ADAM SEDGWICK, M.A., (continued 3)

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. 8a).

Indeed, as can be seen from fig. 8b, 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 8b).

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.

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. 8b, 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. 8a, 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 12a; and from these it will also be clear, especially from fig. 11a, 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. 12a), 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[TN1] 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.

Just before the appearance of the external gills this part of the digestive canal commences to atrophy. It begins to do so close to the terminal vesicle, which, however, still remains as or more conspicuous than it was before. The lumen of the canal becomes smaller and smaller, and finally it becomes a solid string of cells, and these also soon become indistinguishable and not a trace of the canal is left.

Almost the whole of it has disappeared before the vesicle begins to atrophy, but very shortly after all trace of the rest of the canal has vanished the terminal vesicle also vanishes. This occurs just about the time or shortly after the appearance of the external gills—there being slight differences probably in this respect in the different species.

In this history there are two points of especial interest:

(1) The terminal vesicle.

(2) The disappearance of a large and well-developed portion of the alimentary canal.

The interest in the terminal vesicle lies in the possibility of its being some rudimentary structure.

In Osseous fishes Kupffer has described the very early appearance of a vesicle near the tail end, which he doubtfully speaks of as the allantois.The figure he gives of it in his earlier paper (Archiv. für Micro. Anat. Vol. II. pl. xxiv, fig. 2) bears a very strong resemblance to my figures of this vesicle at the time when the hind end of the alimentary canal is commencing to disappear; and I feel fairly confident that it is the same structure as I have found in the Dog-fish: but until the relations of the Kupffer's vesicle to the alimentary canal are known, any comparison between it and the terminal vesicle in the Dog-fish must be to a certain extent guess-work.

I have, however, been quite unsuccessful in finding any other vesicular structure which can possibly correspond to the so-called allantoic vesicle of Osseous fish.

The disappearance of a large portion of the alimentary canal behind the anus is very peculiar. In order, however, to understand the whole difficulties of the case I shall be obliged to speak of the relations of the anus of the Dog-fish to the anus of Rusconi in the Lamprey, &c.

In those vertebrates whose alimentary canal is formed by an involution, the anus of Rusconi represents the opening of this involution, and therefore the point where the alimentary canal primitively communicates with the exterior. When, however, the anus of Rusconi becomes closed, the wall of the alimentary canal still remains at that point in close juxtaposition to the surface, and the new and final anus is formed at or close to that point. In the Dog-fish, although the anus of Rusconi is not present, still, during the closing of the alimentary canal, the point which would correspond with this becomes marked out by the alimentary canal there approaching the surface, and it is at this point that the involution to form the true anus subsequently appears.

The anus in the Dog-fish has thus, more than a mere secondary significance. It corresponds with the point of closing of the primitive involution. If it was not for this peculiarity of the vertebrate anus we would naturally suppose, from the disappearance of a considerable portion of the alimentary canal lying behind its present termination, that in the adult the alimentary canal once extended much farther back than at present, and that the anus we now find was only a secondary anus, and not the primitive one. It is perhaps possible that this hinder portion of the alimentary canal is a result of the combined growth of the tail and the persisting continuity (at the end of the body) of the epiblast with the hypoblast.

Whichever view is correct, it may be well to mention, in order to shew that the difficulty about the anus of Rusconi is no mere visionary one, that Götte (Untersuchung über die Entwicklung der Bombinator igneus, Archiv. für Micro. Anat., vol. V. 1869) has also described the disappearance of the hind portion of the alimentary canal in Batrachians, a rudiment (according to him) remaining in the shape of a lymphatic trunk.

It is, perhaps, possible that we have a further remnant of this hind portion of the alimentary canal amongst the higher vertebrates in the allantois.

Organs developed from the Digestive Canal.

In reference to the development of the liver, pancreas, &c., as far as my observations have at present gone, the Dog-fish presents no features of peculiar interest. The liver is developed as in the Bird, and independently of the yolk.

There are, however, two organs derived from the hypoblast which deserve more attention. Immediately under the notochord, and in contact with it (vide Pl. 3, fig. 10; Pl. 4, figs. 11 and 12, x), a small roundish (in section) mass of cells is to be seen in most of the sections.

Its mode of development is shewn in fig. 10, x. That section shows a mass of cells becoming pinched off from the top of the alimentary canal. By this process of pinching off from the alimentary canal a small rod-like body close under the notochord is formed. It persists till after the appearance of the external gills, but later than that I have not hitherto succeeded in finding any trace of it.

It was first seen by Götte (loc. cit.) in the Batrachians, and he gave a correct account of its development, and added that it became the thoracic duct.

I have not myself worked out the later stages in the development of this body with sufficient care to be in a position to judge of the correctness of Götte's statements as to its final fate. If it is true that it becomes the thoracic duct it is very remarkable, and ought to throw some light upon the homologies of the lymphatic system.

Some time before the appearance of the external gills another mass of cells becomes, I believe, constricted off from the part of the alimentary canal in the neighbourhood of the anus, and forms a solid rod composed at first of dark granular cells lying between the Wolffian ducts. I have not followed out its development quite completely, but I have very little doubt that it is really constricted off from a portion of the alimentary canal chiefly in front of the point where the anus appears, but also, I believe, from a small portion behind this.

Though the cells of which it is composed are at first columnar and granular (fig. 12, su, r), they soon begin to become altered, and in the latter stage of its development the body forms a conspicuous rounded mass of cells with clear protoplasm, and each provided with a large nucleus. Later still it becomes divided into a number of separate areas of cells by septa of connective tissue, in which (the septa) capillaries are also present. Since I have not followed it to its condition in the adult, I cannot make any definite statements as to the fate of this body; but I think that it possibly becomes the so-called suprarenal organ, which in the Dog-fish forms a yellowish elongated body lying between the two kidneys.

The development of the Wolffian Duct and Body and of the Oviduct.

The development of the Wolffian duct and the Oviduct in the various classes of vertebrates is at present involved in some obscurity, owing to the very different accounts given by different observers.

The manner of development of these parts in the Dog-fish is different from anything that previous investigators have met with in other classes, but I believe that it gives a clearer insight into the true constitution of these parts than vertebrate embryology has hitherto supplied, and at the same time renders easier the task of understanding the differences in the modes of development in the different classes.

I shall commence with a simple description of the observed facts, and then give my view as to their meaning. At about the time of the appearance of the third visceral cleft, and a short way behind the point up to which the alimentary canal is closed in front, the splanchnopleure and somatopleure fuse together opposite the level of the dorsal aorta.

From the mass of cells formed by this fusion a solid knob rises up towards the epiblast (Pl. 4, fig. 11b, ov), and from this knob a solid rod of cells grows backwards towards the tail (fig. 11c, ov) very closely applied to the epiblast. This description will be rendered clear by referring to figs. 11b and c. Fig. 11b is a section at the level of the knob, and fig. 11c is a section of the same embryo a short way behind this point. So closely does the rod of cells apply itself to the epiblast that it might very easily be supposed to be derived from it. Such, indeed, was at first my view till I cut a section passing through the knob. In order, however, to avoid all possibility of mistake I made sections of a large number of embryos of about the age at which this appears, and invariably found the large knob in front, and from it the solid string growing backwards.

