Chapter V: Introduction: 1 (4)

What I have called the second stage corresponds to a period in which no trace of the embryo is to be seen. In the third stage the embryonic end of the blastoderm projects outwards to form a structure which I shall speak of as the "embryonic rim," and in the fourth and last stage a distinct medullary groove is formed. For a considerable period during the second stage the segmentation cavity remains of about the same size; during the third stage it begins to be encroached upon, and becomes smaller both absolutely, and relatively to the increased size of the germ.

The segmentation cavity of the Dog-fish most nearly agrees with that of Osseous fishes in its mode of formation and relation to the embryo.

Dog-fish resemble Osseous fish in the fact that their embryos are entirely formed from a portion of the germ which does not form part of the roof of the segmentation cavity, so that the cells forming the roof of the segmentation cavity take _no share_ at any time in the formation of their embryos. They further agree with Osseous fish (always supposing that the descriptions of Oellacher, _loc. cit._, and Götte, _Archiv. für Micr. Anat._ Bd. IX. are correct) in the floor of the segmentation cavity being formed at one period by yolk. Together with these points of similarity there are some important differences.

(1) The segmentation cavity in the Osseous fish from the first arises as a cavity between the yolk and the blastoderm, and its floor is never at any period covered with cells. In the Dog-fish, as we have said above, both in the earlier and later periods the floor is covered with cells.

(2) The roof in the Dog-fish is _invariably_ formed by the epiblast and a row of flattened lower layer cells.

According to both Götte and Oellacher the roof of the segmentation cavity in Osseous fishes is in the earlier stages formed _alone_ of the two layers which correspond with the single layer forming the epiblast in the Dog-fish. In Osseous fishes it is very difficult to distinguish the various layers, owing to the similarity of their component cells. In Dog-fish this is very easy, owing to the great distinctness of the epiblast, and it appears to me, on this account, very probable that the two above-named observers may be in error as to the constitution of its roof in the Osseous fish. With both the Bird and the Frog the segmentation cavity of the Dog-fish has some points of agreement, and some points of difference, but it would take me too far from my present subject to discuss them.

When the segmentation cavity is first formed, no great changes have taken place in the cells forming the blastoderm. The upper layer--the _epiblast_--is composed of a single layer of columnar cells, and the remainder of the cells of blastoderm, forming the lower layer, are of a fairly uniform size, and polygonal from mutual pressure. The whole edge of the blastoderm is thickened, but this thickening is especially marked at its embryonic end.

This thickened edge of the blastoderm is still more conspicuous in the next and second stage (Pl. 3, fig. 3).

In the second stage the chief points of progress, in addition to the increased thickness of the edge of the blastoderm, are--

(1) The increased thickness and distinctness of the epiblast, caused by its cells becoming more columnar, though it remains as a one-cell-thick layer.

(2) The disappearance of the cells from the floor of the segmentation cavity.

The lower layer cells have undergone no important changes, and the blastoderm has increased very little if at all in size.

From Pl. 3, fig. 3, it is seen that there is a far larger collection of cells at the embryonic than at the opposite end.

Passing over some rather unimportant stages, I will come to the next important one.

The general features of this (the third) stage in a surface view are--

(1) The increase in size of the blastoderm.

(2) The diminution in size of the segmentation cavity, both relatively and absolutely.

(3) The appearance of a portion of the blastoderm projecting beyond the rest over the yolk. This projecting rim extends for nearly half the circumference of the yolk, but is most marked at the point where the embryo will shortly appear. I will call it the "embryonic rim."

These points are still better seen from sections than from surface views, and will be gathered at once from an inspection of Pl. 3, fig. 4.

The epiblast has become still more columnar, and is markedly thicker in the region where the embryo will appear. But its most remarkable feature is that at the outer edge of the "embryonic rim" (_er_) it turns round and becomes continuous with the lower layer cells. This feature is most important, and involves some peculiar modifications in the development. I will, however, reserve a discussion of its meaning till the next stage.

The only other important feature of this stage is the appearance of a layer of cells on the floor of the segmentation cavity.

Does this layer come from an ingrowth from the thickened edge of the blastoderm, or does it arise from the formation of new cells in the yolk?

It is almost impossible to answer this question with certainty. The following facts, however, make me believe that the newly formed cells do play an important part in the formation of this layer.

(1) The presence at an earlier date of almost a row of nuclei under the floor of the segmentation cavity (Pl. 3, fig. 1).

(2) The presence on the floor of the cavity of such large cells as those represented in fig. 1, _bd_, cells which are very different, as far as the size and granules are concerned, from the remainder of the cells of the blastoderm.

On the other hand, from this as well as other sections, I have satisfied myself that there is a distinct ingrowth of cells from the embryonic swelling. It is therefore most probable that both these processes, viz. a fresh formation and an ingrowth, have a share in the formation of the layer of cells on the floor of the segmentation cavity.

In the next stage we find the embryo rising up as a distinct body from the blastoderm, and I shall in future speak of the body, which now becomes distinct as the embryo. It corresponds with what Kupffer (_loc. cit._) in his paper on the "Osseous Fishes" has called the "embryonic keel." This starting-point for speaking of the embryo as a distinct body is purely arbitrary and one merely of convenience. If I wished to fix more correctly upon a period which could be spoken of as marking the commencing formation of the embryo, I should select the time when structures first appear to mark out the portion of the germ from which the embryo becomes formed; this period would be in the Elasmobranchii, as in the Osseous fish, at the termination of segmentation, when the want of symmetry between the embryonic end of the germ and the opposite end first appears.

