Book - The development of the chick (1919) 2
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Lillie FR. The development of the chick. (1919) Henry Holt And Company New York, New York.
- 1 Part I The Early Development to the End of the Third Day
- 2 Chapter II The Development Prior to Laying
Part I The Early Development to the End of the Third Day
Chapter II The Development Prior to Laying
During the growth period the germinal vesicle has increased to an enormous size (455 x 72 ju in an ovum 37 mm. in diameter, Fig. 8). It lies in contact with the vitelline membrane. The margins of the lenticular nucleus are folded into the interior in such a way that sections give an effect of rod-shaped bodies springing from the membrane which were doubtfully interpreted as
Fig. 8. — Vertical section of germinal vesicle of hen's egg after Sonnenbrodt. Size of egg 37 mm. in diameter; size of nucleus 455Mx72/i.
chromosomes by Holl. The real chromosomes are however in the center in the form of double rods (Fig. 8). The maturation divisions of the hen's egg have not been described, but we have fortunately a very good account of the maturation and fertilization of the pigeon's egg by E. H. Harper, which furnishes the basis of the following description:
The wall of the germinal vesicle begins to break down in ovarian eggs of about 18.75 mm. diameter, the full size of the egg of the pigeon being about 25 mm. Part of the fluid contents of the germinal vesicle flows out and forms a layer outside the disintegrating wall (Fig. 9). The chromosomes and nucleoli form a group near the center of the upper plane surface of the germinal
Fig. 9. — Vertical section of the germinal vesicle and part of the germinal disc of an ovarian ovum | inch in diameter; pigeon, x 385. (After Harper.) Chr., Chromosomes. Gr., Granulosa. G. V., Wall of germinal vesicle.
vesicle. The first maturation spindle is formed before ovulation, containing eight quadruple chromosomes (tetrads). The spindle is still in the equatorial plate stage when the ovum is grasped by the mouth of the oviduct (Fig. 10). The bulk of the substance of the germinal vesicle soon forms a yolk-free cone extending from the maturation spindle deep into the superficial i _^
yolk. The outer end of the Fig. 10. — Vertical section of the germinal spindle is in almost imme- ^li^c of the pigeon's egg showing the diate contact with the SUr- ^""^ maturation spindle. The egg was c e .. T.I clasped hy the funnel of the oviduct.
lace 01 the ovum. In the o -^ oaaa .vr* u x
8.oO P.M. X 2000. (After Harper.)
later stages of formation of m. Sp. 1, First maturation spindle. Tetr., the first polar body each Tetrad.
tetrad, or quadruple chromosome, separates into two dyads or double chromosomes, and the members of each pair of dyads separate and approach opposite ends of the spindle (anaphase).
Thus at each end of the spindle there are eight dyads. Those at the outer end then enter a Httle bud of jorotoplasm projecting above the surface of the germinal disc, and this bud with the dyads is cut off as the first polar body, which lies in a depression of the germinal disc beneath the vitelline membrane (Fig. 11). Eight dyads, therefore, remain within the germinal disc.
A second maturation spindle is then formed almost immediately, apparently without the intervention of a resting stage of the nucleus, and takes a radial position similar to that occupied by the first, with the dyads forming an equatorial plate (Fig. 11).
Fig. 11. — Second maturation spindle and first polar body of the pigeon's egg; a combination of two sections. 8.15 p.m. x 2000. (After Harper.) m. Sp. 2, Second maturation spindle, p. b. 1, First polar body. v. M., Vitelline membrane.
Each dyad then divides along the preformed plane of division, and the daughter-chromosomes diverge towards opposite poles of the spindle. The outer end of the second maturation spindle then enters a superficial bud of the protoplasm of the germinal disc similar to that of the first maturation spindle; and this bud together with the contained chromosomes becomes cut off as the second polar body.
The result of these processes of maturation is the formation of three cells, viz., the two polar bodies and the mature egg. The polar bodies are relatively very minute and soon degenerate completely.
After the formation of the second polar body there remain in the egg eight chromosomes, each of which represents one quarter of an original tetrad. These form a small resting nucleus known as the egg-nucleus or female pronucleus. It is many times smaller than the original germinal vesicle (Fig. 12), and it rapidly withdraws from the surface of the egg to a deeper position near the center of the germinal disc. (Concerning the
Fig. 12. — Egg nucleus (female pronucleus) and polar bodies
of the pigeon's egg. (After Harper.) 8.30 p.m. x 2000.
