hyomandibular cleft auditory vesicle aortic archill anL int. nnyfal optic cup /lens epiphysis choroid fissure prosencephalon. bulbo-conus arteriosus atrium lateral mesoderm marginof amnion lateral body fold neural tube ,29th somite omphalomesenterif artery caudal fold Pig. 34. — Dextro-dorsal view ( X 14) of entire embryo of 29 somites (about 55 hours incubation). At this stage flexion has involved the body farther caudally as well as in the brain region. It is especially marked at about the level of the heart in the region of transition from myelencephalon to spinal cord. Since this is the future neck region of the embryo the flexure at this level is known as the cervical flexure (Fig. 34). STRUCTURE OF FIFTY-HOUR CHICKS 95 II. The Nervous System Growth of the Telencephalic Region. — The completion of torsion in the head region causes rapid changes in the configura- tion of the brain as seen in entire embryos from 40 to 50 hours of incubation. The same fundamental regions can, however, be identified throughout this range of development. The an- terior part of the brain has undergone rapid enlargement. A slight constriction in the dorsal wall (Fig. 35) indicates the impending division of the prosencephalon into telencephalon and diencephalon. Except for its considerable increase in size no important changes have taken place in the telencephalic region. The Epiphysis. — In the mid-dorsal waU of the diencephalic region a small evagination has appeared. This evagination is the epiphysis (Fig. 34 and 35). It is destined to become dif- ferentiated into the pineal gland of the adult. The Infimdibulum and Rathke's Pocket. — In the floor of the diencephalon the infundibular depression has become deepened and Hes close to a newly formed ectodermal invagination known as Rathke's pocket (Fig. 35). The epithelium of Rathke's pocket is destined to be separated from the superficial ectoderm and to become permanently associated with the infundibular portion of the diencephalon to form the hypophysis or pituitary body. The Optic Vesicles. — The optic vesicles have undergone changes which completely alter their appearance. In 33-hour chicks they are spheroidal vesicles connected by broad stalks with the lateral walls of the diencephalon (Fig. 21). At this stage the lumen of each optic vesicle (opticoele) is widely con- tinuous with the lumen of the prosencephalon (prosoccele) (Fig. 28, A). The constriction of the optic stalk which begins to be apparent in 38-hour embryos (Fig. 22) is much more marked in 55-hour chicks. The most striking and important advance in their develop- ment is the invagination of the distal ends of the single-walled optic vesicles to form double walled optic cups (Fig. ^6, B). The concavities of the cups are directed laterally. Mesially the cups are continuous with the ventro-lateral walls of the diencephalic region of the original prosencephalon over the 96 EARLY EMBRYOLOGY OF THE CHICK narrowed optic stalks. The invaginated layer of the optic cup is termed the sensory layer because it is destined to give rise to the sensory layer of the retina. The layer against which prosencephalon choroid fissure Rathke's pocket ventral aortic root- anterior intestinal portal mesencephalon cut ectoderm metencephalon anterior cardinal vein myelencephalon neuromere of myelencephalon auditory vesicle aortic arches I. II, III. pharynx duct of Cuvier posterior cardinal vein dorsal aorta omphalomesenteric vein cut splanchnopleure cut somatopleure roots of oinphal( mesenteric artery lateral mesoderm om phjjomesenter ic artery posterior intestinal portal Fig. 35. — Diagram of dissection of chick of about 50 hours. (Modified from Prentiss.) The splanchnopleure of the yolk-sac cephalic to the anterior in- testinal portal, the ectoderm of the left side of the head, and the mesoderm in the pericardial region have been dissected away. A window has been cut in the splanchnopleure of the dorsal wall of the mid-gut to show the origin of the omphalomesenteric arteries. the sensory layer comes to lie after its invagination is termed the pigment layer because it gives rise to the pigmented layer of the retina. The double-walled cups formed by invagination, STRUCTURE OF FIFTY-HOUR CHICKS 97 are also termed secondary optic vesicles in distinction to prim- ary optic vesicles, as they are called before the invagination. The formerly capacious lumen of the primary optic vesicle is visceral furrow sero- amniotic cavity fore-gut intra-embryonic coelom dorsal mesocardium sero-amniotic raphe extra-embryonic coelom epi-myocardium of ventricle somatopleure splanchnopleure vitelline vessels extra-embryonic coelom atnum neural crest dorsal aorta post, cardinal v. •embryonic coelom intra-embryonic coelom omphalomesenteric vein lateral amniotic fold amnion serosa mesonephric duct mesonephric tubule mid-gut lateral body fold telline vessels mesonephric duct '' mesonephric tubule post, cardinal dorsal aorta Fig. 36. — Diagrams of transverse sections of 5S-hour (30-somite) chick. The location of the sections is indicated on an outline sketch of the entire embryo. practically obliterated in the formation of the optic cup. What remains of the primary opticoele is now but a narrow space be- 98 EARLY EMBRYOLOGY OF THE CHICK tween the sensory and the pigment layers of the retina (Fig. 36, B). ■ Later when these two layers fuse this space is entirely obliterated. While the secondary optic vesicles are usually spoken of as the optic cups, they are not complete cups. The invagination which gives rise to the secondary optic vesicles, instead of be- ginning at the most lateral point in the primary optic vesicles, begins at a point somewhat toward their ventral surface and is directed mesiodorsad. As a result the optic cups are formed without any lip on their ventral aspect. They may be likened to cups with a segment broken out of one side. This gap in the optic cup is the choroid fissure (Fig. 35). In Figure 36, By a section is shown which passes through the head of the embryo on a slight slant so that the right optic cup, being cut to one side of the choroid fissure appears complete while the left optic cup being cut in the region of the fissure shows no ventral lip. The infolding process by which the optic cups are formed from the primary optic vesicles is continued to the region of the optic stalks. As a result the optic stalks are infolded so that their ventral surfaces become grooved. Later in develop- ment the optic nerves and blood vessels come to lie in the grooves thus formed in the optic stalks. The Lens. — The lens of the eye arises independently of the optic vesicles, from the superficial ectoderm of the head. The first indications of lens formation appear in chicks of about 40 hours as local thickenings of the ectoderm immediately over- lying the optic vesicles. These placodes of thickened ectoderm sink below the general level of the surface of the head to form small vesicles which extend into the secondary optic vesicles. Their opening to the surface is rapidly constricted and even- tually they are disconnected altogether from the superficial ectoderm. At this stage the opening to the outside still persists although it is very small (Fig. 36, B, right eye). In sections which do not pass directly through the opening, the lens vesi- cle appears completely separated from the overlying ectoderm (Fig. 36, B, left eye). The derivation of the lens from a placode of thickened epi- thelium which sinks below the general surface, and eventually loses its connection with the superficial ectoderm, is strikingly similar to the early steps in the derivation of the auditory STRUCTURE OF FIFTY-HOUR CHICKS 99 vesicle. But these primordia once separated from the ectoderm follow divergent lines of differentiation leading to adult condi- tions which are structurally and functionally totally unlike. The origin of these two structures from cell groups similarly folded off from the same germ layer, but which once established undergo each their own characteristic differentiation, exempli- fies a sequence of events so characteristic of developmental processes in general as to call for at least a comment in passing. The Posterior Part of the Brain and the Cord Region of the Neural Tube. — Caudal to the diencephalon the brain shows no great change as compared with the last stages considered. The mesencephalon is somewhat enlarged and the constrictions separating it from the diencephalon cephalically and the metencephalon caudally are more sharply marked. The meten- cephalon is more clearly marked off from the myelencephalon and its roof is beginning to show thickening. In the myelen- cephalon the neuromeric constrictions are still evident in the ventral and lateral walls (Figs. 34 and 35). The dorsal wall has become much thinner than the ventral and lateral walls (Fig. 36, A and B) and shows no trace of division between the neuromeres. In the cord region of the neural tube the lateral walls have become thickened at the expense of the lumen so that the neural canal appears slit-like in sections of embryos of this age (Fig. 36, £) rather than elliptical as it is immediately after the closure of the neural folds. At this stage the closure of the neural tube is completed throughout its entire length. The last regions to close were at the cephaHc and caudal ends of the neural groove. In younger stages where they remained open these regions were known as the anterior neuropore and the sinus rhomboidalis, respectively. The Neural Crest.— In the closure of the neural tube the superficial ectoderm which at first lay on either side of the neural groove, continuous with the neural plate ectoderm^ becomes fused in the mid-line and separated from the neural plate to constitute an unbroken ectodermal covering (Cf. Figs. 17, Bf and 28, B). At the same time the lateral margins of the neural plate become fused to complete the neural tube. There are cells lying originally at the edges of the neural folds which are not involved in the fusion of either the superficial ectoderm lOO EARLY EMBRYOLOGY OF THE CHICK or the neural plate. These cells form a pair of longitudinal aggregations extending one on either side of the mid-dorsal line in the angles between the superficial ectoderm and the neural tube (Fig. 37, A). With the fusion of the edges of the neural folds to complete the neural tube, and the fusion of the superficial ectoderm dorsal to the neural tube, these two longi- tudinal cell masses become for a time confluent in the mid-line neural tube neural tube Pig. 37. — Drawings from transverse sections to show origin of neural crest cells. The location of the area drawn is indicated on the small sketch to the left of each drawing. A, anterior rhombencephalic region of 30-hour chick; B, posterior rhomb- encephalic region of 36-hour chick; C, mid-dorsal region of cord in 5S-hour chick. (Fig. 37, B). But because this aggregation of cells arises from paired components and soon again separates into right and left parts it is to be considered as potentially paired. On account of its position dorsal to the neural tube it is known as the neural crest. The neural crest should not be confused with the margin of STRUCTURE OF FIFTY-HOUR CHICKS lOI the neural fold with which it is associated before the closure of the neural tube. The margin of the neural fold involves cells which go into the superficial ectoderm and into the neural tube, as well as those which are concerned in the formation of the neural crest. When first established the neural crest is continuous antero- posteriorly. As development proceeds, the cells of the neural crest migrate ventro-laterally on either side of the spinal cord (Fig. 37, C), and at the same time become segmen tally clus- tered. The segmentally arranged cell groups thus derived from the neural crest give rise to the dorsal root ganglia of the spinal nerves, and in the head region to the ganglia of the sensory cranial nerves. (For a later stage of the dorsal root ganglia see Figure 44.) in. The Digestive Tract The Fore-gut. — The manner in which the three primary regions of the gut-tract are estabUshed has already been con- sidered in a general way (see Chapter XI and Fig. 31). In 50 to 55-hour chicks the fore-gut has acquired considerable length. It extends from the anterior intestinal portal cephalad almost to the infundibulum (Fig. 35). As the first region of the tract to be established, the fore-gut is naturally the most advanced in differentiation. We can already recognize a pharyngeal and an oesophageal portion. The pharyngeal region lies ventral to the myelencephalon and is encircled by the aortic arches (Fig. 35). The pharynx is somewhat flattened dorso-ventrally and has a considerably larger lumen than the oesophageal part of the fore-gut (Cf . Fig. 36, B and C). The Stomodaeum.^There is at this stage no mouth opening into the pharynx. However, the location where the opening will be formed is indicated by the approximation of a ventral outpocketing near the anterior end of the pharynx, to a depres- sion formed in the adjacent ectoderm of the ventral surface of the head (Fig. 35). The ectodermal depression, known as the stomodaeum, deepens until its floor lies in contact with the ento- derm of the pharyngeal out-pocketing (Fig. 35). The thin layer of tissue formed by the apposition of the stomodaeal ecto- derm to the pharyngeal entoderm is known as the oral plate. I02 EARLY EMBRYOLOGY OF THE CHICK Later in development the oral plate breaks thiough bringing the stomodaeum and the pharynx into open communication. Growth of surrounding structures deepens the original stomodaeal depression, and it becomes the oral cavity. The region of the oral plate in the embryo becomes, in the adult, the region of transition from oral cavity to pharynx. The Pre-oral Gut. — It will be noted by reference to Figure 35 that the oral opening is not established at the extreme cephalic end of the pharynx. The part of the pharynx which extends cephalic to the mouth opening is known as the pre-oral gut. After the rupture of the oral plate, the pre-oral gut eventually disappears, but an indication of it persists for a time as a small diverticulum termed SeesselFs pocket(Cf.Figs. 35 and 43). The Mid-gut. — Although the mid-gut is still the most ex- tensive of the three primary divisions of the digestive tract, it presents little of interest. It is nothing more than a region where the gut still lies open to the yolk. It does not have even a fixed identity. As fast as any part of the mid-gut acquires a ventral wall by the closing-in process involved in the progress of the subcephalic and subcaudal folds it ceases to be mid-gut and becomes fore-gut or hind-gut. Differentiation and local specializations appear in the digestive tract only in regions which have ceased to be mid-gut. The Hind-gut. — The hind-gut first appears in embryos of about 55 hours (Fig. 35). The method of its formation is similar to that by which the fore-gut was estabhshed. The sub-caudal fold undercuts the tail region and walls off a gut pocket. The hind-gut is lengthened at the expense of the mid-gut as the sub-caudal fold progresses cephalad