I described in the last stage the appearance of the "embryonic rim." It is in the middle point of this, where it projects most, that the formation of the embryo takes place. There appear two parallel folds extending from the edge of the blastoderm towards the centre, and cut off at their central end by another transverse fold. These three folds raise up, between them, a flat broadish ridge, "_the embryo_" (Pl. 3, fig. 5). The head end of the embryo is the end nearest the centre of the blastoderm, the tail end being the one formed by its (the blastoderm's) edge.

Almost from its first appearance this ridge acquires a shallow groove--the medullary groove (Pl. 3, fig. 5, _mg_)--along its middle line, where the epiblast and hypoblast are in absolute contact (vide fig. 6_a_, 7_a_, 7_b_, &c.) and where the mesoblast (which is already formed by this stage) is totally absent. This groove ends abruptly a little before the front end of the embryo, and is deepest in the middle and wide and shallow behind.

On each side of it is a plate of mesoblast equivalent to the combined vertebral and lateral plates of the Chick. These, though they cannot be considered as entirely the cause of the medullary groove, may perhaps help to make it deeper. In the parts of the germ outside the embryo the mesoblast is again totally absent, or, more correctly, we might say that outside the embryo the _lower layer cells_ do not become differentiated into hypoblast and mesoblast, and remain continuous only with the lower of the two layers into which the _lower layer cells_ become differentiated in the body of embryo. This state of things is not really very different from what we find in the Chick. Here outside the embryo (_i.e._ in the opaque area) there is a layer of cells in which no differentiation into hypoblast and mesoblast takes place, but the layer remains continuous rather with the hypoblast than the mesoblast.

There is one peculiarity in the formation of the mesoblast which I wish to call attention to, _i.e._ its formation as two lateral masses, one on each side of the middle line, but not continuous across this line (vide figs. 6_a_ and 6_b_, and 7_a_ and 7_b_). Whether this remarkable condition is the most primitive, _i.e._ whether, when in the stage before this the mesoblast is first formed, it is only on each side of the middle line that the differentiation of the lower layer cells into hypoblast and mesoblast takes place, I do not certainly know, but it is undoubtedly a very early condition of the mesoblast. The condition of the mesoblast as two plates, one on each side of the neural canal, is precisely similar to its embryonic condition in many of the Vermes, _e.g. Euaxes_ and _Lumbricus_. In these there are two plates of mesoblast, one on each side of the nervous cord, which are known as the _Germinal streaks_ (Keimstreifen) (vide Kowalevsky "Würmern u. Arthropoden"; _Mém. de l'Acad. Imp. St Petersbourg_, 1871).

From longitudinal sections I have found that the segmentation cavity has ceased by this stage to have any distinct existence, but that the whole space between the epiblast and the yolk is filled up with a mass of elongated cells, which probably are solely concerned in the formation of the vascular system. The thickened posterior edge of the blastoderm is still visible.

At the embryonic end of the blastoderm, as I pointed out in an earlier stage, the epiblast and the lower layer cells are perfectly continuous.

Where they join the epiblast, the _lower layer cells_ become distinctly divided, and this division commenced even in the earlier stage, into two layers; a lower one, more directly continuous with the epiblast, consisting of cells somewhat resembling the epiblast-cells, and an upper one of more flattened cells (Pl. 3, fig. 4, _m_). The first of these forms the hypoblast, and the latter the mesoblast. They are indicated by _hy_ and _m_ in the figures. The hypoblast, as I said before, remains continuous with the whole of the rest of lower layer cells of the blastoderm (vide fig. 7_b_). This division into hypoblast and mesoblast commences at the earlier stage, but becomes much more marked during this one.

In describing the formation of the hypoblast and mesoblast in this way I have assumed that they are formed out of the large mass of lower layer cells which underlie the epiblast at the embryonic end of the blastoderm. But there is another and, in some ways, rather a tempting view, viz. to suppose that the epiblast, where it becomes continuous with the hypoblast, in reality becomes involuted, and that from this involuted epiblast are formed the whole mesoblast and hypoblast.

In this case we would be compelled to suppose that the mass of lower layer cells which forms the embryonic swelling is used as food for the growth of the involuted epiblast, or else employed solely in the growth over the yolk of the non-embryonic portion of the blastoderm; but the latter possibility does not seem compatible with my sections.

I do not believe that it is possible, from the examination of sections alone, to decide which of these two views (viz. whether the epiblast is involuted, or whether it becomes merely continuous with the lower layer cells) is the true one. The question must be decided from other considerations.

The following ones have induced me to take the view that there is no involution, but that the mesoblast and hypoblast are formed from the lower layer cells.

(1) That it would be rather surprising to find the mass of lower layer cells which forms the "embryo swelling" playing no part in the formation of embryo.

(2) That the view that it is the lower layer cells from which the hypoblast and mesoblast are derived agrees with the mode of formation of these two layers in the Bird, and also in the Frog; since although, in the latter animal, there is an involution, this is not of the epiblast, but of the larger cells of the lower pole of the yolk, which in part correspond with what I have called the lower layer cells in the Dog-fish.

