Paper - Contributions to the embryology of the marsupialia 4-4
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Hill JP. The Early Development of the Marsupialia, with Special Reference to the Native Cat (Dasyurus Viverrinus). (1910) Quart. J. Micro. Sci. 56(1): 1-134.
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Chapter IV. - Growth of the Blastocyst and Differentiation of the Embryonal Ectoderm and the Entoderm
1. Growth of the Blastocyst
In the preceding chapter we have seen that the cleavage process in Dasyurus results in the formation of a small spherical vesicle, about '4 mm. in diameter, Avhich consists, internally to the investment formed by the apposed zona and shell-membrane, simply of a cellular wall, unilaminar throughout its entire extent, and enclosing a fluid-filled cavity normally devoid of any cellular elements. The stage of the just completed blastocyst is followed by a period of active growth of the same, and it is a noteworthy featui'e in the development of Dasyurus that during this time the blastocyst undergoes no essential structural change, but remains unilaminar until it has reached a diameter of from 4'5 to 5'5 mm. Even during cleavage, the egg of Dasyurus increases in diameter, partly owing to the thickening of the shell membrane, partly, and, indeed, mainly, as the result of the accumulation of uterine fluid under pressure within the egg-envelopes, but the increase due to these causes combined is relatively insignificant, being only about '1 mm. As soon, however, as the cellular wall of the blastocyst is completed, rapid growth sets in, under the influence of the hydrostatic pressure of the fluid, which tensely fills the blastocyst cavity, with the result that the small relatively thick-walled blastocyst becomes convei'ted into a large extremely thin-walled vesicle, but beyond becoming very attenuated, the cellular wall during this period of actjve growth uudei'goes no essential change, and retains its unilaminar character until the blastocyst, as already mentioned, has reached a diameter of from 4'5 to 5‘5 mm. In vesicles of about this size there become differentiated from the formative cells of the upper hemisphei-e the embryonal ectoderm and the entoderm, and this latter layer then gradually spreads round inside the non-formative (extraembiwonal ectodermal) layer of the lower hemisphere so as to form a complete lining' to the blastocyst, whicli thereby becomes bilaminar. Sucli a marked enlargement of the blastocyst prior to the differentiation of the embryonal ectoderm and entoderm as is here described for Dasyurus does not apparently occur, so far as known, in other Marsupials : in Perameles, for example, the embryonal ectoderm and the entoderm are in process of differentiation in vesicles a little over 1 mm. in diameter (v. p. 77), in Macropus these two layers are already fully established in a vesicle only *8 mm. in diameter (v. p. 79), and much the same holds good for Ti'ichosurus and Petrogale. It is pai'alleled by the marked growth which in the Monotremes follows the completion of the blastocyst and which precedes the appearance of embryonal diffei-entiatiou. It must be remembered, however, that the growing blastocyst in the Monotreme is bilaminar and not unilaminar as in Dasyurus, owing to the fact that the entoderm is established as a complete layer at a very much earlier period than is the case in the latter. I am nevertheless inclined to regard the attainment by the Dasyurus blastocyst of a large size, prior to the differentiation of the embi'yonal ectoderm and the entoderm, as a more primitive condition than that found in other Marsupials. The pronounced hypertrophy which the uteri of Dasyurus undergo during the early stages of gestation, an hypertrophy which appears to be proportionately greater than that met with in other forms,^ is no doubt to be correlated with the presence in them of such a considerable number of actively growing blastocysts.
Selenka states (Heft 5, p. 180) that he examined seven blastocysts of Dasyurus “-f mm.†in diameter, taken from a female fifteen days after copulation. He describes their structure as follows : “ Man unterscheidet (1) eine sehr zarte aussere, homogene Haut (Granulosamembran), (2) darmiter ein Lagei' von Ektodermzellen, welche im Gebiete des Embryonalschildes prismafcich, am gegeniiberliegenden Pole nahezu kubisch, im iibrigen abgeplattet erscbeinen, (3) ein inneres zusammenliangeudes Lager von abgeflacbten Entodermzellen.†This description, apart from the reference to the thin shell-membrane, is entirely inapplicable to blastocysts of Dasyurus of the mentioned size which I have studied.
Por example, the uteri of a female (5, 18 . vii . '01) from which 1
obtained twenty-one normal vesicles, 4'5-6 mm. in diameter, with the embryonal area definitely established, measured as follows : Left uterus, 4'5 X 4'7 X 1'4 cm. (fourteen vesicles) ; right uterus, 4'5 X 4'2 x 1‘45 cm. (seven vesicles and one shrivelled).
I have examined a practically complete series of vesicles of Dasyinms ranging from '4 mm. to 4 mm. in diameter and all of them without exception are unilaminar.
Of vesicles under 1 mm. diameter I possess serial sections of more than two dozen, I'anging from '5 mm. to '8 mm. in diameter, and obtained from three different females. These differ structurally in no essential respect from the just completed blastocysts. A surface view of a blastocyst '6 mm. in diameter is shown in fig. 63, PI. 6; in this the difference in the cytoplasmic chai'acters of the cells of opposite hemispheres is clearly brought out, the non-formative cells of the lower hemisphere having much more marked perinuclear zones of dense cytoplasm (deutoplasm) than the formative cells of the upper hemisphere ; moreover, the former cells tend to be of larger superficial extent than the latter. Pig. 34, PI. 3, represents a section of a blastocyst '57 mm. in diameter, and fig. 35 a section of one '73 mm. in diameter. These blastocysts differ in no essential way from the '43 mm. blastocyst represented in fig. 33. As in the latter, the cellular wall is unilaminar throughout, but both it and the shell-membrane have undergone considerable attenuation. Moreover in these blastocysts, apart from the clue afforded by the shrivelled yolk^body, it is practically impossible to determine from the sections which is morphologically the upper hemisphere and which the lower. In fig. 36, from a '6 mm. blastocyst, on the other hand, the cells of the hemisphere opposite the yolk-body (y.b.) are larger than those of the hemisphei'e adjacent to which that body is situated. In the '57 mm. blastocyst the shell-membrane has a thickness of ‘0052 mm., in the -73 mm. blastocyst it measures -0045 rnrh., and in a -84 mm. blastocyst •0026 mm. The zona is now no longer recognisable as an independent membrane. In blastocysts of this stage of growth a variable number of small spherical cells or cellfragments are frequently met with in the blastocyst cavity, usually lying in contact Avith the inner aspect of the cellular wall (fig. 34, i.c.). In some blastocysts such structures are absent, in others one or two may be present, in yet others numbers of them may occur. They raa,y be definitely nucleated, but this is exceptional; more usually they contain one or more deeply staining granules (of chromatin ?), or are devoid of such. They ai'e of no morphological importance, and I think thei*e can be no doubt that they represent cells or fragments of cells which have been separated off from the cellular wall during the process of active growth. They are of common occurrence in later blastocysts, and it is possible the so-called “ yolk-balls †observed by Selenka in Didelphys are of the same nature.
