In Figure 9m, for about thirty sections (one tenth the entire length of the embryo), behind the section represented in the last figure, there is a very gradual change in the embryo, converting the deep groove, mg in Figure 9l, into the shallow slit, pg, Figure 9m.
There is no line of demarcation between the typical medullary groove region of Figure 9l and the equally typical primitive groove region represented in Figure 9m. As was noted in the preceding stage, the medullary folds are quite continuous with the folds of the primitive streak, and the medullary groove with the primitive groove; so that, unless we take the dorsal opening of the neurenteric canal as the point of separation, there is no line of division between these structures. The entoderm (en) and the lateral regions of the ectoderm (ec) and mesoderm (mes) in Figure 9m are about as they were in Figure 9l, but in the middle line is seen a compact mass of cells forming the primitive streak (ps), with the shallow primitive groove (pg) on the dorsal side. The cells on each side of the primitive groove and for a short distance below it are compact, as is shown in the figure, but as we pass ventrally and laterally they become looser and more angular to form the lateral sheets of mesoblast (mes), very much as is the case in the chick and other forms. For a few sections posterior to the one shown in Figure 9m the primitive streak may be seen, then it disappears, and only the ectoderm and entoderm remain as thin sheets of tissue above the yolk.
STAGE VII
Figures 10 AND 10a (Plates XV., XVI.)
Although of practically the same size as the preceding, this stage has advanced sufficiently in development to warrant a description.
The medullary folds are apparently still slightly open for the greater part of their length, though they are evidently fused together in the head region, except at the extreme end. Transverse sections, however, of Figure 12, in which the medullary folds, from the dorsal aspect, seemed open (mg) as in Figure 10, have shown that these folds are fused throughout their length.
The first cerebral vesicle (v′) is clearly indicated as an enlargement of the anterior end of the nervous system, and a slight enlargement (v″) posterior to the first probably represents the second cerebral vesicle.
There are now eight pairs of somites (s).
The head-fold (h) now shows in both dorsal and ventral views, appearing in the former, when viewed by transmitted light, as a lighter, circular area on either side of the body, just posterior to the hinder edge of the amnion.
The head-fold of the amnion (a) has extended about twice as far backward as it did in the preceding stage.
Owing to the opacity caused by the medullary folds being in contact along the middle line, the notochord is no longer visible in surface views.
The head at this stage begins to push down into the yolk in a strange way that will be described later.
STAGE VIII
Figures 11-11k (Plates XVI., XVII., XVIII.)
This stage is about one fourth longer than the preceding. The medullary canal is enclosed throughout its entire length, though it appears in surface view (Fig. 11) to be open in the posterior half (mc) of the embryo. An enlargement of this apparently open region at the extreme posterior end (pg) is probably caused by the remains of the primitive groove or the neurenteric canal, and a slight opacity at the same point may be caused by the primitive streak. The anterior end of the neural tube is bent in a ventral direction (v), as in the preceding stage. The somites (s) now number fifteen pairs; they are somewhat irregular in size and shape.
The head-fold is not so striking a feature as in the preceding stage. The head-fold of the amnion (a) now covers nearly two thirds of the embryo. The heart (ht) is seen as a dark, rounded object projecting to the right side of the neural canal, just anterior to the first somite. The vitelline blood-vessels are just beginning to form, but are not shown in the figure.
The depression of the anterior region that was noted in the preceding stage has advanced so far that a considerable part of the embryo now projects forward under the blastoderm. In some cases it is almost concealed in a dorsal view; in other cases it may easily be seen through the transparent membranes, especially after clearing.
In opening eggs of this stage one is at first apt to underestimate the size of the embryos, since the anterior part of the embryos cannot be seen until after they are removed from the yolk and are viewed from the ventral side.
The embryo from which the series of transverse sections of this stage was made, while of the same state of development as that shown in Figure 11, was more fully covered by the blastoderm than is shown in the surface view in question.
Figure 11a passes through the tip of the head. Dorsal to the embryo is the ectoderm and a thick mass of yolk (y). The amnion (a) is seen as an irregular membrane which entirely surrounds the head. The medullary canal (mc) is entirely closed except at the extreme anterior end, which is bent downward so that the opening is on the ventral side. The nervous (nl) and epidermal (ep) layers of the ectoderm are in contact throughout, but are clearly distinguishable because of the difference in the compactness of their cells.
In Figure 11b is represented a section, behind the preceding, which passes through the posterior tip of the turned-under anterior end (mc′). Here the medullary canal is closed both above (mc) and below (mc′). The amnion (a) has about the same appearance as in the more anterior section, but there is here a considerable space, filled with mesoblast (mes), between the nervous (nl) and epidermal (ep) layers of ectoderm.
Figure 11c is twenty sections, about one tenth the length of the embryo, posterior to the one last described. The large mass of overhanging yolk (y) is still present, as is also the amnion (a), though the latter no longer passes entirely around the embryo; only the true amnion could be made out. The thickened walls of the medullary canal have reduced that cavity to a narrow, Y-shaped slit (mc). The notochord (nt) is very slender in this region, compared to its diameter farther toward the posterior end. The enteron (ent) is a large cavity, whose wall is made up of loosely arranged cells except around a median, ventral depression where the cells are more compact. This depression may be traced through ten or fifteen sections and may represent the beginning of the thyroid gland, though this point was not worked out with certainty. Surrounding the notochord and enteron is a loose mass of typical, stellate mesoblast cells (mes), which are cleft on either side to form the anterior limit of the body cavity (bc). Between the body cavity below and the enteron above, on each side, is a small blood-vessel (bv) which when followed caudad is found to open ventrally and medially into the anterior end of the heart.
Figure 11d is about a dozen sections posterior to the preceding. The appearance of the overhanging yolk (y), of the amnion (a), and of the notochord (nt) is about as in the more anterior section. The medullary canal (mc) is a straight, vertical slit, and the depression in the floor of the pharynx (ent) is much more shallow. The body cavity (bc) is much larger and extends across the mid-ventral line beneath the heart (ht), which is cut through its middle region. The heart may be traced through about twenty sections (one tenth the length of the embryo); its mesoblastic wall (mes′) is thin and irregular, and is lined by a distinct endothelium (en′) whose exact origin has not yet been worked out.