If the view be accepted that it is from the lower layer cells that the hypoblast and mesoblast are formed, it becomes necessary to explain what the continuity of the hypoblast with the epiblast means.

The explanation of this is, I believe, the keystone to the whole position. The vertebrates may be divided as to their early development into two classes, viz. those with _holoblastic ova_, in which the digestive canal is formed by an _involution_ with the presence of an "_anus of Rusconi_."

This class includes "Amphioxus," the "Lamprey," the "Sturgeon," and "Batrachians."

The second class are those with _meroblastic ova_ and no _anus of Rusconi_, and with an alimentary canal formed by the infolding of the sheet of hypoblast, the digestive canal remaining in communication with the food-yolk for the greater part of embryonic life by an umbilical canal.

This class includes the "Elasmobranchii," "Osseous fish," "Reptiles," and "Aves."

The mode of formation of the alimentary canal in the first class is clearly the more primitive; and it is equally clear that its mode of formation in the second class is an adaptation due to the presence of the large quantity of food-yolk.

In the Dog-fish I believe that we can see, to a certain extent, how the change from the one to the other of these modes of development of the alimentary canal took place.

In all the members of the first class, viz. "_Amphioxus_," the "Lamprey," the "Sturgeon," and the "Batrachians," the epiblast becomes continuous with the hypoblast at the so-called "anus of Rusconi," and the alimentary canal, potentially in all and actually in the Sturgeon (vide Kowalevsky, Owsjannikow, and Wagner, _Bulletin der Acad. d. St Petersbourg_, Vol. XIV. 1870, "Entwicklung der Störe"), communicates freely at its extreme hind end with the neural canal. The same is the case in the Dog-fish. In these, when the folding in to form the alimentary canal on the one hand, and the neural on the other, takes place, the two foldings unite at the corner, where the epiblast and hypoblast are in continuity, and place the two tubes, the neural and alimentary, in free communication with each other[14].

Footnote 14: This has been already made out by Kowalevsky,
"Würmern u. Arthropoden," _loc. cit._

There is, however, nothing corresponding with the "anus of Rusconi," which merely indicates the position of the involution of the digestive canal, and subsequently completely closes up, though it nearly coincides in position with the true anus in the Batrachians, &c.

This remarkable point of similarity between the Dog-fish's development and the normal mode of development in the first class (the holoblastic) of vertebrates, renders it quite clear that the continuity of the epiblast and hypoblast in the Dogfish is really the remnant of a more primitive condition, when the alimentary canal was formed by an involution. Besides the continuity between neural and alimentary canals, we have other remnants of the primitive involution. Amongst these the most marked is the formation of the embryonic rim, which is nothing less than the commencement of an involution. Its form is due to the flattened, sheet-like condition of the germ. In the mode in which the alimentary canal is closed in front I shall shew there are indications of the primitive mode of formation of the alimentary canal; and in certain peculiarities of the anus, which I shall speak of later, we have indications of the primitive anus of Rusconi; and finally, in the general growth of the epiblast (small cells of the upper pole of the Batrachian egg) over the yolk (lower pole of the Batrachian egg), we have an example of the manner in which the primitive involution, to form the alimentary canal, invariably disappears when the quantity of yolk in an egg becomes very great.

I believe that in the Dog-fish we have before our eyes one of the steps by which a direct mode of formation comes to be substituted for an _in_direct one by involution. We find, in fact, in the Dog-fish, that the cells from which are derived the mesoblast and hypoblast come to occupy their final position in the primitive arrangement of the cells during segmentation, and not by a subsequent and secondary involution.

This change in the mode of formation of the alimentary canal is clearly a result of change of mechanical conditions from the presence of the large food-yolk.

Excellent parallels to it will be found amongst the Mollusca. In this class the presence or absence of food-yolk produces not very dissimilar changes to those which are produced amongst vertebrates from the same cause.

The continuity of the hypoblast and epiblast at the embryonic rim is a remnant which, having no meaning or function, except in reference to the earlier mode of development, is likely to become lost, and in Birds no trace of it is any longer to be found.

I will not in the present preliminary paper attempt hypothetically to trace the steps by which the involution gradually disappeared, though I do not think it would be very difficult to do so. Nor will I attempt to discuss the question whether the condition with a large amount of food-yolk (as seems more probable) was twice acquired--once by the Elasmobranchii and Osseous fishes, and once by Reptiles and Birds--or whether only once, the Reptiles and Birds being lineal descendants of the Dog-fish.

In reference to the former point, however, I may mention that the Batrachians and Lampreys are to a certain extent intermediate in condition between the _Amphioxus_ and the Dog-fishes, since in them the yolk becomes divided during segmentation into lower layer cells and epiblast, but a modified involution is still retained, while the Dog-fish may be looked upon as intermediate between Birds and Batrachians, the continuity at the hind end between the epiblast and hypoblast being retained by them, though not the involution.

It may be convenient here to call attention to some of the similarities and some of the differences which I have not yet spoken of between the development of Osseous fish and the Dog-fish in the early stages. The points of similarity are--(1) The swollen edge of the blastoderm. (2) The embryo-swelling. (3) The embryo-keel. (4) The spreading of the blastoderm over the yolk-sac from a point corresponding with the position of the embryo, and not with the centre of the germ. The growth is almost nothing at that point, and most rapid at the opposite pole of the blastoderm, being less and less rapid along points of the circumference in proportion to their proximity to the embryonic swelling. (5) The medullary groove.