This string is the commencement of the Oviduct or Müller's duct, which in the Dog-fish as in the Batrachians is the first portion of the genito-urinary system to appear, and is in the Dog-fish undoubtedly at first solid. All my specimens have been hardened with osmic acid, and with specimens hardened with this reagent it is quite easy to detect even the very smallest hole in a mass of cells.

As a solid string or rod of cells the Oviduct remains for some time; it grows, indeed, rapidly in length, the extreme hind end of the rod being very small and the front end continuing to remain attached to the knob. The knob, however, travels inwards and approaches nearer and nearer to the true pleuro-peritoneal cavity, always remaining attached to the intermediate cell mass.

At about the time when five visceral clefts are present the Oviduct first begins to get a lumen and to open at its front end into the pleuro-peritoneal cavity. The cells of the rod are first of all arranged in an irregular manner, but gradually become columnar and acquire a radiating arrangement around a central point. At this point, where the ends of all the cells meet, a very small hole appears, which gradually grows larger and becomes the cavity of the duct (fig. 12, ov). The hole first makes its appearance at the anterior end of the duct, and then gradually extends backwards, so that the hind end is still without a lumen, when the lumen of the front end is of a considerable size.

At the front knob the same alteration in the cells takes place as in the rest of the duct, but the cells become deficient on the side adjoining the pleuro-peritoneal cavity, so that an opening is formed into the pleuro-peritoneal cavity, which soon becomes of a considerable size. Soon after its first formation, indeed, the opening becomes so large that it may be met in from two to three consecutive sections if these are very thin.

Thus is formed the lumen of the Oviduct. The duct still, at this age, ends behind without having become attached to the cloaca, so that at this time the Oviduct is a canal closed behind, but communicating in front by a large opening with the pleuro-peritoneal cavity.

It has during this time been travelling downwards, and is now much nearer the pleuro-peritoneal cavity than the epiblast.

It may be well to point out that the mode of development which I have described is really not very different from an involution, and must, in fact, be only looked upon as a modification of an involution. Many examples from all classes in the animal kingdom could be selected to exemplify how an involution may become simply a solid thickening. In the Osseous fish nearly all the organs which are usually formed by an involution have undergone this change in their mode of development. I shall attempt to give reasons later on for the solid form having been acquired in this particular case of the Oviduct.

At about the time when a lumen appears in the Oviduct the first traces of the Wolffian duct become visible.

At intervals along the whole length, between the front and hind ends of the Oviduct, involutions arise from the pleuro-peritoneal cavity (fig. 12a, pwd) on the inside (nearer the middle line) of the Oviduct. The upper ends of these numerous involutions unite together and form a string of cells, at first solid, but very soon acquiring a lumen, and becoming a duct which communicates (as it clearly must from its mode of formation), at numerous points with the pleuro-peritoneal cavity. It is very probable that there is one involution to each segment of the body between the front and hind ends of the Oviduct. This duct is the Wolffian duct, which thus, together with the Oviduct, is formed before the appearance of the external gills.

For a considerable period the front end of the Oviduct does not undergo important changes; the hind end, however, comes into connection with the extreme end of the alimentary canal. The two Oviducts do not open together into the cloaca, though, as my sections prove, their openings are very close together. The whole Oviduct, as might be expected, shares in the general growth, and its lumen becomes in both sexes very considerably greater than it was before.

It is difficult to define the period at which I find these changes accomplished without giving drawings of the whole embryo. The stage is one considerably after the external gills have appeared, but before the period at which the growth of the olfactory bulbs renders the head of an elongated shape.

During the same period the Wolffian duct has undergone most important changes. It has commenced to bud off diverticula, which subsequently become the tubules of the Wolffian body (vide fig. 13, wd). I am fairly satisfied that the tubules are really budded off, and are not formed independently in the mesoblast. The Dog-fish agrees so far with Birds, where I have also no doubt the tubules of the Wolffian body are formed as diverticula from the Wolffian duct.

The Wolffian ducts have also become much longer than the Oviduct, and are now found behind the anus, though they do not extend as far forward as does the Oviduct.

They have further acquired a communication with the Oviduct, in the form of a narrow duct passing from each of them into an Oviduct a short way before the latter opens into the cloacal dilatation of the alimentary canal.

The canals formed by the primitive involution leading from the pleuro-peritoneal cavity into the Wolffian duct have become much more elongated, and at the same time narrower. One of these is shewn in fig. 13, pwd.

Any doubt which could possibly be entertained as to the true character of the ducts whose development I have described is entirely removed by the development of the tubules of the Wolffian body. In the still later stage than this further proofs are furnished involving the function of the Oviduct. At the period when the olfactory lobes have become so developed as to render the head of the typical elongated shape of the adult, I find that the males and females can be distinguished by the presence in the former of the clasping appendages[16]. I find at this stage that in the female the front ends of the Oviducts have approached the middle line, dilated considerably, and commenced to exhibit at their front ends the peculiarities of the adult. In the male they are much less conspicuous, though still present.

At the same time the tubules of the Wolffian body become much more numerous, the Malpighian tufts appear, and the ducts cease almost, if not entirely, to communicate with the pleuro-peritoneal cavity. I have not made out anything very definitely as to the development of the Malpighian tufts, but I am inclined to believe that they arise independently in the mesoblast of the intermediate cell mass.

The facts which I have made out in reference to the development of the Wolffian duct, especially of its arising as a series of involutions from the pleuro-peritoneal cavity, will be found, I believe, of the greatest importance in understanding the true constitution of the Wolffian body. To this I will return directly, but first wish to clear the ground by insisting upon one preliminary point.

From their development the Oviduct and Wolffian body appear to stand to each other in the relation of the Wolffian duct being the equivalent to a series, so to speak, of Oviducts.

I pointed out before that the mode of development of the Oviduct could only be considered as a modification of a simple involution from the pleuro-peritoneal cavity. Its development, both in the Birds and in the Batrachians as an involution, still more conclusively proves the truth of this view.

The explanation of its first appearing as a solid rod of cells which keeps close to the epiblast is, I am inclined to think, the following. Since the Oviduct had to grow a long way backwards from its primitive point of involution, it was clearly advantageous for it not to bore its way through the mesoblast of the intermediate cell mass, but to pass between this and the epiblast. This modification having been adopted, was followed by the knob forming the origin of the duct coming to be placed at the outside of the intermediate cell mass rather than close to the pleuro-peritoneal cavity, a change which necessitated the mode of development by an involution being dropped and the solid mode of development substituted for it, a lumen being only subsequently acquired.

In support of the modification in the development being due to this cause is the fact that in Birds a similar modification has taken place with the Wolffian duct. The Wolffian duct there arises differently from its mode of development in all the lower vertebrates as a solid rod close to the epiblast[17], instead of as an involution.

If the above explanation about the Oviduct be correct, then it is clear that similar causes have produced a similar modification in development (only with a different organ) in Birds; while, at the same time, the primitive mode of origin of the Oviduct (Müller's duct) has been retained by them.

The Oviduct, then, may be considered as arising by an involution from the pleuro-peritoneal cavity.

The Wolffian duct arises by a series of such involutions, all of which are behind (nearer the tail) the involution to form the Oviduct.

The natural interpretation of these facts is that in the place of the Oviduct and Wolffian body there were primitively a series of similar bodies (probably corresponding in number with the vertebral segments), each arising by an involution from the pleuro-peritoneal cavity; and that the first of these subsequently became modified to carry eggs, while the rest coalesced to form the Wolffian duct.

