Paper - On the blastodermic vesicle of sus scrofa domesticus
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Weysse AW. On the blastodermic vesicle of sus scrofa domesticus. (1894) Proc. Amer. Acad. of Arts and Science 3: 283-321.
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Contributions From The Zoological Laboratory Of The Museum Of Comparative Zoology, Under The Direction Of E. L. Mark, XLIII.
- 1 On The Blastodermic Vesicle Of Sus Scrofa Domesticus
- 1.1 I. Introduction
- 1.2 II. Description of the Embryos
- 1.3 III. Historical and Theoretical
- 1.4 Literature Cited
- 1.5 Explanation of Plates
On The Blastodermic Vesicle Of Sus Scrofa Domesticus
By A. W. WeyssE.
Communicated by E, L. Mark. Received August 10, 1894.
In the latter part of the summer of 1893 the opportunity was opened to me of obtaining young embryos of Sus scrofa domesticus. So I let the work rest which I had in hand on the development of some other mammals and began investigations on this. I was fortunate enough to secure a number of embryos several days younger than the youngest of the six, from fourteen to fifteen days old, which Keibel (93) has recently described so elaborately ; and since these present phenomena of development unusual in the ontogeny of the Mammalia, it has seemed best to record them, in the hope that they may possibly lead to similar discoveries in allied forms.
The incompleteness of our knowledge of the early stages in the embryology of the higher viviparous animals is due largely, of course, to the difficulty of securing sufficient material in successive stages of growth, and there is the further difficulty that it is impossible to determine in the great majority of cases the exact age of the embryos, since spermatozoa may remain for several hours, days, or even months, within the body of the female before fertilization takes place. And further, it has long been kuown that, especially during the earlier phases of development, ova which were apparently fertilized at the same time grow at very different rates, so that within one uterus we may find embryos illustrating several stages of ontogeny. For these reasons I cannot give exact ages for the embryos I am about to describe, though in almost every case I can give the period which has elapsed between cvitus and the time when the sows were killed and the embryos obtained.
The present paper is based on the results obtained by the study of thirty embryos taken from four sows. In all I had nine sows served at different times, and these were killed at from nine to eleven days after coitus. Only three of the nine proved to be pregnant at the time they were killed, and of these three, two were killed ten days and the other eleven days after copulation. Each of the two sows killed on the tenth day contained eleven embryos in varying stages of development, though all the embryos in one were much less advanced than those in the other, as I conclude from the difference in the size both of the embryos and of the cells of which they are composed, and from the difference in the size and structure of the germinal disks, The third sow, killed on the eleventh day, contained only four embryos, two of which were as old as the oldest of those from the first two sows; the other two were younger. The remaining four embryos of the thirty were found in a sow which had been served before coming under my control, and were apparently in about the same stage ontogenetically as some of the older embryos in the first two cases mentioned above. The uterus in which these last embryos lay, contained six in all, but two were so badly folded as to be unavailable as far as the study of the germinal disk was concerned. These embryos were obtained by opening a large number of apparently empty uteri; in this way I have found about 3% of the individuals examined pregnant, but all of the embryos with the exception of the six just mentioned were in a much more advanced stage of devolopment, so that they afford no information on the subject matter of the present article, and I shall reserve the consideration of them for a future paper.
In the case of the first uteri which I opened, I followed with some variations one of the methods which Keibel (’93) has described in his work on the pig. When the uterus had been removed from the animal, it was cut open along the side opposite the mesometrium and placed in a flat-bottomed glass dish containing Kleinenberg’s picro-sulphuric mixture and resting on a black tile. Thon, by carefully spreading out the complicated folds of the inner wall of the uterus and genily agitating them, the embryos readily floated out into the fluid, and could be distinguished at once against the black surface of the tile. They were then carefully removed on a spatula to a smaller vessel of the picrosulphuric mixture, where they remained several hours and were then transferred to 70% alcohol, and after twelve or fourteen hours to 90% alcohol, which was gently warmed and changed several times until all trace of the acid had been washed out. But this method had some minor disadvantages which seemed avoidable; the action of the acid mixture on the instruments used, and the staining of the hands, were at least undesirable, and to be avoided unless absolutely necessary. Accordingly, in my later work, instead of using Kleinenberg’s mixture as a medium for floating out the embryos from the uterus, I employed normal salt solution (0.75% NaCl in water), at a temperature of about 40° C. Keibel (93) says that he did not use this because it is said to injure the embryos, and Bonnet (84) found that, if embryos lay a long time in this solution, they became swollen. This is doubtless true, if they remain in the fluid as long as is necessary to detach such complicated embryos as Keibel worked with, some of which were more than a meter long and greatly folded amongst the plications of the uterine wall. The same may be true for the embryos of the sheep, — upon which Bonnet worked, — which at a corresponding stage of development closely resemble those of the pig, but in the case of such small embryos as those which I have been concerned with the very short time during which it is necessary for them to remain in the salt solution has absolutely no effect either on the general form of the embryo or on the histological conditions, as my sections clearly demonstrate. As soon as the embryos were floated out from the uterus, they were at once transferred as before to Kleinenberg’s picro-sulphuric mixture, All the embryos which I have studied have been fixed in this fluid. Had my supply of material been larger, I should have employed several other fixing reagents, but my work on young embryos of other mammals, as well as the results of the experiments of other embryologists, gave me complete confidence in the reliability of this reagent for the material in hand. The results in this case were in the highest degree satisfactory.
After the specimens had been entirely freed from acid, drawings of them as opaque objects seen against a black background were made with the aid of an Abbé camera lucida; they were then returned to 70% alcohol, and from this transferred to Kleinenberg’s alcoholic hematoxylin (70%) diluted with twice its volume of his solution of calcium chloride and alum. Here they remained for a few hours, the time varying according to the size of the embryos, and were then transferred to a 0.1% solution of hydrochloric acid in 70% alcohol, the process of decolorizing being carefully watched under the microscope until the object had attained the proper color. It was then placed in 70% alcohol containing a slight trace of ammonia, which made the stain permanent by neutralizing the acid. I have found that washing simply in neutral alcohol, though it be never so carefully done, will not always prevent an ultimate fading of this stain; the addition of ammonia is an absolute essential if one wishes to be perfectly sure that the sections will not fade. Objects which I have treated thus have always preserved their color. In my work on the pig I have not as yet used carmine stains. On the embryos of both the rat and the mouse IJ have obtained very brilliant results, both with borax carmine and with hydrochloric carmine, but, so far as I could see, they had no advantage over hematoxylin. The last is valuable because of its high alcoholic grade, and because, when properly decolorized, it becomes a most highly differential stain for these embryonic tissues, Small semi-transparent objects, like the embryos of the pig at this stage of development, can readily be decolorized ¢m toto, for the extent of the decolorizing can be determined by the aid of the microscope ; but opaque objects, like the uterus of the mouse, have to be decolorized largely after sectioning.
When the embryos had been stained and decolorized, they were cleared in chloroform, embedded in paraffine, and cut on the MinotZimmerman microtome into sections 10 thick. They were then spread on the surface of distilled water, which rested on a thin film of albumen affixative covering the slide. The slide was gently warmed in an alcohol flame, until the sections became perfectly smooth. Finally the water was removed, the paraffine dissolved out in xylol, and the sections mounted in Canada balsam.
I have given this rather extended account of my technique in order to show that there has been no lack of care in this direction which could affect the histological condition of my specimens. I will now give a description of the embryos themselves, and then proceed to a consideration of the investigations of other authors, and to the theoretical interpretation of the phenomena described.
II. Description of the Embryos
1. General Characteristics of the Blastodermic Vesicle
The embryos are all in more or less advanced stages of the so-called blastodermic vesicle or didermic blastocyst, as it has been described by Balfour (’81), Van Beneden (’80), Hubrecht (90), Bonnet (91), and others. They consist in general of at least two well defined layers, —an outer of more or less isodiametric cells, and an inner, in contact with the outer, of greatly flattened cells, which are very much larger than those of the outer layer. There is no trace of cells lying between these two layers at any point. At one region of the embryo, the outer layer of cells is thickened, forming the germinal disk. For the sake of convenience this region may be spoken of as the germinal or embryonic area, and the rest of the vesicle as the extra-germinal or extra-embryonic region. In most cases there is evidence of a third layer of cells, which is outside the two layers just mentioned, and is in a more or less disintegrated condition. I shall later refer to this more at length.
It has not seemed to me necessary to give figures of the whole blastocyst. In each case it consists of a hollow vesicle with a double wall, which if fully distended would be about spherical. I have never found the vesicles completely distended, and often they are greatly folded, so that it is not always possible to make sections in just the plane one wishes ; for if the germinal disk lies on the edge of a fold, it is usually necessary to cut at right angles to the fold in order to avoid getting sections oblique to the surface of the disk. Something of an idea of the general appearance of the vesicle can be gained from Bonnet’s (84, Taf. IX. Fig. 2) figure of a sheep embryo of the thirteenth day, which has the same general characters as the pig embryos which I have studied.
The germinal disk of this stage can be detected by the naked eye, even before staining, as a very small opaque white spot on the surface of the vesicle. The smallest vesicle which I have examined is about 1 mm. in diameter, while the largest is about 4.5 mm. In the latter there is no trace whatever of the beginning of the excessive elongation which takes place before the fourteenth day, and has been well figured by Keibel (93); this growth undoubtedly takes place very rapidly, for Bonnet (91) has estimated that in the sheep, where a similar though less extensive growth occurs, the embryo must elongate at the rate of more than I cm. per hour, and that in the pig the growth is still more rapid. The germinal disk varies in size from 0.1 mm. in the smallest to 0.265 mm. in the largest embryo here considered.