In external appearance the early embryos of Dog-fish and Teleostei are very similar; some of my drawings could almost be substituted for those given by Oellacher. This similarity is especially marked at the first appearance of the medullary groove. In the Dog-fish the medullary groove becomes converted into the medullary canal in the same way as in Birds and all other vertebrates, except Osseous fishes, where it comes to nothing, and is, in fact, a rudimentary structure. But in spite of Oellacher's assertions to the contrary, I am convinced from the similarity of its position and appearance to the true medullary groove in the Dog-fish, that the groove which appears in Osseous fishes is the true medullary groove; although Oellacher and Kuppfer appear to have conclusively proved that it does not become converted into the medullary canal. The chief difference between the Dog-fish and Osseous fish, in addition to the point of difference about the medullary groove, is that the epiblast is in the Dog-fish a single layer, and not divided into nervous and epidermic layers as in Osseous fish, and this difference is the more important, since, throughout the whole period of development till after the commencement of the formation of the neural canal, the epiblast remains in Dog-fish as a one-cell-deep layer of cells, and thus the possibility is excluded of any concealed division into a neural and epidermic layer, as has been supposed to be the case by Stricker and others in Birds.

_Development of the Embryo._

After the embryo has become definitely established, for some time it grows rapidly in length, without externally undergoing other important changes, with the exception of the appearance of two swellings, one on each side of its tail.

These swellings, which I will call the _Caudal lobes_ (figs. 8 and 9, _ts_), are also found in Osseous fishes, and have been called by Oellacher the _Embryonal saum_. They are caused by a thickening of mesoblast on each side of the hind end of the embryo, at the edge of the embryonic rim, and form a very conspicuous feature throughout the early stages of the development of the Dog-fish, and are still more marked in the Torpedo (Pl. 3, fig. 9). Although from the surface the other changes which are visible are very insignificant, sections shew that the _notochord_ is commencing to be formed.

I pointed out that beneath the medullary groove the epiblast and hypoblast were not separated by any interposed mesoblast. Along the line (where the mesoblast is deficient) which forms the long axis of the embryo, a rod-like thickening of the hypoblast appears (Pl. 3, figs. 7_a_ and 7_b_, _ch_ and _ch´_), first at the head end of the embryo, and gradually extends backwards. This is the rudiment of the notochord; it remains attached for some time to the hypoblast, and becomes separated from it first at the head end of the embryo, and the separation is then carried backwards. This thickening of the hypoblast projects up and comes in contact with the epiblast, and in the later stages with bad (especially chromic-acid) specimens the line of separation between the epiblast and the thickening may become a little obscured, and might possibly lead to the supposition that a structure similar to that which has been called the "_axis cord_" was present. In all my best (osmic-acid) specimens the line of junction is quite clear; and any one who is aware how easily two separate masses of cells may be made indistinguishably to fuse together from simple pressure will not be surprised to find the occasional obscurity of the line of junction between the epiblast and hypoblast. In the earlier stage of the thickening there is never in the osmic-acid preparations any appearance of fusion except in very badly prepared ones. Its mode of formation will be quite clear without further description from an inspection of Pl. 3, figs. 7_a_ and 7_b_, _ch_ and _ch´_. Both are taken from one embryo. In fig. 7_b_, the most anterior of the two, the notochord has become quite separated from the hypoblast. In fig. 7_a_, _ch_, there is only a very marked thickening of hypoblast, which reaches up to the epiblast, but the thickening is still attached to the hypoblast. Had I had space to insert a drawing of a third section of the same embryo there would only have been a slight thickening of the hypoblast. In the earlier stage it will be seen, by referring to figs. 6_a_ and 6_b_, that there is no sign of a thickening of the hypoblast. My numerous sections (all made from embryos hardened in osmic acid) shewing these points are so clear that I do not think there can be any doubt whatever of the notochord being formed as a thickening of the hypoblast. Two interpretations of this seem possible.

I mentioned that the mesoblast appeared to be primitively formed as two independent sheets, _split off, so to speak, from the hypoblast_, one on each side of the middle line of the embryo. If we looked upon the notochord as a third _median sheet of mesoblast_, split off from the hypoblast somewhat later than the other two, we should avoid having to admit its hypoblastic origin.

Professor Huxley, to whom I have shewn my specimens, strongly advocates this view.

The other possibility is that the notochord is primitively a true _hypoblastic_ structure which has only by adaptation become an apparently _mesoblastic_ one in the higher vertebrates. In favour of this view are the following considerations:

(1) That this is the undoubtedly natural interpretation of the sections. (2) That the notochord becomes separated from the hypoblast after the latter has acquired its typical structure, and differs in that respect from the two lateral sheets of mesoblast, which are formed coincidently with the hypoblast by a homogeneous mass of cells becoming differentiated into two distinct layers. (3) That the first mode of looking at the matter really proves too much, since it is clear that by the same method of reasoning we could prove the mesoblastic origin of any organ derived from the hypoblast and budded off into the mesoblast. We would merely have to assert that it was really a mass of mesoblast budded off from the hypoblast rather later than the remainder of the mesoblast. Still, it must be admitted that the first view I have suggested is a possible, not to say a probable one, though the mode of arguing by which it can be upheld may be rather dangerous if generally applied. We ought not, however, for that reason necessarily to reject it in the present case. As Mr Ray Lankester pointed out to me, if we accept the hypoblastic origin of the notochord, we should find a partial parallel to it in the endostyle of Tunicates, and it is perhaps interesting to note in reference to it that the notochord is the only _unsegmented_ portion of the axial skeleton.

