Book - Outlines of Chordate Development 4
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Kellicott WE. Outlines of Chordate Development (1913) Henry Holt and Co., New York.
- Outlines of Chordate Development: 1. Amphioxus | 2. Early Frog | 3. Later Frog Organogeny | 4. Early Chick - Embryonic Membranes and Appendages | 5. Later Chick - Organogeny | 6. Early Mammal - Embryonic Membranes and Appendages | Figures
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- 1 Chapter 4 The Early Development Of The Chick The Embryonic Membranes And Appendages
- 1.1 Introduction
- 1.2 The Egg And Its Production
- 1.3 The Formation Of The Embryo
- 1.4 The Early Development Of The Embryo
- 1.5 The Embryo Of About Thirty Hours
- 1.6 The Separation of the Embryo from the Extra-Embryonic Structures
- 1.7 The Establishment of the External form of the Embryo
- 1.8 The Embryonic Membranes and Appendages
Chapter 4 The Early Development Of The Chick The Embryonic Membranes And Appendages
MANY of the great embryological classics are based upon the development of the chick. The pioneer works of Harvey, of Malpighi, of Wolff, Pander, and Von Baer, bear witness to the fact that answers to many of the fundamental problems of the science of embryology were sought in the development of Ihis form; and from its study came many of the long dominant conceptions of the process, as well as the morphology, of development.
Today, when the development of the chick is better known, as a whole, than that of -perhaps any other vertebrate, it remains an extremely important subject, not so much because of its historical importance, nor because of its very great practical convenience on account of its abundance, ease of manipulation, and freedom from seasonal limitations in its use, as for other more significant reasons.
The development of the chick is fairly typical of the development of the members of the largest and most important Chordate division, the Sauropsida. And besides, it suggests interpretations of many of the very special features of mammalian development. The egg of the fowl represents the climax of the process of yolk accumulation, which begins in Amphioxus and steadily increases through the Ganoids, Amphibia, and Elasmobranchs. Consequently we find here, in pronounced form, many interesting phases of the influence of deutoplasm upon development. Indeed so great is the accumulation of yolk here, that it remains no longer contained within the limits of the embryo proper, and instead of exercising a retardative influence upon the rate of development, it becomes so related to the embryo that development is greatly hastened. In this form the presence of the great mass of yolk results chiefly in an extensive modification of the external form of the embryo, particularly during its early phases. The embryo develops for some time as a flat disc, upon the surface of the yolk mass, so that it gives a sort of map-like spherical projection of a Chordate embryo. And the morphological separation of embryo and yolk, freeing the former from the influence of the inert deutoplasm, enables the young chick to proceed to a remarkably advanced stage of development during the three weeks of its brief embryonic life.
Moreover, the chick embryo possesses, in comparatively simple form, certain embryonic membranes and appendages, which, in the Mammal, are highly specialized and come to play an important role in the modification of embryonic form, and an essential role in relating the embryo to the walls of the maternal cavity in which it develops, a relation that is singly the most important distinction of Mammalian development.
As an introduction to the subject we may outline, in a few words, the more striking points in the life of the young chick. Fertilization is internal, following a process of copulation, and after laying, the eggs are brooded by the mother during their three weeks incubation. This ensures, besides protection, the temperature necessary for development. Normally this is about 38 C.; development ceases entirely at temperatures above 41, and becomes very slow when it falls to 25 or even to 28. Newly laid eggs may remain alive, however, without undergoing any advance, for a considerable period at much lower temperatures than these; development then proceeds when the temperature is raised.
The processes of maturation, fertilization, cleavage, and blastula formation are completed before the ovum leaves the body of the parent, while the egg is passing down the oviduct. Thus the hen's egg, as laid, may be roughly compared to the seed of a plant, in which a simple embryo is already formed and surrounded with nutritive material for its later development.
The chief steps in the formation of the definitive embryo, occur during the first day of incubation, and during the second day a complicated series of folds appear, which largely effect a separation of the embryo from the yolk-mass, with which it then remains in connection by a narrow stalk. At the same time a very extensive circulatory system develops, putting the embryo into relation with its outlying food supply (Fig. 118). Development now becomes very rapid since, through the morphological separation of embryo and yolk, the usual retardative effect of the latter is obviated, while an efficient physiological connection is established through the precocious appearance of the circulation.
During the second and third days of incubation, appear the embryonic membranes and appendages, which provide for respiration, extend the nutritive surfaces, and afford the spaces within which the embryo may fully extend. In general the development of the embryo occurs progressively from the anterior end, posteriorly. Thus the brain and other structures of the head are very large and in a fairly advanced stage of development, while the posterior part of the trunk and the tail are still in process of formation. The heart, which appears very far forward, is also a very prominent feature of the early embryo. From about the fifth day the development and enlargement of the body region reduce the relative prominence of the head region, and the typically bird-like form of the embryo is acquired about the eighth day.
On the twentieth day, usually, the chick makes a small perforation through the shell and begins to breathe with its lungs, and on the following day the young chick breaks entirely from the shell. The chick is representative of those birds whose young are " precocious," for it is able to run about actively, to pick up food, and to lead a generally active life, within a few hours after hatching.
The Egg And Its Production
We shall find it convenient to describe first the general structure of the hen's egg in its newly laid condition, although this is not the true ovum; for the "egg" is not laid for some time, usually twenty-one to twenty-three hours, after fertilization, and during these hours the process of cleavage is completed, gastrulation and germ layer formation are well advanced, so that the "egg" already contains a multicellular embryo.
The true ovum, or egg cell proper, is the large yolk, or vitellus, surrounded by a thin but rather tough vitelline membrane (Fig. 85). The extreme animal pole is nearly free from yolk and appears at the time of laying as a circular whitish area, the blastoderm, 3-4 mm. in diameter: this pole is the less dense and is therefore turned upward when the vitellus is free to rotate. The blastoderm of this stage will be fully described later, but we may now distinguish in it two regions, a central translucent area pellucida, and a whitish peripheral area opaca. The great mass of the ovum or vitellus is composed of the deutoplasm or "yolk," of which two forms are present, known as white and yellow yolk. The white yolk occupies the region just beneath the blastoderm, and extends thence as a flask-shaped mass, to
Fig. 85. Semidiagrammatic illustration of the hen's egg at the time of laying A. Entire "egg." Modified from Marshall. B. Diagram of a vertical section through the vitellus or ovum proper, showing the concentric layers of white and yellow yolk, o, Air chamber; ac, chalaziferous layer of albumen; ad, dense layer of albumen; a/, fluid layer of albumen; b, blastoderm; c, chalaza; I, latebra; nl, neck of latebra; P, nucleus of Pander; pv, perivitelline space; smi, inner layer of shell membrane; smo, outer layer of shell membrane; v, vitellus or "yolk" ; vm, vitelline membrane; wy, layers of white yolk; yy, layers of yellow yolk.
the center of the whole vitellus; surrounding this the deutoplasm is arranged in several concentric layers, thick layers of the yellow yolk alternating with thinner strata of white yolk (Fig. 85, B). These two forms of yolk differ in physical characteristics other than color, and also in chemical composition; both are made up of yolk spheres or plates, which in the white yolk are smaller and quite variable in size and form.
Surrounding the vitellus or egg cell, are several nutritive and protective egg membranes, all of the tertiary class, i.e., formed by the accessory reproductive organs, the vitellus alone having been formed within the ovary; there are no secondary or chorionic membranes. Immediately surrounding the vitellus is the " white' 7 or albumen. The chemical composition of this is quite complex, various albumens forming the predominating constituents. Two denser, opaque, twisted cords, the chalazce, extend through the albumen from opposite sides of the vitellus, toward the apices of the shell. These are continuous with a very thin, dense layer of albumen surrounding the vitellus, the chalaziferous layer. Outside of this the albumen forms a rather thick dense layer, and superficially there is a still thicker layer of more fluid albumen. In the hard-boiled egg the albumen can often be seen to be laid around the vitellus in spiral sheets.
The entire ovum is enclosed in a definite ovoid shell, very resistant to pressure applied gradually, though easily broken by a sharp blow. The shell is covered superficially by a thin structureless cuticle perforated by numerous pores. The chief substance of the shell is composed of loosely arranged particles of the carbonates and phosphates of calcium and magnesium, in an organic matrix. The inner surface of the shell is formed by a thinner but denser layer of inorganic salts. The dried shell is very porous and affords an easy pathway for the passage of gases and water vapor.
Lining the shell is the tough shell membrane. This is a double sheet of fibrous connective tissue; at the blunter end of the shell its two layers are separated by an air space, which is often of considerable size in eggs that have been laid for some time.
The Reproductive Organs of the Fowl
A better understanding of the structure of the egg can be had from the study of its formation, but first we must review the main facts regarding the re productive system of the fowl. These organs are asymmetrically developed, those of the right side having degenerated to functionless vestiges; the left ovary and oviduct correspondingly become very large.
Fig. 86. The reproductive system of the fowl. After Duval (Coste). The figure shows two eggs in the oviduct, whereas normally only one egg is in the oviduct at a time, b, Blastoderm; c cicatrix; cl, cloaca; da, dense layer of albumen;/, empty egg follicle from which the ovum has escaped; g, glandular portion of oviduct; i, isthmus; m, mesovarium; 01-04, ovarian ova in various stages of growth; O\, ovum in upper end of oviduct; Oz, ovum in middle portion of oviduct (the oviduct has been cut open to show the structure of this ovum) ; os, ostium or infundibulum; ov, ovary containing ova in various stages of growth; r, rectum; u, uterus; v, vitellus; w, ventral body wall, opened and reflected.
The ovary (Fig. 86) appears to be composed of a mass of globules of varying size, suspended from the dorsal abdominal wall by a double peritoneal fold known as the mesovarium (cf. mesentery). These globules are partly grown, immature ova, and in the adult hen they vary in size from a simple cell up to the full-sized vitellus. The oviduct is a large, rather thickwalled, muscular tube, considerably convoluted and showing a well-marked regional differentiation. Its upper end opens out of the body cavity from the region of the ovary, while its lower end discharges directly into the cloaca. Three general regions are to be distinguished : the first, or oviduct proper, is the most extensive and is itself divisible into three sections. The abdominal opening of the oviduct is a wide, flaring, funnel-shaped opening called the ostium, or infundibulum, or sometimes the fimbriated opening, from its fringe-like margin; its walls are thin and muscular, and it is lined internally with cilia. The ostium leads directly into the long convoluted glandular portion, and this is followed by the short third section of the true oviduct, the isthmus. The second general region is the so-called uterus, a dilated portion, also with glandular walls. The short terminal region is a thin- walled vagina, which opens into the cloaca just dorsally to the opening of the rectum. The functional characteristics of these regions will be described in the following paragraphs.
The Formation of the Egg and its Early Development
The early developmental history of the egg may conveniently be described in three periods: (A) ovarian development to the time of ovulation; (B) from ovulation through fertilization; (C) from the beginning of cleavage to the time of laying.
A. THE HISTORY OF THE OVARIAN OVUM TO OVULATION
The rhythm of egg production in the domestic fowl is unusual in that, as a rule, a long period of egg formation and laying, extending over several months, is followed by a relatively brief period of nearly or quite complete cessation. This is quite in contrast to the great majority of animals, in which a large number of ova are produced within a very brief period, a condition probably correlated with the very large size of the ova, for the formation of even a single egg requires a considerable expenditure of energy and substance. Moreover, there is space in the organs of reproduction for but a very few ova of such large dimensions. Such a succession in the formation of the ova makes it possible to observe, in a single ovary, most of the steps in the formation of the fully grown ovum.
The first phase of oogenesis, the multiplication of the oogonia, occurs during the embryonic life of the chick, and is practically completed by the time of hatching. All of the ova produced later, during the period of adult life, are thus in the form of primary oocytes at the "birth" of the chick. Surrounded by the non-germinal cells of the "germinal" epithelium, the oogonia or primitive ova multiply rapidly. Some of them leave the epithelium and migrate into the stroma of the ovary where they degenerate. The remaining oogonia, which commence to enlarge while still continuing their multiplication, together with the rapidly proliferating epithelial cells, then form elongated strands or cords, extending from the epithelium into the stroma. Soon these definitive oogonia cease multiplication and are then to be termed primary oocytes (Fig. 87, A). The strands then break up into cell groups or "nests," each consisting of a single primary oocyte surrounded by a number of the original epithelial cells; these latter take up a definite epithelial arrangement around the oocyte, and thus form the primitive egg follicle (granulosa cells). This arrangement of the cells occurs a few days after hatching. The structure of the oocyte follicle at this age is shown in Fig. 87, B.