Figure 11e is just back of the heart, and shows in its place the two vitelline veins (vv). The depression in the floor of the enteron (ent) is entirely distinct from the one that has been mentioned above, and is simply the posterior limit of the head-fold of the entoderm; the fifth section posterior to this shows where this depression opens ventrally to the yolk sac. The other structures shown in the figure are not markedly different from what was seen in Figure 11d.
Figure 11f is about one tenth the length of the embryo posterior to Figure 11e. The chief differences here noticed are in the enteric and cœlomic cavities. The former is no longer enclosed, a dorsal fold in the entoderm being all that remains of the cavity that was seen in the more anterior figures, while the latter is here reduced to a narrow cleft between the somatic and splanchnic mesoblast. A thickening of the mesoblast on either side of the notochord, especially on the left, represents a mesoblastic somite. The medullary canal (mc) is more open than in the more anterior sections.
For about one third of the length of the embryo posterior to Figure 11f there is a gradual flattening, in a dorso-ventral direction, with loss of the amnion, until the condition represented in Figure 11g is reached. The most striking feature of this region is the great thickness of the ectoderm (ec), which is still made up of scattered, irregular cells. In the middle line, directly over the medullary canal (here a nearly cylindrical tube), is a sort of break in the ectoderm, as though there had not been a complete fusion of the epidermal layer when the nervous layer came together on the closure of the medullary groove. This break in the ectoderm may be followed back to the region of the primitive streak, and will be mentioned again. As has been noted, the medullary canal (mc) is nearly circular in cross-section, and is closely underlaid by the notochord (nt), which is several times the diameter that it was in more anterior sections. The mesoblast (mes) is a comparatively thin layer, intermediate in thickness between the ectoderm and entoderm. It shows laterally a slight separation to form the body cavity.
Figure 11h is about ten sections posterior to Figure 11g, and differs from it chiefly in that the notochord (nt) is continuous with the lower side of the medullary canal (mc), though still distinct from the underlying entoderm (en).
Figure 11i, four sections farther from the head, shows the same greatly thickened ectoderm (ec) with the same break (ec′) in the middle line. The section is posterior to the notochord and passes through the anterior edge of the blastopore or, as it may now perhaps better be called, the neurenteric canal. The cells of the medullary wall are continuous with those of the entoderm. The mesoderm (mes) is still distinct from the other germ layers.
Figure 11j is the next section posterior to the one just described and differs from it only in showing the actual opening of the neurenteric canal (nc) into the medullary canal (mc). The medullary canal extends, with gradually diminishing caliber, for about fifteen sections posterior to the point at which the neurenteric canal empties into it. The mesoblast (mes) is so closely attached to the lower wall of the neurenteric canal that it seems to be actually continuous with it.
For a considerable distance posterior to the end of the medullary canal we find the structure similar to that shown in Figure 11k, which is about the twentieth section posterior to Figure 11j. The break (ec′) in the ectoderm is here seen as a compact group of cells which at first glance seem to be continuous with a rounded mass of cells below (ps). Examination under greater magnification, however, shows that the two groups of cells are distinct. As the sections are followed back of this region, the upper mass of cells (ec′) gradually disappears, and after its disappearance the lower mass (ps), which is already continuous with the mesoderm (mes) on either side, becomes continuous with the under side of the ectoderm. The mass of cells (ps) is apparently the primitive streak, though it is distinct from the ectoderm for a considerable distance posterior to the neurenteric canal. Just what may be the meaning of the thickened ridge of ectoderm (ec) it is difficult to determine.
STAGE IX
Figures 12-12g (Plates XVIII., XIX.)
The entire length of the embryo proper is 6.5 mm. from the extreme posterior end to the region of the midbrain (v²), which now, on account of the cranial flexure, forms the most anterior part of the body. Besides being slightly longer than the preceding stage, the embryo has increased in thickness, especially in the anterior region, where the enlargement of the cerebral cavity is considerable.
Body torsion has begun (Fig. 12), so that the anterior third of the embryo now lies on its right side, while the rest of the body is still dorsal side up. The direction of body torsion does not seem to be as definite as it is in the chick, some alligator embryos turning to the right side, others to the left. Clarke has illustrated this fact in his alligator figures. He says (17) that embryos lie “more frequently on the left, but often on the right side.”
The head is distinctly retort-shaped, and at the side of the forebrain (v′) a small crescentic thickening is the optic vesicle (e). The auditory vesicle, though of considerable size, does not show in this surface view. The head-fold (h) extends for about one third the length of the entire embryo, though its exact limit is difficult to determine in surface view. There is no sign of a tail-fold.
About seventeen pairs of somites are present.
The amnion extends over the anterior two thirds of the embryo.
The above-mentioned increase in the diameter of this embryo over that of the preceding is evident when the first two transverse sections of this stage are compared with the corresponding sections of the earlier stage; in the middle and posterior regions there is not very much difference in size.
Figure 12a passes through the region of the forebrain. This end of the embryo lies on its side, as was noted above and as may be recognized from the relative positions of the head and the overlying yolk (y). The great size of this and the following figure is due partly to the increase in size mentioned above and partly to the fact that the sections pass through the region of cranial flexure. The present figure (12a) represents the brain cavity as large and dumbbell-shaped, with comparatively thick walls of compactly arranged cells. The ventral end of this cavity (fb) is cut anterior to the region of the optic vesicles, while the dorsal end (mb) may perhaps be called the midbrain. In the sections that follow this one the two cavities are distinct from each other. The medullary canal, as was stated above, is now completely enclosed, except for the ventral opening of the neurenteric canal, to be presently noticed. Surrounding the brain is a considerable mass of mesoblast (mes). It is composed of the typical stellate cells. The ectoderm (ec) is made up of the same irregularly and loosely arranged cells that have been seen in earlier stages; it is of unequal thickness in different regions, the thicker parts being at the sides. The amnion (a) has the usual appearance, and in this region of course completely surrounds the embryo.
Figure 12b is ten sections posterior to the section just described. The width of the embryo is greater in this region, but the dorso-ventral diameter is about the same as in the more anterior section.