In describing the embryos more at length I shall speak of the layer of nearly isodiametric cells as the ectoderm, and of the inner layer of flattened cells as the entoderm; the relation of these to the outer disintegrating layer, which with Rauber (’75) is best designated as the “ Deckschicht,” I shall discuss later. The two prominent layers (ectoderm and entoderm) are distinguished from each other not only by the shape of their component cells, but also by the shape of their nuclei, and by the chromatic reaction of their protoplasm. The nuclei of the extra-germinal region of the ectoderm are nearly always perfectly spherical; those of the germinal disk, especially in later stages of development, are more often slightly elongated or ellipsoid, with the longest axis at right angles to the surface of the disk; there are significant exceptions to this last rule which will be considered later. The entodermal nuclei in the extra-germinal region are generally flattened parallel to the surface of the embryonic vesicle ; those in the region of the disk also have this shape in the younger embryos, but in the older stages they are spherical, and the cells in which they lie are essentially isodiametric. Furthermore, when properly decolorized the hematoxylin gives three distinct shades of blue to the embryonic cytoplasm. The extra-germinal ectoderm is stained a light blue, the ectoderm of the germinal disk is sharply marked off from this by a deeper shade, and the cytoplasm of the entodermal cells stains uniformly a still deeper blue. This differentiation in color, which is often of very great value, does not appear before the embryo is decolorized, and it disappears if the process of decolorizing is carried too far, for in that case the whole vesicle becomes a uniform light blue. In almost every instance, these three shades of blue are manifest in my preparations. The chromatic substance of the nucleus stains deeply throughout the vesicle, both in resting nuclei and in those undergoing karyokinetic changes. In the latter, when cut at the proper angle, the nuclear spindle and the protoplasmic radiations around the centrosomes can be clearly seen. It should further be noted, that this stain brings out the cell walls with great distinctness, especially in the ectoderm. In the entoderm the cells are extremely attenuated at their regions of contact, appearing spindle-shaped in section, the nucleus occupying the swollen portion, so that the dividing walls are apparent in the region of the germinal disk only, where the axes of the cell are nearly equal. In my oldest embryos there is no sign of the formation of mesoderm, or of the medullary groove. With this description of the embryonic vesicle in general, I now proceed to the consideration of the more detailed structure of several embryos which I have selected for illustration here. I have chosen these because they show in a typical manner the phenomena which all present, and with drawings from them I can explain intelligibly any variations from the type.
2. Detailed Account of Observations on the Germinal Disk
(Plate II Figs. 7, 8, and 9.)
The first embryo which I shall describe is represented by drawings (Plate II. Figs. 7, 8, and 9) of three sections of the germinal disk. This embryo was taken from a sow killed ten days after coitus, which contained eleven embryos. The embryo in question was with one exception the smallest found, but it should be noted here that the ratio between the size of the embryo and that of the germinal disk is not constant for different embryos; and furthermore, that the ratio of the size of the disk to its degree of development varies; e. g. Figs. 11, 12, and 13, Plate II., were drawn from an embryo much larger than that from which Figs. 7, 8, and 9 were made; the two embryos came from the same uterus, and yet the former disk is slightly smaller than the latter; on the other hand, it represents a much later stage of development, as I shall show hereafter, and this fact is doubtless sufficient to account for the difference in size. The whole vesicle from which Figs. 7, 8, and 9 were drawn measured about 1.25 mm. in diameter. As I have already said, the vesicles are flattened out and greatly wrinkled, so that such measurements are at best approximate only, though they serve to give a general idea of the relative sizes of the embryos. The germinal disk, which lies on a part of the vesicle that is not folded, is slightly elliptical in outline, the chief and transverse axes being about 0.11 mm. and 0.167 mm. long respectively ; the sections were made nearly at right angles to the shorter axis, which, as I think I can show later, represents the chief axis of the future embryo.
The ectoderm consists of the characteristic large cells with spherical on the inner surface of the ectodermal layer, and are more numerous in the region of the germinal disk than in the extra-germinal area, but they everywhere retain their elongated flattened outline. The germinal disk was cut into ten sections, of which Figs. 7 and 8 represent respectively the second and third, and Fig. 9, the sixth. According to my method of orientation the two former are at the posterior end of the future embryo, the latter nearer the anterior end. It will be seen from the figures that the ectodermal cells of the germinal disk are slightly elongated in a direction at right angles to the surface of the disk, and show a tendency to arrange themselves in two interlocking layers. At the margin of the disk the transition from disk ectoderm to extragerminal ectoderm is abrupt, but the general characteristics of the cells in the two regions is such as to suggest an identity of origin at least, — the differences being merely the slight variation in the chromatic affinity of the cytoplasm, which I have already mentioned, and the change in shape, which would necessarily attend a difference in cell arrangement. The exposed surface of the disk is pretty uniformly even, except at one very significant point. Two sections through this point are shown in Figs. 7 and 8, Plate IJ., where in the centre of each section a cell is seen to project above the general level of the disk, and in Fig. 8 to be slightly cut off from it. The same appearance is present in the two sections which intervene between Fig. 8 and Fig. 9, and the whole projection running through the four sections contains three cells, which are without any question true ectodermal cells of the germinal disk, which have assumed this new position. These cells represent a very early stage in the development of a structure which I shall later designate as the bridge, and for that reason I shall call these bridge cells. It should be noted here that there is no evidence whatever of a third layer of cells, i. e. a “ Deckschicht,” outside the ectoderm in the region of the germinal disk, though in the extra-germinal region there are a few widely separated cells attached to the outer surface of the ectoderm; these differ from the ectodermal cells in having very small nuclei; they resemble the cells of the entoderm in general outline. By an enumeration of the nuclei I have ascertained approximately the number of cells in the area of the germinal disk, which is as follows: cells in the ectoderm proper, 188; in the entoderm, 48; in the bridge, 3. The presence and position of this bridge supply the criterion on which I have determined the chief axis and its poles for the futureembryo. It should be further noted here, that in the two sections immediately succeeding Fig. 9, i. e. sections seven and eight of the series through the disk, there is a slight elevation of the ectodermal cells at the margin of the disk on either side. The significance of this fact will appear in my further description of the bridge. Moreover, in sections seven, eight, and nine of the disk there is a distinct groove or furrow in the upper surface of the ectoderm ; this runs along the median line, and is therefore in a line continuous with that of the bridge cells described above. And now a word as to similar stages in embryos which I have not figured here.
The ten other embryos which came from the same uterus as the one I have just described vary in size from 1 mm. to 1.9 mm. in diameter, and represent very diverse stages of development. Only two of these are in about the same stage as the one mentioned above, the others being clearly much more advanced, and of these two I consider one further developed than the other, since it has many more bridge cells on the disk. The younger embryo measured 1.4 mm. in diameter, while its germinal disk was 0.11 mm. in diameter and very nearly circular in outline. An enumeration of the nuclei shows approximately 117 cells in the ectoderm proper, 33 in the entoderm, and 6 in the bridge. It is somewhat difficult to determine with absolute certainty the number of cells in the bridge in this case, for some cells are just in process of passing over from the true ectoderm of the disk to the bridge; the direction of the spindle in the karyokinetic figures makes this point indisputable. The mass of bridge cells appears, as in the specimen already described, nearer one margin of the germinal disk, and for that reason I term the margin near the bridge cells the posterior end of the embryo. Anterior to this point there is the same lateral or marginal uprising which I mentioned in the first case. The entoderm consists of greatly flattened cells with widely separated nuclei, but so far as I can determine they are all connected by delicate protoplasmic masses, which often appear like a thin membrane lying across the rounded inner boundaries of the ectodermal cells. One may therefore pass through several sections without finding an entodermal nucleus on a certain area of the embryonic vesicle, but I should not feel justified in assuming that the entoderm fails to cover any portion of the interior surface of the ectoderm of this vesicle.
The second embryo to which I referred in connection with this was but 1 mm. in diameter, and therefore the smallest in my collection. The germinal disk is, however, clearly in a more advanced stage of development, for the reason which I have already given, and the vesicle also has a larger number of entodermal cells, so that there can be no doubt in this case that they form a complete layer on the inside of the ectoderm. On the outside of the vesicle are a very few “‘ Deckschicht ” cells. Beyond these facts the embryo presents no characteristic differences from the embryos already described.
(Plate I. Fig. 1, Plate II. Fig. 10.)
I will now take up a stage which shows a distinct advance in the development of the embryo. Fig. 1, Plate I., shows the portion of the blastodermic vesicle containing the germinal disk. This embryo came from the uterus of the sow which was served before coming under my control, so that I do not know the time which elapsed between coitus and the killing of the animal. The stage of development, however, clearly places it at this point in my series. The whole vesicle, which was somewhat wrinkled, was about 2.65 mm. in diameter, while the germinal disk measured 0.205 mm. in its longest axis and 0.18 mm. in its transverse axis, thus having an elliptical or slightly ovate outline, which is shown in the figure. The drawing (Fig. 1) was made from the whole object before staining, and is represented as seen by reflected light against a black background. JIn sections the entoderm shows the characteristic spindle-shaped cells in the extra-germinal region, while in the area covered by the germinal disk it consists of cells lying closely together and showing sharply defined cell boundaries. On the outside of the blastodermic vesicle are a large number of “ Deckschicht ” nuclei in the extra-germinal region, all with little chromatic substance and with ill defined cell boundaries. But it is the ectoderm of the germinal disk which presents the most interesting phenomena in this embryo. Fig. 10, Plate II., shows a median longitudinal section through the disk shown in Fig. 1, Plate I. Here the general surface of the disk is seen to be somewhat depressed, thus leaving a raised margin, while at one pole of the longest axis there is a large upgrowth or overgrowth of ectodermal cells forming the bridge to which I have already referred. The beginning of the formation of this overgrowth is at what I hope to establish as the posterior end of the germinal disk. The bridge consists at this stage of practically two layers of cells, which are essentially the same in structure as the remaining ectodermal cells of the germinal disk. Clearly this overgrowth is only a later stage in the growth of the structure which was seen in its first stage of development in Figs. 7 and 8, Plate II., and the raised lateral margins seen here I hold to be comparable with the raised margins described in the previous embryos, while the depressed area seems to be due to a broadening of the median groove of the vesicle, described in detail above. It may be noted here that the germinal disk is unusually large for a bridge which has developed to so small an extent, and furthermore the bridge is more widely separated from the underlying ectoderm than it 1s in many embryos of this stage.