If we admit that the Wolffian duct is formed by the coalescence of a series of similar organs, we shall only have to extend the suggestion of Gegenbaur as to the homology of the Wolffian body in order to see its true nature. Gegenbaur looks upon the whole urinogenital system as homologous with a pair of segmental organs. Accepting its homology with the segmental organs, its development in Elasmobranchii proves that it is not one pair, but a series of pairs of segmental organs with which the urinogenital system is homologous. The first of these have become modified so as to form the Oviducts, and the remainder have coalesced to form the Wolffian ducts.

The part of a segmental organ which opens to the exterior appears to be lost in the case of all but the last one, where this part is still retained, and serves as the external opening for all.

Whether the external opening of the first segmental organ (Oviduct) is retained or not is doubtful. Supposing it has been lost, we must look upon the external opening for the Wolffian body as serving also for the Oviduct. In the case of all other vertebrates whose development has been investigated (but the Elasmobranchii), the Wolffian duct arises by a single involution, or, what is equivalent to it, the other involutions having disappeared. This even appears to be the case in the Marsipobranchii. In the adult Lamprey the Wolffian duct terminates at its anterior end by a large ciliated opening into the pleuro-peritoneal cavity. It will, perhaps, be found, when the development of the Marsipobranchii is more carefully studied, that there are primitively a number of such openings[18]. The Oviduct, when present, arises in other vertebrates as a single involution, strongly supporting the view that its mode of formation in the Dog-fish is fundamentally merely an involution.

The duct of the testes is, I have little doubt, derived from the anterior part of the Wolffian body; if so, it must be looked upon as not precisely equivalent to the Oviduct, but rather to a series of coalesced organs, each equivalent to the Oviduct. The Oviduct is in the Elasmobranchii, as in other vertebrates, primitively developed in both sexes. In the male, however, it atrophies. I found it still visible in the male Torpedos, though much smaller than in the females near the close of intra-uterine life.

Whether or not these theoretical considerations as to the nature of the Wolffian body and Oviduct are correct, I believe that the facts I have brought to light in reference to the development of these parts in the Dog-fish will be found of service to every one who is anxious to discover the true relations of these parts.

Before leaving the subject I will say one or[TN2] two words about the development of the Ovary. In both sexes the germinal epithelium (fig. 13) becomes thickened below the Oviduct, and in both sexes a knob (in section but really a ridge) comes to project into the pleuro-peritoneal cavity on each side of the mesentery (fig. 13, pov). In both sexes, but especially the females, the epithelium on the upper surface of this ridge becomes very much thickened, whilst subsequently it elsewhere atrophies. In the females, however, the thickened epithelium on the knob grows more and more conspicuous, and develops a number of especially large cells with large nuclei, precisely similar to Waldeyer's (loc. cit.) primitive ova of the Bird. In the male the epithelium on the ridge, though containing primitive ova, is not as conspicuous as in the female. Though I have not worked out the matter further than this at present, I still have no doubt that these projecting ridges become the Ovaries.

The Head.

The study of the development of the parts of the head, on account of the crowding of organs which occurs there, always presents greater difficulties to the investigator than that of the remainder of the body. My observations upon it are correspondingly incomplete. I have, however, made out a few points connected with it in reference to some less well-known organs, which I have thought it worth while calling attention to in this preliminary account.

The continuation of the Pleuro-peritoneal Cavity into the Head.

In the earlier part of this paper (p. 86) I called attention to the extension of the separation between somatopleure and splanchnopleure into the head, forming a space continuous with the pleuro-peritoneal cavity (Pl. 3, fig. 8a, pp); this becomes more marked in the next stage, and, indeed, the pleuro-peritoneal cavity is present for a considerable time in the head before it becomes visible elsewhere. At the time of the appearance of the second visceral cleft it has become for the most part atrophied, but there persist two separated portions of it in front of the first cleft, and also remnants of it less well marked between and behind the two clefts. The visceral clefts necessarily divide it into separate parts.

The two portions in front of the first visceral cleft remain very conspicuous till the appearance of the external gills, and above the hinder one of the two the fifth nerve bifurcates.

These two are shewn as they appear in a surface view in fig. 14, pp. They are in reality somewhat flattened spaces, lined by a mesoblastic epithelium; the epithelium on the inner surface of the space corresponding to the splanchnopleure, and that on the outer to the somatopleure.

I have not followed the history of these later than the time of the appearance of the external gills.

The presence of the pleuro-peritoneal cavity in the head is interesting, as shewing the fundamental similarity between the head and the remainder of the body.

The Pituitary Body.

All my sections seem to prove that it is a portion of the epiblastic involution to form the mouth which is pinched off to form the pituitary body, and not a portion of the hypoblast of the throat. Since Götte (Archiv. für Micr. Anat. Bd. IX.) has also found that the same is the case with the Batrachians and Mammalia, I have little doubt it will be found to be universally the case amongst vertebrates.

Probably the observations which lead to the supposition that it was the throat which was pinched off to form the pituitary body were made after the opening between the mouth and throat was completed, when it would naturally be impossible to tell whether the pinching off was from the epiblast of the mouth involution or the hypoblast of the throat.

The Cranial Nerves.

The cranial nerves in their early condition are so clearly visible that I have thought it worth while giving a figure of them, and calling attention to some points about their embryonic peculiarities.

From my figure (14) it will be seen that there is behind the auditory vesicle a nervous tract, from which four nerves descend, and that each of these nerves is distributed to the front portion of a visceral arch. When the next and last arch (in this species) is developed, a branch from this nervous mass will also pass down to it. That each of these is of an equal morphological value can hardly be doubted.

The nerve to the third arch becomes the glosso-pharyngeal (fig. 14, gl), the nerves to the other arches become the branchial branches of the vagus nerve (fig. 14, vg). Thus the study of their development strongly supports Gegenbaur's view of the nature of the vagus and glosso-pharyngeal, viz. that the vagus is a compound nerve, each component part of it which goes to an arch being equivalent to one nerve, such as the glosso-pharyngeal.

Of the nerves in front of the auditory sac the posterior is the seventh nerve (fig. 14, VII). Its mode of distribution to the second arch leaves hardly a doubt that it is equivalent to one such nerve as those distributed to the posterior arches. Subsequently it acquires another branch, passing forwards towards the arch in front.

The most anterior nerve is the fifth (fig. 14, V), of which two branches are at this stage developed. The natural interpretation of its present condition is, that it is equivalent to two nerves, but the absence of relation in its branches to any visceral clefts renders it more difficult to determine the morphology of the fifth nerve than of the other nerves. The front branch of the two is the ophthalmic branch of the adult, and the hind branch the inferior maxillary branch. The latter branch subsequently gives off low down, i.e. near its distal extremity, another branch, the superior maxillary branch.

In its embryonic condition this latter branch does not appear like a third branch of the fifth, equivalent to the seventh or the glosso-pharyngeal nerves, but rather resembles the branch of the seventh nerve which passes to the arch in front, which also is present in all the other cranial nerves.

Modes of Preparation.

Before concluding I will say one or two words as to my modes of preparation.

I have used picric and chromic acids, both applied in the usual way; but for the early stages I have found osmic acid by far the most useful reagent. I placed the object to be hardened, in osmic acid (half per cent.) for two hours and a half, and then for twenty four in absolute alcohol.

I then embedded and cut sections of it in the usual way, without staining further.