The overlying yolk and blastoderm are not shown in any figure of the series except the first. In this figure the forebrain (fb) and midbrain (mb) are widely separated instead of being connected, as in the preceding figure, where the section passed through the actual bend of the cranial flexure. The anterior and ventral part of the cranial cavity, the forebrain (fb), is nearly circular in outline. It exhibits on one side a well-marked optic vesicle (ov), which is sufficiently advanced in development to show a rudimentary optic stalk. The outer wall of the optic vesicle is in close contact with the superficial ectoderm, which shows as yet no sign of the formation of a lens vesicle. The plane of the section being probably not quite at right angles to the long axis of the embryo, the optic vesicle of one side only was cut. The wall of this part of the forebrain is of about the same thickness and appearance as in the preceding stage. The other cerebral cavity (mb) of this section is probably the hinder part of the midbrain, though it may be the anterior part of the hindbrain; there is no sharp line of demarcation between these regions of the brain. This cavity (mb) is much smaller in section than the forebrain; its walls are of about the same thickness.
Ventral to the midbrain is the anterior end of the notochord (nt), surrounded by the mesoblast. At various places throughout the mesoblast irregular open spaces may be seen; these are blood-vessels. The ectoderm (ec) and amnion (a) have about the same appearance as in the preceding figure, though the former seems somewhat thinner.
Figure 12c is just back of the bent-under forebrain represented in the preceding figure and in front of the main body of the heart. The plane of the section not being at right angles to the long axis of the body (as was mentioned above), the figure is not bilaterally symmetrical. The neural canal, since the section passes through the auditory vesicles, may here be called the hindbrain (hb). It has an almond-shaped cavity, surrounded by a wall of medium thickness. In close contact with the wall of the hindbrain, on each side, is the inner side of the auditory vesicle (o), which is seen as a deep, wide-mouthed pit in the superficial ectoderm. On the right side of the section the auditory pit is cut through its middle region; it is simply a thickened and condensed area of the ectoderm which has been invaginated in the usual way. Directly beneath the hindbrain is the notochord (nt), on each side of which, in the mesoblast, is the dorsal aorta (ao), or rather the continuation of the aorta into the head. Beneath these structures and extending from one side of the section to the other is the pharynx (ph); its lining wall is fused on each side with the ectoderm, but there is no actual opening to the exterior. These points of contact (g) between entoderm and ectoderm are of course the gill clefts; they are not yet visible from the outside. The roof of the pharynx is flat and comparatively thin, while the floor is thickened and depressed to form a deep, wide pit, traceable through six or eight sections. This pit may be the thyroid gland already noticed in the preceding stage. Below the main cavity of the pharynx and close to each side of the thyroid rudiment just mentioned is a large blood-vessel (tr). These two vessels when traced posteriorly are found to be continuous with the anterior end of the heart, and hence may be called the truncus. They were noticed in Figure 11c, bv. The ectoderm surrounding the lower side of the embryo was so thin and indistinct that it could scarcely be distinguished from the mesoderm of that region. The amnion (a) is still a continuous envelope entirely surrounding the embryo.
Figure 12d, about twenty sections posterior to Figure 12c, is in the posterior heart region. The spinal cord (sc), as might be expected, is smaller than in the more anterior region, but is otherwise not markedly different from what was there seen. The notochord (nt) also has the same appearance as before. The enteron (ent) shows of course in this region no gill clefts; it is a small, irregular cavity with thicker walls than in the figure just described. The ventro-lateral depression is entirely distinct from the depression that was called the thyroid rudiment in the preceding figure. Dorsal to the enteron are the two dorsal aortæ (ao), now smaller and more ventral to the notochord than in the preceding figure. Ventral to the enteron is the large heart (ht), projecting below the body cavity, which is no longer enclosed. The mesodermic wall (mes′) of the heart is still comparatively thin and is separated by a considerable space from the membranous endocardium (en′). The extent and shape of the heart are shown in the surface view of this stage. On the right side of the section the body cavity extends to a point nearly opposite the middle of the spinal cord, considerably dorsal to the notochord, while on the left side the dorsal limit of the body cavity is scarcely level with the lower side of the notochord. Between the dorsal end of the body cavity and the side of the spinal cord, on the left, is a dense mass of mesoblast (s), one of the mesoblastic somites. A few sections either anterior or posterior to the one under discussion will show the condition of the two sides reversed—that is, the body cavity will extend to the greater distance on the left and will be interrupted by a mesoblastic somite on the right. It is evident, then, that the upper angle of the body cavity is extended dorsally as a series of narrow pouches between the somites. The mesoblast that lines the body cavity, the splanchnopleure (sm) and somatopleure (so), is somewhat denser than the general mass of mesoblast, so that these layers are quite distinct, the former (sm) extending around the enteron (ent) and heart (ht), and the latter (so) being carried dorsalward as the mesoblastic part of the amnion (a). The amnion may be traced through about 130 of the 200 sections into which this embryo was cut.
Figure 12e is nearly one fourth the length of the embryo posterior to Figure 12d; it is approximately in the middle region. The diameter of the embryo has been gradually decreasing until now it is very much less than in the head region. The section being behind the head-fold the entoderm (en) is nearly flat and the enteron is quite unenclosed. The canal of the spinal cord (sc) is smaller in proportion to the thickness of its walls, and the notochord (nt) is somewhat larger than in the preceding sections. In proportion to its extent, the ectoderm is very thick. Under the notochord the dorsal aortæ (ao) are seen as two large, round openings in the mesoblast. On the left side the section passes through the center of a somite and shows a small, round myocœl (myc). The mesoblastic layer of the amnion (so) is distinct throughout from the ectoblastic layer (a).
The most important structures to be here noted are the first rudiments of the Wolffian ducts (wd). They are seen in the present section as lateral ridges of mesoblast projecting outward and upward toward the ectoblast, which suddenly becomes thin as it passes over them. These ridges or cords of mesoblast are as yet quite solid. They arise suddenly at about the eightieth section of the series of two hundred and may be traced through about forty sections, or one fifth of the length of the embryo. Their exact length is difficult to determine because, while their anterior ends are blunt and sharply defined, they taper so gradually posteriorly that it is hard to tell just where they end. They apparently originate anteriorly and gradually extend toward the tail. In a slightly younger embryo the rudimentary Wolffian duct could be seen as a still smaller rod of cells extending posteriorly for a few sections, from the seventy-fifth section of a series of about two hundred. In the particular series under discussion the left rudimentary Wolffian duct was about one fifth longer than the right one.