Whether the strong _à priori_ difficulties of the hypoblastic origin of the notochord are sufficient to counterbalance the natural interpretation of my sections, cannot, I think, be decided from the single case of the Dog-fish. It is to be hoped that more complete investigations of the Lamprey, &c., may throw further light upon the question.

Whichever view of the primitive origin of the notochord is the true one, its apparent origin is very instructive as illustrating the possible way in which an organ might come to change the layer to which it primarily belonged.

If the notochord is a true mesoblastic structure, it is easy to be seen how, by becoming separated from the hypoblast a little later than is the case with the Dog-fish, its mesoblastic origin would become lost; while if, on the other hand, it is primitively a hypoblastic structure, we see from higher vertebrates how, by becoming separated from the hypoblast rather earlier than in the Dog-fish, viz. at the same time as the rest of the mesoblast, its primitive derivation from the hypoblast has become concealed.

The view seemingly held by many embryologists of the present day, that an organ, when it was primitively derived from one layer, can never be apparently formed in another layer, appears to me both unreasonable on _à priori_ grounds, and also unsupported by facts.

I see no reason for doubting that the embryo in the earliest periods of development is as subject to the laws of natural selection as is the animal at any other period. Indeed, there appear to me grounds for the thinking that it is more so. The remarkable differences in allied species as to the amount of food-yolk, which always entail corresponding alterations in the development--the different modes of segmentation in allied species, such as are found in the Amphipoda and Isopoda--the suppression of many stages in freshwater species, which are retained in the allied marine species--are all instances of modifications due to natural selection affecting the earliest stages of development. If such points as these can be affected by natural selection I see no reason why the arrangement of individual cells (or rather primitive elements) should not also be modified; why, in fact, a mass of cells which was originally derived from one layer, but in the course of development became budded off from that layer and entered another layer, should not by a series of small steps cease ever to be attached to the original layer, but from the first moment it can be distinguished should be found as a separate mass in the second layer.

The change of layers will, of course, only take place where some economy is effected by it. The variations in the mode of development of the nervous system may probably be explained in this way.

If we admit that organs can undergo changes, as to the primitive layer from which they are derived, important consequences must follow.

It will, for instance, by no means be sufficient evidence of two organs not being homologous that they are not developed from the same layer. It renders the task of tracing out the homologies from development much more difficult than if the ordinary view of the invariable correspondence of the three layers throughout the animal kingdom be accepted. Although I do not believe that this correspondence is invariable or exact, I think that we both find and should expect to find that it is, roughly speaking, fairly so.

Thus, the muscles, internal skeleton, and connective tissue are always placed in the adult between the skin (epidermis) and the epithelium of the alimentary canal.

We should therefore expect to find them, and, as a matter of fact, we always do find them, developed from a middle layer when this is present.

The upper layer must always and does always form the epidermis, and similarly the lower layer or hypoblast must form a part of the epithelium of the alimentary canal. A full discussion of this question would, however, lead me too far away from my present subject.

The only other point of interest which I can touch on in this stage is the commencing closure of the alimentary canal in the region of the head. This is shewn in Pl. 3, figs. 6_a_, 6_b_, 7_b_, _n.a_. From these figures it can be seen that the closing does not take place as much by an infolding as by an ingrowth from the side walls of the alimentary canal towards the middle line. In this abnormal mode of closing of the alimentary canal we have again, I believe, an intermediate stage between the mode of formation of the alimentary canal in the Frog and the typical folding in which occurs in Birds. There is, however, another point in reference to it which is still more interesting. The cells to form the ingrowth from the bottom (ventral) wall of the alimentary canal are derived by a continuous fresh formation from the yolk, being formed around the nuclei spoken of above (vide p. 63 et seq.). All my sections shew this with more or less clearness, especially those a little later than fig. 6_b_, in which the lower wall of the alimentary canal is nearly completed. This is the more interesting since, from the mode of formation of the alimentary canal in the Batrachians, &c., we might expect that the cells from the yolk would take a share in its formation in the Dog-fish. I have not as yet made out for certain the share which is taken by these freshly formed cells of the yolk in the formation of any other organ.

By the completion of its lower wall in the way described, the throat early becomes a closed tube, its closing taking place before any other important changes are visible in the embryo from surface views.

A considerable increase in length is attained before other changes than an increase in depth of the medullary groove and a more complete folding off of the embryo from the blastoderm take place. The first important change is the formation of the protovertebræ.

These are formed by the lateral plates of mesoblast, which I said were equivalent at once to the vertebral and lateral plates in the Bird, becoming split by transverse divisions into cubical masses.

At the time when this occurs, and, indeed, up till a considerably later period, the mesoblast is not split into somatopleure and splanchnopleure, and it is not divided into vertebral and lateral plates. The transverse lines of division of the protovertebræ do not, however, extend to the outer edge of the undivided lateral plates.

