Development of the Digestive Ca.n.a.l of the American Alligator.
by Albert M. Reese.
In a previous paper (6) the writer described the general features in the development of the American Alligator; and in other papers special features were taken up in more detail.
In the present paper the development of the enteron is described in detail, but the derivatives of the digestive tract (liver, pancreas, lungs, etc.) are mentioned only incidentally; the development of these latter structures may be described in a later paper.
No detailed description of the histological changes taking place during development has been attempted, though a brief description of the histology is given for each stage discussed.
The material upon which this work was done is the same as that used for the preceding researches. It was collected by the author in central Florida and southern Georgia by means of a grant from the Smithsonian Inst.i.tution, for which a.s.sistance acknowledgment is herewith gratefully made.
Various methods of fixation were employed in preserving the material. In practically all cases the embryos were stained in toto with Borax Carmine and on the slide with Lyon"s Blue. Transverse, sagittal, and horizontal sections were cut, their thickness varying from five to thirty microns, depending upon the size of the embryos.
The first indication of the formation of the enteron is seen in the very early embryo shown, from the dorsal aspect, in figure 1. The medullary folds and notochord are evident at this stage, but no mesoblastic somites are to be seen.
A sagittal section of approximately this stage, shown in figure 1A, represents the foregut, _fg_, as a shallow enclosure of the anterior region of the entoderm, while the wide blastopore, _blp_, connects the region of the hindgut with the exterior. No sign of a tail fold being present, there is, of course, no real hindgut. The entoderm, which has the appearance of being thickened because of the fact that the notochord has not yet completely separated from it, is continuous, through the blastopore, with the ectoderm. Posterior to the blastopore the primitive streak, _ps_, is seen as a collection of scattered cells between the ectoderm and the entoderm, apparently formed by proliferation from the ventral side of the ectoderm.
A slightly later stage is shown in figure 2, a dorsal view of an embryo with five pairs of mesoblastic somites. A sagittal section of this stage is shown in figure 2A. The foregut is here more inclosed, and the notochord, _nt_, having separated from the entoderm, _en_, is seen as a distinct layer of cells extending from the foregut to the blastopore.
A transverse section through the headfold of this stage is shown in figure 2B. The foregut is seen as a wide cavity, _ent_, depressed dorsally, apparently, by the formation of the medullary groove and the notochord; it is wider laterally than in a dorso-ventral direction, and its walls are made up of about three layers of closely arranged, irregular cells; the wall is somewhat thinner on the dorsal side, just below the notochord.
Figure 3 is a dorsal view of the next stage to be described; about fifteen pairs of somites are present.
Figure 3A is a transverse section through this embryo near the anterior end of the enteron, _ent_, which cavity, cephalad to this region, is bluntly pointed. As seen in the figure the enteron is here wide from side to side, and is depressed dorso-ventrally except for a wide groove in the ventral wall. This groove is lined with rather more closely arranged cells, and marks the region where the mouth will break through at a somewhat later stage. A short distance caudad to this region the groove disappears and the pharynx is reduced to a shallow slit extending almost to the superficial ectoderm on either side; then the slit-like pharynx becomes suddenly reduced in a lateral and increased in a dorso-ventral direction, to a.s.sume the outline shown in figures 3B and 3C. At a point about one-third of the length of the embryo from the tip of the head, the enteron opens to the yolk-sac, so that what now may be called the foregut has this considerable extent. There is, however, not the slightest indication of a tail-fold, so that there is no inclosed hindgut at all. As is shown in figure 3D, the neurenteric ca.n.a.l, _nc_, still opens ventrally, though the medullary ca.n.a.l, _mc_, has now no dorsal opening to the exterior. The medullary ca.n.a.l continues for a short distance (about fifteen sections of five microns thickness) posterior to the opening of the neurenteric ca.n.a.l.
Figure 4 is a surface view of the next stage to be described. There are here about twenty pairs of somites, though the exact number cannot be determined. Although not visible externally in the surface view shown, the gill clefts are beginning to form, and the first one opens to the exterior as will be seen in sections of another embryo of this stage.