and is also lengthened by its own growth caudad. It shows no local specializations until later in development. IV. The Visceral Clefts and Visceral Arches At this stage the chick embryo has unmistakable visceral arches and visceral clefts. Although only transitory, they are morphologically of great importance not only from the com- parative view point, and because of their significance as struc- tures exemplifying recapitulation, but also because of their STRUCTURE OF FIFTY-HOUR CHICKS 103 participation in the formation of the embryonic arterial system, of some of the ductless glands, of the eustachian tube, and of the face and jaws. The visceral clefts are formed by the meeting of ectodermal depressions, the visceral furrows, with diverticula from the lateral walls of the pharynx, the pharyngeal pouches. During most of the time the visceral furrows are conspicuous features in entire embryos, they may be seen in sections to be closed by a thin double layer of tissue composed of the ectoderm of the floor of the visceral furrow and the entoderm at the distal ex- tremity of the pharyngeal pouch (Fig. 36, A). The breaking through of this thin double layer of tissue brings the pharyngeal pouches into communication with the visceral furrows thereby establishing open visceral clefts. In birds an open condition of the clefts is transitory. In the chick the most posterior of the series of clefts never becomes open. Although some of the clefts never become open and others open for but a short time the term cleft is usually used to designate these structures which are potentially clefts, whether open or not. The position of the visceral clefts is best seen in entire em- bryos. They are commonly designated by number beginning with the first cleft posterior to the mouth and proceeding caudad. The first post-oral cleft appears earliest in develop- ment and is discernible at about 46 hours of incubation. Vis- ceral cleft II appears soon after, and by 50 to 55 hours three clefts have been formed (Fig. 34). Between adjacent visceral clefts, the lateral body walls about the pharynx are thickened. Each of these lateral thickenings in the mid-ventral line meets and merges with the corresponding thickening of the opposite side of the body. Thus the pharynx is encompassed laterally and ventrally by a series of arch-like thickenings, the visceral or gill arches. The visceral arches like the visceral clefts are designated by number, beginning at the anterior end of the styles. Visceral arch I lies cephalic to the first post-oral cleft, between it and the mouth region. Because of the part it plays in the formation of the mandible it is also designated as the mandibular arch. Visceral arch II is fre- quently termed the hyoid arch, and visceral cleft I, because of its position between the mandibular and hyoid arches, is known as the hyomandibular cleft. Posterior to the hyoid arch the I04 EARLY EMBRYOLOGY OF THE CHICK visceral arches and clefts are ordinarily designated by their post-oral numbers only. There are other structures which are just beginning to be differentiated in the pharyngeal region and fore-gut of embryos of this stage, but it seems better to consider them in connection with later stages when their significance will be more readily grasped. V. The Circulatory System The Heart. — In embryos of 30 to 40 hours incubation we traced the expansion of the heart till it was bent to the right of the embryo In the form of a U-shaped tube (Figs. 19, 21, 23). The disappearance of the dorsal mesocardium except at its li posterior end, leaves the mid-region of the heart lying unat- ' tached and extending to the right, into the pericardial region of the coelom. The heart is fixed with reference to the body of the embryo at its cephalic end where the ventral aortic roots lie embedded beneath the floor of the pharynx, and caudally in the sinus region where it is attached by the omphalomesenteric veins, by the ducts of Cuvier, and by the persistent portion of the dorsal mesocardium. During the period between 30 and 55 hours of incubation the heart itself is growing more rapidly than is the body of the embryo in the region where the heart lies. Since its cephalic and caudal ends are fixed, the unattached mid-region of the heart becomes at first U-shaped and then twisted on itself to form a loop. The atrial region of the heart is forced somewhat to the left, and the conus region is thrown across the atrial region by being twisted to the right and dorsally. The ven- tricular region constitutes the loop proper (Cf. Figs. 22, 29 and 34). This twisting process reverses the original cephalo- caudal relations of the atrial and ventricular regions. Before the twisting, the atrial region of the heart was caudal to the ventricular region as it is in the adult fish heart. In the twist- ing of the heart the atrial region, by reason of its association with the fixed sinus region of the heart, undergoes relatively little change in position. The ventricular region is carried, over the dextral side of the atrium and comes to lie caudal to it, thus arriving in the relative position it occupies in the adult heart. The bending and subsequent twisting of the heart lead toward STRUCTURE OF FIFTY-HOUR CHICKS IO5 its division into separate chambers. As yet, however, no indi- cation of the actual partitioning off of the heart is apparent. It is still essentially a tubular organ through which the blood passes directly without any division into separate channels or currents. The Aortic Arches. — In 33 to 38 hour chicks the ventral aortae communicate with the dorsal aortae over a single pair of aortic arches which bend around the anterior end of the pharynx (Figs. 23 and 24) . With the formation of the visceral arches new aortic arches appear. The original pair of aortic arches comes to lie in the mandibular arch, and the new aortic arches are formed caudal to the first pair, one pair in each visceral arch. In chicks of 50 to 55 hours, three pairs of aortic arches have been established and a fourth is usually beginning to form (Figs. 34, 35, and 36, A and 5). The Fusion of the Dorsal Aortae. — The dorsal aortae arise as vessels paired throughout their entire length (Fig. 23). As development progresses they fuse in the mid-line to form the unpaired dorsal aorta familiar in adult anatomy. This fusion takes place first at about the level of the sinus venosus and progresses thence cephalad and caudad. Cephalically it never extends to the pharyngeal region. Caudally the whole length of the aorta is eventually involved. At this stage the fusion has progressed caudad to about the level of the 14th somite (Figs. 34, 35, 36). The Cardinal and Omphalomesenteric Vessels. — The rela- tionships of the cardinal veins and the omphalomesenteric vessels are little changed from the conditions in 40 to 50 hour chicks. The posterior cardinals have elongated, keeping pace with the caudal progress of differentiation in the mesoderm. They lie just dorsal to the intermediate mesoderm in the angle formed between it and the somites (Fig. 36, D). The entrance of the omphalomesenteric veins into the sinus venosus, and the origin of the omphalomesenteric arteries from the dorsal aortae show little change from conditions familiar from the study of younger embryos. VI. The Differentiation of the Somites When the somites are first formed they consist of a nearly solid mass of cells derived from the dorsal mesoderm (Fig. sSj A). The cells composing them show a more or less radial io6 EARLY EMBRYOLOGY OF THE CHICK neural fold ectoderm of head somite intermediate mesoderm somatic mesoderm coelom splanchnic mesoderm entoderm epithelial layer of somite core of somite pronephric tubule (intermediate mesoderm) somatic mesoderm coelom iplanchnic mesoderm entoderm D epithelial layer of somite cavity of somite core of somite migrating cells posterior cardinal vein mesonephric duct mesonephric tubule coelom dorsal aorta dorsal ganglion (neural crest) myotome dermatome sclerotome myocoele posterior cardinal vein mesonephric duct mesonephric tubule dorsal aorta intra-embryonic coelom extra-embryonic coelom Pig. 38. — Drawings from transverse sections to show the differentiation of the somites. A, second somite of 4-somite chick; B, ninth somite of 12-somite chick; C, twentieth somite of 30-somite chick; D, seventeenth somite of 33-somite chick. STRUCTURE OF FIFTY-HOUR CHICKS 107 arrangement. In the center of the somite a cavity is usually discernible. This cavity is at first extremely minute. In somites which have been recently formed it may be altogether wanting. As the somite becomes more sharply marked ofif the radial arrangement of the outer zone of cells appears more definitely (Fig. 38, B). The boundaries of the central cavity are con- siderably extended but its lumen is almost completely filled by a core of irregularly arranged cells. In sections which pass through the middle of the somite, this central core of cells is seen to arise from the lateral wall of the somite where it is continuous with the intermediate mesoderm. A little later in development the outer zone of cells on the ventro-mesial face of the somite loses its originally definite boundaries and becomes merged with the central core of cells. This ill-defined cell aggregation, known as the sclerotome, be- comes mesenchymal in characteristics, and extends ventro- mesiad from the somite of either side toward the notochord (Fig. 2)^, C and D). The cells of the sclerotomes of either side continue to converge about the notochord and later take part in the formation of the axial skeleton. Duting the formation of the sclerotome the dorsal part of the original outer cell-zone of the somite has maintained its definite boundaries and epithehal characteristics. The part of this outer zone which lies parallel to the ectoderm is known as the dermatome (Fig. 38, C and D). It later becomes asso- ciated with the ectoderm and forms the deeper layers of the integument, the ectoderm giving rise to the epithelial layer only. The dorso-mesial portion of the outer zone of the somite be- comes the myotome. It is folded somewhat laterad from its original position next to the neural tube (Fig. 2>^, C) and comes to lie ventro-mesial to the dermatome and parallel to it (Fig. 38, D). (A later stage in the differentiation of the somite is shown in Figure 44) . The portion of the original cavity which persists for a time between the dermatome and myotome is termed the myocoele. The myotomes undergo the most extensive growth of any of the parts of the somite, giv- ing rise eventually to the entire skeletal musculature of the body. Io8 EARLY EMBRYOLOGY OF THE CHICK VII. The Urinary System In the section-diagrams of Figure 36, Z) and E, certain parts of the urinary system which have been established in chicks of 50 to 55 hours will be found located and labeled. The urinary system is relatively late in becoming- differentiated. Only a few of the early steps in its formation can at this time be made out. Many structures which later become of great importance are not represented even by primordial cell aggregations. Ex- cept for those well grounded in comparative anatomy, any logical discussion of the structures which have appeared must anticipate much that occurs later in development. Consider- ation of the mode of origin and significance of the nephric organs appearing at this stage has, therefore, been deferred. CHAPTER XIII THE DEVELOPMENT OF THE CHICK DURING THE THIRD AND FOURTH DAYS OF INCUBATION 1. External Features. Torsion; flexion; the visceral arches and clefts; the oral region; the appendage buds; the allantois. II. The Nervous System. Summary of development prior to the third day; the formation of the telencephaHc vesicles; the diencepha- lon; the mesencephalon; the metencephalon; the myelencephalon; the gangUa of the cranial nerves; the spinal cord; the spinal nerve roots. III. The Sense Organs. The eye; the ear; the olfactory organs. IV. The Digestive and Respiratory Systems. Summary of development prior to the third day; the establishment of the oral opening; the pharyngeal derivatives; the trachea; the lung-buds; the oesopha- gus and stomach; the liver; the pancreas; the mid- gut region; the cloaca; the proctodaeum and the cloa- cal membrane. V. The Circulatory System. The functional significance of the embryonic circulation; the vitelHne circulation; the allantoic circulation; the intra-embryonic circulation; the heart. VI. The Urinary System. The general relationships of pronephros, mesonephros, and metanephros; the pronephric tubules of the chick; the mesonephric tubules. VII. The Coelom and Mesenteries. I. External Features Torsion. — Chicks of three days incubation (Fig. 39) have been affected by torsion throughout their entire length. Tor- sion is complete well posterior to the level of the heart but 109 no EARLY EMBRYOLOGY OF THE CHICK the caudal portion of the embryo is not yet completely turned on its side. In four-day chicks the entire body has been turned through 90 degrees and the embryo lies with its left side on the yolk (Fig. 40). myelencephalon ganglion IX visceral cleft II aortic arch IV bulbo-conus arteriosus hyoid arch auditory vesicle / . hyomandibular cleft mandibular arch ganglion V metencephalon ant. cardinal v. mesencephalon horoid fissure — lens sensory layer pigment layer appendage bud posterior appendage bud vitelline artery Pig. 39. -Dextro-dorsal view ( X 14) of entire chick embryo of 36 somites (about three days incubation). Flexion. — The cranial and cervical flexures which appeared in embryos during the second day have increased so that in three-day and four-day chicks the long axis of the embryo shows nearly right-angled bends in the mid-brain and in the neck region. The mid-body region of three-day chicks is slightly concaved dorsally. This is due to the fact that the embryo is still broadly attached to the yolk in that region. By the end of the fourth day the body folds have undercut the embryo so it remains attached to the yolk only by a slender stalk. The yolk-stalk soon becomes elongated allowing the embryo to become first straight in the mid-dorsal region, and then convex STRUCTURE OF FOUR-DAY CHICKS III dorsally. At the same time the caudal flexure is becoming more pronounced. The progressive increase in the cranial, cervical, dorsal, and caudal flexures results in the bending of the embryo on itself so that its originally straight long-axis becomes C-shaped and its head and tail lie close together (Fig. 40). myelencephalon visceral arch III bulbo-conusarteriosus atrium auditory vesicle endolymphatic duct ganglion IX/ / ganglion VII-VIII hyomandi bular cleft mandibular arch ganglion V meten- cephalon mesen- cephalon anterior appendage bud omphalo- mesenteric vein border mesoneph; posterior appendage bud Fig. 40. — Dextral view of entire chick embryo of 41 somites (about four days incubation) . The Visceral Arches and Clefts. — A fourth visceral cleft has appeared caudal to the three that were already formed in 55- hour chicks. The visceral arches are thicker and more conspicu- ous than in earlier embryos. In lightly stained whole-mounts of a