(Plate I. Fig. 2, Plate IV. Figs. 21 and 26.)
In Fig. 2, Plate I., is shown a surface view of a germinal disk from an embryo taken from still another sow, which was killed ten days after copulation. The blastodermic vesicle was almost completely distended, there being but one marked fold, and measured 1.95 mm. in diameter. The germinal disk was about 0.15 mm. in diametet and circular in outline. It was cut into fourteen sections; these were all drawn with the aid of the camera lucida to an enlargement of 275 diameters, and from these drawings a reconstruction of the surface was made and then reduced to an enlargement of 100 diameters, as it appears in Fig. 2. A reconstruction in wax was also made as a surer means of control to guard against any error in the graphic reconstruction. Two sections of this embryo are shown on Plate IV., Figs. 21 and 26, made through the region of the germinal disk and the extragerminal area respectively. The sections are oblique to the anteroposterior axis of the disk, cutting it at an angle of about 45°, and running on the reconstruction drawing from the lower right-hand corner to the upper left, Fig. 2, Plate I. Of the fourteen sections into which the disk was cut, Fig. 21, Plate IV., represents the seventh, and passes through the deeper portion of the depression which lies just beneath the central point of the free margin of the bridge, as seen in Fig. 2. In this specimen the two lateral elevations mentioned in the embryos already described are seen to have increased in size until they have come into contact with the posterior overgrowth, with which they have fused at two points, thus giving the bridge three points of aitachment to the ectoderm proper and leaving three openings from the depression or cavity beneath it to the outside. This bridge, then, has manifestly three points of origin, one posterior and two lateral. Further evidence in corroboration of a similar method of formation will appear in the course of the description of the other embryos.
Before leaving the ectoderm of the germinal disk, it should be added that it is relatively very thick in all these younger embryos, consisting of two or even three interlocking layers of cells, in addition to the bridge cells, which are usually two layers deep. This great thickening of the ectoderm of the germinal disk is not, however, the earliest condition ontogenetically, for in younger stages of development, as, for example, those represented by Figs. 7-10, Plate IL, it is much thinner than in either the stage under discussion or that represented by the figures on Plate III.
The cells of the entoderm are essentially the same in structure in the region of the germinal disk as they were over the same area in the embryo represented in Fig. 1, Plate I., and Fig. 10, Plate II.; but there is a further thickening or increase in number of the entodermal cells immediately surrounding the germinal disk, and extending on all sides of it for a distance about equal to the diameter of the disk itself, so that the diameter of the entodermal thickening is at this stage about three times the diameter of the germinal disk. The normal distribution of the entodermal nuclei in the remainder of the extra-germinal area can be seen from Fig. 26, Plate IV. A possible explanation as to the significance of this thickening of the entoderm has occurred to me, which I will mention in the theoretical considerations concerning the interpretation of the vesicle.
(Plate I. Figs. 3-5; Plate II. Figs. 11-18; Plate III. Figs. 14-19; Plate IV. Fig. 20.)
The next two embryos in the series are shown in Figs. 3 and 4, Plate I. I have given no sections of these, as the surface views show the more important characteristics, and because they are essentially the same as sections of other embryos which I have figured farther on. Fig. 3 shows that the bridge has a nearly circular margin bordering the opening into the cavity beneath. There is no evidence on the surface of the germinal disk or in the sections — which are taken at right angles to the longer (i. e. transverse) axis —as to whether the bridge arose at three points, as mentioned above, and then became one by a fusion of the three parts, or whether it arose as one continuous overgrowth from the margins towards the centre of the disk. The vesicle to which this germinal disk belongs was 2.25 mm. in diameter and very little folded. The disk itself measured 0.19 mm. in its longer axis and 0.15 in its shorter or antero-posterior axis. It will be observed that the elliptical outline of the germinal disk is the same with regard to the chief axis of the future embryo, as in the case of the first embryo described.
Figure 4 represents a disk which is a little older than that shown in Fig. 3. The bridge covers a larger portion of the germinal disk than in the preceding case, and if we assume that the disk had at first the shape shown in Fig. 3, it has begun to elongate in the direction of its chief axis, until it is now nearly circular in outline. The right-hand side of the figure shows a lateral opening into the cavity beneath the bridge, where the lateral overgrowth has not as yet entirely fused with the overgrowth from the posterior end to form a continuous bridge such as exists in Fig. 83. The left-hand side, however, is complete, exactly as in Fig. 8. This germinal disk might be considered as illustrating a stage intermediate between Figs. 2 and 3, so far as the fusion of the various parts of the bridge is concerned; it is, however, clearly more advanced, not alone on account of the greater size of the germinal disk, — for this may vary greatly, as we have already seen, — but because of the greater extent and degree of development of the bridge. The embryonic vesicle in this case. was only slightly folded, nearly circular in outline, and about 2.75 mm. in diameter ; the germinal disk measured 0.2 mm. in diameter.
Figures 11, 12, and 13, Plate II., represent sections through a germinal disk in which the bridge has reached about the same stage of development as in the two cases last described. The embryo from which these three sections were taken came from the same uterus as that of Figs. 7, 8, and 9, and the sow was killed therefore on the tenth day after coitus. This vesicle measured about 1.55 mm. in diameter, while the disk, which was slightly elliptical, measured 0.145 mm. in its longer axis (the sections shown in Figs. 11, 12, and 18 are parallel to this axis), and 0.11 mm. in its shorter axis. This shows, then, a still greater elongation than the preceding germinal disk in the direction of the chief axis of the future embryo. The disk was cut into eleven sections, of which Fig. 11 represents the fifth; it is, therefore, a little to one side of the median plane. This section shows well a phenomenon which is present in all sections of bridges that have developed to some extent, and seems to point to a double origin of this structure. It will be noticed that the extra-germinal ectoderm appears to extend as a continuous layer over the right-hand portion of the disk and to constitute the upper layer of bridge cells, while the lower layer is clearly derived from the true ectoderm of the germinal disk itself, as we can see from the position of the nuclei at the region of contact of the bridge with the underlying ectoderm. It should be observed, however, that this apparent extension of the extra-germinal ectoderm over the ectoderm of the germinal disk occurs in the region of the bridge only. The whole appearance suggests an upfolding of the margin of the disk, which carries both the extra-germinal and the germinal ectoderm with it. Figs. 12 and 13 represent the eighth and ninth sections respectively through the same disk. Fig. 12 is a section at the extreme lateral margin of the opening of the bridge, the cavity beneath it appearing as a somewhat triangular space in the section.
Fig. 13 lies beyond the region of the cavity, and the bridge is here in contact with the underlying ectoderm.
I am so fortunate as to have another embryo from the same uterus from which this came, which is in almost precisely the same stage of development, and is cut very nearly at right angles to the chief axis of the germinal disk. This gives a series of sections transverse to those which I have just described. The general relation of the overgrowth, or bridge, to the underlying ectoderm will be plain, if I describe briefly two or three of the sections. Beginning with the second section in the series, which lies at what I term the posterior end of the embryo, a condition is found which is very like that represented in Fig. 18, Plate IL., consisting of a layer of true, somewhat columnar ectodermal cells, with the long axis perpendicular to the surface of the disk, and overlaid by a layer of bridge cells, slightly elongated in a direction parallel to the surface of the disk. The next section anterior to this shows the bridge passing across the germinal disk from one side to the other, and separated from it in the central region by a cavity ; the structure here resembles an arch spanning the disk from side to side. Taking next a still more anterior section, the bridge is represented by an upfolded region at either side of the germinal disk, overhanging the true ectoderm, and presenting very much the appearance that Fig. 10, Plate II., would present if there were also an upfolding at the right-hand side of the drawing similar to that at the left. The succeeding sections simply show these lateral upfoldings diminishing in size until they disappear near the anterior margin of the germinal disk. Thus this series of sections, together with the series described just before it, gives the basis for a very complete conception of the bridge as it appears at this stage.
Returning now to the figures, we find a condition somewhat more advanced than the preceding, represented by Fig. 5, Plate I. The vesicle from which this germinal disk came was taken from the uterus on the eleventh day after coitus, together with three others, which were all manifestly older. The vesicle was somewhat wrinkled, and measured 3.4 mm. in its longest diameter. The disk lay near one margin in an unwrinkled area, and was ovate in outline, the long axis being 0.23 mm. and the greatest transverse axis 0.2 mm. in length, At the broad or anterior end the crescent-shaped free margin of the bridge appears sharply marked off from the underlying ectoderm by a cavity which extends to within a short distance of the margin of the disk on all sides except at the anterior end, where the bridge is wanting. At this point the ectoderm is slightly thicker than in the region beneath the bridge, and this thickening appears on the surface as a slight elevation, which I have tried to show by the shading in the drawing. This germinal disk illustrates the form and structure of the bridge so clearly and so typically, and represents almost, if not quite, its maximum development as a free and independent structure, that I have thought best to illustrate it more fully by sections than I have done in the case of the preceding embryos.