Figure 12f is just posterior to the head-fold of the amnion, passing, in fact, on the left side through the extreme edge of its lateral fold, which is shown as a upward bend in the ectoblast and somatopleure.
The ectoblast (ec) shows the same remarkable thickening that was noted in the corresponding region of the preceding stage. The spinal cord (sc), notochord (nt), aortæ (ao), and entoderm (en) need no special mention. The mesoderm seems to be separated by unusually wide spaces from both ectoderm and entoderm, and is made up of rather closely packed cells except around the aortæ, where there seems scarcely enough tissue to hold these vessels in place. The body cavity (bc) is large, and a small myocœl (myc) is seen on the left.
Figure 12g is through the neurenteric canal (nc), a distinct opening through the floor of the spinal canal. The section is of course just back of the posterior end of the notochord. The entoderm (en) along the margin of the neurenteric canal is naturally continuous with the wall of the spinal cord (sc). The ectoderm (ec) is thicker than ever, except in the median plane, where it passes over the spinal cord. The mesoblast is more abundant than in the preceding figure, and shows on the left what appears to be a distinct myocœl (myc), though in surface view the mesoblastic somites do not extend this far toward the tail.
STAGE X
Figures 13-13g (Plates XIX., XX., XXI.)
This embryo (Fig. 13) is about 5 mm. in length, and hence is slightly smaller than the preceding stage, though somewhat more advanced in development. The medullary canal is still apparently unclosed for a short distance at the extreme posterior end; this appearance is probably due to the neurenteric canal (nc) and to the thinness of the roof of the medullary canal rather than to any lack of fusion of the medullary folds. The optic vesicle is more distinct than in the preceding stage; a somewhat similar, though smaller, opacity (o) marks the position of the ear. There are now about twenty pairs of somites, though it is difficult to determine their exact number on account of the torsion of the body. The amnion is at about the same stage of development as in Stage IX. The heart (ht) is a large double mass, whose outlines may be dimly seen when the embryo is viewed by transmitted light. The vitelline vessels (vv) are still but faintly outlined in the vascular area; the veins and arteries cannot yet be distinguished from each other. The gill clefts, though not visible externally in the embryo drawn, may be seen in sections of this stage as evaginations of the wall of the pharynx.
The transverse sections of this stage are slightly more advanced in development than was the embryo that has just been described in surface view. Only those sections have been figured which show a decided advance in the development of some special structures over their condition in the preceding stage. The sections of the preceding stages were drawn under a magnification of eighty-seven diameters; those of this and the following stage were drawn under a magnification of only forty-one diameters. All of the figures have been reduced one half in reproduction.
Figure 13a is the most anterior section of this series to be described. On account of the cranial flexure, which causes the long axis of the forebrain to lie at right angles to that of the spinal cord, this section cuts the head region longitudinally. The ectoderm (ec) is of varying thickness, the thickest areas being on each side of the forebrain; it is more compact than in the earlier stages, and, owing to the low magnification under which it is drawn, it is represented here by a single heavy line. Under this magnification only the nuclei of the mesoderm cells (mes) can be seen, so that this tissue is best represented by dots, more closely set in some places than in others. The forebrain is an elongated cavity (fb) with thick, dense walls. Attached to each side of the forebrain is an optic vesicle (ov), which is considerably larger than in the preceding stage. The connection between the cavity of the forebrain and that of the optic vesicle is not seen in this section; it is a wide passage that may be seen in several sections posterior to the one under discussion. The beginning of the invagination of the optic vesicle to form the optic cup may be seen on both sides, but more plainly on the right. On the right side also is noticed a marked thickening of the ectoderm, which is invaginated to form a small pit, the lens vesicle (lv); on the left side the section is just behind the lens vesicle. Above the optic stalk on each side, in the angle between the optic vesicle and the side of the forebrain, is a small blood-vessel (bv). Several other blood-vessels may be seen at various places in the mesoblast, four of them near the pharynx being especially noticeable. The hindbrain (hb) is wider than, but not so deep as, the forebrain; its walls are very thick laterally, but are thin on the dorsal and ventral sides. The dorsal wall is reduced to a mere membrane, which, with the overlying ectoderm, has been pushed into the brain cavity, as is generally the case with such embryos. Close to the ventral wall of the hindbrain the notochord (nt) is seen. The character of the notochord has already begun to change; the cells are becoming rounded and vacuolated, with but few visible nuclei except around the periphery of the notochord. Near the center of the section, close to the ventral end of the forebrain, is the pharynx (ph), cut near its anterior limit; it is here a small, irregularly rectangular cavity with a comparatively thin wall. On the left side of the pharynx the first gill cleft (g) is indicated as a narrow diverticulum reaching toward the ectoderm. A few sections posterior to this one the first gill cleft is widely open to the exterior. As has been said, in the surface view of this stage above described none of the gill clefts showed; so that in this respect at least the sectioned embryo was more nearly of the state of development of the embryo represented in Figure 14, to be described later.
Figure 13b, about forty sections posterior to Figure 13a, passes through the hindbrain in the region of the ears. Being back of the region affected by cranial flexure, this section is of course of much less area than the preceding. The ectoderm shows no unusual features; it is of uniform thickness except where it becomes continuous with the entoderm around the mandibular folds (md); there it is somewhat thickened. The most striking feature of the section is the presence of two large auditory vesicles (o). The section being not quite at right angles to this part of the embryo, the vesicles are not cut in exactly the same plane; the one on the left is cut through its opening to the exterior, while the one on the right appears as a completely enclosed cavity. In a section a short distance posterior to this one the appearance of the vesicles would be the reverse of what it is here. As may be seen in the figure, the vesicles are large, thick-walled cavities lying close to the lateral walls of the hindbrain. The hindbrain itself has the usual triangular cross-section, with thick lateral walls and a thin, wrinkled dorsal wall. Close to the ventral side of the hindbrain lies the notochord (nt), on each side of which, in the angle between the brain and the auditory vesicles, is a small blood-vessel (bv). Ventral to these structures and close to the dorsal wall of the pharynx (ph) are the two large dorsal aortæ (ao). The ventral side of the section passes through the open anterior end of the pharynx (ph). On the left is seen the widely open hyomandibular cleft (g′), between the main body of the section and the mandibular arch (md). On the right side the plane of the section was such that the hyomandibular cleft was not cut through its external opening. In each mandibular fold a large aortic arch (ar) is seen, and also a slight condensation of mesoblast, the latter probably being the forerunner of cartilage.