If we pass now to vesicles from 1 to 3 or 3'5 mm. in diameter, we find the wall still unilaminar, but considerably more attenuated than it is in the blastocysts last referred to. In a vesicle with a diameter of 1'24 mm. the shell-membrane has a thickness of about '0015 mm., whilst the cellular wall has a thickness of only '0045 mm. In a 3'5 mm. vesicle the shell-membi'ane measures about •0012 mm., Avhilstthe cellular wall ranges from •OOlS to •OOIS mm. in thickness. A small portion of the wall of a vesicle, 2^4 mm. in diameter, is shown in PI. 6, fig. 64. In these later vesicles I have failed to detect, either in surface examination of the vesicles in to to or in sections, any regional differences between the cells indicative of a differentiation of the wall into upper or formative, and lower or non-formative, hemispheres. Everywhere the wall is composed of flattened, exti'emely attenuated cells, polygonal in surface view, and all apparently of the same character. It might therefore be supposed that the polarity, which is recognisable in early blastocysts, and which is dependent on the pronounced differences existent between the cells of the upper and lower rings of the 16-celled stage, is of no fundamental importance, since it apparently becomes lost at an early period during the growth of the blastocyst. Such an assumption, however, would be very wide of the maxk, as I hope to demonstrate in the next section of this paper, and, indeed, in view of the facts already set forth, is an altogether improbable one.
Reappearance of Polar Differentiation in the Blastocyst Wall. - Following on the period of what may be termed the preliminary growth of the blastocyst, in the course of which the original polar differentiation in the blastocyst wall apparently becomes obliterated, is an extremely interesting one, during which that differentiation again becomes manifest. In view of the fact (1) that the fourth cleavage in Dasyurus is of the nature of a qualitative cytoplasmic division, and (2) that approximately one half or rather less of the unilaminar vosicle wall is formed from the eight smaller and less yolk-rich cells of the upper ring of the 16-celled stage, and its remainder from the eight larger more yolk-rich cells of the lower ring, it thus becomes a question of the first importance to determine if we can the significance of that differentiation.
Amongst the Eutheria, it has been conclusively shown by various observers (Van Beneden, Heape, Hubrecht, Assheton, and others) that there occurs during cleavage an early separation of the blastomeres into two more or less distinctly differentiated groups, one of which eventually, by a process of overgrowth, completely encloses the other. The peripheral cell-group or layer forms the outer extra-embryonal layer of the wall of the later blastocyst (the trophoblast of Hubrecht, or trophoblastic ectgderm as I prefer to term it). It therefore takes no direct part in the formation of the embryo, and may be distinguished as non-formative. The enclosed cell-group, termed the inner cell-mass or embryonal knot, gives rise, on the other hand, to the embryonal ectoderm as well as to the entire entoderm of the vesicle, and may accordingly be distinguished as formative. May it not be, then, that we have here at the fourth cleavage in Dasyurus a separation of the blastomeres into two determinate cell-groups, respectively formative and non-formative in significance, entirely comparable with, and, indeed, even more distinct than that which occurs during cleavage in the Eutheria ? I venture to think that the evidence brought forward in this paper conclusively justifies an answer in the affirmative to that question.
If we assume that the upper cell-ring of the 16-celled stage in Dasyurus is formative in destiny and the lower cell-ring non-formative, then we might naturally expect to find in the unilaminar wall of the later blastocyst some differentiation indicative of its origin from two distinct cell-groups, and indicative at the same time of the future embryonal and extra-embryonal regions. Now just such a differentiation, does, as a matter of fact, become evident in vesicles 3'5 to 4'5 mm. in diameter. We have already seen that the wall in early blastocysts '4 to '8 mm. in diameter exhibits a wellmarked polar differentiation in correspondence with its mode of oi'igin from the diffei-entiated cell-rings of the 16-celled stage, its upper hemisphere or thereabouts consisting of smaller cells, poor in deutoplasm, its remainder of larger cells, rich in deutoplasm. .In later blastocysts, 1-3 mm. or, more in diameter, it is no longer possible to recognise this distinction - at all events I have failed to observe i't - but if we pass to blastocysts 4-5 mm. in diameter, in which the wall is still unilaminar, we find on careful examination of the entire vesicle under a low power that there is now present a definite continuous line^ which encircles the vesicle in theequatorial region so as to divide its wall into two hemi-, spherical areas (PI. 4, fig. S8,j.L). If we remove and stain; a portion of the wall of such a vesicle, including this line,) and examine it microscopically (figs. ,42-46.), it becomes apparent at once, from the .disposition of the cells on either side of the bns, that we have to do with a sutural line or line of junction produced by the meeting of twp sets- of cells,' which are pursuing their .own, independent courses of growth and division. ; The, cells never cross the demax'cation line from the one side tn the other, but remain strictly confined to their own territory, so that we are justified in regarding the vesicle wall as composed of two independently growing zones. Now tlj^ existence of two such independent zones in the unilaminar wall is, to my mind, only intelligible on the view that they are the products of two originally distinct, predetermined cell'groups, and if this be admitted, then I think we are justified in concluding, in view of the facts already set forth, that tlie two zones in question are derived, the one from the upper cell-ring of the 16-celled stage, the other from the lower ring ; that, in other words, they represent respectively the upper and lower hemispheres of the early blastocysts.
If, now, we find that the embryonal ectoderm and the entoderm arise from one of these two regions of the unilaminar wall, whilst the other directly forms the outer extra-embryonal layer of the later (bilaminar) vesicle, then we must designate the former region as the upper or formative, and the latter as the lower or non-formative. Further, bearing in mind the characters of the cells of the two rings of the 16-celled stage, T think we are justified in holding that the formative region is derived from the ring of smaller, less yolk-rich cells, and the non-formative region from the ring of larger, more yolkrich cells, even if it is impossible to demonstrate an actual genetic continuity between the constituent cells of these two rings and those forming the independently growing areas of the later blastocyst. I have recently re-examined a series of vesicles, measuring 1'5-1'8 mm. in diameter, obtained from a female killed in 1906, and I have so far found it impossible, either in the entire vesicle or in portions of the wall stained and mounted on the flat, to distinguish between the cells over opposite hemispheres. Thus the only actual guide Ave have for the determination of the poles in such vesicles is the yolk-body, and though the latter is liable to- displacement, it is Avorthy of record that I have several times found it in relation to the formative area in vesicles 4‘5-6 mm. in diameter, but never in relation to the non-formative region. This evidence is, therefore, so far as it goes, confirmatory of the conclusion reached above, viz, that the formative hemisphere is derived from the smaller-celled ring of the 16-celled stage. On that conclusion is based my interpretation of the poles in the unsegmented ovum, and of the two cell-rings o£ the 16-celled stage as respectively upper and lower.