The egg cell, both nucleus and cytoplasm, now begins to enlarge and deutoplasmic granules are laid down all around the centrally located nucleus, and throughout the cytoplasm, except in its peripheral region which remains comparatively free from yolk. This peripheral protoplasmic layer is definitely thickened at one point, namely, toward the attached surface of the ovum or follicle, forming there the germinal disc or spot. When the ovum reaches a diameter of something more than 0.5 mm. the nucleus migrates into the germinal disc, where it remains
Fig. 87. Growth stages in the oogenesis of the hen's egg. After Sonnenbrodt. A. Oocyte measuring 0.012 X0.016 mm., the nucleus of which is 0.006 mm. in diameter. B. Oocyte measuring 0.018 X0.028 mm., the nucleus of which is 0.0105 X0.014 mm. Enclosed in follicle. C. Oocyte measuring 0.040 X0.045 mm., the nucleus of which is 0.020X0.022 mm. D. The nucleus only, of an oocyte measuring 5.84 X 6. 16 mm., the nucleus itself measuring 0.214 X 0.238 mm. Total view showing the small chromosomes in the midst of a collection of chromatin nucleoli. E. Vertical section through the nucleus only, of an oocyte, the follicle of which measured 37 mm. in diameter. The nucleus itself is 0.455 mm. in diameter and 0.072 mm. in greatest thickness, c, Chromosomes; cr, extranuclear chromsome-like bodies; /, follicle; ra, nuclear membrane; mf, folds in nuclear membrane; n, nucleus; nu, chromatin nucleolus; ps, pseud ochromosomes; s, centrosphere; v, yolk nucleus or vitellogenous body.
throughout the remainder of its ovarian history (Fig. 88). It should be noted that the eccentricity of the nucleus, which marks the animal pole of the ovum, is toward the attached surface.
The formation and accumulation of yolk now become more rapid. The surface of the ovum is in the form of a zona radiata, through the pores of which nutritive substances diffuse from the follicle cells, which thus function as nurse cells (Fig. 88). The rate of growth of the egg during its final stages may be determined by the arrangement of the concentric layers of white and yellow yolk (Fig. 85, B), which mark daily periodicities in the formation of deutoplasm (Riddle) . Toward the close of the growth period the follicle becomes more clearly membranous, and along the side opposite its attachment, which is comparatively non- vascular, a modified band appears; this is the cicatrix, where the follicle ruptures when the ovum escapes from the ovary.
Fig. 88. Section through the pigeon's ovarian ovum, 1.44X1.25 mm. From Lillie (Development of the Chick), /.s., Stalk of follicle; G.V., germinal vesicle or nucleus; Gr., granulosa cells; I/., latebra; p.P., peripheral protoplasm; pr.f., primordial follicles; Th.ex., theca externa; Th. int., theca internal Y.Y., yellow yolk; Z.r., zona radiata.
Just before the egg leaves the ovary its nucleus, lying flattened against the vitelline membrane, reaches the enormous diameter of about 0.3 mm. It is now known as the germinal vesicle, since the condensation of its small chromatin content leaves the nucleus as a large clear spot (Fig. 87, D, E). The last events before ovulation are the breaking down of the nuclear wall and the formation of the first polar spindle. This rotates into position and the primary oocyte, prepared for its first maturation division, pauses to await ovulation.
B. THE PERIOD FROM OVULATION THROUGH FERTILIZATION
At the time a completed egg is laid, or very shortly thereafter, the region of the ostium or infundibulum of the oviduct, becomes very active and seems to grasp the ovarian follicle containing the primary oocyte, through muscular or ciliary action, or both. The follicle then becomes ruptured, apparently by the pressure exerted by the contraction of the infundibulum, or by its pulling away from the region of the ovary, and the oocyte, contained within the infundibulum, is withdrawn from the follicle, and ovulation is accomplished. In some cases it may be that the follicle is ruptured before it can be grasped by the infundibulum and the freed oocyte is subsequently received by the fimbriated opening. The oocyte always enters the infundibulum with its chief axis transverse to the long axis of the oviduct, and this relation is retained during the entire passage of the ovum down the oviduct.
The spermatozoa, after their receipt by the female, make their way to the extreme upper end of the oviduct, where they collect, remaining alive and capable of functioning for two weeks or more. Upon its entrance into the oviduct, therefore, the primary oocyte becomes bathed in a fluid containing sperm cells, and fertilization immediately ensues. The details regarding the penetration of the spermatozoa are not fully described, but it is known that this occurs immediately after ovulation, while the ovum is in the infundibulum. Polyspermy is normal, five to twenty-four spermatozoa having been counted within a single ovum (Patterson). Entrance of the spermatozoa affords the stimulus to the completion of maturation, which offers no unusual features. After the second maturation division the egg nucleus unites with one of the sperm nuclei and the first cleavage spindle is typically established.
C. FROM THE BEGINNING OF CLEAVAGE TO THE TIME OF LAYING
Before continuing our account of the development of the ovum we must outline the series of events occurring during the passage of the egg down the oviduct; we shall follow the accounts given by Patterson, and Pearl and Curtis.
The first cleavage furrow appears about three hours after ovulation. During this interval the egg has traversed the entire glandular portion of the oviduct, the walls of which have secreted the denser layers of albumen and the chalazse (40 to 50 per cent, of the entire weight of albumen) . The egg is carried along chiefly by peristaltic contraction of the oviducal wall, and as it passes it is rotated about the long axis of the oviduct, so that the germ disc describes a spiral path; this accounts for the spiral arrangement of the albumen around the yolk. During the next hour or so (Pearl) the egg traverses the isthmus, the walls of which secrete the shell membrane over the surface of the dense albumen as the egg enters this region. The fluid layer of albumen is added while the egg is traversing the isthmus and the upper part of the uterus. The formation of this fluid albumen, which passes through the shell membrane already laid down, is completed five to seven hours after the egg enters the uterus (nine to eleven hours after entering the oviduct). Before this the calcareous shell substance has begun to be laid down on the shell membrane. The egg usually occupies twelve to sixteen hours longer in completing its passage through the uterus and vagina. At the end of this time, twenty-one to twenty-seven hours after ovulation, gastrulation has begun and the egg is ready to be laid. If the completely formed egg reaches the vagina during the middle of the day (8 A. M. to 4 P. M.) it may be laid promptly; otherwise it is retained within the vagina until the following day. In the latter case, since development continues during the entire period of its retention, which may be quite prolonged, the embryo may be in a fairly advanced stage when the egg is finally deposited. Thus, variation in the period of retention accounts for the variation in developmental stages observed in different eggs as laid.
Table Showing The Chief Events In The Early History Of The Hen's Egg
Hours after ovulation
Location in oviduct
Action of oviduct
Action of germ disc
Reception of ovum
Maturation and fertilization
Secretion of chalazae, chalaziferous, and dense layers of albumen
First cleavage furrow
3 to 4
Secretion of shell membrane and fluid albumen
Formation of eight cells
4 to 21 (27)
Uterus and vagina
Secretion of shell and fluid albumen. Retention prior to laying
Gastrulation begun, or completed if egg is long retained
We may now return to describe the processes of development occurring during the period prior to laying. The unicellular germ disc consists of a quite definitely circumscribed area at the animal pole of the vitellus. The disc is about 3 mm. in diameter and less than 0.5 mm. in thickness. Beneath, the protoplasm merges with a well-marked region of the white yolk called the nucleus of Pander, which connects with the central white yolk by a narrow stalk called the latebra (Fig. 85, A). In the disc itself two regions may be distinguished, a large central area, which is to form the blastoderm proper, and a narrow marginal area of denser appearance, known as the periblast. Peripherally the periblast continues for some distance, perhaps completely, over the surface of the vitellus as an extremely thin protoplasmic layer.
The first cleavage furrow appears as a short shallow groove, near the middle of the germ disc, in length approximately onehalf the diameter of the disc (Fig. 89, A). Sections show that this cleavage also fails to extend completely through the disc vertically (Fig. 90, A). It is not known that the first cleavage plane coincides with the median sagittal plane of the embryo: indeed it seems probable that, as in other eggs of this extremely meroblastic type, there is no correspondence between these two planes. The position of the main embryonic axis is, however, fairly uniform, though not completely fixed. It lies approximately at right angles to the long axis of the whole egg, the anterior end of the embryo directed to the left, when the sharper end of the egg is held pointing away from the observer. In the few cases actually observed, the first cleavage plane seems to have no definite relation to these axes.
The second cleavage is also vertical, and approximately at right angles to the first, giving four adequal cells, all still incomplete (Fig. 89, B) . About an hour after the first cleavage, the third appears. Typically, though not in a majority of instances, this is a fairly regular cleavage, parallel with the first, and dividing the disc into two rows of four cells each. Frequently this cleavage is more or less irregular in form, and the synchronism of division is lost by this time (Fig. 89, C) . The subsequent stage, consequently, may be said to consist only approximately of sixteen cells. These are very irregular, but the general tendency of the fourth plane is to separate each of the eight cells into a central and a distal cell.
During the appearance of the third and fourth cleavages, the central portion of the germ disc has become divided horizontally by a cleavage plane, connecting the lower margins of the first and second planes. In this way a small group of central cells become completely circumscribed, and are to be distinguished from the marginal cells, which remain incomplete both below and distally, retaining their connection with the periblast (Fig. 90, B) . A definite though narrow space appears, accompanying the horizontal cleavage, which separates the superficial cellular elements from the underlying undivided protoplasm. This space is the beginning of the segmentation cavity or blastoccel; the protoplasm beneath it is termed the central periblast. The original periblast region is now distinguished as marginal periblast. The two periblastic regions retain their connection with one another peripherally, in the deeper region of the marginal cells.
Fig. 89. Cleavage in the hen's egg. Surface views of the blastoderm and the inner part of the marginal periblast only. From Patterson. The anterior margin of the blastodisc is toward the top of the page. A. Two-cell stage. About three hours after fertilization. B. Four cells. About three and one-fourth hours after fertilization. C. Eight cells. About four hours after fertilization.
D. Thirty-four cells. About four and three-fourths hours after fertilization.
E. One hundred and fifty-four cells upon the surface; the blastoderm averages about three cells in thickness at this stage. About seven hours after fertilization, ac, Accessory cleavage furrows; ra, radial furrow; p. inner part of marginal periblast; sac, small cell formed by the accessory cleavage furrows.
Fig. 90. Vertical sections through the chick blastoderm during the process of cleavage. After Patterson. A. Section through the two-cell stage. B. Median section through the thirty-two cell stage. C. Part of a longitudinal section through the sixty-four cell stage, b, Blastoccel or segmentation cavity; c, central cells; i, inner cell cut off by horizontal cleavage plane; I, neck of latebra; m, marginal cell; mp, marginal periblast; n, nucleus; p, first cleavage; v, vitelline membrane.
We should note here a few details regarding the history of the accessory or supernumerary spermatozoa. During the period intervening between fertilization and the early cleavages, these form nuclei which migrate to the outlying portion of the blastodisc. There they may divide once or twice, forming small groups of daughter nuclei. These divisions are sometimes accompanied by slight indications of cytoplasmic division, and the short superficial grooves thus formed are known as the accessory cleavages (Fig. 89) . These are visible during the f ourand eight-cell stages; they are usually radial in direction, lying just across, or without, the margin of the blastodisc. No true cells are formed by such cleavages. The accessory sperm nuclei degenerate rapidly, the accessory cleavages fade out, and by the time thirty-two cells are formed no traces of these structures are left.
During subsequent cleavages the number of central cells increases rapidly, by additions from the dividing marginal cells, and through their own multiplication, which becomes very rapid as the cells diminish in size. Additional horizontal cleavages appear in the central cells, converting the roof of the blastocoel into a layer several cells in thickness (Fig. 90, C). No cells are added to the germ disc from the floor of the segmentation cavity. The marginal cells become greatly shortened through the continued cutting off of central cells, and finally they are limited to the extreme margin of the blastodisc.