E. N., Egg nucleus, p. b. 1, First polar body. p. b. 2, Second polar body. p'v. S., Perivitelline space, v. M., Vitelline membrane.
general theory of the maturation process see E. B. Wilson, "The Cell in Development and Inheritance/' the Macmillan Company, New York.)
The spermatozoa traverse the entire length of the oviduct and are found in the uppermost portion in a fertile hen. The period of life of the spermatozoa w^ithin the oviduct is considerable, as proved by the fact that hens may continue to lay fertile eggs for a period of at least three weeks after isolation from the cock. After the end of the third week the vitality of the spermatozoa is apparently reduced, as eggs laid during the fourth and fifth weeks may exhibit, at the most, abnormal cleavage, which soon ceases. Eggs laid forty days after isolation are certainly unfertilized, and do not develop (Lau and Barfurth). The so-called parthenogenetic cleavage of such eggs is merely a phenomenon of fragmentation of the protoplasm; there is no true cell-division.
The ovum is surrounded immediately after ovulation, that is in the infundibulum, by a fluid containing spermatozoa in suspension. In the egg of the pigeon from twelve to twenty-five spermatozoa immediately bore through the egg-membrane and enter the germinal disc, within which the heads, which represent the nuclei of the spermatozoa, enlarge and become transformed into sperm nuclei (Fig. 13). In the hen's egg five or six usually enter. The fate of the middle piece and tail of the spermatozoa is not known in
birds, but it is improbable that they furnish any definitive morphological element of the fertilized egg. At the time of entrance of the spermatozoa the first maturation spindle is in process of formation ; it lies in the center of a group of granules at the surface of the egg, which is bounded by a non-granular zone of protoplasm, called by Harper the polar ring, in which the sperm-nuclei accumulate. External to the polar ring the protoplasm is granular again (Fig. 14).
The sperm-nuclei remain quiescent while the polar bodies are being formed, and, when the egg nucleus is reconstituted, one of them, which may be called the male pronucleus or primary sperm nucleus, moves inwards and comes into contact with the egg nucleus (Fig. 15). The opposed faces of the conjugating nuclei become flattened together, until the contours form a single sphere, the first segmentation nucleus, in which a partition separates the original components, viz., the sperm and egg nucleus. The partition apparently disappears. However, it is very unlikely that a complete intermingling of the contents of the two germ-nuclei takes place, because in other groups of animals where the processes have been more fully studied, it has been determined that each germ-nucieus forms an independent group of chromosomes of the same number in each.
Shortly after its formation, the first segmentation nucleus prepares for division in the usual karyokinetic way. The first segmentation (or cleavage) spindle thus formed lies near the center of the germinal disc a short distance beneath the surface and its axis is tangential to the surface, or, in other words, at right angles to the axis of the egg. The fertilization may be considered to be completed at this stage.
Fig. 13. — Stages in the transformation of sperm heads into the sperm nuclei from the ovum of the pigeon. x2000. (After Harper.) The order of stages is indicated by the letters a — g.
The entrance of several spermatozoa appears to be characteristic of vertebrates with large ova; thus for instance, it has been described in selachii, some amphibia, reptiles, and birds. Such a condition is known as polyspermy; it is normal in the forms mentioned, but occurs only under abnormal conditions in the
Fig. 14. — Horizontal section of the germinal disc of a pigeon's ovum immediately after ovulation, x 125. (After Harper.) N., Nucleus, probably first maturation spindle, p. r.,
Polar ring. Sp. N., Sperm nuclei.
Fig. 15. — Vertical section of the pigeon's egg showing germ nuclei (pronuclei) in the center of the disc, x 2000. 10.40 p.m. (After Harper.)
great majority of animals. Harper observed that the number of sperm-nuclei formed in the pigeon varied from, twelve to twentyfive in different cases. Only one of these serves as a functional sperm-nucleus; the remainder or supernumerary sperm-nuclei migrate, as though repelled, from the center towards the margins and deeper portions of the germinal disc, where they become temporarily active, dividing and furnishing a secondar}- area of small cells (accessory cleavage) surrounding the true cleavagecells produced by division of the central portion of the disc around the descendants of the segmentation nucleus. It has been supposed by some authors who studied the selachii that the descendants of the supernumerary sperm-nuclei form functional nuclei of the so-called periblast, but this view has been disproved for the pigeon (Blount), in which it can be demonstrated that the supernumerary sperm-nuclei have but a brief period of activity, and then degenerate.