Of vesicles over 1 mm. in diameter, the smallest in which I have been able to detect the sutural line above referred to measure 3'25 mm. in diameter. In three lots of vesicles, 3'5 mm. in diameter from three different females, I have failed to X'ecognise it, whilst in two other lots, respectively 3'75 mm. (average) and 4 mm. in diameter, the line appears to be in course of differentiation as in the 3'25 mm. vesicles. A portion of the wall of one of the 3'5 mm. vesicles just referred to is shown in PL 4, fig. 41, and a portion of the wall of the 3'25 mm. stage, including the sutural line, in fig. 42. Both vesicles were fixed in the same fluid, viz. picro-nitro-osmic acid. Comparison of the two figures reveals the existence, quite apart from the presence of the junctional line in fig. 42, and its absence in fig. 41, of certain more or less obvious differences between them. In fig. 41 the cells are larger, and their cytoplasmic bodies are inconspicuous, being fairly homogeneous and lightly staining. In fig. 42, on the contrary, the cellbodies are strongly marked, the cytoplasm being distinguishable into a lighter-staining peripheral zone, and a much more deeply staining perinuclear zone, showing evidence of intense metabolic activity. This latter zone is more or less vacuolated, and contains, besides larger lightly staining granules, numerous smaller ones of varying size, stained brown by the osmic acid of the fixative. In the 4 ram. vesicles the cells show pi-ecisely the same characters; in the 3'75 mm. vesicles, which were fixed in a picro-corrosive-acetic fluid, the granules ax'e absent from the cytoplasm, otherwise the cells are similar to those of the other two. Mitotic figures are common. The sutural line is recognisable in all three sets of vesicles (3'25, 3'75, and 4 mm.) (fig. but I cannot be certain that it runs con tinuously round, and it appears to have a rather more sinuous course than in later blastocysts. The cells of the two regions of the bliistocyst wall, separated by the sutural line, differ somewhat in tlieir characters. On one side of the line (fig. 42, tr.ect.) the cells appear to be on the whole slightly larger, and of more uniform size than they are on the other, and they also stain somewhat more deeply. Comparison with later blastocysts shows that the region of more uniform • cells is non-formative, that of less uniform, formative. At^this stage, however, the differences between the cells of the two regions are as yet so little pi'onounced that it is practically impossible in the absence of the sutural line to say to which hemisphere an isolated piece of the wall should be referred.
I am inclined to regard the sutui'al line in these vesicles as being in course of differentiation, and judging from the disposition of the cells on either side of it, I think its appearance is to be correlated with the marked increase in the mitotic activity of the cells of the two hemispheres which sets in in vesicles of 3-4 mm. diameter. The preliminary increase in size of the blastocyst up to about the 3 mm. stage might be described as of a passive character, i.e. it does not appear to be effected as the result of the very active division of the wall-cells, but is characterised rather by a minimum of mitotic division and a maximum of increase in surface extent of the cells, due to excessive stretching consequent on the rapid imbibition of uterine fluid. Once, however, the requisite size has been attained, the cells of the unilaminar wall commence to divide activel}', and doubtless as the outcome of that wave of activity, the sutural line makes its appeai-ance between the two groups of independently growing cells.
On the inner surface of the blastocyst wall, especially in the region of the formative hemisphere, there are present in these vesicles numbers of small deeply staining cells of spherical form, and containing osmicated granules similar to those in the wall-cells. They may occur singly or in groups, and appear to me to be of the same nature as the inteimal cells of the earlier blastocyst. In addition to these cells, there are present clusters of cytoplasmic spheres, staining similarly to the spherical cells, and apparently of the nature of fragmentation products formed either directly from the wall-cells or from these internal cells.
2. Differentiation of the Embryonal Ectoderm and the Entoderm
After the preliminary growth in size of the blastocyst is completed, the next most important step in the progressive development of the latter is that just dealt with, involving the appearance of the sutural line, with resulting re-establishment of polar differentiation in the blastocyst wall. Following on that, we have the extremely important period during which the embryonal ectoderm and the entoderm become definitely established.
For the investigation of the earlier phases of this critical period I have had at my disposal a large number of unilaminar blastocysts derived from three females, distinguished in my notebooks as (3, 25 . vii . '01, with fifteen vesicles of a maximum diameter of 4‘5 mm. ; 8 . vii . '99, with twelve vesicles, 4‘6 .mm. in diameter ; and 6 . vii . '04, with twfenty-two vesicles, 4‘5 and 5 mm. in diameter. These three lots of vesicles may for descriptive purposes be designated as '01, '99, and '04 respectively.
The '01 vesicles are distinctly less advanced than the other two. The sutural line is now, at all events, definitely continuous, and can readily be made out in the intact vesicle with the aid of a low-power lens (PI. 4, fig. 38, j.L), but the differences between the cellular constituents of the two hemispheres which it separates are much less obvious than they are in the '99 and '04 vesicles. Here, again, one hemisphere forming half or perhaps rather more of the entire vesicle is distinguished from the other by the greater uniformity and the slightly deeper staining character of its constituent cells (figs. 43 and 44, tr. ecL). This hemisphere, subsequent stages show, is the lower or non-formativ^ hemisphere. It is characterised especially by the striking 'uniformity in the size of its cells. Over the opposite hemisphere, the upper or formative one (figs. 43 and 44, the cells are more variable iu size, the nuclei thus appearing less uniformly and less closely arranged, and they stain,. on the whole, somewhat less deeply than those of the lower hemisphere. The non-formative cells are on the average smaller than the largest of the formative cells, but they are more uniform iu size, and their nuclei thus lie at more regular distances apart, and appear more closely packed. They are also richer in deutoplasmic material, and so stain rather more deeply than the formative cells. Sections show that the cellular wall is unilaminar throughout its extent, and that, whilst it is somewhat thicker than that of 3‘5 mm. vesicles, it is still very attenuated, its thickness, including the shellmembrane, ranging-from ‘004 to '008 mm. I have examined a number of series of sections taken through portions of the wall known to include the sutural line, and find it quite impossible to locate the position of the- latter; indeed, I cannot certainly distinguish between the formative and nonformative regions.
In the blastocyst cavity, lying in contact with the inner surface of the wall, and most abundant in the region of the formative hemisphere, there are present numbers of deeply staining spherical cells with relatively small nuclei similar to those described in connection with the 3'25 mm. vesicles. They occur singly or in groups, and may appear quite normal or may show more or less evident signs of degeneration. Their nuclei may stain deeply and homogeneously, or may be represented by one or two deeply staining granules, vacuoles may occur in their cytoplasm, and spherical cytoplasmic masses of very variable size, with or without deeply staining granules of chromatin) may occur along with them. In sections and preparations of the wall of these, and other 4*5 mm. vesicles there are to be found, in both the formative and non-formative hemispheres, small localised areas from which such spherical cells are being proliferated off in numbers together. PI. 5, fig. 47, from the formative hemisphere of an ^04 vesicle shows One of the most marked examples of such proliferative. activity that I have encountered. A similar but smaller proliferative area occurs on the non-formative hemisphere of the same vesicle.
These spherical cells are, I am convinced, of no morphological importance, and are destined sooner or later to degenerate. They have certainly nothing to do with the entoderm, the parent-cells of that layer arising exclusively from the formative hemisphei'e and not from cells such as these, which are budded ofE from both hemispheres. The fact that they are, in unilaminar vesicles, more numerous over the formative hemisphere may perhaps be taken as an indication of the greater mitotic activity of the formative as compared with the non-formative cells.
The Primitive Entodermal Cells. - Following closely on the stage represented by these '01 blastocysts is the extremely important one constituted by the '99 and '04 vesicles before referred to. This stage is the crucial one in primary germ-layer formation, and marks the transition from the unilaminar to the bilaminar condition, since in it the entodermal cells are not only distinctly recognisable as constituents of the formative region, but are to be seen both in actual process of separation from the latter and as definitely internal cells, frequently provided with, and even connected together by, pseudopodial-like processes of their cell-bodies. Such cells are already present in the '01 vesicles (fig. 71), and probably also in the blastocysts in which the sutural line first makes its appearance, but are much less conspicuous than in these older blastocysts.