About the time two or three hundred cells are formed, intercellular furrows extend out into the marginal periblast (Fig. 89, E). Up to this time both central and marginal periblast have been entirely free from nuclei, but soon these areas, which are still directly continuous, become converted into a nucleated syncytium. The details of this process have not been described for the chick, but are well known in the pigeon (Blount). In the latter form, when the marginal cells have become reduced to an approximately spherical form by the cutting off of central cells, their nuclei continue to divide, while accompanying cytoplasmic divisions are either incomplete or entirely lacking. A large number of free nuclei is thus formed in the margin of the blastodisc. These nuclei, continuing to multiply, wander out into the marginal periblast, converting this into a nucleated though non-cellular tissue. From this region some of the nuclei migrate inward below the blastodisc, converting the central periblast also into a nucleated structure, except in its middle area, above the nucleus of Pander, which appears to remain free from nuclei. The nucleated rim of the periblast forms a part of what is known later as the germ wall.
The circular blastoderm now begins to extend radially, partly on account of the growth of its own cells, and partly (continuing Blount's account) by the addition of cells from the marginal periblast. The region of the original blastodisc now becomes thinner and more transparent, and is known as the area pellucida, while its circular margin, apparently derived largely from the periblast, is called the area opaca. The ring-like periblast continues to grow and to become nucleated more peripherally, at the same time that it is contributing cells to the blastoderm, so that it steadily increases in diameter. The inner nucleated margin of the periblast, which becomes of cellular composition and contributes to the later extra-embryonic tissues, is known as the germ wall (Fig. 91). Later the cells of the blastoderm extend peripherally, overlapping the inner margin of the germ wall, so that there is a narrow region transitional between pellucid and opaque areas. We should not overlook the fact that the lower surface of the periblast is directly continuous with the yolk-mass, and is peripherally continuous with a very thin superficial layer of protoplasm; this latter is sometimes referred to as a part of the germ wall. The thinning of the blastoderm, mentioned above, may be regarded as indicating the completion of the blastula stage and the beginning of gastrulation, to which we may next turn our attention.
In the chick the formation of the primary germ layers, ectoderm and endoderm, i.e., gastrulation proper, is quite easily distinguished from the processes of notogenesis and mesoderm formation. In the following account of gastrulation many details are supplied from Patterson's account of this process in the pigeon, for this period in the development of the chick is incompletely known, although it is clear that the pigeon and chick are in agreement as to essentials. The thinning of the blastoderm through the rearrangement of its cells, continues rapidly as the diameter of the area pellucida increases. It is most marked in a wedge-shaped area in the posterior part of the blastoderm, the most posterior portion of which may come to be only one cell in thickness (Fig. 92, A). In this region the subgerminal or segmentation cavity increases in depth, and around the hinder margin of the thinner area the germ wall (area opaca) appears to be interrupted more or less completely, so that the blastoderm cells border directly upon the yolk.
The first step in the real process of gastrulation is the turning under of a few of the marginal cells in this posterior region of the blastoderm, where it is free from the germ wall. As the involution of superficial cells continues, this margin soon becomes thickened, and since the involuted cells are the rudiment of the endoderm, the thickening represents the lip of the blastopore, which is here reduced to a short crescent, between the two free extremities of the germ wall (Fig. 92, B). Once established, the endoderm rapidly grows forward between the yolk and the ectoderm, as the upper cells of the blastoderm may now be called, extending through the segmentation cavity laterally as well as anteriorly (Fig. 91). During these early stages the endoderm cells are considerably scattered, and are arranged as a solid mass or definite layer only in the region of the blastoporal margin. As the endoderm cells gradually extend across the segmentation cavity, two other processes become apparent (Fig. 92, C). First, the free posterior extremities of the germ wall approach, and finally meet and fuse; this process, is known as the closure of the blastopore, the blastoporal opening being present only virtually, and represented by the region between the extremities of the germ wall. The second process is the beginning of the extension of the blastoderm, or gastrula, from every side save the region of the blastoporal margin. The endoderm cells soon extend out to the margin of the segmentation cavity in every direction except anteriorly.
Fig. 92. Diagrams of reconstructions of the pigeon's blastoderm. From Lillie (Development of the Chick) after Patterson. A. Thirty-one hours after fertilization. B. Thirty-six hours after fertilization. C. Thirty-eight hours after fertilization. E, Gut endoderm; GW, germ wall; O, margin of overgrowth ; PA , outer margin of area pellucida; R, in B, margin of involution; in C, mass of cells left after closure of the blastopore; S, beginning of yolk-sac endoderm; SG, anterior part of subgerminal cavity (blastocoel), as yet free from endoderm; Y, yolk zone; Z, zone of junction. The numbers 1-7 along the line C-D, in A, indicate the number of cells in the thickness of the blastoderm in these regions.
The closure of the blastopore produces a small thickened region, representing the contracted blastoporal margin, which is left just within (anterior to) the germ wall, where this now becomes continuous posteriorly. The process of extension of the blastoderm now involves this region and leaves the contracted blastoporal margin well within the area pellucida.
Normally the egg is laid twenty-two to twenty-three hours after fertilization, in approximately this condition, with gastrulation not quite completed. The structure of the unincubated blastoderm may therefore be described as follows. Three general regions are to be distinguished. The original area pellucida is surrounded by a complete area opaca, and beyond this is a narrow margin where the blastoderm cells are extending over the surface of the yolk. The pellucid area is formed by a layer of ectoderm cells, slightly thickened toward the middle of the area; posteriorly it includes also a sheet of endoderm cells. The endoderm is in the form of a definite layer only toward the posterior margin, elsewhere the endoderm cells are scattered through the segmentation cavity, in the process of migrating toward its anterior margin. A narrow space between the endoderm and yolk, really the remains of the segmentation cavity there, is the rudiment of the archenteron. The area opaca, save in its posterior region, is formed by the thickened margin of the blastoderm resting upon the germ wall and fusing peripherally with it. Posteriorly, where the germ wall is narrower, there is a thickened region of the cellular germ disc which represents the contracted blastoporal margin. In this region the ectoderm and endoderm are continuous.
During the first few hours of incubation, or even before laying in cases where the eggs have been retained for some time longer than usual in the vagina, the endoderm extends completely across the segmentation cavity and becomes organized into a fairly definite layer.
From the preceding description it will be seen that the process of gastrulation in the chick is essentially a process of involution. There is no true invagination, and the process of epiboly is not immediately concerned in the establishment of the primary inner layer. Moreover, the process of epiboly is here greatly limited, being restricted to a narrow posterior section of the blastoderm. In other words, the entire blastoporal region is greatly reduced, doubtless in correlation with the excessive amount of yolk in the ovum.
The formation of the middle germ layer and the chief axial structures of the embryo, is not intimately bound up with the gastrulation process, as in the forms previously described. We may now consider these processes in connection with the general development of the whole embryonic region.
The Formation Of The Embryo
The earliest indication of the true embryo becomes visible during the early hours of incubation, immediately after the entire area pellucida has become two layered, through the complete extension of the endoderm. It appears first as a slightly thickened band, not very well marked, extending directly from a point approximately in the middle of the area pellucida, nearly to its posterior margin. This is called the primitive streak; it is constituted at first solely by a thickening in the ectodermal layer (Fig. 93). Once established, the primitive streak grows very rapidly, chiefly through posterior elongation, the anterior end remaining relatively fixed. The blastoderm, too, shares in this elongation and becomes irregularly oval, and then pear-shaped, the blunter end being anterior.
Fig. 93. Total views of chick blastoderms. A. After Duval (modified) ; B-D, after Lillie. A. Unincubated blastoderm, with primitive streak just forming. B. Primitive streak formed, head process not yet indicated. C. Head process formed; head-fold just commencing. D. Just before the establishment of the first mesodermal somites, ac, Amnio-cardiac vesicle; cr, crescent-shaped thickening at the posterior side of the blastoderm, in the region of endoderm and mesoderm formation; g, primitive groove; hf, head-fold; hp, head process; i, blood islands; m, axial thickening of mesoderm; mp, medullary plate; mr, margin of mesoderm; n, Hensen's node; o, area opaca; p, area pellucida; pa, proamnion; pi, primitive plate; pp, primitive pit; s, primitive streak; t, sinus terminalis (marginal sinus); v, area vasculosa; vi, area vitellina interna.
As the primitive streak elongates a narrow transparent line appears along its middle; this is the primitive groove. Anteriorly the primitive groove terminates in a small depression, the primitive pit, near the anterior end of the primitive streak, where a slight thickening, known as the primitive knot or Hensen's node, is visible (Fig. 97). In front of this another larger though less definite thickening, known as the head process, soon may be made out. Posteriorly the primitive streak is somewhat transversely extended, just within the border of the area pellucida, forming a more or less well marked crescentic area, through which the primitive groove may be continued, giving this a bifurcated appearance here. Between this broadened hinder end of the primitive streak and the margin of the pellucid area, a wide thickened region can sometimes be discerned; this is known as the primitive plate.
Many important structural details of the primitive streak region may be determined through the study of sections. Figures 94, 101 illustrate sections through representative regions of an early primitive streak. The ectoderm is broadly thickened as the rudiment of the medullary plate, and along the mid-line is the primitive streak, soon marked by the rapid proliferation of ectoderm cells, which are thrown off in the space between ectoderm and endoderm. These cells are the rudiment of the mesoderm. The endoderm forms a uniformly thin sheet of cells across the entire blastoderm.
A little later (Fig. 94, B) the primitive groove is indicated, bordered by a pair of primitive folds. Proliferation of mesoderm cells from the inner surface of the ectoderm is now very rapid, and these cells soon migrate distally throughout a large part of the space between the two primary germ layers. Near the midline they become intimately related with the endoderm, often giving the misleading appearance of having been derived directly from that layer (Fig. 101). Soon the cells along the middle of the primitive streak have multiplied so extensively that they form a dense mass, in which clearly marked limits of the germ layers cannot be made out. More laterally, however, the layers are completely distinct. The ectoderm extends out over the germ wall and yolk; the endoderm, now more than one cell thick, reaches only to the germ wall, with which it fuses, the mesoderm forms a loosely arranged sheet of cells, thinning peripherally and extending for a short distance between the ectoderm and germ wall.
The conditions found near the extremities of the primitive streak deserve special mention. Posteriorly, the primitive plate shows a transversely extended region of mesoderm proliferation, which continues across the posterior side of the primitive plate, so that the pellucid area behind the primitive streak is composed of all three germ layers (Fig. 94, C). In the region of the head process, i.e., in front of the primitive pit and streak, conditions are not essentially unlike those found farther back. The primitive groove is absent, and the cells of the three layers are more intimately fused. Relatively little mesoderm is proliferated from the anterior part of the head process, so that in front of this, the blastoderm is for a considerable time composed of only the two primary layers.
Fig. 94. Transverse sections through various levels of the blastoderm at different ages. A. Through the head process of an embryo of about sixteen hours. After Duval. B. Through the primitive streak of an embryo of seventeen and one-half hours. After Lillie. C. Through the primitive plate of an embryo of about the same age as A. After Lillie. ec, Ectoderm; en, endoderm; /, primitive folds; g, primitive groove; gw, germ wall; m, mesoderm; mp, medullary plate; pp, primitive plate.
Surface views of entire blastoderms now show considerable modifications in the structure and relations of the primary pellucid and opaque areas. The outline of the elongated pellucid area becomes irregular; as a whole it is divisible into a posterior darker region where all three layers are present, and an anterior lighter area, crescentic in. form, composed of ectoderm andendoderm only (Fig. 93, B). Later the irregular anterior border of the mesoderm can be seen to advance along the sides of the area pellucida, in front of the level of the head process (Fig. 93, C). Finally the mesoderm extends completely around the margin of the pellucid area, leaving only a small area directly in front of the head process in the two layered condition ; this area is known as the proamnion (Fig. 93, D).
Meanwhile the area opaca has been undergoing very marked modifications. This has broadened rapidly, and in its lateral and posterior regions, where the mesoderm has extended more widely over the germ wall, it has assumed an irregularly mottled appearance. Sections show this to be due to the formation of differentiated cell groups known as blood islands, the beginning of the vascular system. The presence of these blood islands marks that portion of the opaque area known as the area vasculosa (Fig. 93, C, D). This appears first immediately behind the embryo, but rapidly spreads laterally and anteriorly. Peripherally it becomes bounded by a definite blood vessel known as the sinus terminalis. Beyond this the opaque area is formed only of ectoderm and endoderm, extending widely over the yolk and known as the area vitellina. The blood islands are formed of compact cell masses scattered through the deeper portion of the germ wall. The cells composing them have apparently, though not certainly, differentiated in situ; they become covered superficially by a layer of scattered germ wall cells, which comes to be known later as coelomic "mesoderm" (Fig. 95). While this layer, and the blood islands, early become directly continuous with the mesoderm of the pellucid area derived from the primitive streak, it is commonly supposed that this relation is secondary. The blood islands later become hollowed out, their constituent cells forming both the walls and the corpuscular contents of the irregular vascular la ounce. These soon anastomose with one another forming a complex network throughout the area vasculosa; still later this net connects with the vascular structures of the pellucid area and of the embryo. The cellular portion of the germ wall remaining the coelomic " mesoderm" and the blood islands forms the rudiment of the yolk-sac endoderm, the development of which we shall describe presently.