The differences between this mode of formation of the protovertebræ and that occurring in Birds are too obvious to require pointing out. I will speak of them more fully when I have given the whole history of the protovertebræ of the Dog-fish.

I will only now say that I have had in the early stages to investigate the formation of the protovertebræ entirely by means of sections, the objects being too opaque to be otherwise studied.

The next change of any importance is the commencement of the formation of the head. The region of the head first becomes distinguishable by the flattening out of the germ at its front end.

The flattened-out portion of the germ grows rapidly, and forms a spatula-like termination to the embryo (Pl. 3, fig. 8).

In the region of the head the medullary groove is at first totally absent (vide section, Pl. 3, fig. 8_a_).

Indeed, as can be seen from fig. 8_b_, the laminæ dorsales, so far from bending up at this stage, actually bend down in the opposite direction.

I am at present quite unable even to form a guess what this peculiar feature of the brain means. It, no doubt, has some meaning in reference to the vertebrate ancestry if we could only discover it. The peculiar spatula-like flattened condition of the head is also (vide _loc. ant. cit._) apparently found in the Sturgeons; it must therefore almost undoubtedly be looked upon as not merely an accidental peculiarity.

While these changes have been taking place in the head not less important changes have occurred in the remainder of the body. In the first place the two caudal lobes have increased in size, and have become, as it were, pushed in together, leaving a groove between them (fig. 8, _ts_). They are very conspicuous objects, and, together with the spatula-like head, give the whole embryo an almost comical appearance. The medullary canal has by this time become completely closed in the region of the tail (figs. 8 and 8_b_).

It is still widely open in the region of the back, and, though more nearly closed again in the neck, is, as I have said, flattened out to nothing in the head.

The groove[15] between the two caudal lobes must not be confused (as may easily be done) with the medullary groove, which by the time the former groove has become conspicuous is a completely closed canal.

Footnote 15: This groove is the only structure which it seems
possible to compare with the so-called "primitive groove" of
Birds. It is, however, doubtful whether they are really
homologous.

The vertebral plates are not divided (vide fig. 7) into a somatopleuric and splanchnopleuric layer by this stage, except in the region of the head (vide fig. 8_b_, _pp_), where there is a distinct space between the two layers, which is undoubtedly homologous with the pleuro-peritoneal cavity of the hinder portion of the body.

It is probably the same cavity which Oellacher (_loc. cit._) calls in Osseous fishes the pericardial cavity. In the Dog-fish, at least, it has no connection with the pericardium. Of its subsequent history I shall say a few words when I come to speak of the later stages.

The embryo does not take more than twenty-four hours in passing from this stage, when the head is a flat plate, to the stage when the whole neural canal (including the region of the head) is closed in. The other changes, in addition to the closing in of the neural canal, are therefore somewhat insignificant. The folding off of the embryo from the germ has, however, progressed considerably, and a portion of the hind gut is closed in below. This is accomplished, not by a tail-fold, as in Birds, but by two lateral folds, which cause the sides of the body to meet and coalesce below. At the extreme hind end, where the epiblast is continuous with the hypoblast, the lateral folds turn round, so to speak, and become continuous with the medullary folds, so that when the various folds meet each other an uninterrupted canal is found passing round from the neural into the alimentary canal, and placing these two in communication at the tail end of the body. Since I have already mentioned this, and spoken of its significance, I will not dwell on it further here.

The cranial flexure commences coincidently with the closing in of the neural canal in the region of the brain, and the division into fore, mid, and hind brain becomes visible at the same time as or even before the closing of the canal occurs. The embryo has now become more or less transparent, and protovertebræ, of which about twenty are present, can _now_ be seen in the fresh specimens. The heart, however, is not yet formed.

Up to this period, a period at which the embryo becomes very similar in external appearance to any other vertebrate embryo, I have followed in my description a chronological order. I shall now cease to do so, since it would be too long for a preliminary notice of this kind, but shall confine myself to the history of a few organs whose development is either more important or more peculiar than that of the others.

_The Protovertebræ._

I have thought it worth while to give a short history of the development of the protovertebræ, firstly, because it is very easy to follow this in the Dog-fish, and, secondly, because I believe that the Dog-fish have more nearly retained the primitive condition of the protovertebræ than any other vertebrate whose embryology has hitherto been described with sufficient detail.

I intend to describe, at the same time, the development of the spinal nerves.

I left each lateral mass of mesoblast in my last stage as a plate which had not yet become split into a somatic and a splanchnic sheet (Pl. 3, fig. 8_a_, _vp_), but which had become cut by transverse lines (not, indeed, extending to the outer limit of the sheet, but as yet not cut off by longitudinal lines of cleavage) into segments, which I called protovertebræ.

This sheet of mesoblast is fairly thick at its proximal (upper) end, but thins off laterally to a sheet two cells deep, and its cells are so arranged as to foreshadow its subsequent splitting into somatic and splanchnic sheets. Its upper (proximal) end is at this stage level with the bottom of the neural canal, but soon begins to grow upwards, and at the same time the splitting into somatopleure and splanchnopleure commences (Pl. 3, fig. 10, _so_ and _sp_).