The '99 blastocysts are distinctly more advanced than the '01 batch and are just a little earlier than the '04 lot. The former measui'ed, as already mentioned, 4'5 mm. in diameter, the latter 4'5 and 5 mm. (the majority being of the latter size). In my notes, on the intact '99 vesicles I find it stated that one hemisphere, forming rather less than half of the entire extent of the vesicle wall, appeared somewhat denser than the other, the sutural line marking the division between the two. I naturally inferred at the time that the denser hemisphere corresponded to the embryonal region of the
Eutherian blastocyst and the less dense to the extra-embryonal region of the same, but just the reverse proves to hold true for the '04 vesicles, the formative hemisphere in these appearing less dense than the non-formative. I cannot now test my former inference by direct observation since I do not appear to have any of the '99 vesicles left intact, but amongst my in toto preparations of the vesicle wall I find one labelled as from the “ lower pole †which unmistakably belongs to the formative hemisphere, hence I conclude that the denser and slightly smaller region which I originally regarded as formative is really non-formative, a conclusion which brings the '99 vesicles into agreement with the '04 batch.
In these latter vesicles the sutural line and the two regions of the wall can be quite readily made out on careful examination under a low power with transmitted light. The one region appears slightly denser (darker) and has more closely arranged nuclei (i. e. is composed of smaller cells) than the other. On the average this denser region appeal's to be rather the less extensive of the two ; the two regions may be about equal ; on the other hand the denser may be the smaller. Examination of stained preparations of the wall demonstrates that the darker hemisphere is non-formative, the lighter, formative. It would therefore seem that in certain of these '04 vesicles the formative region has grown more rapidly than the non-formative.
In stained preparations of the wall both of the '99 and '04 vesicles, the differences between the two hemispheres are now so well marked that there is no diflBculty in referring even an isolated fragment to its proper region. The non-formative hemisphere differs in no essential way from that of the '01 vesicles, and as in these, is readily distinguishable from the formative by the much greater uniformity in the size and staining properties of its cells (fig. 45), as well as by the fact that there are no primitive entodermal cells such as occur in relation to the formative hemisphere, in connection with it. Its constituent cells are on the average distinctly smaller than the largest of the formative ; their nuclei lie nearer each other, with the result that in surface examination of the blastocyst the non-formative region appears rather denser than the formative. In in toto preparations of the wall the former usually stains darker than the latter (fig. 45), but this is not always the case ; in fig. 46, from an '04 vesicle, there is practically no difference in this respect between the two regions ; in yet others of my preparations of '99 vesicles the formative region has stained more deeply than the nonformative.
The formative hemisphere in the earlier blastocysts of this particular developmental stage was described (ante, p. 51) as differing from the non-formative in that its constituent cells were much less uniform in chai*acter than those of the latter. This same feature, but in much enhanced degree, characterises the formative region of the vesicles under consideration, for it can now be definitely stated that the latter I'egion is constituted by cells of two distinct varieties, viz. (1) moi*e lightly staining cells which form the chief constituent of the formative region, its basis so to speak, and which are on the average larger than those of the other variety, and (2), a less numerous series of cells, distinctly smaller than the largest cells of the former variety, and with denser, more granular and more deeply staining cytoplasm, and frequently met with in mitotic division (cf. PI. 6, fig. 65). The two varieties of cells are intermingled promiscuously, the smaller cells occurring singly and in groups but in a quite irregular fashion, so that here and there we meet with patches of the wall composed exclusively of the larger cells.
The evidence presently to be adduced shows that the larger cells furnish the embryonal ectoderm, and that the smaller cells give origin to the primitive entodermal cells from which the definitive entoderm arises. The smaller cells may therefore be regarded as entodermal mothei'-cells. Whether these latter cells are progressively formed from the larger cells simply by division, or whether the two vaifieties become definitely differentiated from each other at a particular stage in development, must for the present be left an open question. Of the actual existence in tlie unilaminar formative region of these '99 aud '04 blastocysts of two varieties of cells, respectively ectodermal and entodermal in significance, there can be no doubt. In preparations of the formative region, however, whilst one can without hesitation identify certain cells as being in all probability of ectodermal significance and others as prospectively entodermal (cf. figs. 65, 66), it must be admitted that one is often in doubt as to whether one is dealing with small ectodermal cells or with genuine entodermal mother-cells. It is, therefore, hardly to be wondered at that I have not yet been able to satisfactorily determine at what precise period the entodermal mother-cells first become differentiated, though judging from the facts that in the eai-liest vesicles in which the sutural line is recognisable one region of the wall already differs from the other in the less uniform size of its constituent cells, and that internally situated entodermal cells are already present in small numbers in the '01 vesicles (fig. 71), I incline to the belief that it will probably be found to about coincide with the first appearance of the sutural line. To this question I may perhaps be able to return at some future time.
In addition to the presence of these entodei'mal mothercells, which enter directly into its constitution, the formative region of the '99 and '04 blastocysts is. characterised by the occurrence on its inner surface of definitely iuteimal cells, which generally agree with the former cells as regards size and staining properties and are evidently related to them. It is these internally situated cells which directly give origin to the definitive entoderm of the later blastocysts, and one need, therefore, have no hesitation in applying to them the designation of primitive entodermal cells. They are exclusively found in relation to the formative hemisphere, and appear in in toto prepai'atious as flattened, darkly staining cells closely applied to the inner surface of the unilaminar wall, and disposed quite irregularly, singly, and in groups. They vary greatly in number in blastocysts of even the same batch, but on the whole are most abundant in the ^04 series, and they also exhibit a remarkable range of variation in shape. They may have a perfectly distinct oval or rounded outline (figs. 67, 71, 72), or, as is more frequently the case, they may lack a determinate form and appear quite like amoeboid cells owing to their possession of cytoplasmic processes of markedly pseudopodial-like character (fig. 69). Frequently, indeed, the cells are connected together by the anastomosing of these processes, so that we have formed in this way the beginnings at least, of a cellular reticulum (figs. 68, 69,70).