I found it advantageous to cut sections of these embryos immediately after hardening, since if kept for long in the absolute alcohol the osmic acid specimens are apt to become brittle.

LIST OF WORKS REFERRED TO.

Gegenbaur. Anat. der Wirbelthiere, III Heft, Leipzig, 1873.

A. Götte. Archiv. für Micr. Anat., Vol. X. 1873. Der Keim der Forelleneies, Archiv. Für. Micr. Anat., Vol. IX. 1873. Untersuchung über die Entwicklung der Bombinator igneus, Archiv. für Micr. Anat., Vol. V. 1869. Kurze Mittheilungen aus der Entwicklungsgeschichte der Unke, Archiv. für Micr. Anat., Vol. IX. 1873.

Kupffer. Archiv. für Micr. Anat., Vol. II. 1866, p. 473. Ibid. Vol. IV. 1868, p. 209.

Kowalevsky. Entwicklungsgeschichte der Holothurien, Mémoires de l'Acad. Impér. des Sciences de St Petersbourg, vii ser. Vol. XI. 1867.

Kowalevsky, Owsjannikow, und Wagner. Entwicklung der Störe, Bulletin der K. Acad. St Petersbourg, Vol. XIV. 1873.

Kowalevsky. Embryologische Studien an Würmern und Arthropoden, Mémoirs de l'Acad. Impér. des Sciences de St Petersbourg, Vol. XIV. 1871.

E. Ray Lankester. Annals and Mag. of Nat. History, Vol. XI. 1873, p. 81.

W. Müller. Ueber die Persistenz der Urniere bei Myxine Glutinosa, Jenaische Zeitschrift, Vol. VII. 1873.

Oellacher. Zeitschrift für Wiss. Zoologie, Vol. XXIII. 1873.

Owsjannikow. Entwicklung der Coregonus, Bul. der K. Akad. St Petersbourg, Vol. XIX.

Romiti. Archiv. für Micr. Anat., Vol. IX. 1873.

Waldeyer. Eierstock u. Eie.

EXPLANATION OF PLATES 3 AND 4.

COMPLETE LIST OF REFERENCE LETTERS.

al. Alimentary canal. ao. Dorsal aorta. auv. Auditory vesicle. bd. Formative cell probably derived from the yolk. cav. Cardinal vein. ch. Notochord. ch´. Thickening of hypoblast to form the notochord. Eb. Line indicating the edge of the blastoderm. ep. Epiblast. ep´. Epidermis. er. Embryonic rim. es. Embryonic swelling. gl. Glosso-pharyngeal nerve. h. Head. ht. Heart. hy. Hypoblast. ll. Lower layer cells. ly. Line of separation between the blastoderm and the yolk. m. Mesoblast. mc. Medullary canal. mg. Medullary groove. mp. Muscle-plate. mp´. Early formed mass of muscles. n. Peculiar nuclei formed in the yolk. n´. Similar nuclei in the cells of the blastoderm. na. Cells which help to close in the alimentary canal, and which are derived from the yolk. ny. Network of lines present in the food-yolk. ol. Olfactory pit. op. Eye. ov. Oviduct. pn. Pineal gland. pov. Projection which becomes the ovary. pp. Pleuro-peritoneal cavity. pp´. Remains of pleuro-peritoneal cavity in the head. prv. Protovertebræ. pwd. Primary points of involution from the pleuro-peritoneal cavity by the coalescence of which the Wolffian duct is formed. sg. Segmentation cavity. so. Somatopleure. sos. Stalk connecting embryo with yolk-sac. sp. Splanchnopleure. spn. Spinal nerve. sur. Suprarenal body. ts. Caudal lobes. v. Blood-vessel. vg. Vagus nerve. V. Fifth nerve. VII. Seventh nerve. vc, 1, 2, 3, &c. 1st, 2nd and 3rd &c. visceral clefts. vp. Vertebral plates. wd. Wolffian duct. x. Peculiar body underlying the notochord derived from the hypoblast. yk. Yolk spherules.

All the figures were drawn with the Camera Lucida.

Plate 3.

Fig. 1. Section parallel with the long axis of the embryo through a blastoderm, in which the floor of the segmentation cavity (sg) is not yet completely lined by cells. The roof of the segmentation cavity is broken. (Magnified 60 diam.) The section is intended chiefly to illustrate the distribution of nuclei (n) in the yolk under the blastoderm. One of the chief points to be noticed in their distribution is the fact that they form almost a complete layer under the floor of the segmentation cavity. This probably indicates that the cells whose nuclei they become take some share in forming the layer of cells which subsequently (vide fig. 4) forms the floor of the cavity.

Fig. 2. Small portion of blastoderm and subjacent yolk of an embryo at the time of the first appearance of the medullary groove. (Magnified 300 diam.)

The specimen is taken from a portion of the blastoderm which will form part of the embryo. It shews two large nuclei of the yolk (n) and the network in the yolk between them; this network is seen to be closer around the nuclei than in the intervening space. The specimen further shews that there are no areas representing cells around the nuclei.

Fig. 3. Section parallel with the long axis of the embryo through a blastoderm, in which the floor of the segmentation cavity is not yet covered by a complete layer of cells. (Magnified 60 diam.)

It illustrates (1) the characters of the epiblast, (2) the embryonic swelling (es), (3) the segmentation cavity (sg). It should have been drawn upon the same scale as fig. 4; the line above it represents its true length upon this scale.

Fig. 4. Longitudinal section through a blastoderm at the time of the first appearance of the embryonic rim, and before the formation of the medullary groove. (Magnified 45 diam.)

It illustrates (1) the embryonic rim, (2) the continuity of epiblast and hypoblast at edge of this, (3) the continual differentiation of the lower layer cells, to form, on the one hand, the hypoblast, which is continuous with the epiblast, and on the other the mesoblast, between this and the epiblast; (4) the segmentation cavity, whose floor of cells is now completed.

N.B. The cells at the embryonic end of the blastoderm have been made rather too large.

Fig. 5. Surface view of the blastoderm shortly after the appearance of the medullary groove. To shew the relation of the embryo to the blastoderm.

Fig. 6a and b. Two transverse sections of the same embryo, shortly after the appearance of the medullary groove. (Magnified 96 diam.)

a. In the region of the groove. It shews (1) the two masses of mesoblast on each side, and the deficiency of the mesoblast underneath the medullary groove; (2) the commencement of the closing in of the alimentary canal below, chiefly from cells (na) derived from the yolk.

b. Section in the region of the head where the medullary groove is deficient, other points as above.

Fig. 7a and b. Two transverse sections of an embryo about the age or rather younger than that represented in fig. 5. (Magnified 96 diam.)

a. Section nearer the tail; it shews the thickening of the hypoblast to form the notochord (ch´).

In b the thickening has become completely separated from the hypoblast as the notochord. In a the epiblast and hypoblast are continuous at the edge of the section, owing to the section passing through the embryonic rim.

Fig. 8. Surface view of a spatula-shaped embryo. The figure shews (1) the flattened head (h) where the medullary groove is deficient, (2) the caudal lobes, with a groove between them; it also shews that at this point, the medullary groove has become roofed over and converted into a canal.

Fig. 8a. Transverse section of fig. 8, passing through the line a. (Magnified 90 diam.) The section shews (1) the absence of the medullary groove in the head and the medullary folds turning down at this time instead of upwards; (2) the presence of the pleuro-peritoneal cavity in the head (pp); (3) the completely closed alimentary canal (al).