The sections were made in a direction very nearly parallel to the long axis of the germinal disk, which at this stage corresponds to the antero-posterior axis of the embryo. The disk was divided into fifteen sections, and of these, Figs. 14-19, Plate III., and Fig. 20, Plate IV., represent respectively the first, second, fourth, sixth, seventh, eighth, and ninth; the sections beyond these simply present the same phenomena in reversed order. Little need be said in explanation of the first two sections, Figs. 14 and 15, Plate III. Figure 14 is taken at the extreme lateral margin of the disk and is consequently nearer the anterior than the posterior end, because of the ovate outline of the disk. The section immediately preceding this consisted merely of a layer of typical ectodermal cells on the outside with a layer of characteristic entodermal cells a short distance beneath. Figs. 14 and 15 show the great rapidity with which the ectoderm increases in thickness in passing from the lateral margin of the disk towards its centre. The third section in the series has not been represented here, since it shows no new features beyond those represented in Fig. 15, except an extension of the ectodermal cells in a posterior direction. The next section is represented by Fig. 16, in which occurs the first appearance of the cavity which marks off the bridge from the rest of the germinal disk. The bridge itself consists of several irregular layers of cells, the outermost of which is made up of cells considerably flattened in the plane of the germinal disk. As to the general characteristics of the disk in section, it should be noted that the anterior (in the figure, the right-hand) end is thicker and more rounded than the posterior, which is rather attenuated. It will also be seen that the lower or inner boundary of the ectodermal cells of the disk is marked by a relatively sharp line due to the presence of a very delicate membrane. At the margin of the germinal ectoderm, — where, between the extraembryonic ectoderm and entoderm, a space (triangular in section) occurs which completely surrounds the disk, — this membrane loses its connection with the ectoderm of the germinal disk and stretches across the space to meet the extra-embryonic portion of the outer layer at some distance from the margin of the disk. The whole entoderm is normally in close contact with the ectoderm, but in the region of this membrane it seems to be more loosely attached than elsewhere, or else the cells are less resistant, for we often find that here they are somewhat torn away, as though by some mechanical injury, possibly due to the effect of the fixing or the hardening reagents, which necessarily produce a slight shrinking of the vesicle. This membrane will be more fully discussed farther on.
I have not introduced the figure of the next section in the series, since it merely shows a condition intermediate between the preceding and following sections. Fig. 17 passes near the lateral margin of the free edge of the bridge, and shows that the cavity beneath the bridge extends much farther towards the posterior end of the germinal disk, and also that the bridge itself is thinner just at this point than in the preceding and the succeeding sections. By comparing this with both Figs. 18 and 19, it will be readily seen that this diminution in thickness results in a ring or crescent very near the inner margin of the under side of the bridge, which in Fig. 17 is cut nearly longitudinally, and in Figs. 18 and 19 transversely near the posterior point of attachment of the bridge to the underlying ectoderm. Fig. 18 shows the first section which passes through the free anterior margin of the bridge.
Figures 19 and 20 may best be considered together. Fig. 19 represents the section which lies in the median plane of the embryo, and consequently here the free edge of the bridge is farthest removed from the anterior end of the disk. In Fig. 20, Plate IV. the free margin of the bridge has begun to advance towards the anterior end of the disk again. The phenomenon of greatest significance in these sections, however, is found at the point where the bridge comes in contact with the ectoderm of the germinal disk near the posterior pole of the chief axis. In Fig. 19 it will be noticed that the cavity beneath the bridge appears to extend between the cells posteriorly as a narrow opening for a short distance. In Fig. 20 we find a continuous canal passing from the cavity beneath the bridge into the space between the ectoderm and the entoderm of the extra-germinal area just at the margin of the germinal disk. This canal appears to arise in the median plane of the embryo, and to pass between the ectodermal cells into the cavity just mentioned in a direction slightly oblique to that plane. At least that must be the conclusion, provided the sections are exactly parallel to the median plane of the disk; but if the sections were only very slightly oblique, they would make a canal as small as this appear to have an oblique direction, even though it were actually parallel to the chief axis of the embryo. This phenomenon is not confined to this embryo. A precisely similar canal can be traced in the embryo figured on Plate I. Fig. 8, and in two or three other cases there are suggestions of a similar condition, but not sufficiently well marked for me to put much stress upon them. It will be readily seen that unless the section should pass in exactly the right direction, i. e. very nearly through the long axis of the canal, it would be impossible to establish its presence except in extremely thin sections. Its occurrence in Fig. 20 is beyond question; I shall discuss its possible morphological significance later.
The fact that the free surface of the germinal ectoderm shows no trace of cells resembling the ectoderm of the extra-germinal area, such as are present on the upper surface of the bridge, may be mentioned here again in passing.
(Plate I. Fig. 6; Plate IV. Figs. 22-25.)
We now come to the last stage in the history of the bridge. This can best be shown in two phases, the first of which is represented by Figs. 22 and 23, Plate IV. These are from sections of an embryo taken from the same uterus as that represented by Fig. 2, Plate I. The blastodermic vesicle was small in comparison with the size of the germinal disk, being but 3.1 mm. in diameter, while the disk, which was ovate in outline, measured about 0.3 mm. in its long diameter, and in its greatest width 0.29 mm. It was cut into thirty sections in a direction nearly perpendicular to the long (chief) axis. Fig. 22 represents the thirteenth section in the series, which begins at the narrower or posterior end, and Fig. 23 represents the seventeenth, which is therefore somewhat more anterior. Fig. 22 shows that the germinal disk consists of cells whose nuclei lie at varying distances from the surface of the disk, and that it has a rather broad median region pretty clearly marked off from a marginal region on either side, by the fact that it contains more nuclei, and also because the general surface of the median region is here slightly elevated above the lateral portions of the disk. This median elevation is more or less marked in the sections which precede this in the series, maintaining about the same relative extent. In the succeeding sections, however, it is no longer marked, so that the surface of the ectoderm is pretty uniformly flat. At the same time there is to be noticed at the margin a layer of cells cut off from the underlying ectoderm by a narrow cavity. This occurs on each side of the disk, but owing to the obliquity of the sections it appears in Fig. 23 at the left-hand side only, while it is seen at the right as well in the sections immediately following. The stage of development represented by these sections can, I think, be readily shown to be only a more advanced condition of the phenomena presented by the germinal disk of Fig. 5. The change is brought about by a simple obliteration of the cavity between the disk and the bridge, produced by the sinking down of the bridge until it comes in contact with the surface of the disk, with which it fuses. The cavity which appears at the left in Fig. 23, and on the right in succeeding sections, is in all probability the last trace of the marginal groove which I mentioned in the description of the preceding embryo as running around the under surface of the bridge.
Another embryo shows a second and later phase in the disappearance of the bridge. Fig. 6, Plate I., represents the germinal disk of an embryo taken from the same uterus as that of Fig. 5. The blastodermic vesicle was nearly circular in outline, slightly folded, and measured 38.9 mm. in diameter. The germinal disk was distinctly ovate in outline, as shown in Fig. 6, and measured in its long axis 0.265 mm. and in its greatest breadth 0.28 mm., thus exceeding in size the disk of Fig. 5 by just 0.03 mm. in each diameter. Though somewhat smaller than the disk just described, it is clearly older, as the description of the sections will show. It was cut into thirty sections in a transverse direction, i. e. at right angles to the long axis of the embryo. Starting from the broader end of the disk, Fig. 24 represents the fourth section in the series, and Fig. 25 the seventh. Here the only evidence we have that a bridge has been present is in the shape and position of some of the more superficial cells and their nuclei. In Fig. 24 several of the surface cells show the characteristic elongated outline with flattened nuclei which the more superficial cells of the bridge present in its greatest development, and in Fig. 25 two cells near the centre show, for the same reason, an undoubted origin from the bridge.
3. Summary of Observations on the Blastodermic Vesicle of the Pig
I have given above in some detail the principal phenomena which my material presents, and now I wish to give in a more compact form what I take to be the typical changes which occur in the period of development which these embryos cover, and to point out one or two variations from the type which serve to throw some light upon its meaning.
The earliest stage which I have, shows a blastodermic vesicle consisting of a sharply defined inner layer of flattened cells, — the entoderm, — which forms a closed sac. In contact with the outside of this is a layer of nearly isodiametric cells, — the ectoderm, — which at one point is thickened to form the germinal disk, both by an increase in the diameter of the cells at right angles to the surface, and by an increase in the number of cell layers. On the outside of the ectoderm is found here and there a “ Deckschicht”’ cell, apparently in process of disintegration. In short, the blastodermic vesicle has seemingly completed only recently the so-called first phase in mammalian gastrulation, as advocated by Hubrecht (’88 and ’90) and Keibel (’89 and ’93). The germinal disk is slightly elliptical in outline; not far from one pole of the shorter axis a proliferation of ectodermal cells has taken place, so that three cells have come to lie above the general. surface of the disk. Consequently, and for additional reasons which I shall give later, I consider the shorter axis to be the chief axis of the embryo, and the pole where the proliferation of cells takes place the posterior pole. There is, furthermore, anterior to this proliferation, a slight elevation at the two lateral margins of the disk, while along the median line between them there is a depression.
As the embryo develops, the germinal disk grows by a multiplication of cells; the area covered by the disk is, however, augmented slowly, the tendency being, for a certain period, to an increase in thickness. Thus the germinal disk, sections of which are shown in Figs. 14 to 21 on Plates III. and IV., is relatively much thicker than those represented by Figs. 7 to 10 on Plate II. While the disk increases thus in thickness, the proliferation of cells at the posterior end continues, producing a distinct upfolding or overgrowth in that region, and at the same time a similar process has been going on at the two lateral margins. Soon these three overgrowths meet and fuse, forming one continuous bridge, at first attached at only three points, but later coming in contact with the disk at all points of the margin, save the anterior. There is present also a depression on the surface of the ectoderm of the disk immediately beneath the bridge, and the cavity which lies between this surface and the under surface of the bridge is connected by a narrow canal with the cavity which surrounds the disk between the extra-germinal ectoderm and the entoderm. The bridge, furthermore, seems to grow not only by a proliferation of the ectodermal cells of the germinal disk, but also by additions from the adjacent cells of the extra-germinal area.