The question now arises. How do these primitive entodermal cells originate from the small, darkly staining cells of the unilaminar formative region designated in the foregoing as the entodermal mother-cells ? I can find no evidence that the primitive entodermal cells are formed by the division of the mother-cells in planes ta.ngential to the surface ; on the contrary, all the evidence shows that we have to do here with an actual inward migration of the mother-cells, with or without previous mitotic division, such inward migration being the outcome of the assumption by the mother-cells, or their division products, of amoeboid properties ; in other words, the evidence shows that the formation of the entodei'm is effected here not by simple delamination (using that term in the sense in which it was originally employed by Lankester), but by a process involving the inward migration, with or without previous division, of certain cells (entodermal mother-cells) of the unilaminar parent layer, a process comparable with that found in certain Invertebrates (Hydroids) and distinguished by Metschnikoff as '^gemischte Delaminatiou.â€
In this connection it has to be remembered that the cells of the unilaminar wall of the blastocyst are under considei'able hydrostatic pressure, and, in correlation therewith, tend to be tangentially flattened, though the flattening in this stage is much less than in the earlier blastocysts. From a series of measurements made from an '04 vosicle, I find that over the formative region the ratio of the breadth to the thickness of the cells varies Horn 6 : 1 to 2 : 1, and even to 3 : 2. On the whole cells of the type indicated by the ratio 6 : 1 predominate, and we should hardly expect to find such cells dividing tangentially. In fact, the only undoubted examples of such division I have met with occur in the single abnormal vesicle present in the '04 batch. In this particular vesicle, which had a diameter of 3 mm. and was thus smaller than the others, thei'e was present on what appeared to correspond to the formative hemisphere of the normal blastocyst a well-defined and conspicuous ovalish patch, 1'23 x '99 mm. in diameter.^ Sections show that over this area the cells of the unilaminar wall are much enlarged and , more or less cubical in form, their thickness varying from ‘012 to ‘019 mm. These cubical cells exhibit distinct evidence of tangential division, both past and in progress. But in normal vesicles, whilst mitotic figures are quite commonly met with in the cells of the formative region (in which, indeed, they are more numerous than in those of the non-formative region), I have failed to find in my sections after long-continued searching even a single spindle disposed directly at right angles to the shell-membrane ; the mitotic spindles lie disposed either tangentially to the surface or obliquely thereto.
For the determination of the mode of origin of the primitive entodermal cells, it is absolutely necessary to study both in to to preparations of the formative region, i.e. small portions of the unilaminar wall stained and mounted on the flat, and sections of the same. Sections alone are, on the whole, distinctly disappointing so far as the question under discussion is concerned, and, indeed, give one an altogether inadequate idea of the primitive entodeimial cells themselves, seeing that practically all one can make out is that
1 Curiously enough, amongst the '99 vesicles there also occurred a single small one, likewise 3 mm. in diameter, and with a thickened patch 1-28 X 1 mm. in diameter, quite similar in its character to that described in the text. I am as yet uncertain whether the thickened area in these two vesicles represents the whole of the formative hemisphere of normal blastocysts or only a hypertrophied part of the same, or whether, indeed, it may not represent the retarded non-formative hemisphere there are present, in close apposition with the inner surface of the unilaininar wall, small, darkly staining cells, apparently quite isolated from each other and usually of flattened form (figs. 73, 74, 76, ent.). One has only to glance at a wellstained in to to preparation of the formative region (cf. fig. 70) to realise how inadequate such a description of the primitive entoderm cells really is.
Sections nevertheless do yield valuable information on certain points. Besides affording the negative evidence of the absence of tangential divisions and the positive evidence that the primitive entodermal cells are actually internal (figs. 73, 74, 76), they show that growth of the wall iu thickness has already set in, and that it is most marked over the formative region, though the thickness attained by the cells is as yet very unequal (figs. 73-76). Measurements takeu from an '04 vesicle show that over the non-formative region (fig. 77) the cells vary in thickness from *006 to '009 mm., whilst over the formative region the range of variation is greater, viz. from ‘006 to ‘013 mm., so that we may conclude that the latter region is on the average thicker than the former (cf. figs. 73-76, with fig. 77 depicting a small portion of the non-formative region). It is still impossible to determine the position of the sutural line, even in sections of fragments of the wall known to contain it.
The entodermal mother-cells are not very readily recognisable in sections. In fig. 75, however, which is drawn from an accurately transverse section through the formative region of an '04 vesicle, there is depicted what is undoubtedly an entodermal mother-cell {ent.). The interesting point about this particular cell is that its cell-body, whilst still intercalated between the adjoining cells of the unilaminar wall, has extended inwards so as to directly underlie one of the wall-cells. ' Division of such a cell as this would necessarily result in the production of an internally situated cell with all the relations of one of the primitive entodermal type. The inwardly projecting spheroidal cell situated immediately to the left (in the figure) of the one just refeiTed to, I also regard as an entodermal mother-cell. Cells of this type are not infrequently met with in sections; they nsually stain somewhat deeply, and are often found in mitosis.
The evidence obtainable from the study of in to to preparations conclusively proves that some at all events of the primitive entodermal cells are actually derived from the entodermal mother-cells, much in the-way suggested above, whilst others of the primitive entodermal cells are directly formed from mother-cells which bodily migrate inwards.
Fig. 65, PI. 6, represents a small portion of the formative region of an '04 vesicle viewed fPom the inner surface. In the centre of the figure, surrounded by the larger, lighter staining (ectodermal) cells of the wall, is a smaller cell in the telophases of division, the cytoplasm of which is granular and stains deeply. That cell unmistakably forms a constituent of the unilaminar wall. I regard it as an entodermal mothercell. Fig. 66 shows another cell of the same character in the anaphases of division, which likewise forms a constituent of the unilaminar wall, but which differs from the corresponding cell in fig. 65 in that its cytoplasmic body has extended out on one side (lower in the figure), so as to directly underlie part of an adjacent ectodermal cell. In other words we have here a surface view of the condition represented in section in fig. 75, only the entodermal mother-cell depicted therein is not actually in process of division. Fig. 67, taken from the same preparation as fig. 65, shows what I take to be the end result of the division of such a cell as is i-epresented in the two preceding figures. Here we see two small deeply staining cells towards the centre of the figure, which from their disposition and agreement in size and cytological characters are manifestly sister-cells, and the products of division of just such an entodermal mother-cell as is represented in fig. 65, or, better, fig. 66. The one cell (upper in the figure) is more angular in form and manifestly still lies in the unilaminar wall; the other (lower in the figure) is ovalish in form and is no longer a constituent of the unilaminar wall, but is on the contrary a free cell, definitely internal both to the latter and to its sister-cell. It is, in fact, a primitive entodermal cell, as comparison with fig. 68 proves, and that it has been formed by the division of a mother-cell situated in the unilaminar wall can hardly, I think, be doubted. Its sistercell, which is still a constituent of the wall, would presumably have migrated inwai-ds some time later.