three-day chick it is still possible to make out the aortic arches running through the visceral arches. In a chick of four days the visceral arches have become so much thickened that it is very difficult to see the vessels traversing them. The Oral Region. — The cervical flexure presses the pharyn- geal region and the ventral surface of the head so closely to- gether that it is difficult to make out the topography of the oral 112 EARLY EMBRYOLOGY OF THE CHICK region by study of entire embryos. If the head and pharyngeal region are cut from the trunk and viewed from the ventral aspect the relations of the structures about the mouth are well shown (Fig. 41). The mandibular arch forms the caudal boundary of the oral depression. Arising on either side in connection with the mandibular arch are paired elevations, the maxillary processes, which grow mesiad and form the cephalo- lateral boundaries of the mouth opening. The nasal pits appear as shallow depressions in the ectoderm of the anterior part of the head which overhangs the mouth region. Surround- epiphytif lateral telencephalic vesicle Pig. 41. — Drawing to show the external appearance of the structures in the oral region of a four-day chick. Ventral aspect. ing each nasal pit is a U-shaped elevation with its limbs directed toward the oral cavity. The lateral limb of the elevation is the naso-lateral process, and the median limb is the naso-medial process. As development proceeds the two naso-medial proces- ses grow toward the mouth and meet the maxillary pro- cesses which are growing in from either side. The fusion of the two naso-medial processes with each other in the mid-line, and the fusion of each of them laterally with the maxillary process of its own side gives rise to the upper jaw (maxilla). The fusion in the mid-line of the right and left components of the mandibular arch gives rise to the lower jaw (mandible). The Appendage Buds. — Both the anterior and posterior ap- pendage-buds have appeared in embryos of three days. They STRUCTURE OF FOUR-DAY CHICKS II3 are formed by bud-like outgrowths from somites. The anterior appendages arise opposite somites 17 to 19 inclusive, and the posterior appendages arise opposite somites 26 to 32 inclusive. During the fourth day the appendage buds increase rapidly in size and become elongated but otherwise their appearance and their relationships show little change. The Allantois. — The development of the extra-embryonic membranes has already been considered (Chap. XI) and needs no further discussion here. In order to show the embryos more clearly, the extra-embryonic membranes, except for the allan- tois, have been removed from the specimens drawn in Figures 39 and 40. The cut edge of the amnion shows at its anterior attachment to the body, opposite the anterior appendage bud and just caudal to the tip of the ventricle. The allantois in the three-day chick is as yet small and is concealed by the pos- terior appendage buds. In four-day embryos it has undergone rapid enlargement and projects from the umbilical region as a stalked vesicle of considerable size. II. The Nervous System Simmiary of Development Prior to the Third Day. — The earliest indication of the formation of the central nervous sys- tem appears in chicks of 16 to 18 hours as a local thickening of the ectoderm which forms the neural plate (Fig. 11). The neural plate then becomes longitudinally folded to form the neural groove (Figs. 14 and 15). By fusion of the margins of the neural folds, first in the cephalic region and later caudally, the neural groove is closed to form a tube and at the same time separated from the body ectoderm. The cephalic portion of the neural tube becomes dilated to form the brain and the re- mainder of the neural tube gives rise to the spinal cord (Figs. 18 and 21). In its early stages the brain shows a series of enlargements in its ventral and lateral walls, indicative of its fundamental metameric structure. In the establishment of the three vesicle condition of the brain, the lines of demarcation between pros- encephalon, mesencephalon, and rhombencephalon are formed by the exaggeration of certain of the inter-neuromeric constric- tions and the obliteration of others (see Chap. IX and Fig. 20). 114 EARLY EMBRYOLOGY OF THE CHICK The original neuromeric enlargements persist longest in the rhombencephalon. The three-vesicle condition of the brain is transitory. By forty hours the division of the rhombencephalon into meten- cephalon and myelencephalon is clearly indicated (Figs. 20, D and 22). The division of the prosencephalon and the estabhsh- ment of the five-vesicle condition characteristic of the adult brain, does not take place until somewhat later. In chicks of 55 hours (Figs. 34 and 35) the appearance of the cranial flexure has resulted in the bending of the brain so that the entire prosencephalon is displaced ventrad and then toward the heart. At the same time the head of the embryo has under- gone torsion and lies with its left side on the yolk. Although flexion and torsion have thus completely changed the general appearance of the brain as seen in entire embryos, the regions already established in 40-hour chicks are still evident. The prosencephalon has, however, become very noticeably enlarged cephalic to the optic vesicles, and a slight constriction in its dorsal wall indicates the beginning of the demarcation of the telencephalic region from the diencephalic region. The Formation of the Telencephalic Vesicles. — By the end of the third day the antero-lateral walls of the primary fore- brain have been evaginated to form a pair of vesicles lying one on either side of the mid-line (Figs. 39, 41, and 42, B). These lateral evaginations are known as the telencephalic vesicles. The openings through which their cavities are continuous with the lumen of the median portion of the brain are later known as the foramina of Monro. The telencephahc division of the brain includes not only the two lateral vesicles but also the median portion of the brain from which they arise. The teloccele has therefore three divisions, a median, broadly con- fluent posteriorly with the diocoele, and two lateral, connecting with the median through the foramina of Monro (Fig. 42, C). Before the formation of the telencephalic vesicles the most anterior part of the brain lay in the mid-line, but the rapid growth of the telencephalic vesicles soon carries them anteriorly beyond the median portion of the teloccele. The median ante- rior wall of the teloccele which formerly was the most anterior part of the brain, and which remains the most anterior part of the brain lying in the mid-line, is known as the lamina terminalis STRUCTURE OF FOUR-DAY CHICKS "5 (Figs. 42, A, and C, and 43). The telencephalic vesicles become the cerebral hemispheres, and their cavities become the paired lateral ventricles of the adult brain. The hemispheres undergo enormous enlargement in their later development and extend dorsally and posteriorly as well as anteriorly, eventually cover- ing the entire diencephalon and mesencephalon under their posterior lobes. metacoele ( ventricle IV ) thin roof of myelencephalon myelocoele cle IV ) ventral cephalic fold spinal cord recessus opticus lamina termina median telocoele (ventricle III) recessus neuroporicus meso-metenceohalic fold mesocoele (Sylvian aqueduct j location of posterior comm issure mescKliencephalic fold tuberculum posterius diocoele( ventricle III ) epiphysis velum transversum metencephalon mesencephalon gangli ganglion VII VIII lamina .. terminalis /median telocoele ( ventricle III ) foramen of Monro (sylvian aqueduct^ lateral telencephalic vesicle metacoele ^ventricle IV ) myelocoele ( ventricle IV) position of auditory vesicle spinal cord Fig. 42. — Diagrams to show the topography of the brain of a four-day chick. A, plan of sagittal section. The arbitrary boundaries between the various brain vesicles (according to von Kupffer) are indicated by broken lines. B, dextral view of a brain which has been dissected free. C, schematic frontal section plan of brain. The flexures of the brain are supposed to have been straightened before the section was cut. As a matter of convenience in dealing with the morphology of the brain, more or less arbitrary lines of division between the adjacent brain regions are recognized. The division be- tween telencephalon and diencephalon is an imaginary line drawn from the velum transversum to the recessus opticus ii6 EARLY EMBRYOLOGY OF THE CHICK (Fig. 42, A). Velum transversum is the name given to the internal ridge formed by the deepening of the dorsal constriction which was first noted in chicks of 55 hours as indicating the impending division of the primary fore-brain (Fig. 35). The recessus opticus is a transverse furrow in the floor of the brain which in the embryo leads on either side into the lumina of thp optic stalks. The Diencephalon. — The lateral walls of the diencephalon at this stage show little differentiation except ventrally where the mandibular arch Seetiell's pocket Rathke's pocket .^>^ omph. met, vein tneionephros somite dortal aorta tuberculum posteriuB infundibulum allantoic vesicle somite allantoic stallr proctodaeum post- anal gut cloaca — splanchnopleure of yolk sac Fig. 43. — Diagram of median longitudinal section of four-day chick. Due to a slight bend in the embryo the section is para-sagittal in the mid-dorsal region but for the most part it passes through the embryo in the sagittal plane. optic stalks merge into the walls of the brain. The develop- ment of the epiphysis as a median evagination in the roof of the diencephalon has already been mentioned (Chap. XII). Ex- cept for some elongation it does not differ from its condition when first formed in embryos of about 55 hours. The in- fundibular depression in the floor of the diencephalon has be- STRUCTURE OF FOUR-DAY CHICKS II7 come appreciably deepened and lies in close proximity to Rathke's pocket with which it is destined to fuse in the forma- tion of the hypophysis (Fig. 43). Later in development the lateral walls of the diencephalon become greatly thickened to form the thalami, thus reducing the size and changing the shape of the diocoele, which is known in adult anatomy as the third brain ventricle. The anterior part of the roof of the diencephalon remains thin and by the ingrowth of blood vessels from above is. pushed into the third ventricle to form the an- terior choroid plexus. The boundary between the diencephalon and the mesen- cephalon is an imaginary line drawn from the internal ridge formed by the original dorsal constriction between the primary fore-brain and mid-brain, to the tuberculum posterius (Fig. 42, A). The tuberculum posterius is a rounded elevation in the floor of the brain of importance chiefly because it is regarded as marking the boundary between diencephalon and mesen- cephalon. The Mesencephalon.— The mesencephalon as yet shows no specializations, beyond a thickening of its walls. The dorsal and lateral walls of the mesencephalon later increase rapidly in thickness and become the optic lobes (corpora quadrigemina) of the adult brain. The optic lobes should not be confused with the optic vesicles arising from the diencephalon of the embryo. They are entirely different structures. The floor of the mesen- cephalon also becomes greatly thickened and is known in the adult as the crura cerebri. It serves as the main pathway of the fiber tracts which connect the cerebral hemispheres with the posterior part of the brain and the spinal cord. The originally capacious mesocoele is thus reduced by the thickening of the walls about it to a narrow canal (Aqueduct of Sylvius). The Metencephalon. — The boundary between the mesen- cephalon and metencephalon is indicated by the original inter- neuromeric constriction which separated them at the time^of their estabHshment (Cf. Figs. 20 and 42). The caudal boun- dary of the metencephalon is not definitely defined. It is regarded as being located approximately at the point where the brain roof changes from the thickened condition character- istic of the metencephalon to the thin condition characteristic of the myelencephalon. The metencephalon shows practically Il8 EARLY EMBRYOLOGY OF THE CHICK no differentiation in four-day chicks. Later in development there is ventrally and laterally an extensive ingrowth of fiber tracts giving rise to the pons and to the cerebellar peduncles of the adult metencephalon. The roof of the metencephalon undergoes extensive enlargement and becomes the cerebellum of the adult brain. The Myelencephalon. — In the myelencephalon the dorsal wall has become greatly reduced in thickness indicative of its final fate as the thin roof of the medulla. Like the roof of the diencephalon, the roof of the myelencephalon later receives a rich supply of small blood vessels by which it is pushed into the myelocoele to form the posterior choroid plexus (choroid plexus of the fourth ventricle). The ventral and lateral walls of the myelencephalon become the floor and side-walls of the medulla of the adult brain. The Ganglia of the Cranial Nerves. — In the brain region, cells derived from the cephalic portion of the neural crest have be- come aggregated to form ganglia. The largest and the most clearly defined of the gangha present in four-day chicks is the Gasserian ganglion of the fifth (trigeminal) cranial nerve (Fig. 42, B). It lies ventro-laterally, opposite the most anterior neuromere of the myelencephalon. From its cells sensory nerve fibers grow mesiad into the brain and distad to the face and mouth region. In four-day chicks the beginning of the ophthalmic division of the fifth nerve extends from the ganglion toward the eye, and the beginning of the mandibulo-maxillary division is growing toward the angle of the mouth (Fig. 40). Immediately cephahc to the auditory vesicle is a mass of neural crest cells which is the primordium of the ganglia of the seventh and eighth nerves. The separation of this double primordium to form the geniculate ganglion of the seventh nerve and the acoustic ganghon of the eighth nerve begins during the fourth day. Posterior to the auditory vesicle the ganglion of the ninth nerve can be clearly seen even in whole-mounts (Fig. 40). The gangha of the tenth (vagus) nerves can be recognized in sections of chicks at the end of the fourth day but are difficult to make out in whole-mounts. The Spinal Cord. — The spinal cord region of the neural tube when first established, exhibits a lumen which is elliptical in cross section. As development progresses the lateral walls of STRUCTURE OF FOUR-DAY CHICKS 119 the cord become greatly thickened in contrast with the dorsal and ventral walls which remain thin. In this process the lumen (central canal) becomes compressed laterally until it appears in cross section as little more than a vertical slit. The thin dorsal wall of the tube is known as the roof plate; the thin ventral wall as the floor plate; and the thickened side walls as the lateral plates. The Spinal Nerve Roots. — During the fourth day the estab- lishment of the spinal nerve roots has begun. The growth of nerve fibers from the