III. Cleavage of the Ovum
The fertilized ovum is morphologically a single cell, with a single nucleus, the first segmentation nucleus. The living protoplasm is aggregated in the germinal disc, and the remainder of the ovum is an inert mass of food material destined to be assimilated by the embryo which arises from the germinal disc. The first step in the development is a series of cell-divisions of the usual karyokinetic type, restricted to the germinal disc, which rapidly becomes multicellular. As the early divisions take place nearly synchronously in all the cells, there is a tendency for the number of the cells to increase in geometrical progression, furnishing 2-, 4-, 8-, and 16- etc., celled stages; but sooner or later the divisions cease to be synchronous. All of the cells of the body are derived from the germinal disc, and the nuclei of all cells trace their lineage back to the first segmentation nucleus. The supernumerary sperm-nuclei do not take part in the formation of the embryo.
Cell-division is the most conspicuous part of the early development; hence this period is known as the cleavage, or segmentation, period. But it should be remembered first, that cell-division is as constant a process in later embryonic stages as in the cleavage period, and second, that it is probable, though little is known yet about this subject in the bird's egg, that other important phenomena are going on during the cleavage period.
The type of cleavage exhibited by the bird's egg is known as meroblastic, for the reason that only a part of the ovum is concerned, viz., the germinal disc. This is obviously due to the great amount of yolk (see Introduction, pp. 11 and 12).
To understand the form and significance of the cleavage of the bird's egg, it is necessary first of all to gain a clear idea of the structure of the germinal disc and its relations to the yolk. At the time of the first cleavage the germinal disc is round in surface view and about 3 mm. in diameter; the center is white and is surrounded by a darker margin about 0.5 mm. wide. These two zones have been compared to the pellucid and opaque areas of later stages. We shall call the outer zone the periblastic zone, or simply periblast. In section, the germinal disc is biconvex, but the outer surface which conforms to the contour of the entire egg is much less arched than the inner surface. The disc is everywhere separated from the yellow yolk by a layer of white yolk (Fig. 2) ; on the other hand, there is no sharp separation between the disc and the white yolk. The granules of the latter are largest in the deeper layers and there is a gradual transition from them to the smaller yolk-granules with which the disc is thickly charged (Fig. 19). It is practically impossible in a section to say where the protoplasm of the disc ceases; it is indeed probable that it extends some distance into the white yolk both beneath and around the margins of the disc. Thus in Figure 21 a cone, apparently of protoplasm, extends into the neck of the latebra a considerable distance. In other cases it does not extend so far.
The Hen's Egg
The form of cleavage of the hen's egg is illustrated in Fig. 16, A-E. The first cleavage appears in surface view as a narrow furrow extending part way across the germinal disc (Fig. 16 A). According to Patterson it occurs just as the egg is entering the isthmus about three hours after the estimated time of fertilization. While the ends of the first cleavage furrow are still extending towards the periblast, the second division begins. It is a vertical division in each cell like the first, and the two furrows meet the first cleavage furrow at right angles. They may meet the first furrow at approximately the same point, in which case they form an approximately straight hne (Fig. 16 B), or they may meet the first cleavage furrow at separate points, in which case the intervening part of the first furrow becomes bent at an angle, forming a cross furrow. The third set of cleavage planes are vertical like the preceding planes, but they tend to be variable otherwise. In Fig. 16 C there is shown an eight-celled stage in which three of the new furrows are approximately at right angles to the second cleavage plane, but other arrangements are found.
Fig. 16. — Five stages of the cleavage of the hen's egg. (A, B, D and E after KolUker; C after Patterson.)
A. First cleavage furrow (x 14). The egg came from the lower end of the oviduct. . , .
B. Four-celled stage (xl7); from the uterus.
C. Eight-celled stage (x 18). u ^ ^a^
D. Nine central and sixteen margmal cells (x about iOj.
E. Late cleavage stage (x about 22).
Before describing the later cleavage stages, we should note certain important relations of the first four or eight cells: First, these are not complete cells in the sense that they are separate from one another. They are, indeed, areas with separate nuclei marked out by cleavage furrows in a continuous mass of protoplasm. The furrows do not cut through the entire depth of the germinal disc, and the cells are therefore connected below by the deeper layer of the protoplasm; nor do the furrows extend into the peril^last, and all the cells are therefore united at their margins by the unsegmented ring of periblast. Second, according to several observers, the center of the cleavage, i.e., the place where the first two cleavage furrows cross, is sometimes excentric. It was believed by those who emphasized this point, that the displacement is towards the posterior end of the blastoderm; but Coste, for instance, failed to note any excentricity, and Patterson noticed both conditions, and showed that the displacement might even be towards the anterior end of the blastoderm. In the pigeon, according to Miss Blount's observations recorded below, excentricity appears to be exceptional; moreover, the excentric area may bear any relation whatever to the future hind end of the embryo, so that in the pigeon it will not bear the interpretation that has been placed on it in the hen's egg.