It is to be noted that the primitive entodermal cell referred to above and those depicted in figs. 71 and 72 are definitely contoured, ovalish and I'ounded cells, entirely devoid of processes. In these respects they differ markedly from the entodermal cells shown in fig^. 68-70, which are very variable in form owing to their possession of more or less elongated pseudopodial-like processes. It might thex'efore be inferred that the formation of these processes only takes place after the entodermal cells have become definitely internal. Such an inference, however, would be incorrect, for I have abundant evidence showing that such processes may be given off from the entodermal mother- cells whilst they are still constituents of the wall. In in toto preparations, it is often difficult to determine with certainty whether a particular entodermal cell still enters into the constitution of the unilaminar wall or not. In the portion of the formative region of a '04 vesicle depicted in fig. 70, however, I am satisfied that all the entodermal cells therein shown (they are readily distinguishable by their smaller size and more deeply staining character) are, with the possible exception of the one on the extreme right, at least partially intercalated between the larger ectodermal cells of the wall. Some of them are entirely situated in the wall ; others have extended inwards in varying degree so as to partially underlie the ectodermal cells. It is these latter entodermal cells in particular which exhibit the cytoplasmic processes above referred to. As the figure shows, these processes have all the characters of pseudopodia,; they vary in size, form, and number from cell to cell, individual processes may be reticulate and their finer prolongations may anastomose with those of others, and they are formed of cytoplasm, less dense and rather less, deeply staining than that of the cell-bodies from which they arise. Attention may be specially directed to the cell towards the left of the hgure (mai'ked ent.). Here we have an entodermal cell whose cytoplasmic body is evidently still partially intercalated between the cells of the wall, but which is, at the same time, prolonged inwards (towards the left) so as to underlie the adjoining ectodermal cell. From this inward prolongation there are given off two slender processes, one short and tapering, the other very much longer ; this latter, after becoming vei'y attenuated, gradually widens to form an irregular fan-shaped expansion, suckerlike in appearance, and produced into several slender threads, which is situated adjacent to the nucleus of the ectodermal cell on the extreme left. Then from the right side of the same cell there is given off a small inwardly projecting bulbous lobe which may well be the start of just such another process as arises from the left side. Processes of the peculiar sucker-like type just described, formed of a slender elongated stem and a distal expanded extremity from which delicate filamentous prolongations are given off, are abundantly met with in preparations, and strikingly recall the pseudopodia of various Ehizopoda. They are seen in connection with other entodermal cells in fig. 70, and with many of those in fig. 68. I regard them as veritable pseudopodia. Towards the right side of fig. 70 the two entodermal cells there situated stand in direct protoplasmic continuity by means of two slender connecting threads, whilst the upper of these two cells is again joined by a very fine process to the irregular pseudopodial expansion which arises from one of the two entodermal cells situated nearer the middle of the figure, and that same expansion is directly connected with the second of the two entodermal cells just mentioned, so that we have here established the beginning of a cell-network, prior to the complete emancipation of its constituent entodermal elements from the unilaminar wall. We have, then, clear evidence that the entodermal elements in Dasyurus, prior to their separation from the unilaminar formative region are capable of exhibiting amoeboid activity, since not only may they send lobose prolongations of their cytoplasmic bodies inwards below the adjacent ectodermal cells, but they may emit more or less elongated processes of indubitable pseudopodial character, which similarly lie in contact with the inner surface of the wall-cells. Furthermore, we have evidence that these pseudopodial processes may anastomose with each â– other so as to initiate the formation of an entodermal reticulum, whilst the cells from which they arise are still constituents of the unilaminar wall - an especially noteworthy phenomenon. Certain of the primitive entodermal cells, as we have seen, are at first devoid of such processes, but since they all eventually form part of a continuous reticulum, it is evident that the entodermal elements are capable of emitting pseudopodial processes as well after as before their separation from the formative region.
Finally, in view of the fact that the entodermal mothei'-cells depicted in fig. 70 are not actually in process of division, and therein differ from those of figs. 65 and 66, we may conclude that the formation of the primitive entodermal cells is effected either with or without the pi*evious division of the mother-cells.
If we admit, as I think on the evidence we must admit, that the entodermal cells in Dasyurus are endowed with amceboid properties, then Ave are relieved of any further difficulty in regard to the mechanism of their inAvard migration from the unilaminar Avail. Doubtless, in the case of those entodermal mother-cells Avhich do not undergo division, the precocious formation of the above-described pseudopodial processes which spread out from the cells like so many suckers considerably facilitates their direct detachment from amongst the cells of the Avail. In the case of those primitive entodermal cells Avhich originate as the direct products of division of the mother-cells, it no doubt depends on a variety of circumstances (e.g. actual form of the dividing cell, direction of the spindle, etc.) whether they exhibit amoeboid activity precociously (i.e. before their actual i separation), or only at a later period.
The entoderm varies considerably in its degree of differentiation iu different vesicles of this stage, and even in different parts of the formative region of one and the same vesicle. In some vesicles there are relatively few primitive entodermal cells, in othei*s they are much more abundant. Fig. 68, from the formative region of an ^04 vesicle, shows a typical patch of them and illustrates very well the highest stage of differentiation which they attain in these vesicles. The entodermal cells therein depicted all appear to be definitely internal, and it is especially worthy of note that the portion of the unilaminar wall in relation to them is composed exclusively of the larger, lighter staining cells. It is these cells which directly form the embryonal ectoderm of the blastocysts next to be described. The entodermal cells are obviously amoeboid in character (obsei've especially the cells near the middle of the figure), and are in active process of linking themselves together into a cellular reticulum. In fig. 69 is shown a small portion of the formative region of another ^04 vesicle. A single entodermal mother-cell in process of division occurs in position in the unilaminar Avail, which is otherwise composed of ectodermal cells, whilst internally there are present three entodermal cells, already linked together by their pseudopodial processes. ^Jfiie two lowermost cells afford especially striking examples of amoeboid activity, the elongated pseudopodial process of the cell on the left terminating iu a well-marked reticulation in definite continuity Avith the corresponding, but shorter and thicker process of the cell on the right.
3. Establishment of the Definitive Embryonal Area
FolloAving directly on the stage represented by the '04 blastocysts described in the preceding section is one designated in my list as 5, 18 . vii . 01 and referred to here as 5, '01. It comprises twenty-two blastocysts obtained from a female killed fifteen days after coition and all normal, Avith the exception of one Avhich Avas shrivelled, and all in precisely the same stag-e of development. They measured from 4‘5 to 6 mm. in diameter.
In this stage the formative region of the preceding blastocysts has become transformed into the definitive embryonal area (embryonic shield, Hubrecht) as the result of the completion of that process of inward migration of the entodermal mother-cells which we saw in pi-ogress in the vesicles last described, and the consequent establishment of the entoderm as a continuous cell-layer undeidying and independent of, the embryonal ectoderm constituted by the larger passive cells of the original unilaminar formative layer.
In the entii*e blastocyst (PI. 4, fig. 39) the embryonal area is quite obvious to the naked eye as the more opaque, hemispherical region, forming rather less than half the entire extent of the vesicle wall ; the larger remainder of the same is formed by the much more transpai-ent, non-formative or extra-embryonal region. Sections of the entire blastocyst show (1) that the embryonal area is bilaminar over its entii-e extent, its outer layer consisting of embryonal ectoderm, already somewhat thickened, its much thinner inner layer consisting of entoderm, partly still in the form of a cellular reticulum, and (2) that the extra-embryonal region is still unilaminar throughout and composed of a relatively thin layer of flattened cells (extra-embryonal or trophoblastic ectoderm, trophoblast [Hubrecht])^ (PI. 8, fig. 78). The entoderm is co-extensive at this stage with the embryonal ectoderm, and terminates in a wavy, irregularly thickened, free, edge (PI. 5, fig. 49), which over most of its extent either directly underlies or extends very slightly beyond the line of junction between the embryonal and extra-embryonal ectoderm. The junctional line is thus not very easily seen. In fig. 48, however, a small portion of the line shows with sufficient distinctness, I think, to demonstrate its identity with that of the preceding stage.
In consonance witli my conviction that this layer is homologous
both Avith the so-called trophoblast of Eutheria and the exti-a-embryonal ectoderm of Prototheria, and in view of the theoretical signification which Hubrecht now insists should be attached to the term “ trophoblast.†and which I am wholly unable to accept, I venture to suggest as an alteiTiative name for this layer that of “ tropho-ectoderni.