The mouth has now broken through, putting the wide pharynx into communication with the exterior; probably the mouth opening is formed at about the time of the opening of the first gill cleft.
Figure 4A represents a transverse section through the head of an embryo of the approximate age of the one just described; it pa.s.ses through both forebrain, _fb_, and hindbrain, _hb_; through the extreme edge of the optic vesicles, _ov_, and through the anterior end of the notochord, _nt_. It is just cephalad to the anterior end of the pharynx and to the hypophysis. The chief purpose in showing this section is to represent the two large head-cavities, _hc_. The origin of these cavities may be discussed at a later time. They are irregularly oval in cross section, and extend in an antero-posterior direction for a distance about equal to their long axis as seen in cross section. The two cavities project towards each other in the middle line, and are almost in contact with the notochord, in the region figured, but they do not fuse at any point.
These two head-cavities are the only ones to be seen, in this animal, unless the small ev.a.g.i.n.ations from their walls represent other cavities fused with these. Their walls are thin but distinct, and consist of a single layer of cells. These cells are completely filled with their large, round nuclei, so that the wall has the appearance, under higher magnification than is used in this figure, of a band of closely strung, round beads.
Figure 4B represents the eighteenth section caudad to the one just described. It pa.s.ses through that region of the enteron, _ph_, which may be called the pre-oral gut, since it lies cephalad to the now open mouth. Owing to the plane of the section the upper angle of the first gill cleft, _g^1_, is seen on the left, although this would not naturally have been expected in a section through the pre-oral gut. The ev.a.g.i.n.ation to form the hypophysis, _p_, is seen against the floor of the forebrain, _fb_. The wall of this region of the enteron is comparatively thin, and consists of not more than two layers of compactly arranged cells with round nuclei.
Figure 4C is about forty sections caudad to the one just described. It pa.s.ses through the mouth, seen as a vertical opening between the two mandibular arches, _md_. The hyomandibular cleft, _g^1_, the only one which opens to the exterior in this embryo, is very wide, and may be traced through a number of sections; in this section the opening is seen only on the left. The pharynx, _ph_, is very wide; as it is followed caudad its ventral opening is gradually closed by the approach of the two mandibular folds. The dorsal wall of this region of the pharynx is very thin, consisting of a single layer of flat cells with round nuclei; while the ventral wall, leading through the mouth and lining the mandibular folds, is composed of two or three layers of compactly arranged cells.
Figure 4D is through a plane sixteen sections caudad to the last. In this region, which is just caudad to the otic vesicles, the pharynx has still its rectangular outline, and its walls are of the same character as in the preceding figure. The posterior edges of the hyomandibular clefts are seen projecting in a ventro-lateral direction, _g^1_; while dorsal to these are the wider, second pair of clefts, _g^2_. Where the mandibular folds come together posterior to the mouth, they fuse first at their outer or ventral border, which leaves a deep, narrow groove in the anterior floor of the mouth. As this groove is followed caudad its ventral wall is seen to become much thickened, _tg_, to form the _anlage_ of the thyroid gland. In the present section the walls of the groove are just fusing, to cut off the cavity of the gland from the dorsal part of the groove. The next section caudad to this shows the thyroid as a round, compact ma.s.s of cells, with a very small lumen, still closely fused with the bottom of the oral groove. The lumen may, in this embryo, be traced for only a few sections, caudad to which the thyroid is seen as a small, solid ma.s.s of cells unattached to the oral groove. Close to the sides of the thyroid are seen two large blood vessels, _ar_, the mandibular arches, which unite into the single ventral aorta just caudad to the posterior end of the thyroid. High power drawings of the thyroid just described are shown in figures 4E and 4F.
Figure 4G is about fifty-five sections caudad to the preceding figure, and pa.s.ses through the middle region of the heart, _ht_. The enteron, _ent_, is cut caudad to the last gill cleft, but it is nearly as large as in the pharyngeal region described above; its walls are of a more even thickness than in the more anterior sections, though there is an area, just below the aorta, where the wall is still but one cell thick.