There is strong evidence of such a method of development as I have just traced, not only in the figures which I have reproduced here, but also in the case of several embryos in which the disk is much larger than at this stage, and has clearly made a greater ontogenetic advance. In these a large well developed bridge is found, overlying a depression in the germinal disk below, to which it is attached at only three points, one of these being usually larger than the others, and apparently representing the posterior overgrowth, which seems to develop slightly in advance of the lateral proliferations. An anomalous condition of the bridge is interesting. In one embryo measuring 4 mm., with a germinal disk 0.28 mm. in diameter, the lateral proliferations seem to have been entirely suppressed, and we have an overgrowth from only one region, the posterior, extending forward along the median line of the disk.
The fate of the bridge seems to be, that its free anterior margin finally meets the true ectoderm of the disk; the structure then sinks down until it comes in contact with the underlying ectoderm, with which it finally fuses. At the same time the disk increases in area, this being largely due to a rearrangement of the cells of the disk in consequence of the addition received from the bridge. This method of increasing in size at this stage was first suggested to me by the fact that few nuclei are found in a karyokinetic condition. Accordingly, in the case of the two germinal disks represented by Figs. 5 and 6, Plate I., which correspond to the two stages of development in question, I counted the number of nuclei in each, as seen in sections, to determine the numerical relations of the cells. In the disk of Fig. 5 I found 1067 cells, in that of Fig. 6, 992; although embryos in their early development grow at very different rates, still the facts which these numbers present, together with the absence of nuclear figures, would seem to point to a simple rearrangement of existing cells as the principal factor in the increase in area of the germinal disk at this stage, rather than to an active multiplication of cells.
The oldest embryos considered in this paper consist, then, of a blastodermic vesicle, composed of a continuous inner sac of entoderm closely surrounded by a layer of ectodermal cells, which in the germinal disk are thickened into a flat, ovate expanse, without primitive groove or streak, with no signs of any mesoderm, and with a few widely scattered “Deckschicht” nuclei on the extra-germinal area. The entodermal cells are thickened in the region of the germinal disk until they become nearly isodiametric, and they are also thickened, though to a less extent, in an area all around the germinal disk, the diameter of which is about three times as great as that of the disk itself. With this summary I now pass to a consideration of the observations of other investigators on the mammalian blastodermic vesicle, and to the theoretical interpretation of some of the phenomena which occur in the embryo of the pig, as I have described them.
III. Historical and Theoretical
1. Consideration of Observations on the Blastodermic Vesicle in General
On account of the incompleteness of our knowledge of the facts concerning the blastodermic vesicle of the Mammalia, there are naturally several theories with regard to the exact method of its formation, and the interpretation of the vesicle when once it has been formed. Since my own material begins with the completely formed blastodermic vesicle, I cannot from an actual observation of the process of development add anything to our knowledge of the method of it formation ; but the phenomena which the vesicle at this stage presents, taken in connection with the observations of other investigators on mammalian embryology, serve to throw not a little light on several of the mooted points of its development.
Although the accounts of mammalian cleavage are few, and not in accord with one another, it seems to be pretty well established that cleavage results in the formation of a hollow sphere of cells, containing on the inside, at one pole, a more or less irregular mass of cells. These facts have been recorded by various authors ; as, for example, Lieberktihn (’79), Van Beneden (’80), Van Beneden et Julin (’80), Heape (’83), Hubrecht (’90), Duval (91), Robinson (’92), Christiani (792), and others. The method of formation of the germinal layers from these structures is in much dispute, however. I will consider briefly four theories which have been advanced on this subject, upon which my own investigations seem to throw some light; but I would not be understood as trying to make all the observed methods of mammalian development conform to one type,— there certainly are not as yet sufficient data for that; and besides, many reasons exist for supposing that there may be several types of development, conforming to the varied conditions under which the very young embryo is placed in different mammals.
The first theory is that which Van Beneden (’80) formulated for the rabbit. He found a well defined outer layer of cells, just beneath this in the region of the germinal disk a layer of flattened cells lining a limited area, and within this a layer which had extended partially around the inner wall of the outer layer, and these three layers he believed to represent the ectoderm, mesoderm, and entoderm respectively.
Other investigators have found different conditions, however, which disprove this theory. I merely mention it; first, because my own embryos show clearly a marked outer layer, which I have termed ectoderm, lined with a flattened layer, the entoderm, and outside of all, unmistakable “ Deckzellen,” which would have to be derived from the outermost layer of cells at a stage such as Van Beneden described ; and, secondly, because not long ago Duval (’91) found, as described in his work on the rat and the mouse, young embryos to which he gave an interpretation very similar to that of Van Beneden for the rabbit. He describes a hollow vesicle, consisting of an outer layer of nearly isodiametric cells (see Duval, ’91, Plate I, Figs. 73 and 74), with’ a number of larger, somewhat irregular cells inside, attached to the outer layer at one pole. He considers these two sets of cells ectoderm and entoderm respectively. As Duval describes the subsequent development, it is difficult to interpret these otherwise; but it should be remembered that Selenka (’83) considered the outer layer a “ Deckschicht,” and that Robinson (92), working on the same animals, has reached conclusions widely different from those of Duval. I should like further to call attention to Duval’s Figs. 75 and 79, Plate I., which resemble strongly those of other investigators on the rabbit, the mole, and the shrew, and which would seem to represent a vesicle consisting of an outer layer of somewhat flattened ceils, and an inner mass differentiated into two distinct regions, very much as Heape (’83, Plate XXIX. Fig. 20) has shown it in the mole.
The second theory is held by a larger number of investigators, perhaps, than any other. It maintains that the flattened cells of the outer layer become columnar and form the ectoderm of the extragerminal region. The inner mass of cells differentiates into two superposed parts; the inner becomes the entoderm and comes to line the inner surface of the ectoderm; the cells of the outer part become columnar, and, fusing with the cells of the outer layer, form the ectoderm of the germinal disk. Balfour (’81), in conjunction with Heape, thought he was able to trace the actual process of transformation of the flattened into columnar cells. Essentially the same views are advocated by Rauber (75), Lieberkithn (79), Kélliker (80), and Hubrecht (’90).
My material supplies no evidence whatever of any transformation of the outer layer of cells, or “ Deckschicht,” into true ectodermal cells; on the other hand, these “ Deckzellen” show unmistakable signs of disintegration. The embryos represented in part in Figs. 1, 3, and 4, Plate I., have these cells in a better state of preservation than most of the others figured here. Fig. 27, Plate IV., is from a section through the extra-germinal area of the embryo of Fig. 3. Here, at the left and the centre, we see two “ Deckschicht ” cells, which are entirely characteristic, the boundaries rather indistinct, the nuclei round and with very little chromatic substance, the whole cell flattened in the plane of the surface of the ectoderm; these occur all over the vesicle at about the same distance apart. At the right-hand side of the figure I have shown several such cells in contact with one another; this is the only place in my material where I have found this phenomenon ; it suggested a possible earlier condition of these cells. The cell boundaries, however, are very indistinct; the cytoplasm is scarcely stainable at all, while the contents of the nucleus stain a nearly uniform light blue. I am inclined, then, to regard these cells as belonging to a purely transitory layer, which may for a time serve some protective or other function, and then disappears by the disintegration of its elements as it gradually becomes of no further use. Bonnet (’91) says that this layer disappears early in the sheep and in the pig, and he finds no trace of it in the youngest sheep embryo he has described (Bonnet, ’84, Taf. IX. Figs. 2 and 3.) It should be remarked that in my oldest embryos, e. g. Fig. 6, Plate I., there is scarcely any evidence that a “ Deckschicht” has been present, — only here and there a small nucleus attached to the surface of the ectoderm.
The third theory mentioned is that advocated by Minot (’85 and ’89) and at the same time by Haddon (’85 and 87) and later by Keibel (87). This theory starts, like the others, with an outer layer, and at one pole an inner attached mass, and assumes that the whole outer layer is entoderm, while the inner mass differentiates into two superposed layers, an outer, which becomes the true ectoderm when the entoderm outside of it, as a Rauber’s “ Deckschicht,” disappears, and an inner, which becomes the entoderm of the germinal disk. Minot (’89) further suggests a complete inversion of the layers for all placental Mammalia. Larlier stages than mine are necessary for a full discussion of this question, but the three-layer condition which I have found over the greater part of the blastodermic vesicles of the pig, seems to me an insurmountable objection to this theory. If the primary vesicle is of entoderm, and the ectoderm later grows around it to produce the didermic blastocyst, to what origin are we to ascribe the outer layer of ‘ Deckzellen” which I have described ?
The last theory which I shall mention is that suggested by Robinson (92). His studies on the embryos of the rat and the mouse have led him to believe that the portion of the outer layer lying beyond the germinal disk is entoderm, while the ectoderm is limited to the outer layer of the germinal disk region. In the rat and the mouse he does not find the ectoderm overgrowing the entoderm. However correct this theory may be for the rat and the mouse, (my own work on these animals leaves me still undecided on this point,) my investigations on the pig show conclusively that the entodermal vesicle becomes entirely surrounded by ectoderm.