Fig. 8b. Transverse section of fig. 8, through the line b. (Magnified 90 diam.) It shews (1) the neural canal completely formed; (2) the vertebral plates of mesoblast not yet split up into somatopleure and splanchnopleure.

Fig. 9. Side view of an embryo of the Torpedo, seen as a transparent object a little older than the embryo represented in fig. 8. (Magnified 20 diam.) The internal anatomy has hardly altered, with the exception of the medullary folds having closed over above the head and the whole embryo having become more folded off from the germ.

The two caudal lobes, and the very marked groove between them, are seen at ts. The front end of the notochord became indistinct, and I could not see its exact termination. The epithelium of the alimentary canal (al) is seen closely underlying the notochord and becoming continuous with the epiblast at the hind end of the notochord.

The first visceral cleft (1vc) and eye (op) are just commencing to be formed, and the cranial flexure has just appeared.

Fig. 10. Section through the dorsal region of an embryo somewhat older than the one represented in fig. 9. (Magnified 96 diam.)

It shews (1) the formation by a pinching off from the top of the alimentary canal of a peculiar body which underlies the notochord (x); (2) the primitive extension of the pleuro-peritoneal cavity up to the top of the vertebral plates.

Plate 4.

Fig. 11a, b, and c. Three sections closely following each other from an embryo in which three visceral clefts are present; a is the most anterior of the three. (Magnified 96 diam.) In all of these the muscle-plates are shewn at mp. They have become separated from the lateral plates in b and c, but are still continuous with them in a. The early formed mass of muscles is also shewn in all the figures (mp´).

The figures further shew (1) the formation of the spinal nerves (spn) as small bodies of cells closely applied to the upper and outer edge of the neural canal.

(2) The commencing formation of the cells which form the axial skeleton from the inner (splanchnopleuric) layer of the muscle-plate. Sections b and c are given more especially to shew the mode of formation of the oviduct (ov).

In b it is seen as a solid knob (ov), arising from the point where the somatopleure and splanchnopleure[TN3] unite, and in c (the section behind b) as a solid rod (ov) closely applied to the epiblast, which has grown backwards from the knob seen in b.

N.B. In all three sections only one side is completed.

Fig. 12a and b. Two transverse sections of an embryo just before the appearance of the external gills. (Magnified 96 diam.)

In a there is seen to be an involution on each side (pwd), while b is a section from the space between two involutions from the pleuro-peritoneal cavity, so that the Wolffian duct (at first solid) (wd) is not connected as in a with the pleuro-peritoneal cavity. The further points shewn in the sections are—

(1) The commencing formation of the spiral valve (al).

(2) The suprarenal body (sur).

(3) The oviduct (ov), which has acquired a lumen.

(4) The increase in length of the muscle-plates, the spinal nerves, &c.

Fig. 13. Section through the dorsal region of an embryo in which the external gills are of considerable length. (Magnified 40 diam.) The chief points to be noticed:

(1) The formation of the Wolffian body by outgrowths from the Wolffian duct (wd).

(2) One of the still continuing connections (primitive involutions) between the Wolffian duct and the pleuro-peritoneal cavity (pwd).

(3) The oviduct largely increased in size (ov).

N.B. On the left side the oviduct has been accidentally made too small.

(4) The growth downwards of the muscle-plate to form the muscles of the abdomen.

(5) The formation of an outgrowth on each side of the mesentery (pov), which will become the ovary.

(6) The spiral valve (al).

Fig. 14. Transparent view of the head of an embryo shortly before the appearance of the external gills. (Magnified 20 diam.) The chief points to be noticed are—

(1) The relation of the cranial nerves to the visceral clefts and the manner in which the glosso-pharyngeal (gl) and vagus (vg) are united.

(2) The remnants of the pleuro-peritoneal cavity in the head (pp).

(3) The eye (op). The stalk, as well as the bulb of the eye, are supposed to be in focus, so that the whole eye has a somewhat peculiar appearance.

VI. A Comparison of the Early Stages in the Development of Vertebrates[19].

With Plate 5.

If the genealogical relationships of animals are to be mainly or largely determined on embryological evidence, it becomes a matter of great importance to know how far evidence of this kind is trustworthy.

The dependence to be placed on it has been generally assumed to be nearly complete. Yet there appears to be no à priori reason why natural selection should not act during the embryonic as well as the adult period of life; and there is no question that during their embryonic existence animals are more susceptible to external forces than after they have become full grown: indeed, an immense mass of evidence could be brought to shew that these forces do act upon embryos, and produce in them great alterations tending to obscure the genealogical inferences to be gathered from their developmental histories. Even the time-honoured layers form to this no exception. In Elasmobranchii, for instance, we find the notochord derived from the hypoblast and the spinal ganglia derived from the involuted epiblast of the neural canal, whilst in the higher vertebrates both of these organs are formed in the mesoblast. Such instances are leading embryologists to recognise the fact that the so-called layers are not quite constant and must not be absolutely depended upon in the determination of homologies. But though it is necessary to recognise the fact that great changes do occur in animals during their embryonic life, it is not necessary to conclude that all embryological evidence is thereby vitiated; but rather it becomes incumbent on us to attempt to determine which embryological features are ancestral and which secondary. For this purpose it is requisite to ascertain what are the general characters of secondary features and how they are produced. Many vertebrates have in the first stages of their development a number of secondary characters which are due to the presence of food material in the ovum; the present essay is mainly an attempt to indicate how those secondary characters arose and to trace their gradual development. At the same time certain important ancestral characters of the early phases of the development of vertebrates, especially with reference to the formation of the hypoblast and mesoblast, are pointed out and their meaning discussed.

There are three orders of vertebrates of which no mention has been made, viz., the Mammals, the Osseous fishes, and the Reptiles. The first of these have been passed over because the accounts of their development are not sufficiently satisfactory, though as far as can be gathered from Bischoff's account of the dog and rabbit there would be no difficulty in shewing their relations with other vertebrates.

We also require further investigations on Osseous fishes, but it seems probable that they develop in nearly the same manner as the Elasmobranchii.

With reference to Reptiles we have no satisfactory investigations.

* * * * *

Amphioxus is the vertebrate whose mode of development in its earliest stages is simplest, and the modes of development of other vertebrates are to be looked upon as modifications of this due to the presence of food material in their ova. It is not necessary to conclude from this that Amphioxus was the ancestor of our present vertebrates, but merely that the earliest stages of development of this vertebrate ancestor were similar to those of Amphioxus.

The ovum of Amphioxus contains very little food material and its segmentation is quite uniform. The result of segmentation is a vesicle whose wall is formed of a single layer of cells. These are all of the same character, and the cavity of the vesicle called the segmentation cavity is of considerable size. A section of the embryo, as we may now call the ovum, is represented in Plate 5, fig. A I.

The first change which occurs is the pushing in of one half of the wall of the vesicle towards the opposite half. At the same time by the narrowing of its mouth the hollow hemisphere so formed becomes again a vesicle[20].

Owing to its mode of formation the wall of this secondary vesicle is composed of two layers which are only separated by a narrow space, the remnant of the segmentation cavity.