The separation between the two sheets is first visible in its uppermost part, and thence extends outwards. By this means each of the protovertebræ becomes divided into two sheets, which are only connected at their upper ends and outside the region of the body. I speak of the whole lateral sheet as being composed of protovertebræ, because at this time no separation into vertebral and lateral plates can be seen; but I may anticipate matters by saying that only the upper portion of the sheet from the level of the top of the digestive canal, becomes subsequently the true protovertebræ. From this it is clear that the pleuro-peritoneal cavity extends primitively quite up to the top of the protovertebræ; and that thus a portion of a sheet of mesoblast, at first perfectly continuous with the splanchnic sheet from which is derived the muscular wall of the alimentary canal, is converted into a part of the voluntary muscular system of the body, having no connection whatever with the involuntary muscular system of the digestive tract.

The pleuro-peritoneal cavity is first distinctly formed at a time when only two visceral clefts are present. Before the appearance of a third visceral cleft in a part of the innermost layer of each protovertebræ (which may be called the splanchnic layer, from its being continuous with the mesoblast of the splanchnopleure), opposite the bottom of the neural tube, some of the cells commence to become distinguishable from the rest, and to form a separate mass. This mass becomes much more distinct a little later, its cells being characterised by being spindle-shaped, and having an elongated nucleus which becomes deeply stained by reagents (Pl. 4, fig. 11, _mp´_). Coincidently with its appearance the young Dog-fish commences spontaneously to move rapidly from side to side with a kind of serpentine motion, so that, even if I had not traced the development of this differentiated mass of cells till it becomes a band of muscles close to the notochord, I should have had little doubt of its muscular nature. It is indicated in figs. 11, 12, 13, by the letters _mp´_. Its early appearance is most probably to be looked upon as an adaptation consequent upon the respiratory requirements of the young Dog-fish necessitating movements within the egg.

Shortly after this date, at a period when three visceral clefts are present, I have detected the first traces of the spinal nerves.

At this time they appear in sections as small elliptical masses of cells, entirely independent of the protovertebræ, and closely applied to the upper and outer corners of the involuted epiblast of the neural canal (Pl. 4, fig. 11, _spn_). These bodies are far removed from any mesoblastic structures, and at the same time the cells composing them are _not_ similar to the cells composing the walls of the neural canal, and are not attached to these, though lying in contact with them. I have not, therefore, sufficient evidence at present to enable me to say with any certainty where the spinal nerves are derived from in the Dog-fish. They may be derived from the involuted epiblast of the neural canal, and, indeed, this is the most natural interpretation of their position.

On the other hand, it is possible that they are formed from wandering cells of the mesoblast--a possibility which, with our present knowledge of wandering cells, must not be thrown aside as altogether improbable.

In any case, it is clear that the condition in the Bird, where the spinal nerves are derived from tissue of the protovertebræ, is not the primitive one. Of this, however, I will speak again when I have concluded my account of the development of the protovertebræ.

About the same time that the first rudiments of the nerves appear, the division of the mesoblast of the sides of the body into a vertebral and a lateral portion occurs. This division first appears in the region where the oviduct (Müller's duct) is formed (Pl. 4, fig. 11, _ov_).

At this part opposite the level of the dorsal aorta the two sheets, viz. the splanchnic and the somatic, unite together, and thus each lateral sheet of mesoblast becomes divided into an upper portion (fig. 11, _mp_), split up by transverse partitions into protovertebræ, and a lower portion not so split, but consisting of an outer layer, the true somatopleure, and an inner layer, the true splanchnopleure. These two divisions of the primitive plate are thus separated by the line at which a fusion between the mesoblast of the somatopleure and splanchnopleure takes place. The mass of cells resulting from the fusion at this point corresponds with the intermediate cell-mass of Birds (vide Waldeyer, _Eierstock und Ei_).

At the same time, in the upper of these two sheets (the protovertebræ), the splanchnic layer sends a growth of cells inwards towards the notochord and the neural canal. This growth is the commencement of the large quantity of mesoblastic tissue around the notochord, which is in part converted into the axial skeleton, and in part into the connective tissue adjoining this.

This mass of cells is at first quite continuous with the splanchnic layer of the protovertebræ, and I see no reason for supposing that it is not derived from the growth of the cells of this layer. The ingrowth to form it first appears a little after the formation of the dorsal aorta; but, as far as I have been able to see, its cells have no connection with the walls of the aorta.

What I have said as to the development of the skeleton-forming layer will be quite clear from figs. 11 and 12_a_; and from these it will also be clear, especially from fig. 11_a_, that the outermost layer of this mass of cells, which was the primitive splanchnic layer of the protovertebræ, still retains its epithelial character, and so can easily be distinguished from those cells which will form the skeleton. In the next stage which I have figured (fig. 12_a_), this outer portion of the splanchnic layer is completely separated from the skeleton-forming cells, and at the same time, having united below as well as above with the outer (somatic) layer of the two layers of which the protovertebræ are formed, the two together form an independent mass (fig. 12, _mp_), similar in appearance and in every way homologous with the muscle-plate of Birds.

On the inner side of this, which we may now call the muscle-plate, is seen the bundle of earlier-developed muscles (fig. 12, _mp´_) which I spoke of before.