In the ventral wall of this part of the enteron, and, to some extent, in the lateral walls, there seems to be a tendency for the nuclei to become collected toward the side of the wall away from the digestive cavity; this condition cannot be well seen in the figure owing to the amount of reduction in reproduction.
Figure 4H is seventy-nine sections posterior to the last, and pa.s.ses through the foregut, _ent_, just cephalad to the anterior intestinal portal and caudad to the heart. The outline of the enteron is here almost a vertical slit, and the lining entoderm consists, in its dorsal and lateral regions, of a single layer of columnar epithelium, while in its ventral region, where it adjoins the liver trabeculae, it is made up of several layers of cuboidal or irregular cells. The nuclei in the dorsal and lateral regions of the entoderm are arranged in a very definite layer at the basal ends of the cells, though an occasional nucleus may be seen near the center of the layer. The mesoderm that extends ventrad from the mesentery, on each side of the entoderm just described, consists of a thick layer of compactly arranged cells. The ventral end of the entodermal wall is fused with the wall of a small cavity, _li_, which may be traced several sections cephalad to this plane. This cavity is a part of the system of hollow liver trabeculae seen as a group of irregular ma.s.ses of cells ventrad to the enteron at the opening of the anterior intestinal portal. The large blood vessel, _bv_, is the meatus venosus.
Figure 4I is just four sections caudad to the preceding. It pa.s.ses through the anterior intestinal portal, _aip_. The medial liver trabecula into which the enteron was seen to open, in the preceding figure, now opens ventrally to the yolk-sac as the anterior intestinal portal. A few liver trabeculae are to be seen on either side of the portal, but they show no lumena, and may be traced through only a few sections. The extent of this uninclosed region, the midgut, is very difficult to determine with accuracy, but, at this stage, it comprises about one-half of all the sections of the series. The difficulty is due partly to the unavoidable tearing of the tissues in removing the embryo from the yolk-sac, and partly to the indefiniteness of the posterior intestinal portal, where the walls of the enteron are very thin. As seen in figure 4I the location of the anterior intestinal portal is very distinct.
A short distance caudad to the anterior intestinal portal there is constricted off from the roof of the midgut a narrow diverticulum, figure 4J, _i_, the meaning of which is not apparent; it extends through only ten to fifteen sections, tapering caudad till it disappears. The region of the hindgut, at this stage, is about one-fifth of the entire length of the embryo. Its anterior portion is wide and, as has been said, rather indefinite in outline.
Figure 4K represents a typical section through the midgut region of an embryo of about the age of the one from which the preceding figures were drawn. This and the following figures of this stage were drawn from an embryo in which the posterior region was in better condition than in the embryo from which the other figures of the stage were taken. The mesentery, _ms_, is here of considerable length and continues around the yolk in a layer of diminishing thickness. The epithelium of this region of the enteron consists of a single layer of fairly regular cells, which are columnar in the dorsal region, just beneath the mesentery, and cuboidal or even flattened in regions more distant from the median plane.
Figure 4L, through the region of the hindgut, shows at _i_ the completely inclosed intestine; it is a comparatively narrow tube, lined with columnar epithelium outside of which is a dense layer of mesoblast continuous with the mesentery. In the center of the figure the allantois, _al_, is seen as an irregular cavity, lined with a single layer of columnar or cuboidal cells, and surrounded by a thick ma.s.s of loosely arranged, stellate mesoblast cells. The allantois is probably somewhat larger here than in the other embryos used for this stage, in which it was torn away. The tail, _t_, of the embryo is shown at the lower side of the figure, surrounded by the amnion; it is cut in the region of a curve so that the caudal intestine, _i_, is cut longitudinally and has the outline of an elongated ellipse. In this embryo the caudal intestine could be followed to the end of the tail, through several dozen sections; for some distance posterior to the allantois it is extremely narrow, so that its lumen is almost obliterated, and its walls are made up, in any one place, of not more than a dozen cuboidal cells. Towards the posterior end of this region the intestine is considerably enlarged as seen in figure 4L.