At this point we should consider briefly the relation of the primitive streak and associated structures to the rudiments of the true embryo. From what has been said in the early part of this chapter, we should not expect to find that all of the a structures mentioned in the preceding paragraphs will be found to contribute to the formation of the definitive embryo. As a matter of fact, of all the structures so far described, only those in the vicinity of the head process, represent actual embryonic rudiments; these are, that thickening of the ectoderm described as the medullary (neural) plate, and the axial endoderm and mesoderm lying directly beneath it. The substance of the primitive streak, therefore, lies posterior to the embryo proper, and the boundary between the two is indicated by the position of the primitive pit;
As the embryo becomes more definitely established, it elongates posteriorly, while the primitive streak correspondingly shortens. The relation between the two structures is such that the embryo draws its substance from the anterior end of the primitive streak, or, we may say, the materials of the anterior end of the primitive streak are slowly redifferentiated and payed into the hinder end of the embryo. The region where this process of redifferentiation is proceeding is indicated as Hensen's node (primitive knot). Finally the primitive streak is wholly transformed into embryonic structures, but this is not fully accomplished until a relatively late stage in development (second day of incubation). To the question as to how much of the definitive embryo is formed of structures primarily anterior to the primitive streak, no precise answer can be given. In a general way, however, it may be said that the region of the primary location of the primitive pit corresponds roughly with the boundary between hind- and midbrain, and that structures developing anteriorly to this level were related originally to the region of the head process, rather than to the primitive streak.
We are now prepared to understand the essential homologies of the primitive streak, having learned of its relation to the embryo, on one hand, and to the structures of the gastrula, on the other. Theoretically the primitive streak is to be interpreted as the result of a modified process of concrescence (confluence). The chief alterations of the typical process of concrescence (confluence) are apparently due to the relatively enormous mass of yolk, and the consequent modification of the gastrula into a flat sheet, more or less intimately related to the yolk through the germ wall. Thus the endoderm is involuted from only a limited extent of the margin of the blastodisc. The region where ectoderm and endoderm become continuous is the rim of the blastopore; therefore we may say that the rim of the blastopore here is a short crescent at the posterior side of the blastula. The blastoporal opening is consequently represented by a space, really only virtual, between this and the contiguous periblast and yolk. Closure of this vestigial blastopore occurs after the growth of the blastoderm begins, so that when the lips of the blastopore approach and fuse, in the typical manner of confluence, the region of their fusion is limited to the posterior region of the blastopore (Fig. 96). We recognize the primitive streak, therefore, as the fused halves of the blastoporal margin; the primitive groove is consequently to be interpreted as a vestige of the blastopore itself. Later in development we know that the anus develops at the posterior end of the remains of the primitive streak, while from the beginning the primitive pit, at its anterior end, represents the vestige of the neurenteric canal, as described for Amphioxus and the frog.
Fig. 96. Diagrams illustrating the idea of confluence (concrescence) as applied to the chick. From Lillie (Development of the Chick). The central area bounded by the broken line represents the area pellucida; external to this is the area opaca, showing the germ wall (G.W.), zone of junction (Z.J.), and margin of overgrowth (M.O). m.n., Marginal notch.
The so-called head process thus represents, theoretically, the earliest portion of the true embryo to be differentiated out of the primitive streak. As a matter of fact, however, it is difficult to observe a true genetic relation between the primitive streak and the earliest portion of the head process, which seems to form precociously. This theoretical distinction is also the basis for the further distinction between gastral and peristomial mesoderm in the chick. Really such a distinction is not evident here, but it is sometimes useful to regard as gastral, that mesoderm originating in the primary head process, and as peristomial, that arising more posteriorly from the primitive streak proper. Ultimately, of course, the " peristomial" mesoderm becomes "gastral" in its relations to other structures.
The fact that the whole process of gastrulation is itself vestigial, to a certain extent, offers an interpretation of the hidependent formation of the medullary plate, which occurs unusually early, and of the mesoderm, which does not appear until the primitive streak is largely established. This latter fact, together with the development of the mesoderm from cells of ectodermal character, are probably both related to the marked restriction of the endoderm; this is reduced both in extent and in importance, for most of the early functions of the endoderm are here , in correlation with the extreme amount of yolk, taken over by the periblast and the cells of the germ wall.
The Early Development Of The Embryo
In the stage we have just been describing, only the posterior limit of the embryo is definitely marked; elsewhere embryonic and extra-embryonic regions are directly continuous, and no boundaries are indicated. This remains the condition of affairs for a long time in the lateral directions, but anteriorly the limit of the embryo becomes sharply marked out very early. This is accomplished about the twenty-second hour after fertilization, by the formation of what is called the head-fold, a transverse, crescentic fold of both ectoderm and endoderm, extending nearly across the area pellucida, a short distance in advance of the head process (Fig. 97). At first a shallow depression, bordered posteriorly by an elevation of the blastoderm, this soon becomes a deep groove, as if the blastoderm were being tucked in under the anterior end of the medullary plate. In longitudinal section through the medullary plate and head fold, the anterior part of the blastoderm has the appearance of a letter S (Fig. 98). This folding is really due chiefly, or wholly, to the rapid forward growth of the medullary plate, which carries with it the underlying endoderm. Thus two cavities are marked out; one occupies the dorsal limb of the fold, is lined with endoderm, and is an extension of the archenteric space between endoderm and yolk; the other occupies the ventral limb, is lined with ectoderm, and is continuous with the space entirely outside the embryo. The dorsal, endodermally lined cavity is the rudiment of the fore-gut. The lower space is sometimes called the cavity of the head-fold; it is merely an external space lying under the head of the embryo. The boundary between embryonic and
Fig. 98. Median sagittal section through the head end of an embryo with four pairs of somites (twenty-three to twenty-four hours). From Lillie (Development of the Chick), a.i.p., Anterior intestinal portal; Ect. ectoderm; Ent., endoderm; F.G., fore-gut; H.F., head-fold; med. pi., anterior limit of medullary plate; Mes., mesoderm; Mes. H.G., mesodermal head cavity; n. F., neural fold; or. pi., oral plate (oral membrane).
extra-embryonic structures is just along the middle, i.e., most posterior limit, of this latter fold or cavity; at this level the morphologically anterior limit of the medullary plate passes abruptly into the extra-embryonic ectoderm of the blastoderm, in the region of the proamnion.
Laterally the head-fold and fore-gut narrow and finally disappear into the flat blastoderm. The posterior curvatures of the lateral extremities of the head-fold mark the lateral surfaces of the head, but otherwise the lateral limits of the embryo proper are not definitely marked until twenty-two to twenty eight hours later, i.e.j forty-two to fifty hours after fertilization. We may proceed, therefore, to describe the development of the essential structures of the embryo, during the period from the formation of the head-fold up to the time when the embryo begins to be completely marked off from the extra-embryonic region of the blastoderm. We shall describe the history of the mesoderm first, not only because this seems the easiest method of approach, but because the differentiations within the mesoderm afford valuable landmarks in describing other structures. Thus the age of the chick embryo is usually indicated by reference to the number of pairs of mesodermal somites formed.
Fig. 99. Median sagittal section through the head end of a chick with eighteen pairs of somites (about forty hours). From Lillie (Development of the Chick). a.i.p., Anterior intestinal portal; Ao., dorsal aorta; Au., auricle; E.E.B.C., exocoelom (extra-embryonic body cavity); F.B., fore-brain; H.B., hind-brain; H.F.Am., head-fold of amnion; Inf., infundibulum; Isth., isthmus; M.B., midbrain; N'ch., notochord; or.pl., oral plate (oral membrane); P.O., pericardial cavity; Ph., pharynx; Pr'a., proamnion; pr'o.g., preoral gut; Rec.opt., optic recess; S.V., sinus venosus; Tr.A., truncus arteriosus; Ven., ventricle.
While the head-fold is becoming well established (Fig. 100), the mesoderm is in the form of a pair of sheets extending from the sides of the primitive streak (peristomial) and head process (gastral), across the whole extent of the area pellucida to the germ wall, where it is continuous with the vascular area. The inner or axial border of each sheet is considerably thickened, and often distinguished as the vertebral plate, the remaining distal portion being known as the lateral plate. The mesoderm also extends posteriorly from the primitive streak, but at this time there is none in the anterior part of the blastoderm in front of the head process and head-fold (proamnion), although around the margin of the pellucid area it is carried forward as a pair of wing-like extensions, so that in surface view its anterior limit is markedly concave (Fig. 93, D).
Fig. 100. Chick embryo of about twenty hours, showing first intersomitic furrow. Dorsal view. From Lillie (Development of the Chick), a.c.v., Amniocardiac vesicle; a.o., inner margin of area opaca; Ect., ectoderm; Ent. endoderm, H.F., head-fold; i.a./.l., first intersomitic furrow; med.pl, medullary plate; Mes., mesoderm; n.gr., neural groove; Pr'a., proamnion; pr.gr., primitive groove.
In the vicinity of the head process (Fig. 101), the vertebral plates at this stage rapidly lose their definite character and the cells scatter throughout the region, combining with the cells continually being budded off from the walls of the fore-gut, to form the general mesenchyme of the head region. Later scattered cells are added to the mesenchyme directly from the ectoderm of the head region. The history of the mesoderm farther back, just in front of the end of the primitive streak, is much the same as in the body region proper, although all of the embryo thus far developed out of the primitive streak shares in the formation of the head only. Here the vertebral plate thickens still more and its cells become rearranged so as to 5 ^ form a short transverse break in the conJg tinuity of the plate (Fig. 100). On each i | side the cells immediately in front of this 3 | become grouped in a definite mass and *> g form the first pair of mesodermal somites, 3 % continuous anteriorly with the forward ex3^ tensions of the vertebral plates (Figs. 100, % *. 104). This rearrangement of cells continues posteriorly, and soon a second split appears on each side, a short distance behind the first, cutting out a definite block i^. of the cells of the vertebral plate. This is the second pair of somites. Additional 2 1 ^ pairs of somites are blocked out in regular I ^ J fashion, as the embryonic region grows at IJj I the expense of the primitive streak. The 5 ,2 formation of somites is not actually completed until about the fifth day of development, by which time about fifty-two pairs have been formed. The first somites formed remain the most anterior in position. The first four pairs are ultimately in eluded in the hinder part of the head region of the embryo, and although no other definite boundary of the head appears for some time, its future limit may be marked by the position of the fourth somites.
This division of the vertebral plate into somites expresses the primary segmentation or metamerism of the body, and is fundamental, the metameric arrangement of other organs being secondary and dependent upon this. Sections through the somites (Fig. 102) show that their superficial cells are arranged in the form of an epithelium, while the cells of the central parts are loosely and irregularly arranged. This central portion, although only virtually a cavity, is termed the myocoel; it corresponds, theoretically, with the region of the enterocoel of other forms.
Fig. 102. Transverse section through the last somite of a chick of about forty-eight hours (twenty-nine pairs of somites). After Lillie. a, Lateral dorsal aorta; c, crelom; cr, neural crest; my, myotome; n, notochord; nc, nerve cord; ne, nephrotome; so, somatic mesoderm; sp, splanchnic mesoderm; W, Wolffian duct.
The more lateral portions of the mesoderm, the lateral plates, remain unsegmented. They are connected with the somites by an intermediate, transitional band, also unsegmented in the chick, known as the nephrotome or intermediate cell mass. The dorsal and ventral surfaces of the nephrotome are continuous with the corresponding surfaces of the somites, the separation between the two bodies being indicated by a depression resulting chiefly from the thickening of the somites.
The intermediate cell mass in part forms the rudiment of the excretory system, and in part contributes to the formation of mesenchyme: its history will be described in connection with the later stages of development.