Figure 13c passes through the anterior part of the heart about seventy-five sections posterior to Figure 13b. The embryo in this region is narrow but deep (dorso-ventrally), the depth being largely due to the size of the heart. The ectoderm (ec) is considerably thickened on each side of the pharynx (ph); this thickened area may be traced for some distance both anteriorly and posteriorly from this point; its significance could not be determined. The spinal cord (sc) and notochord (nt) need no special description; the former is smaller and the latter larger than in the more anterior sections. The two large blood-vessels (ac) near the spinal cord and notochord are probably the anterior cardinal veins. The aortæ are cut by the plane of this section just anterior to their point of fusion into a single vessel. A few blood corpuscles are seen in the right aorta. The enteron (ent), cut posterior to the region of the gill clefts, is a large elliptical cavity, with its long axis in a transverse position. Its entodermal wall is comparatively thin and smooth, with the cell nuclei arranged chiefly on the outer side, i. e., away from the cavity of the enteron. The body cavity (bc) is here still unenclosed, and its walls, the somatic stalk, are cut off close to the body of the embryo. The heart (ht), the most conspicuous feature of this section, is nearly as large in cross-section as all the rest of the embryo. As seen in such a section it is entirely detached from the body of the embryo, and in this particular case has about the shape of the human stomach. The mesoblastic portion of its wall (mes′) is of very irregular thickness; it forms a dense layer entirely around the outside, except for the pointed dorsal region, and is especially thick along the ventral margin, where it is thrown into well marked folds, the heavy muscle columns. Lining the cavity of the heart is the membranous endothelium (en′), and between this and the dense outer wall just described is a loose reticular tissue with but few nuclei.
As the series is followed toward the tail the sections diminish in size until, at a point about one third the embryo length from the posterior end, they are of scarcely one fourth the area of the sections through the region of the hindbrain.
Figure 13d is about one hundred and twenty-five sections posterior to Figure 13c. Although not so small as the sections that follow it, this section is considerably smaller in area than the one last described. The amnion (a), which was not represented in the last three figures, is very evident here. The spinal cord (sc) is considerably smaller here than in the preceding figure, while the notochord (nt) is not only relatively but actually larger than in the more anterior regions. Beneath the notochord is the aorta (ao), now a single large vessel. The mesoblast on each side of the body is here differentiated into a distinct muscle plate (mp). These muscle plates have very much the appearance of the thickened ectoderm seen in the younger stages of development. At about its middle region (i. e., at the end of the reference line ec) each muscle plate is separated from the overlying ectoderm by an empty space; this space is still more marked in some other series. Ventral to the aorta, and supported by a well marked though still thick mesentery (ms), is the intestine. It is a small, nearly cylindrical tube with thick walls; the splanchnic mesoblast which surrounds it is more dense than the general mass of mesoblast; it was somewhat torn in the section and is so represented in the figure. The urinary organs have made considerable progress since the last stage. In the figure under discussion they are seen as a group of tubules on either side of the aorta. The tubule most distant from the middle line, on each side, is the Wolffian duct (wd). It extends through the posterior two thirds of the embryo and varies in diameter at different points; it is usually lined with a single layer of cubical cells which contain large nuclei. The Wolffian bodies (wt) are a mass of slightly convoluted tubules that may be traced throughout the greater part of the region through which the Wolffian duct extends. These tubules also vary somewhat in diameter, but they are usually of greater caliber than the duct. No actual nephrostomes are to be seen, though the occasional fusion of a tubule with the peritoneal epithelium, as is seen on the left side of the present figure, may represent such an opening.
Figure 13e is about one hundred and forty sections posterior to the section just described. The embryo is here very slender, so that the contrast between this and the first figure (13a) of this stage is remarkable. Except in size, this section does not differ greatly from the preceding. The spinal cord, notochord, etc., are smaller than before, but are of about the same relative size. The mesentery (ms) in the section drawn was torn across, so that the intestine is not represented. Medial to the Wolffian duct is a tubule (wt), which seems to be the same as those which were called Wolffian tubules in the preceding stage, but which may be the beginning of the ureter.
Figure 13f, about two hundred and fifty sections posterior to the last, passes through the extreme posterior end of the embryo. The section is nearly circular in outline and is somewhat larger than the preceding. The amnion (a) completely encircles the embryo. The ectoderm (ec) is of fairly even thickness, and the mesoblast which it encloses is of the usual character. The spinal cord (sc) is nearly circular in outline, as is its central canal. The digestive tract (ent) is larger in section than it was in more anterior regions; it is nearly circular in cross-section and its walls are made up of several layers of cells, so that it resembles to a considerable degree the spinal cord of the same region. In the narrow space between the spinal cord and the hindgut is seen the notochord (nt), somewhat flattened and relatively and actually smaller than in the preceding figure. A few scattered blood-vessels may be seen in the mesoblast at various places.