neuroblasts can only be traced with the aid of special methods of staining. The more general steps in the spinal cord dorsal ganglion dorsal root ventral root spinal nerve neuron of ventral root (motor) Pig. 44. — Drawing to show the structure and relations of a spinal ganglion and the roots of a spinal nerve. The left half of the drawing represents struc- tures as they appear after treatment by the usual nuclear staining method. The right half of the section shows schematically the nerve cells and the fibers grow- ing out from them as they may be demonstrated by the Golgi method. {Nerve cells and fibers after Ramon y Cajdl.) development of the roots of the spinal nerves can, however, be followed in sections prepared by the ordinary methods. In the adult each spinal nerve is connected with the cord by two roots, a dorsal root which is sensory in function and a ven- tral root, which is motor in function. Lateral to the cord the dorsal and ventral roots unite. The spinal ganglion (dorsal root ganglion) is located on the dorsal root between the spinal cord and the point where dorsal and ventral roots unite. Distal to the union of dorsal and ventral roots is a branch, the ramus I20 EARLY EMBRYOLOGY OF THE CHICK communicans, which extends ventrad to a ganglion of the sym- pathetic nerve cord. When first formed from the neural crest cells, the spinal ganglion has no connection with the cord (Fig. 37). The dorsal root is established by the growth of nerve fibers from cells of the spinal ganglion mesiad into the dorsal part of the lateral plate of the cord. At the same time fibers grow distad from these cells to form the peripheral part of the nerve (Fig. 44). The fibers which arise from the dorsal root ganglion conduct sensory impulses toward the cord. Coincident with the establishment of the dorsal root, the ventral root is formed by fibers which grow out from cells located in the ventral part of the lateral plate of the cord (Fig. 44)'. The fibers which thus arise from cells in the cord and pass out through the ventral root, conduct motor impulses from the brain and cord to the muscles with which they are associated peripherally. The sympathetic ganglia arise from cells of the neural crest which migrate ventrally and form cellular masses lying on either side of the mid-line at the level of the dorsal aorta. By the end of the fourth day these cells constitute a pair of cords in which enlargements can be made out opposite the spinal ganglia. These enlargements are the primary sympathetic gangha. Each sympathetic ganghon is connected with the corresponding spinal nerve by a cellular cord which is the primordium of the ramus communicans. The sympathetic ganglia later receive both sensory and motor fibers from the spinal nerve roots by way of the rami communicantes, and from nerve cells in the sympathetic ganglia, fibers extend to the viscera. III. The Sense Organs The Eye. — The primary optic vesicles arise in chicks of about 30 hours as dilations in the lateral wall of the prosencephalon (Figs. 19 and 23). At first the optic vesicles open broadly into the brain, but later constrictions develop which narrow their attachment to the form of a stalk (Fig. 22). In chicks of 55 hours the primary optic vesicles are invaginated to form the double-walled secondary optic vesicles or optic cups. The invagination takes place in such a way that the ventral wall STRUCTURE OF FOUR-DAY CHICKS 121 of the cup is incomplete, the gap in it being known as the choroid fissure (Figs. 35 and 36, B). The lens arises as a thickening of the superficial ectoderm which becomes depressed to form a vesicular invagination ex- tending into the optic cup (Fig. 36, B). ectoderm diocoele mesenchyme concentration of mesenchyme pigment layer sensory layer lens area enlarged in B corneal region optic stalk '•^ M P-gr ^m ' 'ill pigment layer of retina sensory layer of retina developing lens fibers Pig. 45. — Drawings to show structure of the eye of a four-day chick. A, diagram to show topography of eye region; B, drawing to show cellular organization of the pigment and sensory layers of the retina. Abbreviations: mes., mesenchymal cell; p.gr., pigment granule; C, drawing to show cellular organization of the lens. In chicks of four days the choroid fissure has become nar- rowed by the growth of the walls of the optic cup on either side of it (Figs. 40 and 42, B). The orifice of the optic cup becomes 122 EARLY EMBRYOLOGY OF THE CHICK narrowed by convergence of its margins toward the lens (Fig. 45, A). Meanwhile the lens has become freed from the super- ficial ectoderm and forms a completely closed vesicle. Sections of the lens at this stage show that the cells constituting that part of its wall which lies toward the center of the optic cup are becoming elongated to form the lens fibers (Fig. 45, C). At this stage we can identify the beginning of most of the structures of the adult eye. The thickened internal layer of the optic cup will give rise to the sensory layer of the retina (Fig. 45, B). Fibers arise from nerve cells in the retina and grow along the groove in the ventral surface of the optic stalk toward the brain to form the optic nerve. The external layer of the optic cup gives rise to the pigment layer of the retina. Mesenchyme cells can be seen aggregating about the outside of the optic cup. From these the sclera and choroid coat are derived. Some of the mesenchyme makes its way into the optic cup through the choroid fissure and gives rise to the cellu- lar elements of the vitreous body. The comple:?^ ciHary appar- atus of the adult eye is derived from the margins of the optic cup adjacent to the lens. The corneal and conjunctival epi- thelium arise from the superficial ectoderm overlying the eye. Mesenchyme cells which make their way between the lens and the corneal epithelium give rise to the substantia propria of the cornea. The Ear.- — Of the structures taking part in the formation of the ear, the first to appear is the auditory placode. The audi- tory placode is recognizable in 36-hour chicks as a thickened plate of ectoderm. Almost as soon as it appears the placode sinks below the level of the surrounding ectoderm to form the floor of the auditory pit (Fig. 22). By constriction of its open- ing to the surface the epithelium of the auditory pit becomes separated from the ectoderm of the head and comes to lie close to the lateral wall of the myelencephalon (Fig. 36, ^). A tubu- lar stalk, the endolymphatic duct, remains for a time adherent to the superficial ectoderm, marking the location of the original invagination (Fig. 40). The degree of development reached by the ear primordium in four-day chicks gives little indication of the nature of the later processes by which the ear is formed. The auditory vesicle by a very complex series of changes will give rise to the STRUCTURE OF FOUR-DAY CHICKS 1 23 entire epithelial portion of the internal ear mechanism. Nerve fibers arising from the acoustic ganglion grow into the brain proximally and to the internal ear distally establishing nerve connections between them. There is at this stage no indication of the differentiation of the external auditory meatus. The dorsal and inner portion of the hyomandibular cleft which gives rise to the eustachian tube and to the middle ear chamber has not yet become associated with the auditory vesicle. The Olfactory Organs. — The olfactory organs are represented in three-day and four-day chicks by a pair of depressions in the ectoderm of the head. These so-called olfactory pits are located ventral to the telencephalic vesicles and just anterior to the mouth (Figs. 40 and 41). By growth of the processes which surround them, the olfactory pits become greatly deepened. The epitheHum lining the pits eventually comes to lie at the extreme upper part of the nasal chambers and constitutes the olfactory epithelium. Nerve fibers grow from these cells to the telencephalic lobes of the brain to form the olfactory nerves. IV. The Digestive and Respiratory Systems Summary of Development Prior to the Third Day. — The primary entoderm which gives rise to the epithelial Hning of the digestive and respiratory systems and their associated glands becomes estabhshed as a separate layer before the egg is laid. In its early relationships the entoderm is a sheet-like layer of cells lying between the ectoderm and the yolk and attached peripherally to the yolk (Fig. 7). The primitive gut is the cavity bounded dorsally by the entoderm and ventrally by the yolk (Fig, 31, A). Only the part of the entoderm which lies within the em- bryonal area is involv-ed in the formation of the enteric tract. The peripheral portion of the entoderm goes into the formation of the yolk-sac. There is at first ho definite line of demarcation between the entoderm destined to be incorporated into the body of the embryo and that which remains extra-embryonic in its associations. The foldings which appear later separating the body of the embryo from the yolk, establish for the first time the boundaries between intra-embryonic and extra-em- bryonic entoderm (Figs. 30 and 32). 124 EARLY EMBRYOLOGY OF THE CHICK The first part of the gut to acquire a complete entodermic lining is the fore-gut. Its floor is formed by the caudally progressing concrescence of the entoderm which takes place as the subcephalic and lateral body folds undercut the cephalic part of the embryo (Figs. i6 and 31, 5). At a considerably later stage the hind-gut is formed by the progress of the sub- caudad fold (Figs. 35 and 31, C). Between the fore-gut and the hind-gut, the mid-gut remains open to the yolk ventrally. As the embryo is more completely separated from the yolk the fore-gut and hind-gut increase in extent at the expense of the mid-gut. By the fourth day of incubation the mid-gut is re- duced to the region where the yolk stalk opens into the enteric tract (Figs. 31, -D and 43). The Establishment of the Oral Opening. — When first estab- lished the gut ends as a blind pocket both cephalically and caudally. The mouth opening does not appear until the third day, the cloacal opening is not established until much later in incubation. In embryos of 55 hours the processes leading to- ward the establishment of the oral opening are clearly indicated. A mid-ventral evagination of the pharynx is estabhshed im- mediately cephalic to the mandibular arch (Fig. 35). Opposite this out-pocketing of the pharynx, and growing in to meet it, the stomodeal depression is formed. The thin membrane formed by the meeting of the pharyngeal entoderm with the stomodeal ectoderm is known as the oral plate. The communication of the fore-gut with the outside is finally established by the breaking through of the oral plate. The formation of the mouth opening in the manner described does not take place at the extreme anterior end of the fore-gut. A small gut pocket extends cephalic to the mouth. ^ This so- called pre-oral gut rapidly becomes less conspicuous after the breaking through of the oral plate. The small depression which in older embryos marks its location is known as Sees- selFs pocket (Fig. 43). Even this small depression eventually disappears altogether. Its importance lies wholly in the fact that it indicates for some time the place at which ectoderm and entoderm originally became continuous in the formation of the oral opening. The Pharyngeal Derivatives. — Several structures arise in the pharyngeal region which do not become parts of the digestive STRUCTURE OF FOUR-DAY CHICKS 1 25 system. Nevertheless the origin of their epithelial portions from fore-gut entoderm and their early association with this part of the gut tract makes it convenient to take them up in connection with the digestive system. The thyroid gland arises as a median diverticulum from the floor of the pharynx which makes its appearance at the level of the second pair of pharyngeal pouches. Toward the end of the fourth day the thyroid evagination has become saccular and retains its connection with the pharynx only by a narrow open- ing at the root of the tongue known as the thyro-glossal duct (Fig. 43). In mammaha the thyroid is contributed to by pri- mordia which arise laterally from the fourth pharyngeal pouches as well as by a median evagination from the floor of the pharynx. It is possible that evaginations which in the chick arise from the fourth pharyngeal pouches are homologous with the lateral thyroid primordia of mammals. In the chick, how- ever, these evaginations do not form typical thyroid tissue. The thymus of the chick does not appear until after the fourth day of incubation. It takes its origin primarily from divertic- ula arising from the posterior faces of the third and fourth pharyngeal pouches. The original epithelial character of the thymus is soon largely lost in an extensive ingrowth of mesen- chyme and the organ becomes chiefly lymphoid in its histolog- ical characteristics. The Trachea. — The first indication of the formation of the respiratory system- is an outgrowth from the pharynx. In chicks of 3 days a mid- ventral groove is formed in the pharynx, beginning just posterior to the level of the fourth pharyngeal pouches and extending caudad. This groove deepens rapidly and by closure of its dorsal margins becomes separated from the pharynx except at its cephaUc end. The tube thus formed is the trachea, and the opening which persists between the cephal- ic end of the trachea and the pharynx is the glottis (Fig. 43). The original entodermal evagination gives rise only to the epithelial lining of the trachea, the supporting structures of the tracheal walls being derived from the surrounding mesenchyme. The Lung-buds. — The tracheal evagination grows caudad and bifurcates to form a pair of lung-buds. As the lung-buds develop they grow into the loose mesenchyme on either side of the mid-line. The adjacent splanchnic mesoderm is pushed 126 EARLY EMBRYOLOGY OF THE CHICK ahead of them in their caudo-lateral growth and comes to constitute the outer investment of the lung-buds. The ento- dermal buds give rise only to the epithehal Hning of the bronchi, and the air passages and air chambers of the lungs. The connective tissue stroma of the lungs is derived from mesen- chyme surrounding the lung-buds, and their pleural covering from the investment of splanchnic mesoderm. The Oesophagus and Stomach. — Immediately caudal to the glottis is a narrowed region of the fore-gut which becomes the oesophagus, and farther caudally a slightly dilated region which becomes the stomach (Fig. 43). The concentration of mesen- chyme cells about the entoderm of the oesophageal and stomach regions foreshadows the formation of their muscular and con- nective tissue coats (Fig. 46, C). The Liver. — In all vertebrates the Hver arises as a diverticu- lum from the ventral wall of the gut immediately caudal to the stomach region. In chick embryos the liver diverticulum appears just as the part of the gut from which it arises is acquiring a floor by the concrescence of the margins of the anterior intestinal portal. As a result the liver evagination appears for a short time on the Up of the intestinal portal, and grows cephalad