Figure 4M pa.s.ses through the region where both the allantois and the Wolffian ducts open into the hindgut. The union of the allantois and the gut accounts for the elongated outline of the enteron in this section.
The openings of the Wolffian ducts, _wdo_, are seen at the lower end of the section of the enteron. The cells lining the Wolffian ducts are smaller than those lining the enteron. In the lower side of the figure are seen the structures of the tail, including the outline of the tiny caudal intestine, _i_, mentioned above. No sign of a cloacal inv.a.g.i.n.ation could be made out with certainty.
The next stage to be studied is shown in surface view in figure 5.
Figure 5A represents a section through the head region of this embryo.
Owing to the obliquity of the plane of the section the figure is quite asymmetrical. The pharynx, _ph_, is lined with a comparatively thin epithelium and opens, on the left, at two places, one the mouth and the other the second gill cleft, _g^2_. In the dorsal wall of this cleft, as well as in the corresponding wall of the opposite cleft, is seen a thickening of the epithelium; these thickenings, _ty_, are the rudiments of the thymus gland, whose development may be described in detail in another paper. Compared to the size of the gill clefts the cavity of the pharynx is, at this stage, comparatively small.
Followed caudad the pharynx becomes depressed until, in the region shown in figure 5B, it is a mere narrow slit, _g_, extending transversely across the embryo and opening through the gill clefts to the exterior on each side.
Figure 5C pa.s.ses through the posterior region of the pharynx, _ph_, the tip of the forebrain, _fb_, the anterior edge of the heart, _ht_, and the curve of the tail, _t_. The chief point of interest in this section is the thyroid gland, _tg_. It now lies deep in the tissue of the floor of the pharynx, entirely separated from the pharyngeal epithelium. It consists of a compact ma.s.s of cells, now showing a bilobed structure in its anterior end, and extending through about twenty-five ten-micron sections. It is solid throughout most of its extent, but, in the section figured, which is near the anterior end, the lobe on the right side shows a small but distinct cavity scarcely visible in the figure.
Caudad to the region just described the pharynx contracts suddenly to form the oesophagus, a narrow, V-shaped slit, which soon divides into an upper and a lower cylindrical tube, figure 5D, _ent_.
Followed caudad the lower of these tubes divides into the two bronchial rudiments, figure 5E, _lu_, which, in the embryo here figured, extend through nearly one hundred sections. In the region shown in figure 5E the three tubes, _oe_ and _lu_, lie at the angles of an imaginary equilateral triangle, while in the region of the liver, where the bronchial rudiments end, the tubes lie in the same horizontal plane.
A short distance caudad to the ends of the bronchial rudiments the oesophagus turns suddenly ventrad and becomes much enlarged to form the stomach, figure 5F, _i"_, which may be traced through twenty-five or thirty sections in this series. The epithelium of the stomach is fairly thick, and consists of five or six layers of compact, indistinctly outlined cells with spherical nuclei. Ventrad to the stomach is seen, in figure 5F, a section of the duodenum, _i_, which extends, with gradually diminishing caliber, for twenty-five or thirty sections caudad to the posterior limit of the stomach, where it opens to the yolk-sac and is lost.
The section that cut this embryo in the posterior region of the stomach also pa.s.sed through the hindgut in the region of the posterior appendages, figure 5G. There the intestine, _i_, is a distinct, cylindrical tube which extends, with not much variation in caliber, and with little variation in position, from this point to the cloaca.
Followed cephalad, towards the posterior intestinal portal, it gradually diminishes in caliber, as did the foregut on approaching the anterior intestinal portal. The epithelium consists here of three or four layers of compactly arranged cells, and has about the same appearance as in the oesophagus and duodenum.
Figure 5H represents a section through the cloacal region, _cl_, showing the openings into the cloaca of the Wolffian ducts, _wdo_. Just anterior to these openings the cloaca opens ventrally into a small, anteriorly-projecting pouch, the rudiment of the allantois.