A sagittal section of an embryo of this stage, drawn under the same magnification as were the transverse sections, is shown in Figure 13g. The embryo being bent laterally could not be cut by any one plane throughout its entire length, so that only the anterior end is represented in the figure. The amnion (a) may be clearly seen except at certain places where it is closely adherent to the superficial ectoderm. Under the low magnification used the superficial ectoderm cannot be distinguished from the ectoderm of the nervous system. The plane of the section being in the anterior end almost exactly median, this part of the central nervous system is seen as the usual retort-shaped cavity, while in the region back of the brain, where the neural canal is narrow, the section passes through the wall of the spinal cord (sc) and does not show the neural canal at all. The wall of the forebrain (fb) is quite thick, especially at the extreme anterior end; the wall of the midbrain (mb), where the marked cranial flexure takes place, is somewhat thinner, and it gradually becomes still thinner as it is followed posteriorly over the hindbrain (hb). Between the floors of the fore- and hindbrains, in the acute angle caused by the cranial flexure, is the anterior end of the notochord (nt), the only part of that structure that lies in the plane of the section. Ventral and posterior to the notochord is a large cavity, the pharynx (ph), whose entoblastic lining can scarcely be distinguished under this magnification from the surrounding tissues. The stomodeal opening being as yet unformed, the pharynx is closed anteriorly; posteriorly also, owing to the plane of the section, the pharynx appears to be closed, since its connection with the yolk stalk is not shown. In the floor of the pharynx, almost under the reference line ph, a slight depression marks the position of the first gill cleft. In the mesoblast ventral to the pharynx and near the gill cleft just mentioned, a couple of irregular openings represent the anterior end of the bulbus arteriosus, posterior and ventral to which is the heart (ht), a large, irregular cavity. The dorsal aorta (ao) may be seen as an elongated opening in the mesoblast, extending in this section from the middle region of the pharynx to the posterior end of the figure where it is somewhat torn. Two of the eighteen or twenty pairs of mesoblastic somites possessed by this embryo are shown at the posterior end of the figure (s), where the plane of the section was far enough from the median line to cut them.
STAGE XI
Figure 14 (Plate XXI.)
Only the anterior region of this embryo is shown in the figure, which is a ventro-lateral view. While there is some change in the general shape and in parts of the head, the reason for figuring this stage is to show the first gill cleft (g′), which lies at an acute angle to the long axis of the neck behind the eye (e). The cleft is narrow but sharp and distinct in outline; it shows neither in this nor in the following stages the branched, Y-shaped outline mentioned by Clarke.
STAGE XII
Figures 15-15f (Plates XXI., XXII.)
In this stage, also, only the anterior region of the embryo is figured in surface view. The shape of the head is about the same as in the preceding stage, but it is drawn in exact profile. Three gill clefts (g¹⁻³) are now present, and are wide and distinct. The first cleft, as in the preceding stage, lies at an acute angle to the long axis of the pharynx and nearly at right angles to the second cleft. The third cleft sends a wide branch (g⁴) toward the posterior, as has been described by Clarke, from which, or in connection with which according to Clarke, the fourth cleft will develop. All three clefts may be distinctly seen to open entirely through the pharyngeal wall. The outlines of the visceral folds, especially of the mandibular, begin to be apparent. The nasal pit (n) now shows as a round depression in front of the more definitely outlined eye (e). The auditory vesicle (o) is so deep beneath the surface that it may be seen only by transmitted light.
Figures 15a-e represent transverse sections of an embryo of about this general state of development, except that the gill clefts are not so definitely open as in the surface view.
Figure 15a, the most anterior section of the series, passes through the forebrain (fb) in the region of the eyes, and through the hindbrain (hb) anterior to the auditory vesicles. The forebrain is here a large cavity with a dense wall of a comparatively even thickness. Owing probably to the section not being exactly in the transverse plane, the eyes are cut in different regions, that on the left (ov) being cut through its stalk, while that on the right (oc) is cut near its middle region and hence does not show any connection with the forebrain. The almost complete obliteration of the cavity of the optic vesicle to form the optic cup by the invagination of the outer wall of the vesicle is shown on the right side of the section (oc). The lens vesicle (lv) is completely cut off from the superficial ectoderm (ec), which is comparatively thin. The hindbrain (hb) has the usual shape for that structure. Its ventral wall is dense and thick, while its roof is reduced to the usual thin, wrinkled membrane. Close to the floor of the hindbrain lies the notochord (nt), which is large and is distinctly vacuolated. To the right of the hindbrain a large mass of darkly stained cells (cn) is one of the cranial nerves, which is connected with the hindbrain a few sections anterior to the one under consideration. The pharynx (ph), which is cut near its extreme anterior end, is represented by three irregular cavities near the base of the forebrain. Scattered throughout the mesoblast, which makes up the greater part of the section, are numerous blood-vessels (bv).
Figure 15b is twenty sections posterior to Figure 15a and passes through the tip of the bent-under forebrain (fb). On the left the section is anterior to the optic vesicle and barely touches the side of the optic stalk, which is seen as a small lump on the ventro-lateral wall of the brain. On the right the connection of the optic vesicle (ov) with the forebrain is shown. Dorsal to the optic vesicle just mentioned is a markedly thickened and slightly invaginated region of the ectoderm (n); this is the nasal pit; on the left side of the figure the thickening is shown, but the section did not pass through the invagination. The hindbrain (hb) is somewhat narrower than in the preceding figure, but is otherwise about the same; the origin of a cranial nerve is seen on its left side (cn). The notochord (nt) has the same appearance as in the preceding section. A number of blood-vessels may be seen, the pair lying nearest the notochord being the aortæ (ao), while the two other pairs, on either side of the fore- and hindbrains, are the anterior cardinals (ac). The first aortic arches are shown at ar. On the left the section passes through the exterior opening of the first gill cleft (g′), so that the mandibular fold (md) on that side is a distinct circular structure, made of a dense mass of mesoderm surrounded by a rather thick ectoderm. The mesoderm of this fold is especially dense near the center, probably the beginning of the visceral bar. Near the center is also seen the aortic arch that has already been mentioned. On the right the section does not pass through the external opening of the first gill cleft (g′) so that the tissue of the mandibular fold is continuous with the rest of the head. It is of course the slight obliquity of the section that causes the pharynx (ph) to be completely enclosed on the right, while on the left it is open to the exterior both through the gill cleft and between the mandibular fold and the tip of the head. The superficial ectoderm shown here as a heavy black line varies considerably in thickness, being thickest in the region of the nasal pit already mentioned and thinnest over the roof of the hindbrain. The amnion (a) in this, as in the other sections of the series, has the appearance of a thin, very irregular line.
Figure 15c is posterior to the region affected by cranial flexure and so shows only one region of the embryo, that of the hindbrain (hb), which is here of essentially the same structure as above described. On each side of the hindbrain is a large auditory vesicle (o); that on the left is cut through its center and shows the beginning of differentiation, its lower end being thick-walled and rounded, while its upper end is more pointed and has a thin, somewhat wrinkled wall. The notochord (nt) is slightly larger than in the more anterior sections. Numerous blood-vessels (bv, ar) are seen in the mesoblast. The pharynx (ph) is here open ventrally and also through the gill cleft of the left side; on the right side the plane of the section did not pass through the external opening of the cleft. The mesoblast of the visceral folds is much more dense than that of the dorsal region of the section.