I can add little towards determining whether in the pig the extension of the entoderm over the inner surface of the ectoderm takes place by a growth proceeding from the margin of the entodermal portion of the germinal disk, as in the rabbit, mole, etc., or whether this entodermal mass of the germinal disk becomes a hollow vesicle, which reaches the ectodermal wall by a multiplication and expansion of its cells, such as would seem to take place in the hedgehog (Hubrecht ’89, Figs. 7, 8, and 9, Plate XV.) and in the cat (Schiifer ’76, Fig. 1, Plate X.). The youngest embryo which I have described, from which Figs. 7, 8, and 9, Plate II., were made, showed, as I have already said, an area at the end of the vesicle farthest from the germinal disk, where for several sections no entodermal nuclei appeared. If I had had several embryos presenting this same phenomenon, I should be inclined to think that the entoderm was in process of lining the ectodermal vesicle and had not yet completed its work; but,as I said in my description of this embryo, the entodermal cells are so widely separated over the whole vesicle that I do not feel justified in asserting that there is a space really free from entoderm at this stage. Bonnet (’84), in his description of a sheep embryo of thirteen days, finds the entoderm in very much the same condition ; he says: “ Die Keimblase ist, wie auch die Schnitte beweisen, durchweg doppelblattrig. Die Entoblastzellen bilden aber in einiger Entfernung vom Schild keine continuirliche Lage, sondern eine netzformig durchbrochene Membran anastomosirender Zellen von 15-24 » Linge.” Van Beneden (’80) finds that it is absent at one pole of the vesicle, and the same condition has been observed by both Heape (’83, Figs. 20-23, Plate X XIX.) and Keibel (89, Fig. 46 a, Taf. XXIV.). Hubrecht (90) quotes Hensen (’76, Fig. 18, Taf. VIII.) as authority for the presence of the entoderm at this region, and the figure would certainly seem to suggest this; but we should at least not ignore Hensen’s own statement of the case when he says, “ Die innere Keimhaut geht nur iiber das obere Drittheil des Kies ; wenn an andere Stellen die Keimhaut zweischichtig erscheint, so mége man dies aus der Drehung beim Uebergang von der Flachen- in die Kantenansicht erkliren.” The figure, however, does give the appearance of two layers at the region under discussion. While such observations as those of Heape (’83) and of Schafer (’76) would seem to point to two distinct methods of the extension of the entoderm in Mammalia, I cannot affirm with certainty that either occurs in the pig; what evidence I have, I am inclined to interpret in support of the phenomenon as it is said to occur in the rabbit, the mole, etc. ; i. e. as a process of marginal growth from the inner mass of cells of the germinal disk.
The process of entoderm formation from the inner mass of cells would seem to be primarily, then, a separation of certain cells from the general group, and I cannot help drawing attention here to the fact that there may be an homology between this process and the method of entoderm formation described by Robinson and Assheton (91) in the frog; furthermore, the formation of the didermic stage in the rat, as interpreted by Robinson (’92), is comparable with this, for he finds first a mass of irregular cells, in which a cavity develops separating a single layer of cells at one pole from a mass of cells at the other; the single layer he considers ectoderm, and the cells at the opposite pole the entoderm, or, as he calls them, epiblast and hypoblast respectively. I am not, however, sure in the light of the results of Selenka (’83) and Duval (’91), that this interpretation is correct.
Before leaving the general consideration of the blastodermic vesicle, there are two or three other points to which I wish to refer. Schafer (76) found in the embryo of the cat a clearly defined non-cellular membrane over the outer surface of the entoderm in the region of the germinal disk; this he calls the “ membrana limitans hypoblastica,” and compares it to the “ membrana prima” described by Hensen (’76) in the first part of his paper on the rabbit (see Hensen, Fig. 19, Taf. IX.). In the second part of his paper, Hensen figures the membrane as in contact with the entoderm in the region of the primitive streak only, and as then passing over the dorsal side of the mesodermal somites, and coming in contact with the ectoderm laterally (see his Fig. 37, Taf. X.). In Hensen’s earlier figure, and in Schifer’s, this membrane is clearly an entodermal structure, and I hold it comparable to the membrane which I have found between the ectoderm and the entoderm of the germinal disk, which leaves the ectoderm at the margin of the disk and passes off to meet the extra-germinal ectoderm farther on. This membrane would be of great assistance, it seems to me, in determining the origin of the mesoderm within the germinal area, Many authors have not figured this structure, as, for example, Kolliker (’82), Heape (’83), Bonnet (84), Hubrecht (’90), and others. In the rat and the mouse I have noticed a sharp line between ectoderm and entoderm, which is probably the same structure, and it has been figured by other investigators of these animals (see Duval 91, Robinson ’92, and others).
There is another point to which I wish to draw attention, without however attaching too great significance to it. In the description of my younger embryos I mentioned the fact that the germinal disk was elliptical in outline, and that, according to my orientation, the shorter axis of the ellipse lay in the plane of bilateral symmetry of the future animal. Though it may be of little morphological significance, it is certainly very interesting to note that in its earlier stages the blastoderm in teleosts (which must be held to be homologous with the germinal disk of the mammalian embryonic vesicle) is also elliptical in outline, and furthermore that the shorter axis of the ellipse corresponds to the chief axis of the future fish, as established by Agassiz and Whitman (’84). This elliptical outline, which seems to be constant in teleosts (see Ryder ’84, Agassiz and Whitman ’85 and ’89, Wilson 91, etc.), is produced in the first place by the first cleavage plane, which divides the protoplasmic mass at the active pole of the egg into two parts, each circular in outline, so that as they lie side by side the blastoderm is elongated ; this condition persists for some time.
If the first plane of cleavage in the teleost is not identical with the plane of bilateral symmetry, my comparison, of course, has no validity. So far as I am aware, there has been but one series of experiments whose results would seem to disprove this theory; and these were conducted by Miss Clapp (’91), who worked on the eggs of Batrachus tau. These eggs — attached by means of their thick outer membrane to the vessel in which they were placed — were artificially fertilized, and the position of the first plane of cleavage noted. Some seven days later the chief axis of the future fish was clearly visible, and was superposed on the line of direction of the first cleavage plane. In only three cases out of twenty-three did the two lines coincide ; in the rest the second line made a greater or less angle to the right or left of the first, — never greater than 70° however. But I think there is a possible source of error here, which makes my comparison still permissible. The author states that rotation is impossible, since the yolk is attached to the egg membrane at the point where the membrane attaches itself to the vessel, but offers nothing in evidence of this statement. Ryder (86), however, infers that the yolk does not become attached until “after the vitellus has been covered by the blastoderm.” During last summer, when through the courtesy of Dr. Alexander Agassiz I had the privilege of studying several weeks at the Newport Marine Laboratory, I collected material of the cleavage stages of Batrachus tau. This material was fixed without puncturing the egg membrane, and a careful examination of it shows no trace of any attachment of the yolk to the membrane, although the contents of the egg are everywhere in contact with it. It should further be remembered that the contents are in a semi-fluid condition, and during the seven days mentioned abundant opportunity is furnished for a rotation of the yolk within the egg membrane.*
Before leaving this matter of the shape of the germinal disk, I wish to refer to a young disk of the shrew, which Hubrecht (90) has figured (Plate XX XVII. Fig. 17) as elliptical, very much as I have described the disks of the pig. From the position of the figure on the plate I am left to infer that the shorter axis of the disk becomes the principal axis of the embryo. In the later development he finds the disk ovate, but he places the narrow end anterior and the broad end posterior, except in one case (Plate XX XVII. Fig. 21), where the broad end is anterior, just as I have placed it in my descriptions of the pig. This ovate outline, oriented thus, is certainly characteristic of slightly later stages, and has been figured many times; e. g. by Kélliker (’82) in the rabbit, Duval (’89) in the chick, Keibel (93) in the pig, ete.
I now come to the last point which I wish to consider before taking up the interpretation of the bridge. In the embryo represented by Fig. 2, Plate I., and in all the following embryos figured on this plate, there occurs a thickening of the entoderm, not only in the region of the germinal disk, but also in a considerable area immediately surrounding it. By a thickening I do not mean that the entodermal cells have multiplied so as to be superimposed upon one another to form a mass more than one layer of cells deep, but simply an increase
Very recently Morgan, (Experimental Studies on the Teleost Eggs, Preliminary Communication, Anat. Anzeiger, Jahrg. VITI. pp. 808-814, 1898,) working on the eggs of Ctenolabrus and Serranus, has arrived at the conclusion that “ there is no relation whatsoever between the cleavage planes of the egg and the median plane of the adult body.” Dr. Morgan bases this statement on the observation of the axes of twenty-two eggs, and his method of determining the position of the axes appears to be satisfactory. I cannot, however, enter upona fuller consideration of the subject here.
in the number of cells over a certain area, so that they come to lie more closely together, and in consequence give the appearance in surface view of a thickened entodermal area. The question of course concerns the significance of this circum-germinal thickening. An explanation has suggested itself to me, which rests, however, on a theory which I do not feel at all sure is established. The theory concerns the origin of the mesoderm. Hubrecht (’90) gives three sources for the mesoderm : the protochordal plate, the primitive streak (“gastrula ridge” and “ Kopffortsatz”), and “an annular zone of hypoblast situated just outside the limits of the embryonic shield, and thus enclosing — but at the outset independent of — the protochordal plate.” The annular zone according to Hubrecht does not arise until after the first stages in the development of the primitive streak, and therefore is a later differentiation in the hypoblast than is’ the protochordal plate.