toward the fork where the omphalomesenteric veins enter the sinus venosus. As closure of the gut floor is completed, the Kver diverticulum comes to lie in its character- istic position in the ventral wall of the gut. In embryos of four days the original evagination has grown out in the form of branching cords of cells and become quite extensive in mas^ (Fig. 43). In its growth the liver pushes ahead of it the splanchnic mesoderm which surrounds the gut, with the result that the hver from its first appearance is invested by mesoderm. (Fig.46,£). The proximal portion of the original evagination remains open to the intestine, and serves as the duct of the hver. This primitive duct later undergoes regional differentiation and gives rise in the adult to the common bile duct, to the hepatic and cys- tic ducts, and to the gall bladder. The cellular cords which bud off from the diverticulum become the secretory units of the liver (hepatic tubules). The same process of concrescence which closes the floor of the fore-gut involves the proximal portion of the omph3.Io- STRUCTURE OF FOUR-DAY CHICKS I27 mesenteric veins which, when they first appear, lie in the lateral folds of the anterior intestinal portal (Fig. 35). As the intes- tinal portal moves caudad in the lengthening of the fore-gut, the proximal portions of the omphalomesenteric veins are brought together in the mid-line and become fused. The fusion extends caudad nearly to the level of the yolk stalk (Fig. 47). Beyond this point they retain their original paired condition. In its growth the liver surrounds the fused portion of the om- phalomesenteric veins (Figs. 43 and 46, D, and E). This early association of the omphalomesenteric veins with the liver fore-shadows the way in which the proximal part of the afferent vitelline circulation is to be involved in the establishment of the hepatic-portal circulation of the adult. The Pancreas. — The pancreas is derived from evaginations appearing in the walls of the intestine at the same level as the liver diverticulum. There are three pancreatic buds, a median dorsal, and a pair of ventro-lateral buds. The dorsal evagina- tion appears at about 72 hours, the ventro-lateral evaginations toward the end of the fourth day. The dorsal pancreatic bud arises directly opposite the liver diverticulum and grows into the dorsal mesentery (Fig. 43). The ventro-lateral buds arise where the duct of the liver connects with the intestine so that the ducts of the liver and the ventral pancreatic ducts open into the intestine by a common duct (ductus choledochus). Later in development the masses of cellular cords derived from the three pancreatic primordia grow together and become fused into a single glandular mass, but usually two and in rare cases all three of the original ducts persist in the adult. The Mid-gut Region. — In chicks of four days the enteric tract shows no local differentiation from the level of the liver to the cloaca except where the yolk-sac is attached. All of the gut tract between the stomach and the yolk-stalk, and the anterior third of the gut lying caudal to the yolk-stalk is des- tined to become the small intestine. The posterior two-thirds of the hind-gut becomes large intestine and cloaca. The Cloaca. — The beginning of the formation of the cloaca is indicated in chicks of four days incubation, by a dilation of the posterior portion of the hind-gut (Fig. 43). Although ex- tensive differentiations in the cloacal region do not appear 128 ganglion VII-VIII auditoty vesicle EARLY EMBRYOLOGY OF THE CHICK ganglion V myelocoele branch of int. carotid a. anterior cardinal vem branch of ant. cardinal v. metacoele neuromere aortic arch 11 aortic arch "^\ ^V aortic arch IV ^^-^.^^ ^\^J— ^ dorsal ^^ ganglion ^.,.^^^—-5 neural /S^Sm notochord ^ ^F dorsal aorta/ / / ^"^m^ ant. cardinal vV y^ J \ visceral cleft III/ / \ B visceral arch WV \ visceral cleft pericardial region of coelom.^^ trachea^ \ ^^^ oesophagus j^^^^N^^j;^— ^kj neural i^^ tube— ^S ^^^^r 1 pharynx int. carotid a. ant. cardinal v. mesococle ■'?;^ dorul aorta' atnum bulbo-conus arteriosus pharyngeal pouch I mandibular arch 'hyomandibular cleft hyoid arch diocoele ventral body wall ' optic stalk sensory layer of retina pigment layer of retina olfactory pit bulbo-conus arteriosus sinus venosus right duct of Cuvier posterior cardinal v, lung bud pleural region Dof coelom left duct of Cuvier diocoele pericardial region of coelom Fig. 46 STRUCTURE OF FOUR-DAY CHICKS 129 ductus choledochus mesonephric duct dorsal mesentery dorsal ganglion neural tube omphaloniesenteric vein ventricle lateral telencephalic vesicle ectoderm of hea4 G dorsal ganglion post, cardinal v. dorsal aorta mesonephric duct allantoic vein vitelline vessels omphalomesenteric veins entoderm sub^ intestinal vein allantoic vein allantoic stalk allantoic art. coelom post, appendage Fig. 46. — Diagrams of transverse sections of a four-day chick. The location of the sections is indicated on a small outline sketch of the entire embryo. 130 EARLY EMBRYOLOGY OF THE CHICK until later in development, certain of its fundamental relation- ships are established at this stage. The cloaca of an adult bird is the common chamber into which the intestinal contents, the urine, and the products of the reproductive organs are received for discharge. The first appearance of the cloaca in the embryo as a dilated terminal portion of the gut establishes at the outset the relations of cloaca and intestine familiar in the adult. Although the urinary system is not at this stage developed to conditions which resemble those in the adult the. parts of it which have been estabhshed are already definitely associated with the cloaca. The proximal portion of the allantoic stalk which is the homologue of the urinary bladder of mammals opens directly into the cloaca (Fig. 43). When the urinary system of the embryo is considered, we shall see that the ducts which drain the developing excretory organs also open into the cloacal region on either side of the allantoic stalk. There is at this stage but little indication of the for- mation of the gonads. The relation of the sexual ducts to the cloaca can be made out only by the study of older embryos. The Proctodaeum and the Cloacal Membrane. — Indications of the formation of the cloacal opening to the outside appear during the fourth day of incubation. Its establishment is accomplished in much the same manner as the establishment of the oral opening. A ventral out-pocketing of the hind-gut arises just caudal to the point at which the allantoic stalk opens into the cloaca (Fig. 43) . At the same time a depression appears in the overlying ectoderm. The external depression which grows in toward the gut pocket is known as the procto- daeum. The double epithelial layer formed by the meeting of gut entoderm with proctodeal ectoderm is the cloacal mem- brane. The formation of the proctodaeum and the cloacal membrane cleaily indicate the location of the future cloacal opening although an open communication is not established by the rupture of the cloacal membrane until considerably later. The cloacal opening does not form at the extreme pos- terior end of the hind-gut and there is, therefore, a post-anal pocket of the hind-gut suggestive of the pre-oral pocket of the fore-gut. STRUCTURE OF FOUR-DAY CHICKS I3I V. The Circul-\tory System The Functional Significance of the Embryonic Circulation. The arrangement of the embnonic circulation is dimciilt to understand only when its functional significance is overlooked. In the embtyo as in the adult the main circulatory channels lead to and from the centers of metabohc acti\^ty. The circu- lating blood carries material from the organs of digestion and absorption to remote parts of the body; ox\'gen to all parts of the body from the organs which are specially constructed to take up oxygen from the surroimding medium; and waste materials from the places of their h*beration, to the organs through which they are eliminated. The differences between the course of the circulation in the embr\'o and in the adult are due to the fact that their centers of metaboUc activity are differently located. The organs which in the adult cany out such functions as digestion and absorption, respiration, and excretion are ex- tremely complex and highly differentiated structures. They are for this reason slow to attain their definitive condition and do not become functional until toward the close of embryonic life. Moreover the conditions by which the developing adult organs are surrounded during embryonic life are in some in- stances an absolute bar to their becoming functional were they sufficiently developed so to do. Suppose the lungs, for example, were fuUy formed at an early stage of development. The fact that the chick embr\^o is living submerged in the anmiotic fluid would render them as incapable of fxmctioning as the lungs of a man under water. Were the embrj'o dependent on the es- tablishment of the organs which carry on metabolism in the adult, development would be at an impasse. To develop, the embr>'o must have not only the raw food material suppHed it by the mother in the form of yolk, it must have a means of digesting the yolk, absorbing it, and canying it to the places where it can be utilized. The utilization of food material to produce the energy- expressed in growth processes depends on presence of ox\-gen. For growth there must be a means of securing oxygen and canying it, as weU as food, to all parts of the body. Xor can continued growth go on unless the waste products Hberated by the growing tissues are elinunated. At 132 EARLY EMBRYOLOGY OF THE CHICK the outset of its development the embryo must, therefore, establish organs for the digestion and absorption of food, the securing of oxygen, and the elimination of waste products. These organs serve the embryo but temporarily and are dif- ferent in structure and in location from the organs which carry out the corresponding functions in the adult, their nature and location depending on the exigencies of the embryo's living conditions. The main channels of the circulation in young embryos lead to and from their temporary organs of digestion and absorption, respiration, and excretion. The arrangement of the main vessels characteristic of the adult appears only as the organs characteristic of the adult develop. The changes by which the circulatory system acquires its adult arrangement are of neces- sity gradual. Any changes which were sufficiently abrupt to interfere with the circulation would result in disaster for the embryo. Even slight curtailment of the normal blood supply to any region would cause its growth to cease; any marked local decrease in the circulation would result in local atrophy or malformation; complete interruption of any important circula- tory channel, even for a short time, would inevitably mean the death of the embryo. Consequently the arrangement of vessels characteristic of the embryo persists during the forma- tion of the adult organs, and becomes altered only gradually as the adult organs and the vessels associated with them become ready to function. If the various circulatory channels of young chick embryos are considered in the light of their functions, the differences between the embryonic and the adult circulations should not be troublesome. The circulation of young chick embryos in- volves three main arcs of which the heart is the common center and pumping station. One of these circulatory arcs, the vitel- line, carries blood to the yolk-sac where food materials are absorbed and then returns the food-laden blood to the heart for distribution within the embryo. Another arc carries blood to and from the allantois. The distal portion of the allantois lies close beneath the egg shell and the blood circulating in the allantoic vessels is thereby brought into a location where inter- change of gases can be carried on with the air which penetrates the shell (Fig. 30, C and D). It is in the allantoic circulation STRUCTURE OF FOUR-DAY CHICKS 133 that the blood gives off its carbon dioxide and acquires a fresh supply of oxygen. The allantoic circulation is also the em- bryo's means of eliminating nitrogenous waste material from the blood. The remaining circulatory arc is confined to the body of the embryo. The intra-embryonic circulation has many distributing and collecting vessels but all of them are alike in function in that they bring food material to, and carry waste material from, the various parts of the developing body. Nowhere in their course are the vessels of the intra- embryonic circulation involved in adding food material or oxygen to that already contained in the blood they convey, and nowhere do they free the blood from waste materials until well along in development, when the nephroi become functional. In the heart the blood from the three circulatory arcs is mingled. As it leaves the heart the mixed blood is not as rich in food material as the blood coming in through the omphalo- mesenteric veins, nor as free from waste materials and as rich in oxygen as the blood returned over the allantoic veins. Its condition of serviceability to the embryo is, however, constantly maintained at a good average by the incoming viteUine and allantoic blood. There is a tendency among students who have done but little work on the circulation to regard any vessel which carries oxygenated blood as an artery," ailti any vessel which carries blood poor in oxygen and high in carbon dioxide content as a vein. This is not entirely correct even for the circulation of adult mammals on which the conception is based. In com- parative anatomy and especially in embryology it is far from being the case. It is necessary, therefore, in dealing with the circulation of the embryo to eradicate this not uncommon misconception. The differentiation between arteries and veins which holds good for all forms, both embryonic and adult, is based on the structure of their walls, and on the direction of their blood flow with reference to the heart. An artery is a vessel carrying blood away from the heart under a relatively high fluctuating pressure due to the pumping of the heart. Correlated with the pressure conditions in it, its walls are heavily reinforced by elastic and muscle tissue. A vein is a vessel carrying blood toward the heart under relatively low and constan 134 EARLY EMBRYOLOGY OF THE CHICK pressure from the blood welling into it from capillaries. Corre- lated with the pressure conditions characteristic for it, the walls of a vein have much less elastic and muscle tissue than artery walls, and more non-elastic fibers reinforcing them. The Vitelline Circulation. — The earHest indication of blood and blood vessel formation is at the chick's source of food supply. Blood islands appear in the extra-embryonic splanchnopleure pharyngeal pouches I -IV ant. cardinal v aortic arch IV aortic arch I ^disappearing j int carotid a. ext. carotid a. dorsal aorta . mesoncphros ^..Au*^ post, cardinal v hind-KUt ext. iliac artery cloaca — allantoic artery ^ proctodaeum OUMV\iA post -anal gut Pig. 47. — Schematic diagram to show the location of the