Two of the stages in the formation of the secondary vesicle by this process of involution are shewn in Plate X, fig. A II , and A III. In the second of these the general growth has been very considerable, rendering the whole animal much larger than before. The cavity of this vesicle, A III , is that of the commencing alimentary canal whose final form is due to changes of shape undergone by this primitive cavity. The inner wall of the vesicle becomes converted into the wall of the alimentary canal or hypoblast, and also into part or the whole of the mesoblast.

During the involution the cells which are being involuted undergo a change of form, and before the completion of the process have acquired a completely different character to the cells forming the external wall of the secondary vesicle or epiblast. This change of character in the cells is already well marked in fig. A II. It is of great importance, since we shall find that some of the departures from this simple mode of development, which characterise other vertebrates, are in part due to the distinction between the hypoblast and epiblast cells appearing during segmentation, and not subsequently as in Amphioxus during the involution of the hypoblast.

Kowalevsky (Entwicklungsgeschichte des Amphioxus) originally believed that the narrow mouth of the vesicle (according to Mr Lankester's terminology blastopore) became the anus of the adult. He has since, and certainly correctly, given up this view. The opening of the involution becomes closed up and the adult anus is no doubt formed as in all other vertebrates by a pushing in from the exterior, though it probably corresponds in position very closely with the point of closing up of the original involution.

The mode of formation of the mesoblast is not certainly known in Amphioxus; we shall find, however, that for all other vertebrates it arises from the cells which are homologous with the involuted cells of this animal.

Since food material is a term which will be very often employed, it will be well to explain exactly the sense in which it will be used. It will be used only with reference to those passive highly refractive particles which are found embedded in most ova.

In some eggs, of which the hen's egg may be taken as a familiar example, the yolk-spherules or food material form the larger portion of the ovum, and a distinction is frequently made between the germinal disc and the yolk.

This distinction is, however, apt to lead to a misconception of the true nature of the egg. There are strong grounds for believing that the so-called yolk, equally with the germinal disc, is composed of an active protoplasmic basis endowed with the power of growth, in which passive yolk-spherules are embedded; but that the part ordinarily called the yolk contains such a preponderating amount of yolk-spherules that the active basis escapes detection, and does not exhibit the same power of growth as the germinal disc.

With the exception of mammals, whose development requires to be more completely investigated, Amphioxus is as far as we know the only vertebrate whose ovum does not contain a large amount of food material.

In none of these (vertebrate) yolk-containing ova is the food material distributed uniformly. It is always concentrated much more at one pole than at the other, and the pole at which it is most concentrated may be conveniently called the lower pole of the egg.

In eggs in which the distribution of food material is not uniform segmentation does not take place with equal rapidity through all parts of the egg, but its rapidity is, roughly speaking, inversely proportional to the quantity of food material.

When the quantity of food material in a part of the egg becomes very great, segmentation does not occur at all; and even in those cases where the quantity of food yolk is not too great to prevent segmentation the resulting segmentation spheres are much larger than where the yolk-granules are more sparsely scattered.

The Frog is the vertebrate whose development comes nearest to that of Amphioxus, as far as the points we are at present considering are concerned. But it will perhaps facilitate the understanding of their relations shortly to explain the diagrammatic sections which I have given of an animal supposed to be intermediate in its development between the Frog and Amphioxus. Plate 5, fig. B I, represents a longitudinal section of this hypothetical egg at the close of segmentation. The lower pole, coloured yellow, represents the part containing more yolk material, and the upper pole, coloured blue, that with less yolk. Owing to the presence of this yolk the lower pole even at the close of segmentation is composed of cells of a different character to those of the upper pole. In this respect this egg can already be distinguished from that of Amphioxus, in which no such difference between the two poles is apparent at the corresponding period (Plate 5, fig. A I).

The segmentation cavity in this ovum is not quite so large proportionately as in Amphioxus, and the encroachment upon it is due to the larger bulk of the lower pole of the egg. In fig. B II the involution of the lower pole has already commenced; this involution is (1) not quite symmetrical, and (2) on the ventral side (the left side) the epiblast cells forming the upper part of the egg are growing round the cells of the lower pole of the egg or lower layer cells. Both of these peculiarities are founded upon what happens in the Frog and the Selachian, but it is to be noticed that the change from the lower layer cells being involuted towards the epiblast cells, to the epiblast cells growing round the lower layer cells, is a necessary consequence of the increased bulk of the latter.

In this involution not only are the cells of the lower pole pushed on, but also some of those of the upper or yellow portion; so that in this as in all other cases the true distinction between the epiblast and hypoblast does not appear till the involution to form the latter is completed. In the next stage, B III, the involution has become nearly completed and the opening to the exterior or blastopore quite constricted.

The segmentation cavity has been entirely obliterated, as would have been found to be the case with Amphioxus had the stage a little older than that on Plate 5, A III, been represented. The cavity marked (al), as was the case with Amphioxus, is that of the alimentary canal.

The similarities between the mode of formation of the hypoblast and alimentary canal in this animal and in Amphioxus are so striking and the differences between the two cases so slight that no further elucidation is required. One or two points need to be spoken of in order to illustrate what occurs in the Frog. When the involution to form the alimentary canal occurs, certain of the lower layer cells (marked hy) become distinguished from the remainder of the lower layer cells as a separate layer and form the hypoblast which lines the alimentary canal. It is to be noticed that the cells which form the ventral epithelium of the alimentary canal are not so soon to be distinguished from the other lower layer cells as those which form its dorsal epithelium. This is probably a consequence of the more active growth, indicated by the asymmetry of the involution, on the dorsal side, and is a fact with important bearings in the ova with more food material. The cells marked m and coloured red also become distinguished as a separate layer from the remainder of the hypoblast and form the mesoblast. The remainder of the lower layer cells form a mass equivalent to the yolk-sac of many vertebrates, and are not converted directly into the tissues of the animal.

Another point to be noticed is the different relation of epiblast cells to the hypoblast cells at the upper and lower side of the mouth of the involution. Above it, on its dorsal side, the epiblast and hypoblast are continuous with one another. On its ventral side they are primitively not so continuous. This is due to the epiblast, as was before mentioned, growing round the lower layer cells on the ventral side, vide B II, and merely remaining continuous with them on the dorsal. The importance of these two points will appear when we come to speak of other vertebrates.

The next animal whose development it is necessary to speak of is the Frog, and its differences from the mode of development are quite easy to follow and interpret. Segmentation is again not uniform, and results in the formation of an upper layer of smaller cells and a lower one of larger; in the centre is a segmentation cavity. The stage at the close of segmentation is represented in C I. From the diagram it is apparent that the lower layer cells occupy a larger bulk than they did in the previous animal (Plate 5, B I), and tend to encroach still more upon the segmentation cavity, otherwise the differences between the two are unimportant. There are, however, two points to be noted. In the first place, although the cells of the upper pole are distinguished in the diagrams from the lower by their colour, it is not possible at this stage to say what will become epiblast and what hypoblast. In the second place the cells of the upper pole or epiblast consist of two layers—an outer called the epidermic layer and an inner called the nervous. In the previous cases the epiblast consisted of a single layer of cells. The presence of these two layers is due to a distinction which, arising in most other vertebrates late, in the Frog arises early. In most other vertebrates in the later stages of development the epiblast consists of an outer layer of passive and an inner of active cells. In the Frog and other Batrachians these two layers become distinguished at the commencement of development.