Concerning this third source there has been much dispute. Bonnet (84) has found it in the sheep, and Robinson (’92) in the rat and the mouse. But many authors find no evidence of such an origin for any part of the mesoderm, as, for example, Kélliker (’82), Heape (83), Fleischmann (’89), Keibel (’91 and ’93), Hertwig (’93), and very many others. It has occurred to me that this circum-germinal thickening might be the first evidence of a later (ontogenetically) formation of mesoderm in this region. Since Keibel’s (793) youngest pig embryo has the mesoderm already well advanced in development and covering the area in question in two layers, somatic and splanchnic, it is impossible to say what the stages intermediate between his and mine may have been, and I merely mention the above suggestion as a possible explanation of an interesting phenomenon. There is, as I have already said, no trace in my specimens of mesoderm in any region of the vesicle, and no sign, either in surface view or in section, of a thickening of entodermal cells in any part of the germinal disk, like the protochordal plate which Hubrecht (90) describes for the shrew. To make sure that this was not merely a subjective impression I enumerated the nuclei in each section of the germinal disk of Fig. 6, Plate I., beginning at the broader end, and the result was as follows: 3, 8, 10, 10, 14, 15, 18, 18, 20, 23, 28, 24, 23, 25, 24, 31, 30, 23, 22, 17, 15, 12, 10, 9, 10, 11, 6. A comparison of these figures with the shape of the disk will show an almost uniform distribution of entodermal cells.
2. Interpretation of the Bridge
T now have to consider the interpretation of the structure which I have called the bridge. There are two structures which have been described in vertebrate ontogeny with which it may perhaps be possible to compare it. One of these has been figured by Heape (’83, Plate XXIX. Figs. 20-28) in the blastodermic vesicle of the mole. He here shows a thickened ectoderm in the region of the germinal disk, with a layer of entoderm beneath but not extending far beyond it, and above it a cavity (his “secondary cavity ”) which is roofed over by a bridge of cells from the “ Deckschicht,” or, as he calls it, the “outer layer.” The history of the structure, as he gives it, is briefly this. The blastodermic vesicle consists of a closed sac of flattened cells, the outer layer, and of a mass of rounded cells within at one pole, the inner mass. The latter differentiates into two parts, which become ectoderm and entoderm. The ectoderm becomes continuous at its margin with the outer layer, from which it is separated over its central area by a shallow cavity. The ectoderm increases in extent and becomes somewhat cup-shaped, so that the cavity increases in depth, but it is filled with amceboid cells derived from the outer layer. Later the ectoderm of the disk flattens out, and the cells of the outer layer above it fuse with it and become a part of the true germinal ectoderm. The interpretation which Heape puts on these phenomena is, that they are a transitory representation of the inversion of the germinal layers which is carried to such a great extent in some rodents. I see no reason why Heape’s observations and conclusions are not entirely correct. A similar phenomenon has been figured by Hubrecht (’89, Plate XVI.) in the hedgehog. Here, however, the portion of the outer layer, or “trophoblast,” which 1s separated from the germinal disk ectoderm, does not become fused with the disk later, but remains in contact with the uterine mucosa. This roof-like structure, where it comes in contact with the ectoderm of the disk, is clearly continuous with the cells of the “trophoblast ” and also with those of the disk, so that Hubrecht’s (89) Fig. 20 B, Plate XVI., resembles my figures of sections through the posterior attachment of the bridge in the pig.
The condition in the pig, although it seems at a casual glance to resemble the structure in the mole embryo, is not directly comparable with it. In the first place, the structure in the mole forms from the beginning an uninterrupted covering to the ectoderm of the germinal disk, and continues to do so through the subsequent development, up to its complete obliteration through fusion with the disk. During the whole process no stage occurs where there is any trace of an overgrowth, such as I have found in the pig, or of an external opening into the cavity which lies between the two layers in question. Moreover, this cavity in the mole is largely filled with a loosely arranged mass of ameeboid cells, except in one case, where, as Heape (’83, Plate X XIX. Fig. 25) says, their absence is due to mechanical injury in the process of preparing the sections. Furthermore, it may be noted that this bridge-like structure appears at a much earlier ontogenetic stage in the mole than in the pig. In the former the entoderm covers the area of the germinal disk only; in the latter I find a complete didermic vesicle when the first trace of the bridge cells appears. It is true, we need not be surprised to find an inversion of the layers in other mammals than those in which it has already been established, especially in the light of Mall’s (93) paper on a human embryo of the second week, in which he explains the conditions as the result of inversion ; but, for the reasons I have given, I do not consider the bridge in the pig as even a potential inversion, — the less so as the explanation I am about to offer is to my mind a much more satisfactory interpretation of the phenomena. If it should be urged that the bridge is homologous with the roof-like structure over the germinal disk of the hedgehog (Hubrecht ’89), it would be necessary to assume the existence of a potential opening through this structure into the cavity beneath it from the very beginning of its formation, but the facts, as recorded for the hedgehog, seem to furnish no grounds for this assumption. Moreover, in this animal the roof-like structure never comes in contact with the ectoderm of the germinal disk, but contributes to the formation of the placenta, a very different fate from that of the bridge in the pig.
I am inclined to compare this bridge with the overgrowth which occurs in the development of Amphioxus just after gastrulation and the elongation of the embryo have taken place. We kncw through the investigations of Kowalevsky (’67 and ’76) and of Hatschek (’81) that at this time there is a sinking of both ectoderm and entoderm along one side of the gastrula, the future dorsal or neural side of the animal, which forms the so called medullary plate, and at about the same time a proliferation of cells begins at the posterior margin of the blastopore, and, growing forward over the blastopore and the medullary plate, meets lateral elevations on either side, and fusing with them forms a continuous roof over the dorsal depression, with an opening at the anterior end, which persists for a considerable time as the neuropore. The neural tube is later formed from the medullary plate, which is in this way cut off from the rest of the ectoderm, but the overgrowth itself takes no part in the formation of the tube, as we see clearly from Kowalevsky’s (76) Figs. 11, 12, and 13, Taf. XV., where the cells of the medullary plate grow across under the roof, and thus separate the roof from the lumen of the neural tube. Kowalevsky says on this point: “Die Riickenrinne, obgleich von aussen vollstiindig bedeckt, innen — unter der Haut — noch offen ist. Die Querschnitte der Gastrula und der zuletzt angefiihrte Querschnitt auf der Fig. 11 erkliren uns diese Erscheinung ganz einfach. Querschnitte der etwas weiter ausgebildeten Larve, Figg. 12 u. 13 zeigen uns nun, dass die oberen Riinder der Medullarplatten sich bald verbinden, anfangs vermittelst einer sehr feinen und platten Briicke, welche sich aber bei den weiter ausgebildeten Larven bedeutend verdickt und so ein Verhiltniss annimmt, wie bei dem ausgewachsenen Amphioxus.”
Now the development of the bridge in Sus scrofa domesticus, as I have traced it in the present paper, corresponds in many important points with the development of the dorsal overgrowth in Amphioxus. In the first place, it begins by a marked proliferation of cells at one pole of the chief axis of the germinal disk, and at the same time by a slight elevation laterally on either side of this axis and nearer the opposite pole. The median growth is more rapid than the lateral growths, and gives a condition like that of Fig. 1, Plate I., which is much the same as that figured by Kowalevsky (76, Taf. XV. Fig. 3) and by Hatschek (’81, Taf. III. Fig. 87) for Amphioxus. The overgrowth continues in the direction of the median axis, and the three parts fuse to form the continuous bridge, as already described for the subsequent stages, represented by the figures on Plate I. These correspond with the phenomena as they have been described by Kowalevsky in Amphioxus: “Die jetzt beginnende Schliessung der Rtickenfurche geht hier, so wie bei den anderen Wirbelthieren, von hinten aus, wobei die ganz hinteren Riinder, welche die Einsttilpungséffnung riickwiirts begrenzten, sich aufheben, eine Art Dach iiber diese Oeffnung bilden und immer mehr und mehr nach vorne wachsend und mit den seitlichen Randern der Riickenfurche verschmelzend den Riicken, resp. das Nervenrohr, zu bilden beginnen.”
It is for these reasons that I have oriented my embryos in the way already described. The opening into the depression below the bridge corresponds, I believe, to the neuropore of Amphioxus, and I am inclined to carry the homology of the two forms still further, and suggest that the canal which I have shown in Fig. 20, Plate IV., corresponds to part of the neurenteric canal of Amphioxus.
There are several objections to this theory which suggest themselves at once. In the first place, as to the structure of the two overgrowths ; that in Amphioxus is figured as consisting of but one layer of ectodermal cells; the bridge in the pig, on the contrary, is from two to three layers of cells thick. This seems to me but a minor point, however, readily explained by the morphological differences between the larva of Amphioxus and the blastodermic vesicle of the Mammalia. In the former case there is no part which may not be said to develop directly into some important tissue or organ of the adult animal; in the latter, it is essentially the germinal disk only of which this is true. Here, then, we have a germinal and an extragerminal region, and in the process of rapidly increasing the number of cells in the disk the extra-germinal cells of the ectoderm, as well as the cells of the disk, apparently contribute to the development of the bridge, thus producing an overgrowth two or three cells thick. Again, it may be urged that I have not shown a blastopore around which the overgrowth should take place. I have found in my youngest embryos no trace of an opening through the germinal disk which could be compared with wnat Hertwig (93) says may possibly be a blastopore, as figured by Heape (’83), Selenka (’86~87), and Keibel (’89). But the blastopore in Amphioxus becomes the neurenteric canal, which leads from the posterior end of the cavity beneath the overgrowth into the gastrula cavity. It is at just this point in several of my embryos that I find a canal leading from the cavity beneath the bridge to the cavity located at the margin of the disk, between ectoderm and entoderm. To be a true neurenteric canal, it should be continued into the gastrula cavity, i. e. the cavity within the entoderm ; but on account of the loose connection of the entodermal cells, it is impossible here to trace any such passage. I do not, however, think we should be justified in asserting on this account that it does not exist. There ought, however, to be at least a fusion of the two primary germ layers in this region, such as always occurs, I believe, in the case of a true neurenteric canal.