more prominent internal organs of the four-day chick. Except for the omphalomesenteric arteries and veins paired structures are represented only on the side toward the observer. of the yolk-sac toward the end of the first day of incuba- tion, and rapidly become differentiated to form vascular endo- thehum enclosing central clusters of primitive blood corpuscles (Fig. 25). By extension and anastomosing of neighboring islands a plexus of blood channels is formed in the yolk-sac. Further extension of the vitelUne plexus brings it into communi- cation with the omphalomesenteric veins which have been de- veloped in the embryo as caudal extensions of the heart (Fig. 21). STRUCTURE OF FOUR-DAY CHICKS .135 Toward the end of the second day of development the om- phalomesenteric arteries establish communication between the dorsal aortae and the vitelHne plexus. (See Chap. X and Figs. 29 and 35.) There is now a system of open channels lead- ing from the embryo to the yolk-sac, and back again to the embryo. With the completion of these channels the heart begins to pulsate, circulation of the blood is thereby estabhshed, and the Pig. 48. — Diagram to show course of vitelline circulation in chick of about four days. (After Lillie.) For the intra-embryonic vessels see Fig. 47. Abbre- viations; A, dorsal aorta; A.V.V., anterior vitelline vein; L.V.V., lateral vitelline vein; M.V., marginal vein (sinus terminalis); P.V.V., posterior vitelline vein; V.A., vitelline artery. The direction of blood flow is indicated by arrows. blood cells formed in the yolk-sac are for the first time carried into the body of the embryo. The course of the vitelline circulation in chicks of four days is shown diagrammatically in Figures 47 and 48. Circulating Oi 136 EARLY EMBRYOLOGY OF THE CHICK in the rich plexus of small vessels on the yolk, the blood finally makes its way either directly into one or another of the larger vitelline veins, or to the sinus terminalis which acts as a collecting channel, and then over the sinus terminalis to one of the vitel- line veins. The vitelline veins converge toward the yolk-stalk where they empty into the omphalomesenteric veins. The omphalomesenteric veins at first paired throughout their entire length have been brought together proximally by the closure of the ventral body wall and become fused to form a median vessel within the body of the embryo. It is through this vessel that the vitelline blood eventually reaches the heart. In the heart the blood of the vitelline, intra-embryonic, and allantoic circulations is mingled. The mixed blood passes out by the ventral aorta and the aortic arches into the dorsal aorta. Leaving the dorsal aorta through the vitelline arteries the blood is returned to the yolk-sac. It should not be inferred that the blood stream ''picks up" deutoplasmic granules and carries them to the embryo. The acquisition of food material by the blood depends on the activ- j ities of the entodermal cells lining the yolk-sac. These cells secrete digestive enzymes which break down the deutoplasmic granules. The liquified material is then absorbed by the yolk- sac cells and transferred to the blood. The blood carries the food material in soluble form to the embryo where it is finally assimilated. The Allantoic Circulation. — The allantoic arteries arise by the prolongation and enlargement of the segmental vessels arising from the aorta at the level of the allantoic stalk. Their size increases rapidly as the allantois increases in extent. From them the blood is distributed in a rich plexus of vessels which ramify in the mesoderm of the allantois (Fig. 47). The situation of the allantois directly beneath the porous shell is such that the blood can carry on interchange of gases with the outside air (Fig. 30, D). It is in the rich plexus of small allantoic vessels where the surface exposure is very great that the blood gives off its carbon dioxide and takes up oxygen. At a later stage of development the ducts of the embryonic excretory organs open into the allantoic stalk near its cloacal end. As the excretory organs become functional the allantoic vesicle becomes the repository for the nitrogenous waste mate- STRUCTURE OF FOUR-DAY CHICKS 137 rials eliminated through them. The watery portion of the waste materials is passed off by evaporation. The remaining soHds are deposited in the allantoic vesicle. They accumulate in the extra-embryonic portion of the allantois and there remain until that portion of the allantois is discarded at the close of embryonic Hfe. The blood from the allantois is collected and returned to the heart over the allantoic veins. From the distal portion of the allantois the smaller veins converge and unite into two main vessels, right and left, which enter the body of the embryo with the allantoic stalk (Fig. 46, H). After their entrance into the body the allantoic veins extend cephalad in the lateral body walls (Figs. 47 and 46, H to D). They enter the sinus venosus on either side of the entrance of the omphalomesenteric vein. The Intra-embryonic Circulation. — The earUest vessels of the intra-embryonic circulation to appear are the large vessels communicating with the heart. In chicks of 33 hours the ventral aorta leads off from the heart cephalically and bifur- cates ventral to the pharynx giving rise to a single pair of aortic arches. The aortic arches pass dorsad around the antero- lateral walls of the pharynx and are continued caudally along the dorsal wall of the gut as the paired dorsal aortae (Fig. 23). When, toward the end of the second day of incubation, vis- ceral clefts and visceral arches appear, the original pair of aortic arches comes to lie in the mandibular arch. In each of the visceral arches posterior to the mandibular, new aortic arches are formed connecting the ventral aortae with the dorsal aortae. By 55 hours three pairs of aortic arches are present and a fourth is beginning to form (Fig. 35). At about this stage extensions of the dorsal aortic roots grow out anteriorly. The vessels thus derived extend cephalad in close association with the brain as the internal carotid arteries. In a later stage vessels arise from the ventral aortic roots and grow cephalad as the external carotid arteries (Fig. 47). By the end of the fourth day of incubation two more pairs of aortic arches have appeared posterior to the four formed in 55 to 60-hour chicks. From their first appearance the fifth aortic arches are very small and they soon disappear altogether. The first and second pairs of aortic arches have by this time suffered a great diminution in size which is indicative of their 138 EARLY EMBRYOLOGY OF THE CHICK final disappearance. In many embryos of this age the first arches, and in a few the second also, have disappeared alto- gether. This leaves only the third, fourth, and sixth pairs of aortic arches. These arches persist intact for some time, and parts of them remain permanently, being incorporated in the formation of the aortic arch and the main vessels arising from it, and in the roots of the pulmonary arteries. In reptiles, birds, and mammals the main adult vessels which connect the heart with the dorsal aorta are derived from the fourth pair of aortic arches of the embryo. The paired condi- tion of these arches persists as the adult condition in reptiles, but in birds and mammals one of the arches degenerates before the end of embryonic fife. In birds the left arch degenerates leaving the right one as the adult aortic arch; in mammals the right arch degenerates leaving the left as the aortic arch of the adult. The dorsal aortae, at first paired, later become fused to form a median vessel. The fusion begins at about the level of the sinus venosus and progresses cephalad and caudad (Fig. 35). Fusion extends cephalad but a short distance, never involving the region of the aortic arches. Caudally the aortae eventually become fused throughout their entire length. Early in development the aorta gives rise to a segmentally arranged series of small vessels which extend into the dorsal body wall. At the level of the anterior appendage buds a pair of the segmental arteries become enlarged and extend into the wing buds as the sub-clavian arteries. Coincident with the development of the allantois, segmental vessels opposite the allantoic stalk become enlarged and extend into it as the allan- toic arteries. The external iliac arteries to the posterior ap- pendage buds arise as branches of the allantoic arteries close to their origin from the aorta (Fig. 47) . The three main arteries which in the adult supply the ab- dominal viscera are represented in four-day chicks only by the omphalomesenteric arteries. The omphalomesenteric arteries arise as paired vessels (Fig. 35), but in the closure of the ventral body wall of the embryo they are brought together and fused to form a single vessel which runs in the mesentery from the aorta to the yolk-stalk (Fig. 47). With the atrophy of the yolk-sac the proximal part of the omphalo-mesenteric artery persists as STRUCTURE OF POUR-DAY CHICKS 1 39 the superior mesenteric of the adult. The coeliac and the inferior mesenteric arteries arise from the aorta independently at a later stage. The cardinal veins are the principal afferent systemic vessels of the early embryo. They appear toward the end of the second day as paired vessels extending anteriorly and posteriorly on either side of the mid-line. At the level of the heart the anterior and posterior cardinal veins of the same side of the body become confluent in the ducts of Cuvier and turn ventrad to enter the sinus venosus (Figs. 24 and 35) . Chicks of four days show little change in the relationships of the cardinal veins (Fig. 47). Later in development the proximal ends of the anterior cardinals become connected by the formation of a new transverse vessel and empty together into the venous atrium of the heart. Their distal portions remain in the adult as the principal afferent vessels (jugular veins) of the cephalic region. The posterior cardinals lie in the angle between the somites and the lateral mesoderm (Fig. 36, D, E). When the mesone- phroi develop from the intermediate mesoderm, the cardinal veins lie just dorsal to them throughout their length (Figs. 52, C and 46, E to H). In young embryos the posterior cardinals are the main afferent vessels of the posterior part of the body. Later in development they are replaced by a new vesssel, the inferior vena cava. The changes by which posterior cardinals become reduced and broken up to form small vessels with new associations, belong to stages of development beyond the scope of this book. The Heart. — The heart in adult vertebrates is a ventral unpaired structure. Its origin in the chick from paired primor- dia is correlated with the way the young embryo lies spread out on the yolk surface. When the ventral body wall is completed by the folding together of layers which formerly extended to right and left over the yolk, the paired primordia of the heart are brought together in the mid-Hne. Their fusion establishes the heart as an unpaired structure lying in the characteristic ventral position (see Chap. IX and Figs. 26 and 27). After the fusion of its paired primordia the heart is a nearly straight, double-walled tube (Figs. 49, A and 19). The primor- dial endocardium of the heart has the same structure and arises in the same manner as the endothelial walls of the primitive 140 EARLY EMBRYOLOGY OF THE CHICK embryonic blood vessels with which it is directly continuous. The epi-myocardial layer of the heart is an outer investment which surrounds and reinforces the endocardial wall. As development progresses the epi-myocardium becomes greatly thickened and is finally differentiated into two layers, a heavy muscular layer, the myocardium, and a thin non-muscular covering layer, the epicardium. In the apposition of the paired primordia of the heart to each other the splanchnic mesodeim from either side of the body comes together dorsal and ventral to the heart. The double- layered supporting membranes thus formed are known as the dorsal mesocardium and the ventral mesocardium, respectively (Fig. 26). The ventral mesocardium disappears shortly after its formation, leaving the heart suspended in the body cavity by the dorsal mesocardium (Fig. 26 E, D). Somewhat later the dorsal mesocardium also disappears except at the caudal end of the heart. Thus the heart comes to lie in the pericardial cavity unattached except at its two ends. The cephalic end of the heart remains fixed with reference to the body of the embryo where the ventral aorta lies embedded ventral to the floor of the pharynx, and the caudal end of the heart is fixed by the persistent portion of the dorsal mesocardium and the omphalomesenteric veins. The straight tubular condition of the heart persists but a short time. The unattached ventricular region becomes dilated and is bent out of the mid-line toward the embryo's right while the fiLxed bulbo-conus arteriosus and the sinus venosus are held in their original median position (Fig. 49, A-E). This bending of the heart to form a U-shaped tube begins to be apparent in embryos of 30 hours and becomes rapidly more conspicuous, until by forty hours the ventricular region of the heart lies well to the right of the embryo's body (Cf. Figs. 21 and 22). The bending of the heart to the side involves a considerable factor of ''mechanical expediency." The initiation of the bending process depends on the fact that the heart is becoming elongated more rapidly than is the chamber in which it lies fixed by its two ends. The fact that the bending takes place to the side rather than dorsally or ventrally may be attributed to STRUCTURE OF FOUR-DAY CHICKS 141 the impediment offered to its dorsal bending by the body of the embryo, and to its ventral bending by the yolk. The lateral bending of the heart attains its greatest extent at about 40 hours of incubation. At this stage torsion of the body of the embryo changes the mechanical limitations in the heart region. As the embryo comes to lie on its left side the heart is no longer pressed against the yolk (Cf. Figs. 21 and 29). As a result the bend begins to swing somewhat ventrad and Hes less closely against the body of the embryo (Figs. 49 and 50, At about this stage of development a new factor affects the changes in the shape of the heart. The closed part of the U-shaped bend is forced caudad and at the same time becomes twisted on itself to form a loop (Figs. 49, F-I and 50, F-I). In the formation of the loop the atrial region is forced sHghtly to the left {i.e., toward the yolk) and the conus is thrown across the atrial region by being bent to the right {i.e., away from the yolk) and then caudad. The ventricular region constitutes the closed end of the loop. This twisting process reverses the original cephalo-caudal relations of the atrial and ventricular regions. The atrial region which was at first caudal to the ventricle now lies cephalic to it as in the adult heart. The atrial region and the ventricular region which formerly were continuous without any line of demarcation, are by this time beginning to be marked