The lateral plate very early becomes separated into two distinct sheets by the development of a wide cavity within it. This cavity is the coelom, which is here, as in the frog, to be described as a schizocoel. The outer or upper sheet of lateralmplate cells is the somatic mesoderm; this unites with the overlying ectoderm to form the somatopleure (Figs. 102, 105, 108). The inner or lower sheet is the splanchnic mesoderm; this unites with the underlying endoderm to form the splanchnopleure. The somatic and splanchnic layers of mesoderm remain united proximally, in the region of the intermediate cell mass. The somatopleure, splanchnopleure and coelorn, later become separated into embryonic and extra-embryonic regions, but during these early stages they form continuous structures extending laterally out to the germ wall, and anteriorly into the head region. From the embryonic portions of these structures develop, respectively, the body wall, the gut wall and vascular organs, and the pericardial, pleural, and peritoneal cavities: their extra-embryonic portions give rise to the embryonic membranes and appendages, and to the extra-embryonic portions of the vascular system and ccelom (exocoelom).
Fig. 103. Ventral views of the head ends of chick embryos. From Lillie (Development of the Chick). A. Embryo with five pairs of somites (about twenty- three hours). B. Embryo with seven pairs of somites (about twentyfive hours), a.c.v., Amnio-cardiac vesicle; a.i.p., anterior intestinal portal; End'c.s., endocardial septum; F.G., fore-gut; Ht., heart; My'C., myocardium; N'ch., notochord; N'ch.T., anterior tip of notochord; n.F., neural fold; op.Ves., optic vesicle; p.C., parietal cavity (coelomic); Pr'a., proamnion; s.2, s.4, second and fourth mesodermal somites; V.o.m., omphalo-mesenteric vein.
The history of the mesoderm and coelom in the region of the head-fold deserves a special word. The coelom very early enlarges, either side of the head region, forming a pair of large spaces called the ammo-cardiac vesicles. These grow inward toward the head-fold, and by the time this is well established (4-6 pairs of somites) they push into the lower limb of the headfold, between its ectodermal and endodermal layers (Fig. 105). Here they finally meet and fuse, forming a median coelomic space bounded, of course, by a mesodermal wall. This is the rudiment of the pericardial cavity, and its formation and subsequent enlargement, bring about a wide separation of the ectoderm and endoderm, or as we may now say, of the somatopleure and splanchnopleure, of the head-fold, the latter being carried much the farther posteriorly (Figs. 98, 99). The later development of this region and of the vascular system in general, may more conveniently be postponed, until after an account of the history of the ectodermal and endodermal layers during these early stages.
We left the embryonic endoderm as a thin sheet of cells extending forward from the primitive streak, and we had described the important events connected with the formation of the fore-gut from its anterior margin (Figs. 97, 98). In the region between the fore-gut and the primitive streak, i.e., in the remainder of the embryonic region thus far formed, the endoderm and mesoderm are united in a secondary median fusion continuous with the cells of the primitive knot. Very soon the mesoderm and endoderm separate again and the median cells, then associated with the mesoderm, become arranged as a small longitudinal rod, the notochord (Fig. 102). Since the germ layers are fused in the primitive knot, and redifferentiate anteriorly out of it, it is of little consequence whether the notochord is regarded as of mesodermal or endodermal origin; it is more closely associated with cells which later are clearly mesodermal. As the primitive streak shortens and the primitive knot moves posteriorly, the notochord continues to differentiate, and so to elongate posteriorly.
Fig. 104. Chick embryo with three pairs of somites (about twenty-three hours). Dorsal view. From Lillie (Development of the Chick), a.c.v., Amniocardiac vesicle; a.o., inner margin of area opaca; F. G., fore-gut; N'ch., notochord; n.F., neural fold; pr.gr., primitive groove; s.l, s.2, s.3, first, second and third somites.
The cavity of the fore-gut is the rudiment of the pharynx. It remains a wide but shallow cavity throughout these early stages, open posteriorly, by way of the anterior intestinal portal, upon the surface of the yolk, where the endoderm remains spread out as a thin flat sheet (Figs. 97, 98, 103). Near the anterior extremity of the head-fold, the endoderm lining the pharynx comes ventrally into contact with the ectoderm, and later fuses with it. This forms the oral plate, which becomes the inner wall of the stomodceum; it is perforated early the third day of incubation. There is a pair of lateral extensions of the fore-gut, where its walls come into contact with the ectoderm, marking the positions of the future first gill pouches.
The rudiments of the central nervous system are the most conspicuous structures of the chick embryo during its early development. We have already noted the formation of the medullary plate, a broad ectodermal thickening developing precociously, before any other part of the embryo is definitely established (Figs. 94, 101). The medullary plate is converted into the medullary tube in the usual manner. A medullary groove appears soon after the head-fold becomes marked; the marginal medullary folds then become elevated and grow rapidly so that they form a pair of high conspicuous ridges on the surface of the anterior part of the blastoderm. They extend posteriorly, gradually diminishing in height, until finally they sink into the general level of the blastoderm in the region of the primitive knot. The formation of the medullary folds and central nervous system in general, as of the somites, notochord, and other parts of the embryo, is progressive posteriorly, so that steps in the formation of all these parts can be read in any series of transverse sections extending from the primitive knot anteriorly.
The elevated medullary folds, or neural folds, soon bend over toward one another and first meet a short distance back from the anterior limit of the head; this is known to be the region of the future mid-brain (Fig. 105). Here the folds soon fuse together forming a superficially continuous sheet of ectoderm, and an underlying medullary or neural tube. From this point the fusion extends very rapidly posteriorly, very slowly anteriorly. The point where the final closure occurs anteriorly, and consequently the final separation from the superficial ectoderm, is known as the neuropore; it is the region later distinguished as the lamina terminalis, and is regarded as the morphologically anterior limit of the brain. Topographically, however, this is not the most anterior part of the central nervous system, for during these early stages the rudiment of the brain grows forward and downward, in front of the fore-gut, so that its anterior end becomes bent like a crook. Its floor remains closely applied to the roof of the fore-gut, its extension being due to the growth of the anterior and dorsal regions chiefly (Fig. 99). Thus the morphologically anterior end of the brain really comes to lie on its antero-ventral aspect.
Certain details in connection with the neural folds and their closure deserve special mention. The actual crests of the neural ridges are flattened, and when the ridges fold together these flattened surfaces form a broad vertical contact. The neural tube is formed by the fusion of the lower or inner margins of these surfaces; the upper margins fuse across the mid-line, restoring a continuous superficial ectoderm. The cells left between the upper and lower margins, derived approximately from the very apices of the neural ridges, are thus left between the neural tube and the surface ectoderm; these are the neural crests (Fig. 102). They do not fuse across the midline, but remain as a pair of longitudinal bands along the dorsolateral surfaces of the neural tube. The neural crests are the rudiments of the ganglia of the cranial and spinal nerves; they are not uniformly developed throughout their extent and may be better marked in some sections than in others.
The anterior portion of the neural tube expands very considerably. Its most anterior region expands transversely, the lateral extensions thus formed representing the rudiments of the optic vesicles (Figs. 106, 107). This is obviously, therefore, the region of the primary fore-brain or prosencephalon, which now includes the entire anterior portion of the nerve tube. Its posterior limit is marked by a slight constriction just back of the optic vesicle rudiments. A little later (9-10 pairs of somites) another constriction appears marking the posterior limit of the mid-brain or mesencephalon. The third section of the brain, the hind-brain or rhombencephalon is much the longest section of the brain (Fig. 107). It is marked by a series of irregular constrictions, ultimately five in number forming six segments, or neuromeres, in this region. No posterior limit of the hind-brain can be made out in these early stages, but from later development it is known that all in front of the fourth mesodermal somites really belongs to the head, and consequently this level may be taken to mark approximately the posterior limit of the brain. The hind-brain narrows posteriorly and the remainder of the neural tube is the rudiment of the spinal cord, which remains narrowed and approximately uniform in diameter.
We may now return to a description of the formation and development of the embryonic vascular system, postponed from an earlier page. The formation of the vascular rudiments in the area opaca continues, hi the manner already described (Fig. 95), and when the extra-embryonic ccelom forms, dividing the mesoderm into somatic and splanchnic layers, the bloodvessels remain associated with the latter, i.e., with the splanchnopleure (Fig. 105, A). The vessels of the pellucid area develop first from its margin, near the area opaca, and gradually extend toward the embryo. They first appear shortly after the head-fold, just as the somites begin to be cut out. The formation of the conspicuous blood islands does not occur in the pellucid portion of the blastoderm; only the tubular vessels develop here. Thus the cellular or corpuscular elements of the early vascular system arise only in the area opaca, and most extensively in its posterior region. Following Riickert, we may say that the vessels of the pellucid area are formed by the rearrangement of small groups of cells in the splanchnic mesoderm of the area. These first form short sections of tubular vessels which very soon connect with the more peripheral vessels of the opaque area. In this way a continuous vascular network develops centripetally, finally reaching the embryo about the time six pairs of somites are formed.
Soon after this, vessels appear in the embryo itself. The first of these are the paired dorsal aortce of the body region (Fig. 105). These are to be regarded as enlarged and straightened inner (axial) margins of the vascular network of the pellucid area. Posteriorly they diverge widely, passing as the vitelline arteries, into the general vascular net (Fig. 107). Anteriorly they become prolonged forward toward the head region, where they connect with a pair of vessels differentiated within the mesenchyme of the head.
The heart is clearly a specialized portion of the system of vessels. Like the dorsal aortae, the rudiment of the heart is paired. When the vessels grow into the head region, which is now elevated above the level of the general splanchopleure, they are related to the ventral, rather than to the dorsal region (Fig. 105). The amnio-cardiac vesicles (see above) become vascularized in the same manner as the rest of the pellucid area, and a pair of ventral aortce is formed beneath the fore-gut (Fig. 103). Posteriorly to the opening of the fore-gut (anterior intestinal portal) these vessels diverge and pass into the vascular net as the rudiments of the vitelline veins. Reference to the transverse section illustrated in Fig. 105, A, shows that beneath the fore-gut the mesoderm has a considerable vertical extent. This is commonly regarded as splanchnic mesoderm, although it is a region where somatic and splanchnic layers become continuous. The paired rudiments of the heart pass along the inner or axial surface of this vertically extended mesoderm, and thus come into close relation, shortly fusing together to form a median, thin-walled tube. This becomes the endothelial lining of the heart; the muscular wall of the heart is formed by the addition of an external layer of mesoderm; for the splanchnic mesoderm of each side forms a fold around the endothelial rudiment (Fig. 105, B). These folds then approach and fuse across the mid-line, both above and below the endothelial tube. For a brief period after their fusion they remain in the midline forming a dorsal mesocardium, connecting the heart with the tissues just beneath the fore-gut, and a ventral mesocardium, extending from the heart to the splanchnopleure. The ventral mesocardium breaks away almost immediately after its formation, while the dorsal mesocardium remains for a time, but then disappears, except at the anterior and posterior extremities of the heart. The heart is then left as a short median tube consisting of endothelial and rudimentary muscular layers, freely suspended in a cavity which is the beginning of the pericardial cavity. Anteriorly it is continuous with a short pair of vessels extending into the head-fold; these are the ventral aortse mentioned above. Posteriorly the heart is directly continuous with the vitelline veins.
Fig. 106. Chick embryo with seven pairs of somites (about twenty-five hours). Dorsal view. From Lillie (Development of the Chick), o.c.s., Anterior cerebral suture; ceph. Mes., cephalic mesoderm; F.G., fore-gut; N'ch., notochord; n. T., neural tube; op.Ves., optic vesicle; Pr'a., proamnion; pr. str., primitive streak; s. 2, s. 7, pecond and seventh somites; V.o.m.j omphalomesenteric vein.
The dorsal aortse have meanwhile extended forward into the head, connecting with their cephalic sections which have developed directly within the mesenchyme of the region. These now unite with the ventral aortaa (Fig. 107, B) around the sides of the anterior end of the fore-gut; these connections are the rudiments of the first, or mandibular aortic arches. Thus the embryonic circulatory system is established and connected with the extra-embryonic vessels.
The Embryo Of About Thirty Hours
(10-12 Pairs Of Somites)
We may summarize, now, the events of this early period of development by mentioning the structures of the chick embryo at about its thirtieth hour of incubation, when it includes ten to twelve pairs of mesodermal somites (Figs. 107, 108).