Figure 15d, as is evident, is a section through the region of the heart, which appears as three irregular cavities (ht) with fairly thick mesoblastic walls (mes′) lined with endothelium (en′). The body wall, though consisting of but little besides the ectoderm (ec), completely surrounds the heart, and the pericardial or body cavity thus formed extends dorsally as a narrow space on either side of the foregut, giving the appearance of a rudimentary mesentery, though no especial development of such a structure would naturally be expected in this region of the embryo. The foregut (ent) is a moderately large cavity lined with a very distinct entoderm of even thickness. Dorsal to the foregut are three large blood-vessels, a median, and now single, dorsal aorta (ao), and a pair of cardinal veins (cv). The notochord (nt) is small and is flattened against the ventral side of the spinal cord (sc), which latter structure needs no special mention. The muscle plates (mp) are considerably elongated, so that they now extend ventrally to a point slightly below the upper angles of the body cavity.
Figure 15e is through the middle region of the embryo, and, owing to the curvature of the body, is not an exact dorso-ventral section; this accounts, in part at least, for the unusual diameter in a dorso-ventral direction of the aorta (ao), which is very large in proportion to the other structures. The posterior cardinal vein is shown on the left, but not on the right. The relative sizes of the spinal cord (sc) and notochord (nt) are very different from what was seen in the preceding figure. In this section the spinal cord is considerably smaller than in the preceding, while the notochord is very much larger; in fact the notochord here seems abnormally large when compared to corresponding sections of other series. It is true, however, that while the spinal cord has been diminishing in diameter the notochord has been increasing. The spinal cord, notochord, and dorsal aorta are all so large that they are flattened against each other, the pushing in of the ventral side of the spinal cord being even more marked than is shown in the figure. On either side of the spinal cord a large spinal ganglion (sg) is seen, closely wedged in between the spinal cord and the adjacent muscle plate (mp). As in the preceding stage, there is a marked space between the muscle plate and the adjacent ectoderm (ec). The somatic mesoblast at the upper angle of the unenclosed body cavity is thickened on each side and somewhat bulged out by the Wolffian body to form what might be termed a Wolffian ridge (wr). In the mid-ventral line is the considerably developed mesentery (ms), from which the intestine has been torn. The Wolffian bodies now consist, on each side, of a group of five or six tubules (wt) of various sizes, near which in a more ventro-lateral position, close to the upper angle of the body cavity, is the more distinct Wolffian duct (wd). The allantois is fairly large by this time, and may be seen in the most posterior sections as an irregular, thick-walled outgrowth from the hindgut.
A horizontal section through the anterior end of an embryo of this age is shown in Figure 15f. While enclosed of course in the same membranous amnion (a), the pharyngeal region of the section is separated by a considerable space from the more anterior region where the section passes through the forebrain (fb) and eyes. The spinal cord (sc), notochord (nt), muscle plates (mp), aortæ (ao), and anterior cardinal veins (ac) need no special description. The appearance of the pharynx (ph), with its gill clefts and folds, is quite similar to that of the corresponding structures in the chick. None of the four clefts (g¹⁻⁴) show, in the plane at which the section was cut, any connection with the exterior; in fact the fourth cleft (g⁴) would scarcely be recognized as a cleft if seen in this section alone. One or two of the more anterior clefts are open to the exterior. Three pairs of aortic arches are seen, and each visceral fold has a central condensation of mesoblast.
STAGE XIII
Figures 16-16g (Plates XXII., XXIII.)
The embryo (Fig. 16) now lies on one side, body torsion being complete. The curvature of the body is so marked that the exact length is difficult to determine. The eye (e) and ear (o) have about the same superficial appearance as in the preceding stage. The nose is not shown in this figure. About thirty somites are present; the exact number cannot be determined in surface view. The amnion is complete, though not shown in the figure, and the tail (t) is well formed. The umbilical stalk was torn in the removal of the embryo, so that it is not shown in the figure. The dim outline of the now convoluted heart may be seen if the “cleared” embryo be viewed by transmitted light; it is not shown in the figure. The allantois (al) is a rounded sac of considerable size just anterior to the tail. Four gill clefts (g¹⁻⁴) are now present; the most posterior one is more faint than is represented in the figure, and it could not be definitely determined from a surface view whether or not it opened to the exterior. The mandibular fold (md) is now fairly well outlined, but there is as yet no sign of the maxillary process.
Figure 16a is the most anterior of a series of transverse sections made of an embryo of the approximate age of the surface view just described; it passes through the tip of the forebrain (fb) and shows the nasal pit (n) of the right side. The great thickening of ectoderm in the region of the nasal invagination is represented by a solid line. Owing to the obliquity of the section, the left nasal pit was not cut. The mesoblast is quite dense and contains two or three small blood-vessels near the roof of the brain. The plane of this section, owing to the cranial and body flexure, cut the embryo also in the region of the pharynx; this part of the section was, as a matter of convenience, omitted from the drawing.
Figure 16b is in reality more anterior in position, considering the entire embryo, than the preceding; but the region of the embryo represented is more posterior, so that it is described at this point. The greatly elongated outline of the brain is due to its being cut through the region of flexure, so that the forebrain (fb), or, perhaps, midbrain, is shown at one end, and the hindbrain (hb) at the other. The walls of these cavities are somewhat wrinkled and irregular and their constituent cells are beginning to show slight differentiation, though this is not shown in the figure. On the left side are seen a couple of darkly stained masses; one is the origin of a cranial nerve (cn); and the other is one of the auditory vesicles (o), which is still more irregular in outline than it was in the preceding stage. The only blood-vessels to be seen are a few very small ones that lie close to the wall of the brain. The ectoderm is quite thin at all points.