off from each other by a constriction (Fig. 49, /, a.v.). As both the atrium and the ventricle be- come enlarged, this constriction is accentuated (Fig. 49, L, a. v.). The constricted region is now termed the atrio-ventricular canal. During the fourth day the bulbo-conus arteriosus becomes closely applied to the ventral surface of the atrium. As the atrium grows it tends to expand on either side of the depression made in it by the pressure of the bulbo-conus (Figs. 49, J-L and 50 J-L). These lateral expansions of the atrium are the first indication of the division of the atrium into right and left chambers which are later completely separated from each other. At the same time a sHght longitudinal groove appears in the surface of the ventricle (Fig. 49, L, i.v.) which indicates the beginning of the separation of the ventricle into right and left chambers. The division of the bulbo-conus to form the 142 EARLY EMBRYOLOGY OF THE CHICK root of the adrta and the pulmonary artery does not appear until a later stage of development. During the changes in the external shape of the heart which have been described, the whole heart has come to occupy a more caudal position with reference to other structures in the M lomitc* Pig. 49. — Ventral views of the heart at various stages to show its changes of shape and its regional differentiation. All the drawings were made from dissections with the aid of camera lucida outlines. The outer of the two layers shown is the epi-myocardium ; the inner, the endocardium. In the stages repre- sented in Figs. E-H torsion of the embryo's body is going on at the level of the heart. Since torsion involves the more cephalic regions first and progresses caudad the transverse axis of the body of the embryo is at different inclinations to the yolk at the cephalic end and at the caudal end of the heart. In drawing these figures their orientation was taken from the body at the level of the conus region of the heart, the sinus region therefore appears inclined. Abbreviations: a.v., constriction between atrium and ventricle; i.v., interventricular groove. embryo. When the heart is first formed it lies at the level of the rhombencephalon. As development progresses it moves STRUCTURE OF FOUR-DAY CHICKS 143 farther and farther caudad until at the end of the fourth day it Hes at the level of the anterior appendage buds. Being un- attached to the body, the ventricular region of the heart is carried farthest caudad (Cf. Figs. 19, 29, 34, and 40). The changes which take place in the heart wall can be seen best in sections. The endocardium in the heart of a four-day D38t 16 I K 76 HOVtS 3t tomitct Fig. 50. — Dextral views of the same series of hearts shown in ventral view in Pig. 49. The heart drawings in Figs. 49 and 50 should be compared with actual specimens or with drawings of entire embryos of corresponding age for the relation of the heart to the body of the embryo. chick is still a single cell layer lining the lumen. The original epi-myocardium at this stage can be differentiated into an inner myocardial portion and an outer epicardial portion. The myocardium has become greatly thickened and the cells in it are elongated and beginning to show the histological character- 144 EARLY EMBRYOLOGY OF THE CHICK istics of developing muscle cells. Their arrangement in bun- dles which project toward the lumen fore-shadows the formation of the muscle bands (trabeculae carneae) which ridge the inner wall of the adult heart. The cells of the epicardial portion of tlie epi-myocardium are becoming flattened to form the epi- thelial and connective tissue covering of the heart (epicardium) . Lying between the endocardium and the myocardium in the region of the atrio- ventricular canal and of the opening of the ventricle into the bulbo-conus, there are loosely aggregated cells which are mesenchymal in characteristics. These cells constitute what is called endocardial cushion tissue. They later take part in the formation of the septa which divide the heart into chambers and in the formation of the connective tissue frame-work of the cardiac valves. VI. The Urinary System The General Relationships of Pronephros, Mesonephros and Metanephros. — In the development of the urinary system of birds and mammals there are formed in succession three dis- tinct excretory organs, pronephros, mesonephros, and meta- nephros. The pronephros is the most anterior of the three, and the first to be formed. It is wholly vestigial, appearing only as a slurred-over recapitulation of structural conditions which exist in the adults of the most primitive of the vertebrate stock. The mesonephros is homologous with the adult excre- tory organs of fishes and amphibia. It makes its appearance in the embryo somewhat later than the pronephros, and is formed caudal to it. The mesonephros is the principal organ of excretion during early embryonic life, but it also disappears in the adult except for parts of its duct system which become associated with the reproductive organs. The metanephros is the most caudally located of the excretory organs, and the last to appear. It becomes functional toward the end of em- bryonic life when the mesonephros is disappearing, and per- sists permanently as the functional kidney of the adult. Figure 51 shows schematically some of the main steps in the embryological history of the nephric organs, which it will be helpful to have in mind before taking up in detail any of the phases of their formation in the chick. The pronephros, meso- nephros and metanephros are all derived from the intermediate STRUCTURE OF FOUR-DAY CHICKS 145 mesoderm, and are all composed of units which are tubular in nature. In the different nephroi these tubules vary in struc- tural detail but their functional significance is in all cases much the same. They are concerned in collecting waste materials from the capillary plexuses which are developed in connection with them. In the accompanying diagrams conventionahzed fr- pronephric tubules pronephric tubules (degenerating) ^^ mesonephric 1?? pronephric tubules with l/^j^ duct nephrostomes /'^^^ — mesonephric tubules mesonephric tubules without nephrostom i mesonephric duct I li mesonephric •=:> i j] tubules ^"nv- - ; and duct degenerating mesonephric duct metanephric duct cloaca Fig. 51. — Schematic diagrams to show the relations of pronephros, meso- nephros, and metanephros at various stages of development. For explanation see text. tubules have been drawn to represent the three nephric organs. No pretense is made of representing either the exact shape or the actual number of the tubules. In the first stage represented (Fig. 51, ^) only the pronephros has been established. It consists of a group of tubules empty- ing into a common duct, called the pronephric duct. The pro- 10 146 EARLY EMBRYOLOGY OF THE CHICK nephric ducts of either side are formed first at the level of the pronephric tubules and then extend caudad, eventually reach- ing and opening into the cloaca (See arrows in Fig. 51, A). As the pronephric ducts are extended caudal to the level at which pronephric tubules are formed they come in close prox- imity to the developing mesonephric tubules. In their growth the mesonephric tubules extend toward the pronephric ducts and soon open into them (Fig. 51, B). Meanwhile the pro- nephric tubules begin to degenerate. Thus the ducts which originally arose in connection with the pronephros are appro- priated by the developing mesonephros. After the degenera- tion of the pronephric tubules these same ducts are called the mesonephric ducts because of their new associations (Fig. 51, C). At a considerably later stage outgrowths develop from the mesonephric ducts near their cloacal ends (Fig. 51, C). These outgrowths form the ducts of the metanephroi. They grow cephalo-laterad and eveiitually connect with the third group of tubules developed from the intermediate mesoderm, the metanephric tubules (Fig. 5 1 , Z>) . With the establishment of the metanephroi or permanent kidneys the mesonephroi begin to degenerate. The only parts of the mesonephric system to persist, except in vestigial form, are some of the ducts and tubules which in the male are appropriated by the testis as a duct system. The Pronephric Tubules of the Chick. — The pronephros in the chick is represented by tubules which first appear at about 36 hours of incubation. The pronephric tubules arise from the intermediate mesoderm, or nephrotome, lateral to the somites. They are paired, segmen tally arranged structures, a tubule appearing on either side opposite each somite from the fifth to the sixteenth. Transverse sections passing through the loth to 14th somites of an embryo of about 38 hours show the proneph- ric tubules favorably. Each tubule arises as a solid bud of cells organized from the intermediate mesoderm near its junction with the lateral mesoderm (Fig. 52, ^). At first the free ends of the buds grow dorsad, passing close to the posterior cardinal veins. Later the end of each tubule is bent caudad coming in contact with the tubule lying posterior to it. In this manner the distal ends of the tubules give rise to a continuous cord of cells, the primordium of the pronephric duct. The pair of cell STRUCTURE OF FOUR-DAY CHICKS 147 cords thus formed continue to extend caudad beyond the pronephric tubules and soon become hollowed out to form open ducts. When they eventually reach the level of the cloaca they turn ventrad and open into it. The significance of the rudimentary structures in the chick which represent pronephric tubules, can be most readily understood by comparing them with fully developed and func- tional pronephric tubules. Figure 52, B, shows the scheme of ntermediate mesoderm dorsal aorta ^ coelom , notochord X somite /^ dorsal aorta v/^ post, cardinal ^^K ^ ^ vein k^\/^ ^ mesonephric W^^^^ duct ^^^ mesonephric PP- S5-IOO. Extra-embryonic Membranes Danchakoff, Vera, 191 7. The Position of the Respiratory Vascular Net in the Allantois of the Chick. Am. Jour. Anat., Vol. 21, pp. 407-420. Duval, M., 1884. Etudes histologiques et morphologiques sur les annexes des embryoAS d'oiseaux. Jour, de I'anat. et de la phys., T. XX. Lillie, F. R., 1903. Experimental Studies on the Development of the Organs in the Embryo of the Fowl (Gallus domesticus). i. Experiments on the Amnion and the Production of Anamiote Embryos of the Chick. Biol. Bull., Vol. V, pp. 92-124. Popoff, D., 1894. Die Dottersackgefasse des Huhnes. Wiesbaden. Shore, T. W., and Pickering, J. W., 1889. The Proamnion and Amnion in the Chick. Jour, of Anat. and Phys., Vol. XXIV, pp. 1-2 1. Stuart, T. P. A., 1899. A Mode of Demonstrating the Developing Membranes in the Chick. Jour. Anat. and Phys., Vol. XXV, pp. 299-300. l6o EARLY EMBRYOLOGY OF THE CHICK The Ductless Glands Atwell, W. J., and Si tier, Ida, 191 8. The Early Appearance of the Anlagen of the Pars Tuberalis in the Hypophysis of the Chick. Anat. Rec, Vol. 15, pp. 181-187. Poll, H., 1906. Die vergleichende Entwickelungsgeschichte der Nebennieren systeme der Wlrbeltiere. Hertwig, O., Handbuch der Vergleichenden und Experimentellen Entwickelungslehre der Wirbeltiere. (Edited by Hertwig, written by numerous collaborators.) Fischer, Jena. Bd. Ill, Teil i, K. II, 2. Soulie, A. H., 1903. Recherches sur le d^veloppement des capsules surrenales chez les vert6br6s superi^urs. Jour, de I'anat. et physiol., T. XXXIX, pp. 197-293. Verdun, M. P., 1898. Sur les d^riv^s branchiaux du poulet. Comptes rendus See. Biol., Tom. V. Anomalies Alsop, Florence M., 1919. The Effect of Abnormal Temperatures upon the Developing Nervous System in the Chick Embryos. Anat. Rec, Vol. 15, pp. 307-323- Glaser, O., 1913. On the Origin of Double-yolked Eggs. Biol. Bull., Vol, 24, pp. 175-186. Mitchell, P. C, 1891. On a Double-chick Embryo. Jour, of Anat. and Physiol., Vol. 25, pp. 316-324. Pohlman, A. G., 1920. A Consideration of the Branchial Arcades in Chick Based on the Anomalous Persistence of the Fourth Left Arch in a Sixteen-day Stage. Anat. Rec, Vol. 18, pp. 159-166. O'Donoghue, C. H., 1910. Three Examples of Duplicity in Chick Embryos with a Case of Ovum in Ovo. Anat. Anz., Bd. 37, pp. 530-536. Stockard, Charles R., 1914. The Artificial Production of Eye Abnormalities in the Chicken Embryo. Anat. Rec, Vol. 8, pp. 33-42, Tannreuther, G. W., 1919. Partial and Complete Duplicity in Chick Em- bryos. Anat. Rec, Vol. 16, pp. 355-367. INDEX To facilitate the use of this book in connection with others in which the termi- nology may dififer somewhat, many synonyms which were not used in the text have been put into the index and cross-referenced to the alternative terms used in this book. For example, WolflSan body, a term not used in this text, is fre- quently applied to the mesonephros. It appears in the index thus: Wolflfian body (= mesonephros, q.v.). Both figure and page references are given in the index. The figure references are preceded by the letter f . Accessory cleavage, 19 Accessory coverings of ovum, f. 3, 10 Acoustico-facialis ganglion ( = gang- lion complex of VII and VIII cranial nerves) f. 40, 118 Acoustic ganglion, f. 42, 118, 123 Air space, f. 3, 12 Albumen, f. 3, 10 Albumen-sac, f. 30, f. 32, 84, 87 Alecithal ovum (see isolecithal). Allantoic, circulation (see circulation). diverticulum, f. $$, 90 stalk, f. 33, f. 43, 90 vesicle, f. 30, f. 32, f. 33, f. 40, 90, 113 Allan tois, fate of, 137 formation of, f. 33, 90 function of, 90, 137 relations of, f. 30, f. 32 Amnion, formation, f. 30, f. 32, 86, 87 fuaiction of, 86 muscle fibers of, 86 relations of, f. 30, f. 32 Amnion, false, 92 Amnio-cardiac vesicles, 49 Amniotic, cavity, f. 30, f. 32, 87 fluid, 86 folds, f. 30, f. 32, 87 raphe, 87 Anal plate (see cloacal membrane). Animal pole, 8 Anterior horns of mesoderm, f . 1 2 Anterior intestinal portal, f. 16, f. 17, f. 31, 46, 57, 69 Anterior neuropore, f. 19, 55, 99 Aortas dorsal, formation of, 73 fusion of, 105, 138 position of, f. 23, f. 24, f. 35, f. 47 Aorta, ventral, f. 23, f. 24, f. 35, f. 47, f. 73,- los, 137 Aortic arches, fate of, 138 formation of, 105 position of, f. 24, f. 35, f. 47 Aortic roots, dorsal, f. 34, f. 47, 137 ventral, f. 23, f. 35, f. 47, 72, 137 Appendage buds, anterior, f. 39, f. 40, 112 posterior, f. 39, f. 40, 112 Aqueduct of Sylvius, f. 42, 117 Area opaca, f. 11, f, 13, 24, 36 vasculosa, f. 15, f. 17, 51 vitellina, f. 15, f. 17, 51 Area pellucida, f. 11, f. 13, 24 Area vasculosa, 51, 58 Arteries, allantoic, f. 47, 138 aortic (see aorta) carotid, ext. f. 47, 137 carotid, int. f. 47, 137 coeliac, 139 definition of, 133 iliac, f. 47, 138 mesenteric, 139 omphalomesenteric, f. 29, f. 47, 78, los, 138 pulmonary, 138 segmental, 138 sub-clavian, 138 vitelline, f. 48, 135 Atrium, f. 23, f. 49, f. 50, 72, 104, 141 Atrio-ventricular constriction, f. 49, 141 11 161 l62 INDEX Auditory, ganglion (see acoustic), nerve, 123 pit, f. 22, 65 placode, 65, 122 vesicle, f. 36, f. 40, 65, 122 Bile duct, common, 126 Blastocoele, f. 6, 21, 23 Blastoderm, f. 6, 20, 24 zones of, f. 7, 24 Blastodisc, 16 Blastomere, 16 Blastopore, f. 6, 22 closure of, 26 concrescence of, f. 9, 28 formation of, in birds, f . 