The vesicle wall in all my sections of this stage appears to be somewhat thinner than that of the '04 blastocysts, but apart from this apparently variational difference the present blastocysts are almost exactly intermediate between the latter and those next to be described.
The embryonal ectoderm (fig. 78, emb. ect.) appears in section fairly uniformly thickened, though its cells are still of the flattened type. In surface view in in toto preparations (cf . fig. 48), they exhibit the same polygonal form and lightly staining qualities as the larger cells of the formative region of the '04 blastocysts, which we have already identified as prospective embryonal ectodermal cells. The junctional line between the embryonal ectoderm and the extra-embryonal is now for the first time readily distinguishable in sections (fig. 78). The extra-embryonal ectoderm (tropho-ectoderm) (PI. 5, figs. 48 and 49, PI. 8, fig. 78, tr. ect.) differs in no essential respect from the corresponding layer in the '04 blastocysts.
The entoderm in these blastocysts is exceedingly closely adherent to the inner surface of the embryonal ectoderm and cannot be removed therefrom by artificial means. It varies slightly in its character in different vesicles and in different parts of its extent in the same vesicle. Mostly it appears as a continuous thin cell-layer (figs. 49 and 78, ent.), but here and there patches occur in which the cells form a reticulum quite similar to that shown in fig. 68 of the preceding stage.
The next stage (designated in my list as 8 . vi . 01), and the last of Dasyurus that need be described in the present communication, comprises eleven vesicles (5-5'5 mm. in diameter), in which the embryonal area is conspicuous and distinctly in advance of that of the preceding vesicles, but is still devoid of any trace of embryonal differentiation (PI. 4, fig. 40; PI. 8, fig. 79).
The embryonal area is hemispherical in form (its greatest diameter varying' from 3'5 to 4 mm.) in all except two of the blastocysts, in which it is elongate, with longer and shorter diameters. It occupies about a third or less of the entire extent of the vesicle wall, and thus appears relatively smaller than that of the preceding (.5, '01) vesicles. The entoderm now extends for a distance of about 1 mm. beyond the limits of the area, so that in the entire vesicle (fig. 40) three zones differing in opacity are distinguishable, viz. the dense hemispherical zone at the upper pole, constituted by the embryonal area; below that, a less dense, narrow annular zone, formed of extra-embryonal ectoderm and the underlying peripheral extension of the entoderm ; and finally, the still less dense hemispherical area, forming the lower hemisphere of the blastocyst and constituted, solely by extra-embryonal ectoderm. Thus approximately the upper half of the blastocyst is bilaminar, the lower half unilaminar. Sections show that the embryonal ectoderm (fig. 79, emh. ect.) is now a quite thick layer of approximately cubical cells, whilst the extraembryonal ectoderm {tr. ect.) is formed of relatively thin flattened cells. The line of junction between the two is perfectly obvious, both in sections (fig. 79) and in surface view (PI. 5, fig. 50). The embryonal ectodermal cells, though much thicker than the extra-embryonal, are of less superficial extent; their nuclei therefore lie closer together than those of the latter, moreover they are larger, stain more deeply, and are more frequently found in division, all of which facts testify to the much greater growth-activity of the embryonal as compared with the exti-a-embryonal ectoderm at this stage of development (cf . fig. 50, emh. ect. and tr. ect.-, in the preparation from which this micro-photograph was made the entoderm underlying the embryonal ectoderm has been removed, whilst it is still partially present over the extra-embryonal ectoderm).
The entoderm (fig. 79, ent.) over the region of the embrvonal area is readily separable as a quite thin membrane, and is then seen to consist of squamous cells, polygonal in outline, and either in direct apposition by their edges or connected together by minute cytoplasmic processes. Beyond the embryonal area, liowever, its peripliei'al extension below the extra-embryonal ectoderm is much less easily separable in the intact condition (cF. fig. 50), because oF its greater delicacy due to the fact that it has here largely the form of a cellular reticulum. In this extra-embryonal region the entodermal cells are frequently found in mitosis. Ic would appear, then, that the entoderm is first laid down in the region of the embi'yonal area as a cellular reticulum, which later becomes ii'ansformed into a continuous cell-membrane, and that its peripheral extension over the inner surface of the extraembryonal ectoderm is the result of the growth and activity oF its own constituent cells.
This peripheral growth continues until there is formed eventually a complete entodermal lining to the blastocyst cavity. The rate of growth appears to be somewhat variable. In a series of primitive streak vesicles (6-6'75 mm. in diameter) the lower third oF the wall is, I find, still unilaminar. In another series of vesicles of the same developmental stage (4'5-6 mm. in diameter) a unilaminar area is present at the lower pole, varying from I x ‘5 mm. in diameter to as much as 4 mm. Even in vesicles 7-7'5 mm. in diameter a unilaminar patch may still occur at the lower pole, but in vesiqles 8'5 mm. in diameter (stage of fiat embryo) the entodermal lining appears always to be complete.
The Origin of the Entoderm in Eutheria. - The remafkable facts relative to the origin of the entoderm in Dasyurus which I have been able to place on record in the jireceding pages, thanks to the large size attained by the blastocyst prior to the differentiation of the formative germlayers and to the circumstance that the formative cells are not arranged, as they are in Eutheria, in the form of a more or less compact cell-mass, but constitute a thin unilaminar cell-layer of relatively great extent which can easily be cut up with scissors, and which, after staining and mounting on the fiat can be examined under the highest powers, throw, it seems to me, a new and unexpected light on the mammalian entoderm, and at the same time help to fill the considerable gap whicli has hitherto existed in our knowledge of its early ontogenesis. Although the mode of origin of the entoderm in Dasyurus would appear, in the present state of our knowledge, to find its closest parallel, not amongst vertebrates, but in certain invertebrates (cf . the mode of origin of the entodermal cells from the wall of the blastula in Hydra as described by Brauer^), the observations of Assheton ('94) on the early history of the entoderm in the rabbit, when viewed in the light of the foregoing, seem to me to afford ground for the belief that phenomena comparable with those hei'e recorded for Dasyui'us will eventually be recognised as occurring also in Eutheria.
Hubrecht ('08), in his recent treatise on early Mammalian ontogeny, deals very briefly with the question of the origin of the entoderm in the latter group, merely stating that “ from the inner cell-mass arises by delamination a separate lower layer which we designate as the entoderm of the embryo. These entoderm cells wander in radial direction along the inner surface of the trophoblast, which in many cases is thus soon transformed into a didermic structure.
. . . When the entoderm has separated off by delamina tion from the embryonic knob, the remaining cells of the latter form the ' embryonic ectoderm, which is thus situated between the entoderm and the trophoblast.