In the next stage (C II) we find that the involution to form the alimentary canal has commenced (al), but that it is of a very different character to the involution in the previous case. It consists in the growing inwards of a number of cells from the point x (C I) towards the segmentation cavity. The cells which grow in this way are partly the blue cells and partly the smaller yellow ones. At first this involuted layer of cells is only separated by a slit from the remainder of the lower layer cells; but by the stage represented in C II this has widened into an elongated cavity (al). In its formation this involution pushes backwards the segmentation cavity, which finally disappears in the stage C III. The point x remains practically stationary, but by the general growth of the epiblast, mesoblast and hypoblast, becomes further removed from the segmentation cavity in C II than in C I. On the opposite side of the embryo to that at which the involution occurs the epiblast cells as before, grow round the lower layer cells. The commencement of this is already apparent in C I, and in C II the process is nearly completed, though there is still a small mass of yolk filling up the blastopore. The features of this involution are in the main exaggerations of what was supposed to occur in the previous animal. The asymmetry of the involution is so great that it is completely one-sided and results, in the first instance, in a mere slit; and the whole process of enclosing the yolk by epiblast is effected by the epiblast cells on the side of the egg opposite to the involution.

The true mesoblast and hypoblast are formed precisely as in the previous case. The involuted cells become separated into two layers, one forming the dorsal epithelium of the alimentary canal, and a layer between this and the epiblast forming the mesoblast. There is also a layer of mesoblast accompanying the epiblast which encloses the yolk, which is derived from the smaller yellow cells at y (C I). The edge of this mesoblast, m´, forms a thickened ridge, a feature which persists in other vertebrates.

It is a point of some importance for understanding the relation between the mode of formation of the alimentary canal in the Frog and other vertebrates to notice that on the ventral surface the cells which are to form the epithelium of the alimentary canal become distinguished as such very much later than do those to form its dorsal epithelium, and are derived not from the involuted cells but from the primitive large yolk-cells. It is indeed probable that only a very small portion of epithelium of the ventral wall of the mid-gut is in the end derived from these larger yolk-cells. The remainder of the yolk-cells (C III, and C II, yk) form the yolk mass and do not become directly formed into the tissues of the animal.

In the last stage I have represented for the frog, C III , there are several features to be noticed.

The direct connection at their hind-ends between the cavities of the neural and alimentary canals is the most important of these. This is a result of the previous continuity of the epiblast and hypoblast at the point x, and is a feature almost certainly found in Amphioxus, but which I will speak of more fully in my account of the Selachian's development. The opening of the blastopore called the anus of Rusconi is now quite narrowed, it does not become the anus of the adult. It may be noticed that at the front end of the embryo the primitive dorsal epithelium of the alimentary canal is growing in such a way as to form the epithelium both of the dorsal and ventral surfaces of the fore-gut.

In spite of various features rendering the development of the Frog more difficult of comprehension than that of most other vertebrates, it is easy to see that the step between it and Amphioxus is not a very great one, and will very likely be bridged over at some future time, when our knowledge of the development of other forms becomes greater.

From the Frog to the Selachian is a considerable step, but I have again hypothetically sketched a type intermediate between them whose development agrees in some important points with that of Pelobates fuscus as described by Bambeke. The points of agreement, though not obvious at first sight, I shall point out in the course of my description.

The first stage (D I), at the close of segmentation, deserves careful attention. The segmentation cavity by the increase of the food yolk is very much diminished in size, and, what is still more important, has as it were sunk down so as to be completely within the lower layer cells. The roof of the segmentation cavity is thus formed of epiblast and lower layer cells, a feature which Bambeke finds in Pelobates fuscus and which is certainly found in the Selachians. In the Frog we found that the segmentation cavity began to be encroached on by the lower layer cells, and from this it is only a small step to find these cells creeping still further up and forming the roof of the cavity. In the lower layer cells themselves we find an important new feature, viz. that during segmentation they become divided in two distinct parts—one of these where the segments owing to the presence of much food yolk are very large, and the other where the segments are much smaller.

The separation between these two is rather sharp. Even this separation was foreshadowed in the Frog's egg, in which a number of lower layer cells were much smaller and more active at the two sides of the segmentation cavity than elsewhere. The segmentation cavity at first lies completely within the region of the small spheres. The larger cells serve almost entirely as food yolk. The epiblast, as is normal with vertebrates, consists of a single layer of columnar cells.

In the next stage (D II) the formation of the alimentary canal (al) has commenced, but it is to be observed that there is in this case no true involution.

As an accompaniment to the encroachment upon the segmentation cavity, which was a feature of the last stage, the cells to form the walls of the alimentary canal have come to occupy their final position during segmentation and without the intermediation of an involution, and traces only of the involution, are to be found in (1) a split in the lower layer cells which passes along the line separating the small and the large lower layer cells; and (2) in the epiblast becoming continuous with the hypoblast on the dorsal side of the mouth of this split. It is even possible that at this point a few cells (though certainly only a very small number) of those marked blue in D I become involuted. This point in this, as in all other cases, is the tail end of the embryo. The other features of this stage are as follows:—(1) The segmentation cavity has become smaller and less conspicuous than it was. (2) The epiblast cells have begun to grow round the yolk even in a more conspicuous manner than they did in the Frog, and are accompanied by a layer of mesoblast cells which again becomes thickened at its edge. The mesoblast cells in the region of the body are formed in the same way as before, viz. by the separation of a layer to form the epithelium of the alimentary canal, the other cells remaining as mesoblast; and as in the Frog, or in a more conspicuous manner, we find that the dorsal surface only of the alimentary cavity has a wall formed of a distinct layer of cells, but on the ventral side the cavity is at first closed in by the large spheres of the yolk only. The formation of the alimentary canal by a split and not by an involution is exactly what Bambeke finds in Pelobates.

The next stage, D III, is about an equivalent age to C III in the Frog. It exhibits the same connection between the neural and the alimentary canals as was found there.

The alimentary canal is beginning to become closed in below, and this occurs near the two ends earlier than in the middle. The cells to form the ventral wall are derived from the large yolk-cells. The non-formation of the ventral wall of the alimentary canal so soon in the middle as at the ends is an early trace of the umbilical canal found in Birds and Selachians, by which the alimentary tract is placed in communication with the yolk-sac. The segmentation cavity has by this stage completely vanished, and the epiblast with its accompanying mesoblast has spread completely round the yolk material so as to form the ventral wall of the body.

Though in some points this manner of development may seem to differ from that of the Frog, there is really a fundamental agreement between the two, and between this mode of development and that of the Selachians we shall find the agreement to be very close.

After segmentation we find that the egg of a Selachian consists of two parts—one of these called the germinal disc or blastoderm, and the other the yolk. The former of these corresponds with the epiblast and the part of the lower pole composed of smaller segments in the last-described egg, and the latter to the larger segments of the lower pole. This latter division, owing to the quantity of yolk which it contains, has not undergone segmentation, but its homology with the larger segments of the previous eggs is proved (1) by its containing a number of nuclei (E I, n), which become the nuclei of true cells and enter the blastoderm, and (2) by the presence in it of a number of lines forming a network similar to that of many cells. The segmentation cavity, as before, lies completely within the lower layer cells.

The next stage, E II, is almost precisely similar to the second stage of the last egg. As there, the primitive involution is merely represented by a split separating the yolk and the germinal disc, and on the dorsal side alone is there a true cellular wall for this split, and at the dorsal mouth of the split the alimentary epithelium becomes continuous with the epiblast.