7, 26 homologies of, 23 Blastula, 20, 21, 24 Blood, as a carrier of food, 79, 132 oxygenation of, 78, 133 Blood cells, origin of, f. 25, 66 Blood islands, differentiation of, f. 25, 66,67 formation of, f , 25, 51 location of, f. 15, f. 17 Blood-vessels, formation of, f. 25, 66, 72 (see also arteries and veins). Body cavity (see coelom) , Body folds, f. 30, f. 32, 80 Bowman's capsule, 148 Brain, first differentiation of, 53 neuromeric structure of, 59 primary vesicles, f . 20, 54, 60 secondary vesicles, f. 42, 63, 114 ventricles of, f. 42, 115 Branchial arches (see visceral arches). Bulbo-conus arteriosus, f . 23 , f . 49, f. 50, 72, 141 Bulbus arteriosus (see bulbo-conus). Capsule of Bowman, 148 Caudad, usage of term, 5 Caudal, usage of term, 5 Caudal fold, f. 31, 81 Caudal flexure, 1 1 1 Central canal of spinal cord, 119 Cephalad, usage of term, 5 Cephalic, usage of term, 5 Cephalic limiting fold, 80 Cephalic mesoderm, 40, 50 Cephalic neural crest, f. 22, loi Cerebellar peduncles, 118 Cerebellum, 118 Cerebral ganglia (see ganglia, cranial). Cerebral hemispheres, 115 Cervical flexure, 94, iii Chalaza, f. 3, 10 Chorion, 92 Choroid coat of eye, 122 Choroid fissure of eye, f. 35, f. 42, 98, 121 Choroid plexus, 117, 118 Circulation, allantoic, f. 47, 136 course of embryonic, 78, 132 establishment of, 78 intra-embryonic, f. 47, 137 significance of embryonic, 131 vitelline, f. 48, 68, 77, 134 Cleavage, accessory, 19 discoidal, f. 5, 16 holoblastic, f. 4, 16 meroblastic, f. 4, 16 process of, in birds, f. 5, 16 Cleavage cavity (see blastocoele). Cloaca, f. 31, f. 43» 130 Cloacal membrane, f. 31, 130 Cloacal opening, 130 Coelom, divisions of embryonic, 150 extra-and intra-embryonic, f. 28, f. 30, f. 32, 49, 151 formation of, f, 54, 49> 150 pericardial region of, f. 16, f. 24, f. 26, f. 27, 49» 72, ISO Concrescence, of blastopore, f. 9, 28 of anterior intestinal portal, 69 Conus arteriosus (see bulbo-conus). Conjunctival epithelium, 122 Cornea, 122 Corpora quadrigemina, 117 Corpus vitreum (see vitreous body). Cranial flexure, 75, 11 1 Crura cerebri, 117 Cutis plate (see dermatome). Cystic duct, 126 Deutoplasm, 7 effect of on cleavage, f . 4, 14 effect of on gastrulation, f. 6, 21 Dermatome, f. 38, f. 44, 107 Diencephalon, f. 42, 65, 116 Diocoele (= lumen of diencephalon, q. v.). Dio-mesencephalic boundary, f. 42, 117 INDEX 163 Dio-telencephalic boundary, f. 42, 115 Discoidal cleavage (see cleavage). Dorsad, usage of term, 5 Dorsal aorta (see aorta). Dorsal flexure, in Dorsal mesentery, f. 54, f. 55, 152 Dorsal mesocardium, f. 26, 69, 71, 140, 152 Dorsal nerve roots, f. 44, 119 Dorsal pancreatic bud, 127 Dorsal root ganglia, f. 44, 119 Dorsal, usage of term, 5 Duct of Cuvier (= common cardinal vein, q. v.). Ductus arteriosus (part of aortic arch VI). Ductus choledochus, f. 46 E., 127 Ductus endo-lymphaticus, f. 40, 122 Ductus venosus (= fused portion of omphalomesenteric vein, q. v.). Ear, 122 Ectoderm, derivatives of, 31 establishment of, 23 Egg, membranes, f. 3, 10 ovarian, f. i, 7 shell, 10, 12 structure of at lajdng, f. 3, 11 Embryo, external form of, 93, 109 separation of from blastoderm, f. 30, f. 32, 80 Embryonal area, 42 Embryonic circulation (see circula- tion). Endocardial cushion tissue, f. 46 D, 144 Endocardial primordia, f. 26, f. 27, 69 Endocardium, 143 Endolymphatic duct, f. 40, 122 Entoderm, derivatives of, 32 establishment of, 20, 23 Endothelium, origin of vascular, f. 25, 66 Epicardium, 69, 143 Epichordal portion of brain, 55 Epimyocardium, fate of, 140, 144 formation of, f. 26, f. 27, 69 Epiphysis, f. 35, f. 42, 95, 116 Eustachian tube, 103, 123 Extra-embryonic ccelom (see coelom). Extra-embryonic membranes, f. 30, f. 32, Chap. XI Extra-embryonic vascular plexus (see vitelline circulation and blood-vessels, origin of). Eye, 120 Facial region, f. 41. in Facial nerve (= cranial nerve VII), 118 Falciform ligament, 153 Fertilization, 9 Flexion, 75, no Floor plate of spinal cord, 119 Foramen of Monro, f. 42, 114 Follicle, ovarian, f. i, 7 Fore-brain (see prosencephalon). Fore-gut (see gut). Fovea cardiaca (= anterior intestinal portal q. v.). Frontal process, f. 41 Gall bladder, 126 Gametes, 7 Ganglia, cranial, f. 42, 118 dorsal root (see spinal). spinal, f. 44, 119 sympathetic, f. 44, 120 Ganglion jugulare (= ganglion of cranial nerve X.) f. 42, 118 Gasserian ganglion (= ganglion of cranial nerve V) f. 40, 118 Gastrocoele, f, 6, f. 7, 22, 26 Gastro-hepatic omentum, 153 Gastrulation, Chap. IV effect of yolk on, f. 6, 21 in Amphioxus, 22 in Amphibia, 23 in birds, f. 7, 24 Geniculate ganglion (= ganglion of cranial nerve VII); f.42, 118 Germ cells (see gametes). Germ layers (see ectoderm, entoderm and mesoderm). Germinal disc (see blastodisc). Germinal epithelium of ovary, f, i Germinal vesicle ( = nucleus of ovum, q. v.). Germ wall, 24 Gill arches (see visceral arches). Glomerulus, f. 52, f. 53, 148 Glomus, f. 52 Glossopharyngeal nerve (= cranial nerve IX), f. 42, 118 164 INDEX ' Glottis, 125 Granular zone of follicle, 8 Gut, delimitation of embryonic, 81 fore-, f. 17, f. 31, 46, 57, 84, loi hind-, f. 31, 84, 102, 130 mid-, f. 31, 84, 102, 127 pre-oral, f. 31, 102, 124 primitive, f. 13, f. 31, 36 post-anal, f. 31, 130 Head fold, 43, 80 Head fold of anmion, f. 29, 86 Head process (see notochord). Heart, differentiation of, f. 49, f. 50, 104, 139 establishment of f. 26, f. 27, 57, 68 primordia of, 50, 71 Heart-beat, 72 Hensen's Node, f. 8, f. 11, f. 13, 28 Hepatic duct, 126 Hepatic-portal circulation, 127 Hepatic tubules, 126 Hind-brain (see rhombencephalon). Hind-gut (see gut). Holoblastic cleavage (see cleavage). Homolecithal ova ( = isolecithal, q. v.) . Hyoid arch, f. 39, f. 41, 103 Hyomandibular cleft, f. 34, 103, 123 Hypophysis, 95, 117 Incubation, 12 Infundibulum, f. 35, f. 42, f. 43, 63, 95, 116 Intermediate mesoderm (see meso- derm). • Internal ear, 123 Interventricular sulcus, f. 49, 141 Intestine, 127 Intra-embryonic ccelom (see coelom). Invagination of entoderm (see gastru- lation). Isolecithal ova, 14 Jugular vein (see vein, anterior cardi- nal). Kidney (see metanephros). Lamina terminalis, f. 42, 114 Latebra, f. 3, 12 Lateral body folds, f. 30, 80 Lateral limiting sulci (= lateral body folds, q. V.) Lateral mesoderm (see mesoderm). Lateral plate of spinal cord, 119 Lateral telencephalic vesicles (see telencephalon). Lateral wings or horns of mesoderm, f. 12, 37 Lens, differentiation of, f. 45, 121 fibers, 122 origin of, 98 vesicle, f. 36, 98 Liver, f. 43, f. 46, 126 Lung buds, f. 46, 125 Mandibular arch, f. 36, f. 4I; 103, 112 Mandible, 112 Marginal notch, f. 9 Margin of overgrowth, f. 7, 24 Maturation of gametes, 9 Maxilla, 112 Maxillary process, f. 41, 112 Meatus venosus (= ductus venosus, q. v.). Medulla, ji8 Medullary plate (= neural plate, q. v.). Meroblastic cleavage (see cleavage). Mesencephalon, f. 42, 54, 65, 117 Mesenchyme, 50 Mesenteries, dorsal, f. 54, f. 55, 152 formation of, 150 ventral, f. 54, f.s 5,15 2 Mesoblast (= mesoderm, q. v.). Mesocardium, dorsal, f. 26, 69, 71, 140, 152 ventral, f. 26, 69, 140, 152 Mesocolon, 153 Mesocoele ( = lumen of mesencepha- lon, q. v.). Mesoderm, derivatives of, 32 differentiation of, 37 dorsal, f. 17, f. 29, f. 54, 38, 47 early growth of, f. 12, 37 formation of, f. 10, 30 intermediate, f. 28, f. 54, 47, 144 of the head, 40, 50 regional divisions of, 47 segmental zone of, 40 somatic layer of, f. 28, f. 54, 49, 150 somites of, f. 38, 47, 56, 105 splanchnic layer of, f. 28, f. 54, 49, 66, 150, 152 INDEX i6s Mesodermic somites (see mesoderm). Meso-diencephalic boundary, f. 42, 117 Mesogaster, 153 Meso-metencephalic boundary, f. 42, 117 Mesonephric duct, f. 51, f. 52, f. 53, 146, 149 Mesonephric tubules, f. 51, f. 52, f. 53, 146, 148 Mesonephros, f. 47, 144 Mesothelium (= epithelial layer of mesoderm lining coelom) f . 54 Metamerism, in mesoderm, 40, 47, 48, 150 in nervous system, f . 20, 59 Metanephros, f. 51, 144 Metacoele ( = lumen of metencephalon q.v.) Metencephalon, f. 42, 65, 117 Metanephric duct, f. 51, 146 Metanephric tubules, f. 51, 146 Mid -brain (see mesencephalon). Middle ear, 123 Mid-gut (see gut). Morula, 20, 21 Mouth opening, 112 Muscle plate (see myotome). Myelencephalic tela (= thin roof of myelencephalon) f. 42, 118 Myelencephalon, f. 42, 65, 118 Myeloccele (= lumen of myelen- cephalon q. v.). Myelo-metencephalic boundary, f, 42, 117 Myocardium, 69, 143 Myocoele, 107 Myotome, f. 38, f. 44, 107 Nasal pit (see olfactory pit). Naso-lateral process, f. 41, 112 Naso-medial process, f. 41, 112 Naso-optic groove, f. 41 Neck of latebra, f . 3 Nephric tubules, f. 51, 145 Nephrostome, f. 52, 147 Nephrotomic plate, 48 Nerves, cranial, 118 spinal, f. 44, 119 sympathetic, t2o Neural cagial ( = lumen of neural tube). Neural crest, f. 37, 99, 120 Neural fold, f. 17, 42, 45, 99 Neural groove, f. 17, 42, 44 Neural plate, f. 11, f. 13, 41 Neural tube, 52, 99 Neurenteric canal, 56 Neuromeres, f. 20, 59 Neuropore, anterior, f. 19, 55, 99 posterior, 56 Notochord, f. 11, f. 13, 40, 55 Nucleus of Pander, f. 3, 12 (Esophagus, f. 43, loi, 126 Olfactory nerve (= cranial nerve I), 123 Olfactory pit, f. 40, f. 41, f. 46, 112, 123 Optic chiasma, f . 42 Optic cup, f. 42, 95, 121 Optic lobes, 117 Optic nerve (= cranial nerve II) 98, 122 Optic stalk, f. 45, 98, 122 Optic vesicle, primary, f. 23, f. 28, 54, 62,9s secondary, f. 36, 97, 120 Opticoele (= lumen of primary optic vesicle, q. v.). Oral cavity, 102 Oral opening, 1 24 Oral plate, f. 31, 124 Oral region, f, 41, in Orientation of embryo within egg, f . 30 Otocyst (see auditory vesicle). Ovum, fertilization of. 9 maturation of, 9 ovarian, f. i, 7 Ovulation, 9 Pancreas, f. 43, 127 Pander's nucleus, f. 3, 12 Pellucid area (see area pellucida). Petrosal ganglion (= gangh'on of cranial, nerve IX) f. 42, 118 Periblast, 10 Pericardial region of coelom, f. 24^ f. 27, f. 55, 49, 1^^ 150 Peritoneal region of coelom, 150 Pharyngeal pouches, f. 36, 103 Pharyngeal derivatives, 124 Phar)mx, f. 35, loi Pigment layer of retina, f. 45, 96, 122 Pineal gland, 95 Pituitary body, 95 z66 INDEX Placodes, auditory^ 65, 122 lens, 98 Pleural region of coelom, f. 46D, 150 Plica encephali ventralis (= ventral cephalic fold) f. 42 Pocket, subcaudal, f. 31, 81 subcephalic, f. 31, 47 Rathke'sf. 35, f. 43, 95, 117 Seessell's, f. 43, 102, 124 Polar bodies, 9 Polyspermy, 10 Pons, 118 Post-anal gut (see gut). Posterior appendage bud, f. 39, f. 40, 112 Posterior commissure, f . 42 Posterior intestinal portal, f . 3 1 Posterior neuropore, 56 Post-oral arches, 103 Post-oral clefts, 103 Prechordal portion of brain, 55 Pre-oral gut (see gut). Primitive groove, f. 13, 31 Primitive gut (see gut). Primitive node (= Hensen's node, q. v.). Primitive pit, f. 13, 28 Primitive plate, f. 29 Primitive ridge or fold, f. 13, 28 Primitive streak, as growth center. 33 fate of, 56 formation of, f. 9, f. 10, 28 interpretation of, f. 9, f. 10, 28, 35 location of, f. 8, f. 11, 27 Primordial follicle ( = very young ovarian follicle) f. i Proamnion, f. 12, 37 Proctodaeum, f. 31, 130 Pronephros, f. 51, 144 Pronephric duct, 146 Pronephric tubules of chick, f. 52, 146 Prosencephalon, f. 20, 54, 61, 95, 114 Prosocoele (= lumen of prosen- cephalon, q. v.). Ramus communicans, 119 Rathke's pocket, f. 35, f- 43> 9S» ii7 Recapitulation, 43, 102, 144 Recessus neuroporicus, f. 42 Recessus opticus, f. 42, 115 Reduction division of gametes, 9 Retina, pigment layer of, f. 45, 96, 122 sensory layer of, f. 45, 96, 122 Rhombencephalon, f. 20, 54, 61, 65 Rhombocoele (= lumen of Rhomben- cephalon, q. v.). Roof plate of spinal cord, 119 Sclera of eye, 122 Sclerotomes, f. 38, f. 44, 107 Sections, location of, 4, 34 Seessell's pocket, f. 43, 102, 124 Segmentation, 14 (see also cleavage). Segmentation cavity (see blastocoele). Sensory layer of retina (see retina). Septa of yolk sac, f. 30, 84 Serial sections, 4 Sero-amniotic cavity, f. 30, f. 32, 87 Sero-amniotic raphe, f. 30, f. 32, 87 Serosa, f. 30, f. 32, 86 Sex cells (see gametes) . Shell, f. 3, 10 Shell membranes, f. 3, 10 Sinus region of the heart (see sinus venosus). Sinus rhomboidalis, f. 21, 55, 99 Sinus terminalis (= terminal vein, q. v.). Sinus venosus, f. 23, f. 49, f. 50, 72 Somatic mesoderm (see mesoderm). Somatopleure, f. 17, 49 Somites, diflferentiation of, f. 38, 105 formation of, 56 Spermatozoa, f. 2, 10 Spinal cord, 54, 118 Spinal ganglia (see ganglia). Spinal nerve roots, development of, f. 44, IT9 Splanchnic mesoderm (see mesoderm). Splanchnopleure, f. 17, 49 Stomach, f. 43, 126 Stomodaeum, f. 31, f. 35, loi, 124 Subcaudal space or pocket, f. 31, 81 Subcephalic space or pocket, f. 17, f. 31, 47 Subgerminal cavity (= blastoccele q. v.). Sylvian aqueduct, f. 42, 117 Sympathetic ganglia, f. 44, j2o Sympathetic nerve roots (see ramus communicans). INDEX 167 Tail,!. 39, 81 Tail fold of amnion, f. 32, 87 Telencephalon, later development of, "5 lateral vesicles of, f. 42, 114 median, f. 42, 114 origin of, 65, 95 Teloccele (= lumen of telencephalon, q. v.). Telo-diencephalic boundary, 115 Telolecithal ova, f. 4, 15 Thalami (optici), 117 Theca folliculi, f. i, 8 Thymus, 125 Thyro-glossal duct, f. 43, 125 Thyroid gland, 125 Torsion of embryo, f. 29, 75, 109 Trabeculae carneae, f, 46D, 144 Trachea, f. 43, 125 Trigeminal ganglion (= Gasserian ganglion of cranial nerve V,q.v.). Trigeminal nerve (= Cranial nerve V), 118 Tuberculum posterious, f. 42, 117 Ureter (derived from metanephric duct, q. v.). Vagus nerve (=« cranial nerve X), 118 Vegetative pole, 8 Vein, allantoic, f. 47 cardinal, ant. f. 24, 74, 105, 139 cardinal, common (= Duct of Cuvier) f. 24, f. 47, 74, 105 cardinal, posterior, f. 24, 74, 105, 139 definition of, 133 omphalomesenteric, f. 21, f. 47, 57, 74, 105, 127 terminal (= sinus terminalis), f. 21, f. 48, 136 vena cava, 139 vitelline, f. 48 Velum transversum, f. 42, 115 Ventrad, usage of term, 5 Ventral, usage of term, 5 Ventral aorta (see aorta). Ventral aortic roots (see aortic roots) . Ventral cephalic fold, f . 42 Ventral ligament of liver, 153 Ventral mesentery (see mesenteries). Ventral mesocardium (see meso- cardium). Ventral nerve roots, f. 44, 119 Ventricle, f. 23, f . 49, f. 50, 72, 141 Ventro-lateral pancreatic buds, 127 Visceral arches, f. 34, f. 40, f. 46, 102, III Visceral clefts, f. 34, f. 40, 102, 11 1 Visceral furrows, f. 36, f. 46, 103 Visceral pouches (= pharyngeal pouches),f.36, 103, 125 Vitelline blood-vessels (see arteries and veins) . Vitelline circulation (see circulation). Vitelline membrane, f. i, f. 3, 8. Vitreous body of eye, 122 Wolffian body ( =mesonephros, q. v.). Wolffian duct (= mesonephric duct, q. v.). Yolk, absorption of, 84, 136 effect of on gastrulation, f. 6, 21 effect of on segmentation, f. 4, 14 white, f. I, f. 3, 12 yellow, f. I, f. 3, 12 Yolk duct, 84 Yolk-sac, f. 30, f. 32, 81, 84,86 Yolk stalk, f. 30, f. 31, f. 32, 84 Zona radiata, f. i, 8 Zone of junction, f. 7, 21, 24 Zones of the blastoderm, 24 THE LIBRARY UNIVERSITY OF CALIFORNIA San Francisco Medical Center THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW Books not returned on time are subject to fines according to the Library Lending Code. Books not in demand may be renewed if application is made before expiration of loan period. 14 DAY NOV 22 1970 25w-10.'67(H552584)4128 413^1