The following cleavages (after the eight-celled stage) in the hen's egg are very irregular, but two classes of furrows may be distinguished in surface view: (1) those that cut off the inner ends of the cells, and (2) those that run in a radial direction. The furrows of the first class produce a group of cells that are bounded on all sides in surface view, l)ut these are, at first, still connected below l)y the deeper protoplasm. They may be called the central cells. These are bounded by cells that are united in the marginal periblast, and thus lack marginal boundaries as well as deep boundaries; they may be called the marginal cells (Fig. 16 D). The distinction between central and marginal cells is one of great importance which should be clearly grasped.
In the surface views of later cleavages the following points should be noted: (1) the group of central cells increases by the addition of cells cut off from the inner ends of the marginal cells, and by the multiplication of the central cells themselves; (2) the marginal cells increase by the formation of new radial furrows. The increase of the central cells is much more rapid than that of the marginal cells, and the cells themselves are much smaller than the marginal cells, both because of their mode of origin and also because of their more rapid multiplication. The area of the central cells is also constantly increasing, with consequent reduction of the marginal zone (Fig. 16 E). Emphasis has been laid by several authors on the excentric position of the smallest cells, and the inference has been drawn that these represent the hinder end of the glastodisc. Similar excentricity in the pigeon's egg is without reference to the future embryonic axis (see Fig. 18).
Fig. 16 A. IMedian section of a blastoderm of the hen's egg which showed about 64 cells in surface view (after Patterson). S.c, segmentation cavity.
But the surface views do not show what is going on in the deeper parts of the germinal disc. At the eight-celled stage a narrow space appears in the depth of the central portion of the blastoderm approximately between protoplasm and yolk; this is the segmentation cavity which furnishes a lower boundary to the central cells. In later stages it extends peripherally to the inner margin of the periblast, and thus all of the central cells become completely bounded. A new class of cleavage planes then forms in these cells after the thirty-two-celled stage, horizontal or parallel to the surface; in this way the central part of the blastoderm becomes two cell-layers deep, and later several layers deep. The' segmentation cavity never cuts under the marginal cells, which remain united below and at their margins by the periblast (Fig. 16 A).
In the older accounts of the horizontal cleavages by Kolliker, Duval and others these are represented as forming before the segmentation cavity, thus leaving the deeper cell in continuity ^\ith the yolk. Such cells are then supposed to continue budding off cells from their upper surfaces. But this view has been shown to be incorrect by the observations of ^Miss Blount on the pigeon described below and by Patterson on the hen included above.
The Pigeon*s Egg
The cleavage of the pigeon's egg has been worked out in detail by Miss Blount; as it must be made the basis of the description of the formation of the germinal wall and the germ-layers in the absence of anj^ consistent account for the hen's egg, it will next be described. The fundamental features of the cleavage are the same as in the hen's egg, so that the description need not be repeated.
The feature to be particularly emphasized in the cleavage of the pigeon's egg is the occurrence of a secondary or accessory cleavage in the marginal zone or periblast (Figs. 17 and 18 A). When the origin of these cells is traced it is found that they arise around the supernumerary sperm-nuclei, which accumulate and multiply in the periblast. The complete history of these nuclei has been worked out ])y Harper and Blount, so that there can be no doubt as to their derivation. Another interesting point illustrated by the figures is that the marginal cells have a peripheral wall wherever the accessory cleavage occurs, but between the groups of accessory cleavage cells the marginal cells are continuous with the periblast (Figs. 17 and 18 A,) as they are everywhere in the hen's egg. In a section of a germinal disc, showing the accessory cleavage (Fig. 20), it is seen that the peripheral boundary of the marginal cells cuts under the margin for a considerable distance.