Still another objection may be raised to this theory, and that is the extremely remote relationship existing between Amphioxus and the Mammalia. If the bridge of the pig is really a reappearance of a structure which occurs as far back in phylogeny as the lowest representative of the Vertebrata, why may we not justly look for its presence in intermediate groups? The question is certainly a fair one, and I can only say that, so far as I am aware, no structure having so marked a resemblance to the overgrowth in Amphioxus has been recorded in any intermediate group. I am tempted, however, to call attention here to a parallel case. The early cleavage in mammals, as described especially by Tafani (’89), bears a striking resemblance to the early cleavage of Amphioxus. Why, then, do we not find the same type of cleavage occurring in intermediate groups of vertebrates? Here again it must be admitted that we do not find it, but in this case the reason is plain; it is because the whole structure of the ovum is so changed by the accumulation in it of nutrient material, that cleavage on the type of Amphioxus is impossible, and it is not until we reach the Mammalia that the conditions are such as to admit of total equal cleavage. But is it not for the same reason that the steps which lead up to the formation of the neural tube have been modified, and that the overgrowth in Amphioxus finds its first striking recurrence in the mammalian embryo? The lumen of the canal is often extremely small in other mammals, and has in some cases been represented by a single line, e. g. Robinson (92, Plate XXIII. Fig. 13 A, Plate XXIV. Fig. 15 D, and Plate XXVI. Fig. 17).
A more serious objection exists in the fact that the cavity beneath the overgrowth in Amphioxus becomes the neural canal, whereas, as I have traced it in the pig, it becomes obliterated by a fusion of the bridge with the ectoderm of the disk. I would not be understood as considering the canal which I have compared to the neurenteric canal decisive evidence in favor of this theory ; it is, however, a significant phenomenon occurring in a significant position. To my mind it occupies the position of the neurenteric canal in Amphioxus; that it is certainly the neurenteric canal of the pig, I would not presume to say; the descriptions of this canal in mammals are varied; the bridge and canal which I have described have never before been recorded in the Mammalia, so far as I am aware.
My conclusions as to this bridge may be briefly summarized as follows. Two interpretations of the structure have presented themselves as in some measure probable. The first homologizes it with the thickening of the ‘ Deckschicht” or Rauber’s layer, or, as Heape (’83) calls it in the mole, the outer layer, which through the development of a secondary cavity becomes separated from the true ectoderm of the germinal disk and forms a sort of roof over it. That this structure is not homologous with the bridge seems to me evident from the fact that in the case of the bridge there is always an opening leading from the outside into the cavity beneath the bridge, and this cavity is never filled with amcboid cells as is the cavity in the mole. Furthermore, on the bridge there are found in some cases “ Deckzellen,” —or rather their remains, consisting of homogeneously staining nuclei and little cytoplasm, — which clearly can take no part in the formation of either bridge cells or true ectodermal cells. Again, the roof of overlying cells in the mole makes its appearance and finally fuses with the germinal disk ectoderm at an earlier stage in ontogeny than that at which the bridge in the pig develops.
The second theory, which seems to me the more probable, homologizes the bridge with the overgrowth along the dorsal side of Amphioxus. The reasons for this comparison are that the two structures develop at about the same time in ontogeny in the two cases, i.e. just after the formation of a didermic vesicle; that, further, a process of growth can be traced for the bridge which corresponds closely to the method of growth in the case of the structure under consideration in Amphioxus; and, finally, that there is a median thickening of the germinal disk corresponding topographically to the medullary plate of Amphioxus, a free margin to the bridge corresponding to the neuropore, and a canal at the opposite pole which may, perhaps, be compared with the neurenteric canal of the primitive vertebrate. From my present knowledge of the bridge in the pig, I cannot homologize it with the roof-like structures in the shrew and the mole, and if it is not comparable with the overgrowth in Amphioxus, it seems to me necessary to regard it as a structure hitherto undescribed in vertebrate embryology. Whatever the interpretation of the bridge may be, we have the fact of its existence, and it is reasonable to expect that it will be found in other mammals as well, as, for example, in the sheep, in which the immediately succeeding stages of development are so similar to those of the pig.
I wish to express my thanks to Dr. Alexander Agassiz, to whom I amgreatly indebted for the opportunity of studying at his private laboratory at Newport and for the privilege of using his library, and to Dr. E. L. Mark, who has very kindly followed all my work and examined my preparations with great care, and also to the employees of the abattoir, who have given me much assistance in securing material.
Cambridge, April 18, 1894.
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Bonnet, R. ’84. Beitriige zur Embryologie der Wiederkiuer, gewonnen am Schafei. Arch. f. Anat. u. Phys., Anat. Abth., Jahrg. 1884, pp. 170-230, Taf. [X.—XI. 91. Grundriss der Entwickelungsgeschichte der Haussiugethiere, 282 pp., 201 Abbild. Berlin.
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Explanation of Plates
All the drawings were made with the camera lucida directly from the object itself, with the exception of Fig. 2, Plate L, which is a graphic reconstruction made from sections drawn with the camera. In the case of all transverse sections it is the posterior face of the section that is shown, so that right on the figure corresponds to the right side of the disk. The embryos came from four uteri, and may accordingly be grouped as follows: A, Figs. 1, 8, 4, 10, 27; B, Figs. 2, 21, 22, 23, 26; C, Figs. 5, 6, 14-20, 24, 25; D, Figs. 7, 8, 9, 11, 12, 13.
All figures magnified 100 diameters, and oriented on the plate with the chief axis vertical and the anterior pole towards the top.
A portion of the blastodermic vesicle containing the germinal disk. The posterior overgrowth is marked, and the marginal elevations and median depression are.apparent.
A later stage than the preceding, with the posterior and the lateral overgrowths fused at two points.
One continuous overgrowth or bridge, with no trace remaining of the fusion of parts.
A later stage showing a more extensive bridge with a lateral opening at the right, where the lateral and the posterior overgrowths have not completely fused.
A germinal disk with ovate outline and highly developed bridge. The broad end of the disk is anterior.
Enlarged disk, ovate, with no separate bridge. Sections show that the bridge has fused with the ectoderm of the disk.
AWW.,dél. B Meisel. lith.Boston.
All figures magnified 400 diameters, ectoderm uppermost.
Transverse section of a very young germinal disk, showing one cell at the centre projecting above the general level of the ectoderm, but clearly an ectodermal cell.
Section immediately succeeding that of Fig. 7, and showing essentially the same phenomena.
More anterior section of the same disk showing the even surface of the ectoderm and the general arrangement of the cells. No “ Deckschicht ” cells.
Longitudinal section, nearly median, through the germinal disk of Fig. 1. At the left hand or posterior end is seen an early stage in the development of the bridge, which here consists of two layers of cells, the outermost resembling the extra-germinal ectoderm, the inner the germinal ectoderm. Note the dividing nucleus at the base of the bridge.
A nearly median longitudinal section of another germinal disk in a little later stage of development.
More lateral section through the same embryo as Fig. 11, showing lateral continuity of the bridge and the cavity beneath it.
A still more lateral section of the same disk as the two preceding, in which the cavity has disappeared, but the bridge cells are readily distinguished from the true ectodermal cells of the disk.
All figures magnified 400 diameters, and all drawn from longitudinal sections through the germinal disk of Fig. 5. The anterior end is at the right.
Figure 14. “15. “16. “17. “18. “19.
First section of the disk ; the disk cells first appear near the anterior end on account of the ovate outline of the disk,
Second section in the series; merely shows an extension of the germinal-disk ectoderm.
Fourth section. First appearance of the bridge, marked off by a distinct cavity.
Sixth section through the disk. Extension of the cavity and decrease in thickness of the bridge.
Seventh section, showing free margin of the bridge at the anterior end, with posterior attachment.
Eighth or median section ; here we have the most posterior point of the free margin of the bridge. The cavity beneath extends a short distance towards the posterior pole, between the cells.
Figures 20 and 27 magnified 400 diameters, all others 275 diameters. Ectoderm in every case uppermost.
Figure 20. The ninth section in the same series as those of Plate III. The free margin of the bridge is again nearer the anterior pole, while the cavity beneath extends by a narrow canal completely through the ectoderm of the disk into the extra-germinal space between ectoderm and entoderm.
Somewhat oblique longitudinal section nearly through the centre of Fig. 2, Plate I. The ectoderm of the disk is greatly thickened, the cavity beneath the bridge is rather shallow, but there is a marked depression just in front of the free edge of the bridge. The entoderm is thickened bey ond the area of the disk in the extra-embryonic region.
A nearly transverse section through a much older germinal disk, which shows the decrease in thickness of the ectoderm attending an extension in area and the fusion of the bridge along the median portion.
A slightly more anterior section of the same disk as Fig. 22, where we see at the left lateral margin the fusion of the bridge not quite completed.
Transverse section, the fourth from the anterior end of the germinal disk of Fig. 6, showing the bridge cells in contact with the germinal-disk ectoderm.
Seventh section of same disk as the preceding, showing near the median line the last trace of the fusion of two bridge cells with the ectoderm.
Section through the extra-germinal region of the blastodermic vesicle of Fig. 2, Plate I. On the upper side are afew “ Deckschicht ” cells, flattened and disintegrating; then a layer of typical ectodermal cells, and, below, the normally distributed entodermal cells of this stage.
Section through the extra-germinal region of Fig. 3, PlateI. The “ Deckschicht ” cells are more numerous than in the preceding case, and at one point several are in contact with one another, — an unusual condition. The entodermal cells are more numerous than in the preceding, since this is a later stage of development.