Figure 16c, the largest section of this series, passes through the forebrain in the region of the eyes and through the gill clefts. The forebrain (fb) exhibits on the left a marked thickening of its wall (ch), the edge of the cerebral hemisphere of that side, which is just beginning to develop; on its right side the lower part of the forebrain is connected by a well marked optic stalk (os) with the optic cup (oc), in whose opening lies the lens vesicle (lv), now reduced to a crescentic slit by the thickening of its posterior wall. The mesoblast is more dense in those parts of the section adjacent to the pharynx than in the more distant regions, and the ectoderm thickens in a marked way as it approaches the borders of the pharynx and gill clefts. Only a few small blood-vessels (bv) are to be seen in the region of the forebrain.
Parts of three pairs of clefts (g) are shown in the figure: one pair opens widely on either side, so that there is a large area of the section that is distinct from the two still larger portions and contains a small, thick-walled cavity (g) on the right side; this cavity is a gill cleft that is cut through neither its outer nor its pharyngeal opening.
No structures other than this small portion of a gill cleft and a few blood-vessels are to be seen in this middle region of the section. In the more posterior part of the section, in which the notochord (nt) is located, a pair of curved clefts may be seen, opening entirely through the wall on the left, but closed on the right (g). One distinct pair of aortic arches is shown (ar), and also the dorsal aortæ (ao), which are of very unequal size. The spinal cord (sc) and muscle plates need no special description.
Figure 16d is in the region of the heart (ht) and lungs (lu). The former is an irregular cavity whose walls, especially on the ventral side (mes′), are becoming very thick and much folded. Although thin, the body wall completely surrounds the heart, as would be expected, since this was true of the preceding stage. The lung rudiments (lu) and the foregut from which they have arisen have the same appearance as in the chick; they consist of three small, thick-walled tubes so arranged as to form a nearly equilateral triangle. They are surrounded by a swollen, rounded mass of mesoblast which almost completely fills the surrounding portion of the body cavity (bc). The pleural sides of these crescentic portions of the body (or pleural) cavity—that is, the boundary of the mass of mesoblast just mentioned—are lined with a thickened layer of cells, shown by the solid black lines in the figure. The lung rudiments may be traced through about fifty sections of this series, or about one twelfth of the entire series. At the dorsal angle of the part of the body cavity (bc) just described, near the dorsal aorta (ao), are two dark, granular masses (ge), which, under a higher magnification than is here used, are seen to consist of a small group of blood-vessels filled with corpuscles; although several sections in front of the anterior limits of the kidneys these are evidently glomeruli. They may be traced, though diminishing in size, far toward the tail, in close connection with the Wolffian bodies. At intervals they are connected by narrow channels with the dorsal aorta; no such connection was present in the section drawn. The notochord (nt), spinal cord (sc), muscle plates (mp), and spinal ganglia (sg) need no special mention. The mesoblast is beginning to condense in the neighborhood of the notochord, and the ectoderm is slightly thickened laterally and dorsally.
Figure 16e is in the region of the liver and the Wolffian bodies; it also shows the tip of the ventricular end of the heart. The liver (li) is a large irregular mass, of a blotchy appearance under this magnification, lying between the heart (vn) and the intestine (i). Under greater magnification it is seen to be made up of indefinite strings of cells; and its still wide opening into the intestine may be seen in more posterior sections. The intestine (i), which in this section might be called the stomach, is a fairly large cavity with the usual thick entodermic walls; it is supported by a comparatively narrow mesentery. The body cavity on the side next this mesentery has the same thick lining that was noted in the region of the lungs. The convolutions of the thick peritoneal lining may easily be mistaken in places for parts of the enteron. The Wolffian bodies may be seen as two groups of tubules (wt) in their usual location. The heart is cut through the ventricle (vn), as has been said. The section being at right angles to the long axes of the villi-like growths of the myocardium, the depressions between these mesoblastic cords are seen as a number of small irregular areas, each one lined with its endocardium. The incompleteness of the body wall below the heart is apparently due to an artificial break and not to a lack of fusion. The only point that need be mentioned in connection with the structures of the dorsal part of the section is that the distinctness of the myocœl (myc) on the right side is somewhat exaggerated.
Figure 16f is in the middle region of the embryo where both splanchnopleure and somatopleure are unfused. Owing chiefly to the unclosed condition of the midgut (i) and to the increase in length of the mesentery (ms), the section is quite deep dorso-ventrally. The continuation of the amnion (a) with the somatopleure is of course here evident.
The most striking feature of the section is the marked projection of the Wolffian ridges, though no local enlargements of these ridges indicate the rudiments of the limbs. A large mass of Wolffian tubules (wt) is seen projecting into the upper part of the body cavity on each side; close to each of these masses is the posterior cardinal vein (pc), and between them is the large aorta (ao). The other structures are about as in the preceding section.
Figure 16g represents a sagittal section of the anterior half of the body of an embryo of this or possibly a slightly younger stage of development. The three regions of the brain are clearly indicated, as well as the cavity of the spinal cord (sc). The roof of the hindbrain has been made too thick in the figure; it should be represented by a mere line. A little mesoblast is to be seen at places between the roof of the brain and the superficial ectoderm. A slight invagination of the epithelium (p), between the floor of the brain and the anterior end of the notochord, probably represents the beginning of the hypophysis. No indication of the paraphysis is yet to be seen. Extending from the region of the hypophysis to the posterior end of the section is the notochord (nt); it is much vacuolated and gradually increases in thickness toward the posterior, though its outline is quite irregular; except at the extreme anterior end and at one or two other places, it lies in close contact with the floor of the neural tube. Directly under the notochord lies, in the posterior half of the figure, the large dorsal aorta (ao). The pharynx (ph), opening between the end of the forebrain and the thick mandibular fold (across which opening the amnion, a, of course extends), is a funnel-shaped space which passes out of the plane of the section toward the posterior end of the figure. Its thick endodermal lining extends to the mandibular fold on the ventral side, while on the dorsal side it gradually thins out and becomes continuous with the thin ectoderm that extends over the forebrain. Just back of the mandibular fold is the bulbus (b), and back of that is the edge of the ventricle (vn). Posterior and dorsal to the ventricle the liver (li) is seen as an irregular mass of cells, and dorsal to the liver one of the Wolffian bodies (wt) is cut through its extreme edge.
STAGE XIV
Figures 17-17g (Plates XXIII., XXIV.)