The accessory cleavage becomes manifest at the time of api^earance of the first cleavage plane, and increases in amount up to about the 32-celled stage, and thereafter gradually decreases until it completely disappears (Figs. 18 B, C, and D). The peripheral boundaries of the marginal cells disappear "pari passu, and, when the accessory cleavage is finally wiped out, the marginal cells are everywhere continuous with the periblast, as in the hen's egg (Figs. 18 B and C). In some eggs the accessory cleavage is much more extensive than in others; indeed, in some it appears to be entirely absent, but this is relatively rare. In the stage shown in Fig. 18 B. for instance, there is usually considerable accessory cleavage; but in this egg there is none. The variation is obviously due to variations in the number of supernumerary spermatozoa, such as mav readilv occur.
Fig. 17. — Photograph of an eight-celled pigeon ovum (after Mary Blount). 2.45 a.m. Accessory cleavage (ac. el.) in the marginal zone bounding the segmented area. Vesicles, appearing black in the photograph, are seen on the surface of the yolk beyond the marginal zone of the germinal disc. Orientation as in Fig. 18.
The question arises whether the disappearance of the cellwalls around the sperm-nuclei is caused by degeneration of the latter, or is simply a later syncytial condition in the periblast in which the sperm-nuclei are embedded. There can be little doubt that the former alternative is correct. While in the stages of the accessory cleavage, sperm-nuclei are readily found both in the accessory cleavage-cells and also in the unsegmented periblast (Figs. 19 and 20), they decrease in number as the accessory cleavage planes disappear, and when the latter are entirely lost the periblast is absolutely devoid of nuclei. Fragmentation of the sperm-nuclei is a frequent accompaniment of their disappearance. Thus the accessory cleavage is a secondary and transient feature of the cleavage of the pigeon's egg due to polyspermy. After it has passed, the ovum is in precisely the same condition as the hen's ovum of the same stage of development. In the hen's egg Patterson has shown that there is a very limited and inconspicuous accessory cleavage (see Fig. 16 C) around the fewer supernumerary sperm-nuclei that occur. But most of these nuclei in the hen tend to pass into deeper portions of the disc and there undergo complete fragmentation without producing superficial furrows.
Fig. 18. — Photographs of the cleavage of the pigeon's ovum (after Mary Blount). The figures are so arranged that the axis of the shell is across the page with the large end to the left. The future axis of the embryo is therefore inclined 45° to the margin of the page with the anterior end to the right above.
A. A very regular sixteen-celled stage; accessory cleavage well shown; thouffh not well focused on the lower margin. 3.45 a.m.
B. Approximate thirty-two celled stage. There is no accessory cleavage in this case. The formation of the central from the marginal cells may be readily observed in this figure. 5.15 a.m.
C. Later stage of cleavage. 7.10 a.m.
D. CleavaQ:e at 9.30 a.m. The marginal cells are now becoming separated peripherally from the periblast which has received its nuclei from them.
Fig. 19. — Transverse section of the blastoderm of a pigeon's egg about 8| hours after fertilization (4.45 a.m.). (After Blount.) 1, Accessory cleavage. 2, Migrating sperm-nuclei, a, b, c, d, Cells of primary cleavage.
Fig. 20. — Transverse section of the blastoderm of a pigeon's egg at the end of the period of multiplication of sperm-nuclei, about 10 hours after fertilization (6.30 A.M.). (After Blount.)
1, Accessory cleavage around the sperm-nuclei. 2, Marginal cells; sharply separated from the sperm-nuclei. 3, Central cells. 4, Sperm-nuclei.
Another feature brought out by these photographs requires emphasis. The periblast ring shows no definite outer margin. but beyond the zone of the accessory cleavage there may occur two or three concentric circles variously indicated (Fig. 17). Vacuoles, appearing black in the photographs, are very common in the outer zones. These appearances indicate that the peiiblastic protoplasm extends farther out in the superficial white yolk than is usually believed to be the case; and this suggests an interesting comparison with the teleost ovum, where the periblastic protoplasm surrounds the entire yolk as a ver}- thin layer. Sections confirm the idea that the periblastic protoplasm has an extension beyond the so-called margin of the blastodisc. Some eggs show a more definite margin than others; it may be that there is a periodic heaping of the periblast at the margins, for which again an analogy may be found in teleosts.
Although the smallest cells may be more or less excentric in the segmented germinal disc of the pigeon, their position bears no constant relation to the future embryonic axis. They may lie in this axis in front of or behind the middle, or to the right or left of it (cf. Fig. 18 A-D).