In the freshly opened egg of this age, the circular blastoderm is seen to have extended approximately one-fourth of the way around the vitellus (i.e., its diameter is roughly 25 mm.). The embryo appears as a definite whitish streak, approximately 4 mm. in length, enlarged anteriorly, and posteriorly fading gradually into the surrounding area. Thus by far the greater part of the blastoderm is extra-embryonic. Three definite areas may be distinguished in the extra-embryonic blastoderm (Fig. 108). (1) Immediately surrounding the embryo is the clear area pellucida, now greatly restricted and not very distinct, save in the head region which includes the proamnion. The area opaca is now clearly divided into (2) the area vasculosa and (3) the area vitellina. The vascular area, save for a slight interruption anteriorly, has grown entirely around the embryo. It has extended both peripherally, over the yolk, and centrally encroaching upon the pellucid area. The broad area vitellina is further separable into two regions, an inner band, the vitellina interna, and a narrow area vitellina externa.
Fig. 107. Chick embryo with twelve pairs of somites (about thirty-three hours). From Lillie (Development of the Chick). A. Dorsal view of entire embryo. B. Ventral view of anterior end. A.C.S., Anterior cerebral suture; a.i.p., anterior intestinal portal; Ao., dorsal aorta; F.G., fore-gut; H.B., hindbrain; Ht., heart; M.B., mid-brain; op. Ves., optic vesicle; or.pl. , oral plate; pr.str., primitive streak; s.2, s.12, second and twelfth somites; v.ao., ventral aorta; V.o.m., omphalo-mesenteric vein.
The examination of sections through the blastoderm shows that the areas pellucida and vasculosa are composed of the three germ layers, the area vitellina of ectoderm and endoderm only. Except in the region about the head of the embryo, the area pellucida is extensively vascularized, but the vessels are still comparatively free from corpuscles. The pellucid and opaque areas are further characterized by the presence of extraembryonic coelom between the two sheets of mesoderm (Fig. 108). The vascular area is definitely limited peripherally by the sinus terminalis. The area vitellina externa is the region where the extension of the blastoderm is actually being carried forward. Here are found conditions similar to those described in the early part of this chapter. The extreme margin ("margin of overgrowth " Lillie) is formed by a single layer of superficial cells called "ectoderm," while between this margin and the area vitellina interna is a band (" zone of junction" Lillie) which is the equivalent of the marginal periblast, where the processes of true blastoderm formation are chiefly localized, and where the extra-embryonic portions of the two primary germ layers are being differentiated.
In enumerating the structures of the embryo itself (Fig. 107) we may begin posteriorly and pass forward, since, read in this direction, a series of transverse sections through a single embryo roughly represents the steps in the development of most of the parts fully formed at this time. Posteriorly there is a short primitive streak region with thickened neural plate and primitive groove. Passing anteriorly the neural folds rise and meet forming the neural tube; the notochord becomes distinguishable, and the mesoderm is differentiated into vertebral and lateral plates connected by the intermediate cell mass; the lateral plate is separated into somatic and splanchnic layers by the extraembryonic ccelom. In the trunk and hinder part of the head ten to twelve pairs of mesodermal somites are formed.
The long head region extends forward from about the fourth pair of somites (counting from the anterior end of the series). The head is now almost completely separated from the yolk ventrally, by the endodermal head-fold, while the ectodermal head-fold, which also separates the lateral surfaces of the head from the yolk and blastoderm, has more than half this extent. The brain is the most prominent feature of the embryo, and its primary divisions, fore-, mid-, and hind-brain, are well marked. Neural crests are distinct in sections from the mid-brain posteriorly. The fore-gut, widely open posteriorly upon the yolk by way of the anterior intestinal portal, is the only strictly endodermal structure thus far differentiated. This is the pharyngeal region, and the rudiments of the first or hyomandibular pair of gill pouches are indicated laterally, while in its floor is the oral plate.
The large vitelline veins unite in front of the level of the first somites, forming the heart, which now has both endothelial and muscular layers. The elongated heart lies in the pericardial cavity and is bent to the right. Anteriorly it continues into the diverging ventral aorta), which pass around the sides of the head, dorsally, as the first or mandibular pair of aortic arches; these are continuous with the wide lateral dorsal aorta? which pass backward and become continuous posteriorly with the extra-embryonic vascular network.
From this point onward we shall not attempt to continue a description of the development of the embryo as a whole. Next we shall outline the processes leading to the more complete formation of the embryo, and to its well-marked separation from the yolk and blastoderm on every side. Then, after a consideration of the formation of the embryonic membranes and appendages, we shall give, in the next chapter, a brief resume of the more essential steps in the organogeny of the later embryo. (Throughout this chapter, and more especially this part of it, the student is referred for additional details to such an account as Lillie's "Development of the Chick," and to the literature there cited).
The Separation of the Embryo from the Extra-Embryonic Structures
The remaining primitive streak region loses its typical structure soon after the fortieth hour of incubation (about 18 pairs of somites) (Fig. 112), and thereafter it remains, as a locus of continued cell proliferation known as the tail bud (Fig. 109, A). Shortly after this, about the forty-sixth hour (about 26 pairs of somites) a definite infolding of the blastoderm occurs posteriorly, marking the hinder limit of the embryo. This fold is the tail-fold, in many respects the counterpart of the headfold although appearing so much later. This fold, in which all three of the germ layers are involved (Fig. 109, J5), slowly grows forward forming a hind-gut, comparable with, though much less extensive than, the fore-gut. The tail bud, thus separated from the yolk beneath, continues to elongate slowly for a considerable time. Mesodermal somites continue to be added posteriorly, through the trunk region, which extends as far as the thirty-fifth somite, formed about the seventieth hour, and finally through the incurved tail region (Fig. 109, B). A total of fifty-two pairs of somites have been counted, although some ten of these disappear again during the eighth day of embryonic life, leaving a total of forty-two pairs.
Fig. 109. Sagittal sections through the posterior ends of chick embryos. From Lillie (Development of the Chick). A. Median section through an embryo with about twenty-one pairs of somites (about forty-four hours). B. Lateral section through embryo with about thirty-five pairs of somites (about seventytwo hours). All., allantois; Am., amnion; Am. cav., amnionic cavity; An. pi., anal plate; An. t., anal tube; Ao., dorsal aorta; Bl. v., blood-vessels in wall of allantois; c.C., central canal of spinal cord; CL, cloaca; Ect., ectoderm; Ectam., ectoderm of amnion; E.E.B.C., exoccelom; Ent. endoderm; Mes., mesoderm; Mesam., mesoderm of amnion; N'ch., notochord; n.T., nerve cord; p'a.G., postanal gut; p.i.p., posterior intestinal portal; s.A., segmental arteries, between the somites; So'pl., somatopleure; Spl'pL, Sp'pl., splanchnopleure; T.B., tail bud; t.f.So'pl., tail-fold in somatopleure; t f.Sp'pL, tail-fold in splanchnopleure.
The formation of the tail-fold is but one phase in the general process of the separation of the embryo from the remainder of the blastoderm, and at the same time from the yolk. The formation of the head-fold we have already described. We may recall that this extended backward along each side of the head. Posteriorly this now becomes continued either side of the trunk, by a somewhat similar lateral fold, which finally reaches to the tail-fold. These folds involve both somatopleure and splanchnopleure (Fig. 110). The lateral folds gradually extend inward toward the mid-line beneath the embryo, converting the flat sheet of endoderm into a median fold above the yolk; this is the intestinal groove, the rudiment of the true intestine (Fig. 110). The head- and tail-folds continue to extend posteriorly and anteriorly, respectively, increasing the extent of the foreand hind-gut. These connect with the intestinal groove by the anterior and posterior intestinal portals.
Fig. 110. Diagrammatic transverse section through the region where the gut is open out over the yolk (yolk-stalk umbilicus), in a chick of about fortyeight hours (about twenty-eight pairs of somites). After Duval. a, Dorsal aorta; c, ccelom; ebc, exocoelom; ig, intestinal groove; la, lateral folds of amnion; vv, vitelline vein.
Gradually these folds all approach beneath the embryo, cutting it off from the yolk, and marking a clear distinction between embryonic and extra-embryonic regions. Finally, about the fifth day, the lateral folds of the splanchnopleure fuse together extensively, and the gut is entirely closed off except in a restricted region in the hinder part of the trunk (Fig. 115).
This remains open to the yolk nearly throughout embryonic life. The walls of this open portion of the gut elongate into a tubular stalk known as the yolk stalk. In reality this is double, composed of an outer wall of somatopleure, and an inner tube of splanchnopleure; the former is sometimes known as the somatic stalk, the latter as the splanchnic stalk or yolk stalk proper. The splanchnic stalk is the pathway by which the blood-vessels pass between embryonic and extra-embryonic structures. The narrow space between the two stalks is, of course, coelomic, connecting embryonic or true coelom, and extra-embryonic coelom or exocoelom.
Fig. 111. Ventral view of the anterior end of a chick embryo with sixteen pairs of somites (about thirty-eight hours). From Lillie (Development of the Chick), a.i.p., Anterior intestinal portal; au.P., auditory pit; B.a., bulbus arteriosus; F.B., fore-brain; Inf., infundibulum; op.Ves., optic vesicle; Or.pl., oral plate; Pr'am., proamnion; s.4, fourth somite; Tr.a., truncus arteriosus; v.Ao., ventral aorta; Ven., ventricle; V.o.m., omphalo-mesenteric (vitelline) vein; VII VIII, acustico-facialis ganglion.
The inner limbs of the lateral folds of the somatopleure form the body watt of the embryo, of which we may now describe lateral and ventral surfaces, as well as the dorsal, which has hitherto been the only free surface of the embryo, save in the head and tail.
The Establishment of the External form of the Embryo
Rapid growth and extensive changes in the external form of the embryo have been proceeding, as the embryo has been folded off from the yolk and blastoderm. The embryo of thirty hours was nearly straight and lay flat upon the yolk, save that a simple flexure of the head was caused by the growth of the fore-brain downward in front of the fore-gut. Continued enlargement of the head (brain) leads to two important general modifications of the form of the embryo, first, a twisting of the embryonic body, and second, the formation of certain bendingg or flexures in the axis of the head and trunk. As the cavities of the brain enlarge, a sharp bending of the neural axis appears between the fore- and mid-brain, soon involving the entire mid-brain. The development of this cranial flexure (Fig. 112), combined with the extensive enlargement of the fore-brain, would force this region down into the yolk but for the fact that the head now falls over upon one side, its left, which now comes to lie over upon the yolk. The trunk retains for a time its original position flat upon the yolk, and thus a twist appears just back of the head. This change in the position of the head begins about the thirty-sixth hour (15 pairs of somites), the cranial flexure having appeared somewhat earlier, about the thirtieth hour (10-12 pairs of somites). Gradually the region of the twist moves posteriorly, leaving the more anterior parts thrown over (Figs. 113, 114), and finally, about the close of the fourth day, the entire embryo comes to lie upon its left side. Long before this, about the end of the second day, another flexure of the longitudinal axis appears; this is the cervical flexure. A broad ventral sweep through the entire hind-brain region, extending a short distance into the neck, results in the more complete flexure of the fore-brain, which thus comes to be directed posteriorly rather than ventrally, its true ventral surface approximating the ventral surface of the pharyngeal region. Later the entire trunk becomes arched dorsally, bringing the head and tail fairly close together on the ventral side (the morphologically ventral side now lies turned toward what was the right side, previous to the torsion of the embryo). During the later phases of embryonic development these last flexures disappear to a considerable extent, leaving permanently well-marked only the original cranial flexure.
Fig. 112. Chick embryo with twenty pairs of somites (about forty-three hours). Dorsal view. From Lillie (Development of the Chick). A.o.m., Vitelline artery; au.P., auditory pit; Cr.FL, cranial flexure; D.C., ductus Cuvieri; Dienc., diencephalon; Mesenc., mesencephalon; Metenc., metencephalon; Myelenc. 1, and 2, anterior and posterior divisions of the myelencephalon; Op.Ves., optic vesicle. Ph., pharynx; pr.str., primitive streak; s.2, s.5, etc., second, fifth, etc., somites; Telenc., telencephalon; Vel.tr., velum transversum; Ven., ventricle.
Fig. 113. Chick embryo with twenty-seven pairs of somites (about fortyeight hours). Viewed from above. After Lillie. a, Auricle; am, posterior margin of amnionic folds; c, carotid loop; cf, cranial flexure; d, diencephalon; dC, ductus Cuvieri; g\, g 2 , gs, first, second and third gill clefts; i, isthmus; I, lens; ma, mandibular arch; ms, mesencephalon; mt, metencephalon; o, otocyst (auditory sac); just to the right of the otocyst is a thickening representing the ganglion of the VII and VIII cranial nerves; r, retinal layer; S2, sw, $20, second, tenth, and twentieth somites; t, tail-bud; v, ventricle; va, vitelline artery; vv, vitelline vein; 1, 2, 3, first, second and third aortic arches; V, ganglion of V cranial nerve.