Assheton, in the paper just referred to, has given a careful account of the first appearance of the entodermal cells in the rabbit, and of what he believes to be the mode of their peripheral extension below the trophoblastic wall of the blastocyst. He shows that the inner cell-mass, at first spherical, gradually, as the blastocyst enlarges, fiattens out below the “ covering layer †of the trophoblast until it forms an approximately circular plate “ nowhere more than two cells thick.†During the process of flattening, cells are seen to jut out from the periphery of the mass; these eventually separate, and appear as rounded cells scattered irregularly over the inner surface of the trophoblast and ‘^extending ' ‘ Zeitschi'. f. wiss. Zool.,' Bd. Hi, 1891.
over an arc of about 60° from the upper pole in all directions.†These “ straggling†cells, as Assheton terms them, as well as the innermost cells of the now flattened inner cell-mass, are regarded as hypoblastic and the outermost cells of the same as epiblastic (embryonic epiblast). “The hypoblast, as a perfectly definite layer, is formed by the time the blastodermic vesicle measures '5 mm. in diameter, that is, about the 102nd hour after coition. It is not, however, as yet by any means a continuous membrane ; it is a network or fenestrated membrane. For this reason, in section it appears to be represented by isolated cells lying beneath the embryonic disc (v. fig. 29, Sy.)†(cf. Dasyurus). In considering the question how the peripherally situated (“ straggling â€) entodermal cells, which are undoubtedly derived from the inner cell-mass, “apparently Avander round the inside of the blastodermic vesicle,†he I'eaches the conclusion that this is not the result of amoeboid activity or growth “in the sense of migration †on the part of these cells, but “ is only an apparent growth round produced by the more rapid growth of a zone of the [trophoblastic] wall of the vesicle immediately surrounding the embryonic disc, in which zone the marginal cells of the inner mass lie.†He is unable to find any evidence of the production of pseudopodial processes by these pei'ipheral entodermal cells, the majority of them appearing at first to be quite isolated from each other and approximately spherical. “Certain of the cells here and there are connected by threads of protoplasm, but this, I think, is not a sign of pseudopodic activity, but merely indicates the final stage in division betAveen the tAvo cells.†By the sixth day the hypoblast of the embryonic disc has assumed the lorm of a continuous membrane, composed of completely flattened cells, Avhilst the peripheral hypoblast cells have become more numerous, and “many of them, possibly all of them, are noAV undoubtedly connected by more or less fine protoplasmic threads.†Such, in brief, is Assheton's account of the early history of the entodenn in the rabbit; it presents obvious points of agreement with my own for Dasyunis, and I ventui'o to think the agreement is even greater than would appear from Assheton's conclusions. In adopting- the view that the more active growth of the region of the blastocyst wall immediately surrounding the inner cell-mass is the sole causal agent in effecting the separation and peripheral spreading of the entodermal cells, I cannot but feel, in view of his own description and figures and of my own results, that he has attributed a much too exclusive importance to that phenomenon and a much too passive role to the entodermal cells themselves. In Dasyurus the inward migration and the later peripheral spreading of the entodermal cells is effected without any such marked unequal growth of tlie blastocyst wall as occurs, according to Assheton, in the rabbit, as the direct outcome of their owu inherent activity, and I believe the possession of a like activity characterises the entodermal cells of the rabbit. The evidence of Assheton's own fig. 40, which shows in surface view a portion of the vesicle wall with the peripheral entodermal cells in relation thereto, and which should be compared with my figs. 68 and 69, conclusively demonstrates, to my mind, the possession by these cells of amoeboid properties, and thus support is afforded for the belief that the separation of the entodermal cells from the formative cell group (inner cell-mass) is here also the expression of an actual migration. Whether or not the strands of protoplasm which Assheton ('08, '09) describes as present in the sheep, pig, ferret, and goat, connecting the inner lining of the inner mass to the wall of the blastocyst, and which he interprets as tending “ to show that the inner lining of the inner mass is of common origin with the wall of the blastocyst,†are of any significance in the present connection, I cannot certainly determine.
4. Summary
The results and conclusions set forth in the preceding pages of this chapter may be summarised as follows;
(1) The unilaminar wall of the blastocyst of Dasyurus consists of two regions distinct in origin and in destiny, viz. an upper or formative region, derived from the upper cell-ring of the 16-celled stage, and destined to furnish the embryonal ectoderm and the entoderm and a lower or nonformative region derived fi-om the lower cell-ring of the mentioned stage, and destined to form directly the extraembryonal or trophoblastic ectoderm (tropho-ectoderrn) of the bilaminar vesicle.
(2) The formative region, unlike the non-formative, is constituted by cells of two varieties, viz. : (i) a more numerous series of larger, lighter-staining' cells destined to form the embryonal ectoderm, and (ii) a less numerous series of smaller, more granular, and more deeply staining cells, destined to give origin to the entoderm and hence distinguishable as the entodermal mother-cells.
(3) The entodermal mother-cells, either without or subsequently to division, bodily migrate inwards from amongst the larger cells of the unilamiuar wall and so come to lie in contact with the inner surface of the latter. Tkey thus give origin to the primitive entodermal cells from which the deKnitive entoderm arises. The larger passive cells, which alone form the unilamiuar wall after the inward migration of the entodermal cells is completed, constitute the embryonal ectoderm.
(4) The entodermal cells as well before as after their migration from the unilamiuar wall are capable of exhibiting amoeboid activity and of emitting pseudopodial processes, by tlie anastomosing of which there is eventually formed a cellular entodermal reticulum underlying, and at first coextensive with, the embryonal ectoderm.
(5) The definitive entoderm thus owes its character as a connected cell-layer primarily to the formation of secondaiy anastomoses between the pseudopodial processes emitted by the primitive entodermal cells (or entodermal mothercells).
(6) The assumption by the entodermal cells of amoeboid j^roperties whilst they are still constituents of the unilaminar wall affords an intelligible explanation of the mechaiiisin of their inwai'd migration.
(7) The entoderm is first laid down below the formative or embryonal region of the blastocyst; thence it extends gradually by its own growth round the inner surface of the uuilaramar non-forrnative region so as to form eventually a complete entodermal lining to the blastocyst cavity. In this way the blastocyst wall becomes bilaminar throughout.
(8) The bilaminar blastocyst consists of two reguous, respectively embryonal and extra-embryonal. The embryonal region (embryonal area) is constituted by an outer layer of embryonal ectoderm and the underlying portion of the entoderm, and the extra-embryonal, of the extra-embrvonal or trophoblastic ectoderm (tropho-ectoderm), which is separated from the embryonal by a well-marked junctional line, together with the underlying portion of the entoderm, which is perfectly continuous with that below the embryonal ectoderm.
(9) The formative or embryonal region of the blastocyst in Dasyurus is from the first freely exposed, and at no time daring the developmental period dealt with in this paper does there exist any cellular layer externally to it, i. e. a covering layer of trophoblast (Deckschicht, Kauber's layer) is absent and there is no entypy of the primary germ-layers (cf. p. 111).
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Contents: 1 Review of Previous Observations | 2 The Ovum of Dasyurus | 3 Cleavage and Blastocyst | 4 Blastocyst Growth Ectoderm Entoderm | 5 Early Stages of Perameles and Macropus | 6 Summary and Conclusions | 7 Early Mammalia Ontogeny | Explanation of Plates
Cite this page: Hill, M.A. (2024, April 26) Embryology Paper - Contributions to the embryology of the marsupialia 4-4. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Paper_-_Contributions_to_the_embryology_of_the_marsupialia_4-4
- © Dr Mark Hill 2024, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G