At the eight-celled stage a horizontal fissure begins to appear beneath the central cells (Fig. 19). This marks the full depth of the blastoderm at all stages, and the several-layered condition arises by horizontal cleavages between this and the surface. Comparison of Figs. 19, 20, and 22, drawn at the same magnification, will show that the depth does not increase by addition of cells cut off from below, as was once supposed to be the case in the bird's ovum. The horizontal fissure not only marks the full depth of the blastoderm, but it also indicates the site of the segmentation cavity which arises gradually by accumulation of fluid between the cells and the underlying unsegmented protoplasm and yolk. The segmentation cavity gradually extends towards the margin of the blastoderm, but it is bounded peripherally by the zone of junction between the marginal cells and the periblast.
IV. Origin of the Periblastic Nuclei, Formation of the Germ- Wall
Our knowledge of this part of the subject in the hen's egg is very incomplete, and the various accounts are contradictory. The reason for this is the great difficulty of securing a complete series of stages, and of arranging them in proper sequence. There is no way of timing the development, so that one has to judge the sequence of the stages, all of which come from the utertis, by the degree of formation of the shell, by the size of the cells and by the appearance of the sections. This can be at best only approximate; and, as the securing of any given stage is largely a matter of chance, no one has, as a matter of fact, secured a complete series. In the pigeon, on the other hand, the time since laying the first egg is a fairly exact criterion of the stage of development of the second egg. It has, therefore, been possible to secure a complete series, and the subject has been worked out by Miss Blount, whose publications furnish the basis of the following account.
The periblast ring is entirely devoid of nuclei after the supernumerary sperm-nuclei have degenerated. The marginal cells become greatly reduced in size owing to multiplication and continuous production of central cells, and their nuclei thus approach more and more closely to the periblastic ring. The scene then changes; the marginal cells cease to produce central cells; when their nuclei divide the peripheral daughter-nuclei move out into the periblast, which is thus converted into a nucleated syncytium. The periblastic nuclei multiply rapidly and invade all portions of the periblastic ring, which maintains its original connection with the white yolk. Not only do the periblastic nuclei invade the periblastic ring, but some of them also migrate centrally into the protoplasm forming the floor of the segmentation cavity. They do not, however, reach the center, but leave a non-nucleated sub-germinal area, corresponding approximately to the nucleus of Pander, free from nuclei. The subgerminal syncytium may be known as the central periblast to distinguish it from the marginal periblast. They are, of course, continuous. In sections one has the appearance of nuclei in the yolk, for there is no sharp boundary between peril)last and yolk (Fig. 22). The syncytium, which has received its nuclei from the marginal cells, is the primordium of the germ-wall (Figs. 21, 22, 23, 24).
There is a snarp contrast between the segmented blastoderm and the syncytial periblast not only in structure but also as regards fate. The marginal cells constitute a zone of junction between blastoderm and periblast. Thus in Fig. 22 it will be observed that the large marginal cells on each side are continuous with the periblast, and nuclei are found in the periblast both central and peripheral to the zone of junction. The latter forms a ring around the blastoderm. It persists during the expansion of the blastoderm over the surface of the yolk.
Fig. 21. — Longitudinal section of the blastoderm of a pigeon's egg at the time of disappearance of the sperm-nuclei. On the left (anterior) margin, the marginal cells have become open, that is, continuous with the periblast, as contrasted with Fig. 20. About 11 hours after fertilization (7.00 A.M.). (After Blount.)
1, Marginal cells. 2, Cone of protoplasm. 3, Marginal periblast. 4, Neck of latebra. 5, Yellow yolk.
Fig. 22. — Transverse section through the center of the blastoderm of a pigeon's egg, 14^ hours after fertilization (10.30 a.m.). (After Blount.)
1, Marginal cells. 2, Marginal periblast. 3, Nuclei of the subgerminal periblast.
The blastoderm now begins to expand, owing largely, at first, to additions of cells to its margin cut off from the periblast. The central as well as the marginal periblast contributes to the blastoderm, but the former appears to be rapidly used up. The marginal periblast, which is commonly called the germ-wall from this stage, on the other hand grows at its periphery while it adds cells to the blastoderm centrally, and thus it moves out in the white yolk, building up the margin of the blastoderm at the same time. The original group of central cells appears to correspond approximately to the pehucid area; the additions from the germwall would thus constitute the opaque area.
Some phases of these processes are illustrated in Figs. 23 and 24. In the vertical section. Fig. 23, the surface of the germwall next the blastoderm is indented as though for the formation of superficial cells. Along the steep central margin of the germwall groups of cells are apparently being cut off and added to the cellular blastoderm. In the horizontal section, Fig. 24, the process of cellularization at the central margin of the germ-wall is apparently proceeding rapidly.