Caudad to the openings of the Wolffian ducts the cloaca extends ventrad as a narrow, solid tongue of epithelium towards the exterior, figure 5I, and fuses with the superficial ectoderm at the caudal end of a prominent ridge that lies in the mid-ventral line between the posterior appendages. In this embryo the cloaca has no actual opening to the exterior; the walls of the part that projects towards the exterior are in close contact, except in the region of the openings of the Wolffian ducts, as is shown in figure 5H.
Owing to the coiling of the end of the long tail the plane of the section, as is seen in figure 5I, pa.s.ses through the posterior end of the embryo no less than four times. In the most posterior of these four sections of the tail, beginning slightly caudad to the section here shown, is seen a small cavity which may be called the post-a.n.a.l gut, _pag_. It has thick walls, and extends for about thirty-five sections in the series under discussion. Its lumen is very large in its caudal region, figure 5I, _pag_, and tapers gradually cephalad until it disappears. Posteriorly the post-a.n.a.l gut ends quite abruptly not very far from the extreme tip of the tail.
Figure 5J is a composite drawing from reconstructions of the enterons of two embryos of approximately this stage. One of these reconstructions was plotted on paper from a series of transverse sections; the other was made in wax from a series of sagittal sections. For the sake of simplicity the gill clefts are not represented, and the pharynx, mouth, and liver are represented in outline only. For the same reason the lung rudiment of one side only is shown.
The relative size of the pharynx, _ph_, as seen in the figure, is smaller than it is in reality because of the small dorso-ventral diameter (the only one here shown) compared to the lateral diameter. The end of the lung rudiment, _lu_, is slightly enlarged and lies in a plane nearer to the observer than that of the oesophagus, _oe_, though this is not well shown in the figure.
The oesophagus, _oe_, diminishes slightly in caliber for a short distance caudad to the origin of the lungs, then gradually increases in caliber until it suddenly bends to the side (towards the observer) and merges into the wide stomach, _i"_. The stomach, which is irregularly conical in shape, lies in a place slightly nearer the observer than the end of the lung rudiment mentioned above.
Lying to one side of the stomach and duodenum, and extending cephalad beyond the end of the lung rudiment is the liver, _li_, whose outline is only roughly shown here by the broken line. The stomach opens rather abruptly into the duodenum, _d_, which slopes back towards the plane of the oesophagus (away from the observer).
The projection from the side of the duodenum, _pan_, not well figured here, indicates the position of the pancreas, better shown in the next reconstruction. The duodenum extends only a short distance caudad to this point and then opens, _aip_, to the yolk-sac.
The yolk-stalk, or unclosed region of the enteron, is still of considerable extent, though its exact boundaries are not easy to determine. The distance between the anterior and posterior intestinal portals is approximately shown in the figure under discussion.
The hindgut is cylindrical in cross section and of about the same diameter throughout, except for a slight enlargement in the cloacal region.
The post-a.n.a.l gut is not shown here; it will be described in connection with the next reconstruction where it is figured.
Figure 6 is a surface view in profile of an embryo of the next stage to be studied. The ma.n.u.s and pes are here well developed, and the general development of the embryo is in considerable advancement over the last stage studied.
Figure 6A represents a reconstruction, from a series of transverse sections, of the enteron of an embryo of about the age of the one shown in figure 6. The outlines of the entire embryo, of the eye, _e_, and of the anterior, _aa_, and posterior, _pa_, appendages are shown by broken lines. Its position being coincident with that of the stomach, liver, and pancreas, the anterior appendage can scarcely be seen. The enteron, including one lung only, for the sake of simplicity, is shaded solid black, while the liver and pancreas, with their ducts, are outlines with unbroken lines. As in the preceding reconstruction no attempt is made to show the gill clefts, and only the dorso-ventral profile of the enteron is shown. Caudad to the pharynx, the enteron being more or less cylindrical in section, this profile gives a good idea of its shape, but in the pharyngeal region, where the lateral diameter is so much greater than the dorso-ventral, the reconstruction gives but a poor idea of the size of that part of the enteron.
The widely-open mouth, _m_, leads, with no line of demarkation, into the pharynx, _ph_, which is of irregular outline and, as has been said, of much greater lateral than dorso-ventral diameter.