Fig. 114. Chick embryo with adjacent portion of area vasculosa, with thirtyfive pairs of somites (about seventy- two hours). Dorsal view. From Lillie (Development of the Chick), a. a., 1, 2, 3, 4, First to fourth aortic arches; Am., amnion; AT., branches of vitelline arteries; Atr., auricle; A.V., vitelline artery; B.A., bulbus arteriosus; cere. Fl., cervical flexure; cr. FL, cranial flexure; D.C., ductus Cuvieri; D.V., ductus venosus; Ep., epiphysis; Gn. V., ganglion of V cranial nerve; Isth., isthmus; Jug., external jugular vein; Md., mandibular arch; M.M., maxillo-mandibular branch of V cranial nerve; Myel., myelencephalon; olf.P., olfactory pit; Ophth. ophthalmic branch of V cranial nerve; Ot., otocyst; 8.2, s.10, s.20, etc., second, tenth, twentieth, etc., somites; V., branches of the vitelline veins; V.c.p., posterior cardinal vein; V.umb., umbilical vein; V.V., vitelline vein; V.V.p., posterior vitelline vein; W.B., wing-bud.
The general changes in form and the development of the externally visible organs may best be appreciated by studying such a series of embryos as that illustrated by Duval (Atlas d'embryologie, Paris, 1889; Plates VII-X). We may merely call attention to a few of the more striking features. The foreand hind-limbs appear, at approximately the same time, about the eightieth hour, as bud-like outgrowths from the body wall (Fig, 114). These enlarge rapidly; their segments are distinguishable at the beginning of the seventh day, and during the eighth day they assume the outlines of the wings and legs, During the eighth day too, the heart is drawn within the body wall, the visceral arches and gill clefts disappear, save as they remain represented in the adult by the jaws (mandibular arch) and the external auditory meatus (hyomandibular gill cleft), and the abdominal viscera are much enlarged. The following day the facial portion of the head begins to enlarge, the feather papillae become well marked, and the general musculature begins to develop to such an extent as to affect the general contours of the body.
The Embryonic Membranes and Appendages
We have now to describe the development of certain extremely important structures which are primarily extra-embryonic, and have to do with the protection, nutrition, and respiration of the embryo, rather than with the actual formation of proper organs. These structures are the yolk-sac, the amnion, and the allantois; strictly speaking the allantois arises as an embryonic structure, but it soon becomes extra-embryonic in position, and its functional value is derived from its later relation. These three structures are all characteristic of the embryos of the higher Craniates Reptiles, Birds, and Mammals, and consequently these classes are often grouped together under the term Amniota.
We have seen that at the thirtieth hour of incubation the blastoderm has extended over approximately one- fourth the surface of the yolk. Throughout its pellucid and vascular areas it is divided by the exocoelom, into somatopleure and splanchnopleure (Fig. 108). Its outlying area vitellina for the most part consists of ectoderm and endoderm only, and is bounded peripherally by a region where the further extension of the blastoderm is being effected. The extension of the blastoderm continues in the manner previously described, the differentiation of the mesoderm and the subsequent formation of exoccelom, somatopleure, and splanchnopleure, slowly proceeding centrif ugally . During the fifth day the blastoderm extends completely over the yolk, save for a narrow circular space, opposite the embryo, which remains uncovered until toward the close of embryonic life (Fig. 115). The mesoderm and associated structures of the blastoderm push around more slowly, but finally arrive at the blastodermal margin, which becomes a narrow ring called the yollt-sac umbilicus. The yolk-sac itself is the splanchnopleural portion of the extra-embryonic blastoderm, one surface applied directly to the surface of the yolk, the other forming the inner lining of the exoccelom (Fig. 115). The lower pole of the yolk-sac is open at the yolk-sac umbilicus, and the upper pole is of course directly continuous with the yolk stalk or splanchnic stalk, and therefore with the embryonic midgut (Fig. 115). While the yolk-mass is now practically enclosed in a layer of endoderm (splanchnopleure) it cannot be said to be included within the enteron, for it remains extra-embryonic in position until the very close of embryonic life. The yolk is never taken directly into the embryonic digestive cavity and there absorbed. The endodermal lining of the yolk-sac early becomes differentiated as a yolk-digesting and absorbing organ, and the nutritive materials taken up by it are received into the yolk-sac circulation and carried to the embryo.
FIGS. 115, 116, 117. Diagrams of the relations of the embryonic membranes in the chick. Figures and description from Lillie (Development of the Chick). The ectoderm and endoderm are represented 1 by plain lines; the mesoderm by a cross-hatched line or band. The yolk-sac is represented by broken parallel lines. In Fig. 115 the allantois is represented as a sac. In Figs. 116 and 117, where it is supposed to be seen in section, its cavity is represented by unbroken parallel lines. The stalk of the allantois is exaggerated in all the diagrams to bring out its connection with the embryo.
Fig. 115. Fourth day of incubation. The embryo is surrounded by the amnion which arises from the somatic umbilicus in front and behind; the sero-amniotic connection is represented above the tail of the embryo; it consists at this time of a fusion of the ectoderm of the amnion and chorion. The allantois is represented as a sac, the stalk of which enters the umbilicus behind the yolk-stalk; the allantois lies in the extra-embryonic body-cavity (exoccelom), and itsmesodermal layer is fused with the corresponding layer of the chorion above the embryo. The septa of the yolk-sac are represented at an early stage. The splitting of the mesoderm has progressed beyond the equator of the yolk-sac, and the undivided portion is slightly thickened to form the beginning of the connectivetissue ring that surrounds the yolk-sac umbilicus. The ectoderm and endoderm meet in the zone of junction, beyond which the ectoderm is continued a short distance. The vitelline membrane is ruptured, but still covers the yolk in the neighborhood of the yolk-sac umbilicus. The albumen is not represented in this figure. (For explanation of lettering see Fig. 117.)
Fig. 116. Ninth day of incubation. The yolk-sac umbilicus has become much narrowed, it is surrounded by the mesodermal connective-tissue ring, and by the free edges of the ectoderm and endoderm. The vitelline membrane still covers the yolk-sac umbilicus and is folded into the albumen. The allantoia has expanded around the amnion and yolk-sac and its outer wall is fused with the chorion. It has pushed a fold of the chorion over the sero-amniotic connection, into which the mesoderm has penetrated, and thus forms the upper fold of the albumen-sac. The lower fold of the albumen-sac is likewise formed by a duplication of the chorion and allantois; it must be understood that lateral folds are forming also, so that the albumen is being surrounded from all sides. The stalk of the allantois is exaggerated so as to show the connection of the allantois with the embryo; it is supposed to pass over the amnion, and not through the cavity of the latter, of course. (For explanation of lettering see Fig. 117.)
Fig. 117. Twelfth day of incubation. The conditions represented in Fig 116 are more advanced. The albumen-sac is closing; its connection with the cavity of the amnion by way of the sero-amniotic connection will be obvious. The inner wall of the allantois has fused extensively with the amnion. The umbilicus of the yolk-sac is much reduced, and some yolk protrudes into the albumen (sac of the yolk-sac umbilicus). Alb., albumen; Alb.S., albumen-sac; AIL, allantois; All.I., inner wall of allantois; All.C., allantoic cavity; AILS., allantoic stalk; All. -\- Am., fusion of allantois and amnion; Am., amnion; Am.C., amnionic cavity; Chor., chorion; C.T.R., connective-tissue ling; Ect. t ectoderm; E.E.B.C., exoccelom (extra-embryonic body-cavity); EnL, endoderm; Mes., mesoderm; S.-Am., sero-amnionic connection; S.Y.S.U., sac of the yolksac umbilicus; Umb., umbilicus; V.M., vitelline membrane; Y.S., yolk-sac, Y.S.S., septa of yolk-sac.
The yolk-sac is characterized by a wealth of blood vessels. The vessels of the vascular area, already mentioned, are the beginnings of the yolk-sac circulation. In the rich network of small vessels, larger pathways soon develop; the first of these are the paired anterior vitelline veins, which pass from the anterior margin of the blastoderm directly into the posterior end of the heart (Fig. 118, A). Soon after these become well established, the pair of vitelline arteries develop (about thirty-eight hours) as lateral branches of the dorsal aorta in the middle trunk region (Fig. 118, A, B). These arteries become distributed by large trunks throughout the yolk-sac, and supply its abundant capillaries. The blood re-collects into the marginal sinus or sinus terminalis, and passes thence into the anterior vitelline veins. By the end of the third day the two anterior vitelline veins have fused, just within the sinus terminalis, and between this point and the heart the right vein begins to disappear (Fig. 118, B). Soon it disappears entirely, and for a time the left anterior vitelline vein alone returns the blood to the heart, but before long, venous trunks appear, parallel with the main branches of the vitelline arteries, and by the end of the fourth day (Fig. 118, C) the greater part of the blood returns to the heart through these, the lateral vitelline or omphalo-mesenteric veins, which unite into the ductus venosus immediately before entering the heart. Thus both the supply and the return of the yolk-sac circulation pass along the wall of the yolk stalk or splanchnic stalk.
The digestive and absorptive surface of the yolk-sac becomes enormously increased by a folding process, which begins at the time the larger vessels begin to be marked out. The folding (Fig. 116) finally comes to be very complicated, and accompanying the folds is a continuation of the network of large capillaries which thus come to have an intimate and extensive relation with the nutritive supply. The inner surface of the yolk-sac finally acquires a structure not unlike that of the lung, its spaces or meshes being filled with the yolk material.
The yolk-sac increases in complexity throughout the period of embryonic development. About two days before hatching, while still a comparatively large organ, it is pushed into the body cavity of the embryo by way of the somatic stalk cavity (coelom), apparently through the contraction of the walls of the allantois and amnion. Its entrance is completed a day or so before hatching, and a few days after hatching both the yolk and the yolk-sac itself become completely absorbed into the intestine of the young chick, and the yolk stalk is definitely closed.
Fig. 118. Diagrams of the circulation in the chick embryo and area vasculosa. The vascular network of the area vasculosa is omitted for the most part.
A. Anterior and central parts of the embryo and vascular area at about thirtyeight hours (sixteen pairs of somites). Viewed from beneath. After Popoff.
B. Median and anterior parts of vascular area and embryo at about seventy-two hours (twenty-seven pairs of somites). Viewed from beneath. After Popoff.
C. The main vascular trunks of the fourth day. After Lillie (modified), a, Dorsal aorta; aa, aortic arches (first and second in A, second, third and fourth in C); ac, anterior cardinal vein; al, allantois; au, auricle; b, bulbus arteriosus; dC, ductus Cuvieri; dv, ductus venosus; ec, external carotid artery; h, .heart;
internal carotid artery; la, lateral dorsal aorta; h, left anterior vitelline vein; p, anterior intestinal portal; pc, posterior cardinal vein; pv, posterior vitelline vein; rv, right anterior vitelline vein; s, sinus venosus; t, sinus terminalis; tr, venous trunks of the area vasculosajf, ventricle; va. vitelline artery; vv, vitelline or omphalo-mesenteric vein.
We must again return to the embryo of thirty hours to describe the formation of the amnion, and the associated chorion, which are derived from the somatopleural portion of the extra-embryonic blastoderm. In a general way we may say that these structures arise from the crests of the outer limbs of those folds which cut the embryo off from the extraembryonic blastoderm. The first indication of the amnion is in the blastoderm, just in front of the head of the embryo, in the anterior region of the so-called proamnion, where, about the thirtieth hour, a slight transverse thickening of the ectoderm becomes apparent in sections. This thickening is the beginning of the ectamnion, which plays an important role throughout the formation of the true amnion. The proamnion, it will be recalled, is that part of the blastoderm which for a time remains mesoderm-free. Hence no coelom, somatopleure, and splanchnopleure are present in the region where the amnion makes its first appearance.
Just in front of the head, the region of the ectamnion, endoderm as well as ectoderm, becomes folded upward as the head-fold of the amnion (Fig. 99). This begins to extend backward over the head of the embryo, but before it has gone very far the mesoderm, with the accompanying exoccelom, extends into the fold, and the endodermal layer is withdrawn. The lateral extremities of the head-fold of the amnion turn posteriorly along the sides of the head, as the lateral folds of the amnion. These are somatopleural from their beginning, and may be described as elevations of the blastoderm in the region where the outer limbs of the lateral embryonic folds pass into the general surface of the blastoderm (Fig. 110).