The superficial cells thus added to the margin of the cellular blastoderm become continuous with the ectoderm, and the deeper layers later form the yolk-sac entoderm which becomes continuous with the embryonic entoderm secondarily. We can thus distinguish a syncytial, more peripheral, and a cellular, more central, portion of the germ-wall.
Fig. 24. — Part of the margin of a horizontal section through the blastoderm of a pigeon's egg about 25 hours after fertiHzation (8.50 p.m.). (After Blount.)
1, Periblast nuclei. 2, 3, Cells organized in the periblast. 4, A cell apparently added to the blastoderm from the periblast. 5, Vacuoles.
In later stages the central margin of the syncytial part of the germ-wall becomes much less steep, owing apparently to active proHferation of cells. This is illustrated in Fig. 25. Later yet the external margin extends out peripherally and forms a short projecting shelf, appearing wedge-shaped in section (Figs. 28 A, etc.). This we shall call the margin of overgrowth.
Fig. 25. — Outlines of the margins of transverse sections of the blastoderm of pigeon's eggs; 26 (A), 28 (B), and 32 (C) hours after fertilization. (After Blount.)
Thus we may distinguish the following zones: (1) margin of overgrowth; (2) zone of junction with the yolk (syncytial germwall); (3) the inner zone of the germ-wall, and (4) the original cellular blastoderm (pellucid area) Fig. 29.
V. Origin of the Ectoderm and Entoderm
The ectoderm and entoderm are the primary germ-layers, out of which all organs of the embryo differentiate; hence great importance attaches to the mode of their origin. But until recently it was not possible, in the case of the chick, to decide between three conflicting views. These are: (1) The theory of delamination, viz., that the superficial cells of the segmented blastoderm form the ectoderm and the deeper cells the entoderm; in other words, that the blastoderm splits into the two primary germlayers. This is the oldest view, but it has not lacked support in recent times, e.g., by Duval. (2) The theory of invagination, viz., that the primary entoderm arises as an ingrowth from the margin of the blastoderm. This view, which was supported by Haeckel, Goette, Rauber, and some others, brings the mode of gastrulation in the bird into line with lower vertebrates. (3) A third and relatively recent point of view is that the primary entoderm arises as an ingrowth of cells from the germ-wall, more particularly from the posterior portion. This view, put forward by Nowack, has been adopted in substance by 0. Hertwig (Handbuch der vergl. u. exp. Entwickelungslehre der Wirbeltiere).
The conflict of opinion was due to the fact that the critical stages occur prior to laying, and no one had investigated a complete series of stages until recently. The investigations of J. T. Patterson on the pigeon have, however, cleared the matter up. A very complete series of stages of the pigeon's ovum was studied, with results that are consistent in themselves and that agree with the principles of formation of the primary germ-layers in the lower vertebrates.
The first step in the process of gastrulation, or formation of the primary entoderm, is a thinning of the blastoderm, wliich begins sHghtly posterior to the center and rapidly involves a sector of the posterior third of the blastoderm. This process occurs between twenty and thirty-one hours after fertilization. It is due apparently to the gradual rearrangement of the cells in a single layer. A late stage of this process is shown in Figure 26, which represents a complete longitudinal section through the Ijlastoderm thirty-one hours after fertilization. It will be observed that the anterior portion of the blastoderm is several cells thick (26 A), but as one passes towards the posterior end the number of layers becomes less, and is reduced to a single layer at the extreme posterior end. Here and there, e.g., at X, the arrangement of the cells indicates that cells of the lower layer are entering the upper layer. It is obvious that such a process must result in increase of the diameter of the blastoderm, and Patterson states that the average diameter twentyone hours after fertilization is 1.915 mm. and 2.573 mm. ten hours later. The thinning also involves enlargement of the segmentation cavity, which may now be known as the subgerminal cavity.
Hand in hand with the thinning out there takes place an interruption of the germ-wall at the posterior end, so that in this region the margin no longer enters a syncytium but rests directly on the yolk (cf. anterior and posterior ends of Fig. 26).
Figure 27 is a reconstruction of the stage in question. The germ-wall, represented by the parallel lines, is absent at the posterior end. Here the cells of the blastoderm rest directly on the yolk. The sector bounded by this free margin and the broken line represents the area of the blastoderm that is approximately one cell thick. The figures 2 to 7 indicate regions approximately two to seven cells thick.