The segmentation cavity has become diminished, and round the yolk the epiblast, accompanied by a layer of mesoblast, is commencing to grow. In this growth all parts of the blastoderm take a share except that part where the epiblast and hypoblast are continuous. This manner of growth is precisely what occurs in the Frog, though there it is not so easily made out; and not all the investigators who have studied the Frog have understood the exact meaning of the appearances they have seen and drawn. This similarity of relation of the epiblast to the yolk in the two cases is a further confirmation of the identity of the Selachian's yolk with the large yolk-spheres of the previous eggs.

The next stage, E III, is in many ways identical with the corresponding stage in the last-described egg, and in the same way as in that case the neural and alimentary canals are placed in communication with each other.

The mode in which this occurs will be easily gathered from a comparison of E II and E III. It is the same for the Selachians and Batrachians. The neural canal (nc) is by the stage figured E III , completely formed in the way so well known in the Bird, and between the roof of the canal and the external epiblast a layer of mesoblast has already grown in. The floor of the neural canal is the same layer marked ep in E II , and therefore remains continuous with the hypoblast at x; and when by a simultaneous process the roof of the neural canal and the ventral wall of the alimentary become formed by the folding over of one continuous layer (the epiblast and hypoblast continuous at the point x), the two canals, viz. the neural and alimentary, are necessarily placed in communication at their hind-ends, as is seen in the diagram.

There are several important points of difference between E III and D III. In the first place, owing to the larger size of the yolk mass in E III , the epiblast, accompanied by mesoblast, has not proceeded nearly so far round it as in the previous case. It is also worth notice that at the right as well as at the left end of the germinal disc the epiblast is commencing to grow round the yolk. The yolk has, however, become surrounded to a much smaller extent on the right hand than on the left. Since, in the earlier stage, the epiblast became continuous with the hypoblast at x, it is not from sections obvious how this occurs. I have therefore appended a diagram to explain it (E´). The blastoderm rests like a disc on the yolk and grows over it on all sides, except at the point where the epiblast and hypoblast are continuous (x). This point becomes as it were left in a bay. Next the two sides of the bay coalesce, the bay becomes obliterated, and the effect produced is exactly as if the blastoderm had grown round the yolk at the point x (corresponding with the tail of the embryo) as well as everywhere else. It thus comes about that the final point where the various parts of the blastoderm meet and completely enclose the yolk mass does not correspond with the anus of Rusconi of the Frog, but is at some little distance from the hind-end of the embryo. In other words, the position of the blastopore in the Selachian is not the same as in the Frog.

Another point deserving attention is the formation of the ventral wall of the alimentary canal. This takes place in two ways—partly by a folding-in at the sides and end, and partly from cells formed around the nuclei (n) in the yolk. From these a large portion of the ventral wall of the mid-gut is formed.

The folding-in of the sheet of hypoblast to assist in the closing-in of the ventral wall of the alimentary canal is a consequence of the flattened form of the original alimentary slit which is far too wide to form the cavity of the final canal. In the Bird whose development must next be considered this folding-in is a still more prominent feature in the formation of the alimentary canal. As in the last case, the alimentary canal is widely open in the middle to the yolk at the time when its two ends are closed below and shut off from it; still later this opening becomes very narrow and forms the duct of the so-called umbilical cord which places the yolk-sac in communication with the alimentary canal. As the young animal becomes larger the yolk-sac ceases to communicate directly with the alimentary canal, and is carried about by it for some time as an appendage and only at a later period shrivels up.

The mesoblast is formed in a somewhat different way in the Sharks than in other vertebrates. It becomes split off from the hypoblast, not in the form of a single sheet as in other vertebrates, but as two lateral sheets, one on each side of the middle line and separated from one another by a considerable interval; whilst the notochord is derived not as in other vertebrates from the mesoblast, but from the hypoblast (vide F. M. Balfour, Development of Selachians[21], Journal of Microscopical Science, Oct., 1874).

Between the Selachians and the Aves there is a considerable gulf, which it is more difficult satisfactorily to bridge over than in the previous cases; owing to this I have not attempted to give any intermediate stage between them.

The first stage of the Bird (F I) is very similar in many respects to the corresponding stage in the Selachian. The segmentation cavity is, however, a less well-defined formation, and it may even be doubted whether a true segmentation cavity, homologous with the segmentation cavity in the previously described eggs, is present. On the floor of the cavity which is formed by the yolk are a few larger cells known as formative cells which, according to Götte's observations, are derived from the yolk, in a somewhat similar manner to the cells which were formed around the nuclei in the Selachian egg, and which helped to form the ventral wall of the alimentary canal. Another point to be noticed is that the segmentation cavity occupies a central position, and not one to the side as in the Selachian.

The yolk is proportionately quite as large as in the Selachian's egg, but, as in that case, there can be little or no doubt of its being homologous with the largest of the segmentation spheres of the previous eggs. It does not undergo segmentation. The epiblast is composed of columnar cells, and extends a short way beyond the edge of the lower layer cells.

In the next stage the more important departures from the previous type of development become visible.

The epiblast spreads uniformly over the yolk-sac and not on the one side only as in the former eggs.

This is due to the embryo (indicated in F II by a thickening of the cells) lying in the centre and not at the edge of the blastoderm. A necessary consequence of this is, that the epiblast does not, as in the previous cases, become continuous with the hypoblast at the tail end of the embryo. This continuity, being of no functional importance, could easily be dispensed with, and the central position of the embryo may perhaps be explained by supposing the process, by which in the Selachian egg the blastopore ceases to correspond in position with the opening of the alimentary slit or anus of Rusconi (vide E´), to occur quite early during segmentation instead of at a late period of development. For the possibility of such a change in the date of formation, the early appearance of the nervous and epidermic layers in the Frog affords a parallel.

The epiblast in its growth round the yolk is only partially accompanied by mesoblast, which, however, is thickened at its extreme edge as in the Frog. Owing to the epiblast not becoming continuous with the hypoblast at the tail end of the embryo, the alimentary slit is not open to the exterior. The hypoblast is formed by some of the lower layer cells becoming distinguished as a separate layer; the remainder of the lower layer cells become the mesoblast.

The formation of the mesoblast and hypoblast out of the lower layer cells has been accepted for the Bird by most observers, but has been disputed by several, and recently by Kölliker. These have supposed that the mesoblast is derived from the epiblast. I feel convinced that these observers are in the wrong, and that the mesoblast is genuinely derived from the lower layer cells.

The greater portion of the alimentary cavity consists of the original segmentation cavity (vide diagrams). This feature of the segmentation cavity of Birds sharply distinguishes it from any segmentation cavity of other eggs, and renders it very doubtful whether the similarly named cavities of the Bird and of other vertebrates are homologous. On the floor of the cavity are still to be seen some of the formative cells, but observers have not hitherto found that they take any share in forming the ventral wall of the alimentary canal.

The features of the next stage are the necessary consequences of those of the last.

The ventral wall of the alimentary canal is entirely formed by a folding-in of the sheet of hypoblast.

The more rapid folding-in at the head still indicates the previous more vigorous growth there, otherwise there is very little difference between the forms of the fold at the head and tail. The alimentary canal does not of course, at this or any period, communicate with the neural tube, since the epiblast and hypoblast are never continuous. The other features, such as the growth of the epiblast round the yolk-sac, are merely continuations of what took place in the last stage.