As these folds of the amnion extend posteriorly, the ectamnion serves actively to draw the more lateral regions toward the mid-line where they are fused together, and in this way the lateral folds are gradually drawn up over the embryo (Lillie). This process occurs progressively in a posterior direction. About the time the embryo has thus become covered over as far back as the level of the vitelline arteries (48 hours, 26-28 pairs of somites), a posterior tail-fold of the amnion appears, (Fig. 109, A), similar to its head-fold save that it is somatopleural, i.e., contains coelom and mesoderm, from its commencement. This tail-fold extends laterally and then anteriorly, and soon its extremities meet the lateral folds. About the seventieth hour (35 pairs of somites) the amnionic folds are completely closed together above the embryo; this closure occurs about at the level of the hind-limb buds.
As the somatopleural amnion folds come together above the embryo, the ectodermal and mesodermal layers of each side fuse together and the original contact surfaces break through, so that the coelomic cavities of the amnionic folds become continuous across the mid-line. This results in the formation of two complete membranes, each composed of two cell layers, around the embryo. The inner of these, close to the embryo, is the true amnion, and the space between this and the embryo is the amnionic cavity (Figs. 119, 125). From the method of its formation it will be seen that the amnion is continuous with the somatic stalk, while the amnionic cavity is a space entirely outside the embryo, and has no connection with any other cavity. The outer membrane is now called the chorion. It is, of course, the somatic portion of the blastoderm, and at this time has no direct connection with any other structure. The space between amnion and chorion is exoccelom. This cavity and the amnionic cavity are filled with a watery fluid. Later on, as we shall see, this space is largely occupied by the allantois, which comes into intimate connection with the inner surface of the chorion.
Fig. 119. Transverse section of chick embryo with thirty-five pairs of somites (about seventy-two hours), passing through the region of the twentythird somite. After Lillie. a, Amnion; ac, amnionic cavity; ao, dorsal aortse; c, embryonic ccelom; ch, chorion; d, dermatome; ebc, exocoelom; g, rudiment of spinal ganglion; m, mesonephric tubules; my, myotome; p, posterior cardinal vein; s, sclerotome; sa, sero-amnionic connection; sc, subcardinal vein; so, somatic mesoderm; sp, splanchnic mesoderm; v, vitelline artery; TF.Wolffian duct.
One important fact has not been mentioned thus far. In the region where the amnionic folds finally close together over the embryo, the amnion and chorion remain firmly united (Fig. 119). This forms the sero-amnionic fusion, which affects in an important way the later arrangement of these membranes, as well as the disposition of the allantois, when it extends into this region (Figs. 115, 116).
The regular arrangement of the lateral folds of the amnion is disturbed somewhat by the twisting of the embryo, which is going on as these folds are becoming elevated and fused. As the embryo turns upon its left side the amnionic folds do not similarly alter their morphological relation to what was originally the median plane. That is, they continue to close together along what was originally marked out as the mid-line, and they finally close immediately above the right hind-limb bud. It follows from this that the left lateral fold becomes much the more extensive, passing from the lower (left) side of the embryo, around to the upper (right) side and thus covering the whole morphologically dorsal surface of the embryo, while the right lateral fold covers only a portion of the upper (morphologically right) surface of the embryo. This is correlated with the fact that the right fold is thicker than the left and is usually thrown into irregular transitory folds or wrinkles.
Soon after the amnion is fully formed, muscle fibers are differentiated in its mesodermal layer. Waves of contraction then slowly pass along the amnion and keep up a movement of the amnionic and coelomic fluids. It is also supposed that they keep the embryo in slight motion, preventing adhesions between the embryo and its appendages. The amnion also serves, probably, as a water cushion, protecting the embryo against deforming pressures. The later relations of the amnion to the allantois and the albumen of the egg will be mentioned below.
The allantois is an embryonic appendage of the greatest importance. Unlike the yolk-sac, amnion, and chorion, it is developed from the embryo itself, and not from -the extraembryonic structures. When fully developed it is the most extensive of all these accessory organs, the arrangement of which becomes considerably modified by its growth. Functionally, too, it is of the utmost importance, it is primarily the embryonic respiratory organ, but it serves also as a reservoir for excretory products, and through the development of muscle fibers in its wall it effects certain movements of the embryo, and assists in the final inclusion of the yolk-sac within the embryonic body.
The morphological relations of the allantois to the embryo will be better understood if we recall certain facts regarding the development of the tail. The tail-fold of the blastoderm differs essentially from the head-fold in that the blastoderm where it appears includes mesoderm, and is already differentiated into somatopleure and splanchnopleure (Fig. 109, A). Preceding the formation of the tail-fold of the somatopleure, the splanchnopleure is folded forward beneath the posterior end of the embryo, establishing a short hind-gut. Soon after, the true, or somatopleural, tail-fold pushes inward, a short distance behind the splanchnic fold. The embryonic region immediately anterior (morphologically) to the somatopleural tail-fold is the rudiment of the tail, the tail bud (Fig. 109, B). As this elongates, a posterior extension of the hind-gut continues into it, as the postanal gut, for the anus is formed on the ventral gut is increased by the forward
Fig. 120. Model of part of the side of the base of the tail. The anterior extent of the hind allantoic stalk. After Ravn ( modified). The broken line marks the cavity of the hind-gut and allantoic extension of the splancnnotoic stalk; ap, anal plate; hi, cut surfface of hind-limb bud; o, viteiiine beneath the hind-gut, between the splanchnopleural and somatopleural tail-folds, is mesodermal and is in reality a deep ventral mesentery. The cavity of the hind-gut now pushes downward into this ventral mesentery, forming an elongated depression, just in front of the anal region (Fig. 109, B).
stalk. a .Cut edge of amnion; Dalian- pleura! tail-fold. The region
This groove-like outgrowth is the rudiment of the allantois, which thus makes its appearance about the commencement of the third day of incubation (about 28 pairs of somites). In front of this the hind-gut narrows again; the dorsal portion of this expansion, and the region just posterior to it, form the rudiment of the cloaca (Fig. 120).
The allantois is clearly an outgrowth or appendage of the hind-gut, and consequently its cavity is directly continuous with the enteron; it is lined with endoderm, and its outer wall is mesodermal. Before the close of the fourth day (Fig. 121) the allantois has extended for some distance out into the exocoelom, lying to the right of the tail. Its terminal portion has enlarged into a dilated vesicle connected with the gut by a narrow tubular allantoic stalk. It has an abundant blood supply, derived from a pair of branches of the dorsal aorta, the allantoic arteries; its blood collects into a single allantoic 'vein and is returned to the heart through the large left umbilical vein (Fig. 138).
Fig. 121. Median sagittal section through posterior end of four-day chick. After Gasser (Maurer). al, Allantois; am, amnion (tail-fold) ; c, cloaca; m, cloacal membrane; n, notochord; r, rectum; s, spinal cord; y, wall of yolk-sac (endoderm and splanchnic mesoderm).
Once established, the allantois grows very rapidly through the exoccelom, between the chorion and the yolk-sac and amnion (Figs. 115, 116). Its vesicle enlarges rapidly and by the end of the sixth day has covered the entire embryo; two days later it has passed nearly around the yolk-sac. As the allantois enlarges it effects certain important fusions with other structures. Superficially it fuses with the chorion, the mesodermal layers of the two membranes uniting so intimately that in effect the chorion forms a part of the outer membrane of the allantois. Along the dorsal surface of the embryo the inner wall of the allantois meets the amnion, and the apposed mesodermal layers of these two membranes fuse together, forming a layer of muscle fibers which function for a time similarly to those of the amnion, and then disappear.
A modified region of the allantois should be mentioned here. During the early days of incubation the albumen of the egg condenses toward what may be described as the posteroventral side of the yolk, from which it is separated only by the remains of the vitelline membrane, in the region where the yolk-sac remains open (Fig. 116). About the ninth or tenth day, the albumen, through continued loss of water, forms a relatively small, dense mass, incompletely separated from the yolk by the intervention of the yolk sac (Fig. 117). The allantois reaches the albumen first from the lower side. It is now fused with the chorion throughout its extent, and as it pushes onward, it sends a short fold in between the albumen and the yolk-sac, and a longer limb around the outer surface of the albumen, almost covering it over. These folds of fused chorion and allantois are the beginnings of the albumen sac. The albumen sac is completed by the downgrowth of a similar fold of fused chorion and allantois from the upper side. These folds finally enclose the yolk completely (Fig. 117). The upper fold is not quite like the lower on account of the obstruction offered to the progress of the allantois, by the sero-amnionic fusion. When the rapidly extending allantois reaches this area of fusion of the amnion and chorion, it pushes the chorion out above the fused area, carrying a fold of the chorion before it in its onward extension (Fig. 116). This fold of the chorion, with the allantois within, is similar to the outer limb of the ventral part of the albumen sac, but the inner limb of the latter is consequently not represented on the dorsal side. Dorsally the inner wall of the albumen sac is formed only by the original chorion of the region, the seroamnionic connection being included (Fig. 117). About the time the albumen sac is completed, by the fusion of the membranes forming its outer wall, the sero-amnionic fusion becomes perforated, and the albumenous material partly passes into solution in the amnionic fluid. For the most part, however, the albumen is absorbed through the walls of the albumen sac. This is fully accomplished toward the close of the embryonic period, and the albumen sac then remains as an appendage of the yolk-sac, with which it passes into the body of the embryo at the end of incubation.
The most characteristic feature of the allantois is the extreme vascularity of its outer wall. The withdrawal of the albumen from the greater part of its surface leaves the fused chorion and outer allantoic wall in direct apposition to the shell membrane. The chorionic layers become extremely thin and the shell membrane becomes very porous, so that there is an easy exchange of gases between the outside air and the capillaries of the outer allantoic wall. These capillaries are very wide and extremely abundant, so altogether the allantois is a very efficient respiratory organ.
The inner wall of the allantois, as already mentioned, fuses with the amnion in the region dorsal to the embryo, during the second week of incubation. Here as elsewhere in the inner wall, muscle fibers may develop. Later on, as the period of incubation draws toward its close, it fuses also with the yolksac. The cavity of the allantois remains connected with the cloaca by the tubular allantoic stalk. It is filled with a fluid containing the excretory products, received from the cloaca where the excretory ducts discharge.
Von Baer's account of the principal events associated with the hatching of the young chick, is summarized in the two following paragraphs quoted from Lillie (" Development of the Chick," p. 232).
" About the fourteenth day the growing embryo accommodates itself to the form of the egg so as to lie parallel to the long axis with its head usually toward the broad end near to the airchamber. Sometimes, however, the embryo is turned in the reverse position (Von Baer). The head is bent toward the breast, and is usually tucked under the right wing. Important changes preparatory to hatching take place on the seventeenth to nineteenth days. The fluid decreases in the amnion. The neck acquires a double bend so that the head is turned forward, and, in consequence, the beak is toward that part of the membranes next to the air-chamber. The intestine is retracted completely into the body-cavity, and on the nineteenth day the yolk-sac begins to enter the body-cavity. On the twentieth day the yolk-sac is completely included, and practically all the amniotic fluid has disappeared. The chick now occupies practically all the space within the egg, outside of the air-chamber. The umbilicus is closing over. The ductus arteriosi begin to contract, so that more blood flows through the lungs. The external wall of the allantois fused with the chorion still remains very vascular.
"Now, if the chick raises its head, the beak readily pierces the membranes and enters the air-chamber. It then begins to breathe slowly the contained air; the chick may be heard, in some cases, to peep within the shell two days before hatching, a sure sign that breathing has begun. But the circulation in the allantois is still maintained and it still preserves its respiratory function. When the chick makes the first small opening in the shell, which usually takes place on the twentieth day, it begins to breathe normally, and then the allantois begins to dr}' up and the circulation in it rapidly ceases. It then becomes separated from the umbilicus, and the remainder of the act of hatching is completed, usually on the twenty-first day."
References to the literature are given at the end of Chapter V.
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Outlines of Chordate Development: 1. Amphioxus | 2. Early Frog | 3. Later Frog Organogeny | 4. Early Chick - Embryonic Membranes and Appendages | 5. Later Chick - Organogeny | 6. Early Mammal - Embryonic Membranes and Appendages | Figures
Reference: Kellicott, W. E., (1913) Outlines of chordate development. New York: H. Holt and Company.