Gastrulation begins by an involution or rolling under of the free margin, as though the free edge were tucked in beneath the blastoderm. The involuted edge then begins to grow forward towards the center of the blastoderm, and thus establishes a lower layer of cells, the primary entoderm. As soon as this process is started the margin of the blastoderm begins to thicken, and thus the inner layer of cells (entoderm) and the outer layer of cells (ectoderm) are continuous with one another in a marginal thickening (Fig. 28).
The margin of invagination is known as the Up of the blastopore or primitive mouth; the space between this margin and the yolk is the blastopore, and the space between the entoderm and yolk, derived from part of the subgerminal cavity, is the archenteron or primitive intestine.
Fig. 27. — Diagrammatic reconstruction of the blastoderm of which a longitudinal section is shown in Fig. 26.
C-D., Plane of Fig. 26.
G. W., Germ-wall. 1, 2, 3, 4, 5, 6, and 7 indicate regions of the blastoderm which are approximately from 1 to 7 cells deep respectively. The broken line around 1 indicates the region where the blastoderm is approximately one cell deep, x 27.2. (After Patterson.)
The first stage in the formation of the entoderm is interpreted as involution of the free margin, and this view is supported by the fact, determined by Patterson, that the antero-posterior diameter of the blastoderm is shorter than the transverse diameter during this process, whereas previously the blastoderm was approximately circular. An even stronger support of this view is furnished by experiments which demonstrate that injuries to the margin made just prior to gastrulation appear later in an anterior position in the entoderm (Patterson). But after the margin has thickened the farther extension of the entoderm is due, largely at least, to ingrowth from the marginal thickening.
Patterson also believes that the thickening of the margin is due not so much to multiplication of cells in situ as to immigration of cells from the sides. This view is also supported by experiments.
Fig. 29. — Diagrammatic reconstruction of the blastoderm of a pigeon's egg, 36 hours after fertilization; from the same series as Fig. 28. X 27.2. (After Patterson.)
E., Invaginated or gut entoderm. O., Margin of overgrowth. PA., Outer margin of pellucid area. R., Margin of invagination (dorsal lip of blastopore). S., Beginning of yolk-sac entoderm. Y., Yolk zone. Z., Zone of junction.
The arrows at the posterior margin indicate the direction of movement of the halves of the margin. The circles in the pellucid area indicate yolk masses in the segmentation cavity.
Figure 29 is a reconstruction of a blastoderm in the stage of Fig. 28, that is at the height of gastrulation. The margin of overgrowth (cf. Fig. 28 O) is represented by the area O; the zone of junction by the ruled area Z; the inner portion of the germ-wall by the area with large granules Y. These zones constitute the opaque area. The circles in the pellucid area represent megaspheres, that is yolk-masses cut off from the floor of the subgerminal cavity and lying in the latter (cf. Fig. 28 M). The invaginated entoderm is represented by the crossed area E; the lip of the Vjlastopore, where ectoderm and entoderm are continuous, by the region R.
Fig. 3L — A diagrammatic reconstruction of the blastoderm represented in Fig. 30. (After Patterson.)
R., Mass of cells left after closure of blastopore. S.G., Anterior portion of subgerminal cavity not yet crossed by the entoderm. Other abbreviations as in Fig. 29.
The last three or four hours prior to laying witness the closure of the blastopore. A comparison of Figs. 27 and 29 will show that the blastopore has become considerably narrower in the later stage. It will be observed that the posterior ends of the germ-wall are approaching. Finally they come into contact, and the blastopore is closed. During this process the lip of the blastopore is not cut off externally, but on the contrary comes to lie within the germ-wall at the posterior margin of the pellucid area.
This is illustrated by Figs. 30 and 31, representing a longitudinal section and a reconstruction of a blastoderm three hours before laying. Considering the reconstruction first, it will be noted that the lip of the blastopore, R, now lies within the blastoderm at the posterior margin of the pellucid area. The greater portion of the pellucid area is now two-layered owing to the continued expansion of the entoderm E, which has met and united with the germ-wall at the sides. The section (Fig. 30) passes longitudinally through the center of the blastoderm. The mass of cells at D represents the original lip of the blastopore. It is continuous with the germ-wall behind and with the entoderm in front. The latter is not a continuous layer (Fig. 30 A), and the cells are not coherent. It is probable that the extension of the entoderm is due largely to independent migration of the cells. Subsequently the entoderm cells unite to form a coherent layer of flattened cells. (Chap. IV)
In some cases the closure of the blastopore takes place in such a way as to produce' a marginal notch, which is referred to again in connection with the primitive streak (Chap. IV).