The section represented in fig. 12 is from a very considerably later embryo than that represented in fig. 11, so that the skeleton-forming cells, few in number in the earlier section, have become very numerous in the later one, and have grown up above the neural canal, and also below the notochord, between the digestive canal and the aorta. They have, moreover, changed their character; they were round before, now they have become stellate. As to their further history, it need only be said that the layer of them immediately around the notochord and neural canal forms the cartilaginous centra and arches of the vertebræ, and that the remaining portion of them, which becomes much more insignificant in size as compared with the muscles, forms the connective tissue of the skeleton and of the parts around and between the muscles.

A muscle-plate itself is at this stage (shewn in fig. 12) composed of an inner and an outer layer of columnar cells (splanchnic and somatic) united at the upper and lower ends of the plate, and on the inner of the two lies the more developed mass of muscles before spoken of (_mp´_).

Each of these plates now grows both upwards and downwards; and at the same time connective-tissue cells appear between the plates and epidermis; but from where they come I do not know for certain; very probably they are derived from the somatic layer of the muscle-plate.

While the muscle-plates continue to grow both upwards and downwards, the cells of which they are composed commence to become elongated and soon acquire an unmistakably muscular character (Pl. 4, fig. 13, _mp_).

Before this has occurred the inner mass of muscles has also undergone further development and become a large and conspicuous band of muscles close to the notochord (fig. 13, _mp´_).

At the same time that the muscle-plates acquire the true histological character of muscle, septa of connective tissue grow in and divide them into a number of distinct segments which subsequently form separate bands of muscle. I will not say more in reference to the development of the muscular system than that the whole of the muscles of the body (apart from the limbs, the origin of whose muscular system I have not yet investigated) are derived from the muscle-plates which grow upwards above the neural canal and downwards to the ventral surface of the body.

During the time the muscle-plates have been undergoing these changes the nerve masses have also undergone developmental changes.

They become more elongated and fibrous, their main attachment to the neural tube being still at its posterior (dorsal) surface, near which they first appeared. Later still they become applied closely to the sides of the neural tube and send fibres to it below as well as above. Below (ventral to) the neural tube a ganglion appears, forming only a slight swelling, but containing a number of characteristic nerve-cells. The ganglion is apparently formed just below the junction of the anterior and posterior roots, though probably the fibres of the two roots do not mix till below it.

The main points which deserve notice in the development of the protovertebræ are--

(1) That at the time when the mesoblast becomes split horizontally into somatopleure and splanchnopleure the vertebral and lateral plates are one, and the splitting extends to the very top of the vertebral or muscle-plate, so that the future muscle-plates are divided into a splanchnic and somatic layer, the space between which is at first continuous with the pleuro-peritoneal cavity.

(2) That the following parts are respectively formed by the vertebral and lateral plates:

(_a_) Vertebral plate. From the splanchnic layer of this, or from cells which appear close to and continuous with it, the skeleton, and connective tissue of the upper part of the body, are derived.

The remainder of the plate, consisting of a splanchnic and somatic layer, is entirely converted into the muscles of the trunk, all of which are derived from it.

(_b_) Between the vertebral plate and the lateral plate is a mass of cells where, as I mentioned above, the mesoblast of the somatopleure and splanchnopleure fuse together. This mass of cells is the equivalent of the _intermediate cell_ mass of Birds (vide Waldeyer, _Eierstock und Ei_).

From it are derived the Wolffian bodies and duct, the oviduct, the ovaries and the testis, and the connective tissue of the parts adjoining these.

(_c_) The lateral plate. From the somatic layer of this is derived the connective tissue of the ventral half of the body; the mesoblast of the limbs, including probably the muscles, and certainly the skeleton. From its splanchnic layer are derived the muscles and connective tissue of the alimentary canal.

(3) The spinal nerves are developed independently of the protovertebræ, so that the protovertebræ of the Elasmobranchii do not appear to be of such a complicated structure as the protovertebræ of Birds.

_The Digestive Canal._

I do not intend to enter into the whole history of the digestive canal, but to confine myself to one or two points of interest connected with it. These fall under two heads:

(1) The history of the portion of the digestive canal between the anus and the end of the tail where the digestive canal opens into the neural canal.

(2) Certain less well-known organs derived from the digestive canal.

The anus is a rather late formation, but its position becomes very early marked out by the hypoblast of the digestive canal approaching at that point close to the surface, whilst receding to some little distance from it on either side. The portion of the digestive tract I propose at present dealing with is that between this point, which I will call, for the sake of brevity, the anus and the hind end of the body. This portion of the canal is at first very short; it is elliptical in section, and of rather a larger bore than the remainder of the canal. Its diameter becomes, however, slightly less as it approaches the tail, dilating again somewhat at its extreme end. It is lined by a markedly columnar epithelium. Though at first very short, its length increases with the growth of the tail, but at the same time its calibre continually becomes smaller as compared with the remainder of the alimentary canal.

It commences to become smaller, first of all, near, though not quite, at its extreme hind end, and thus becomes of a conical shape; the base of the cone being just behind the anus, while the apex of the cone is situated a short distance from the hind end of the embryo. The extreme hind end, however, at the same time does not diminish in size, and becomes relatively (if not also absolutely) much larger in diameter than it was at first, as compared with the remainder of the digestive canal. It becomes, in fact, a vesicle or vesicular dilatation at the end of a conical canal.