ADAM SEDGWICK, M.A., (continued 2)

The investigations of Dursy (Der Primitivstreif des Hühnchens, von Dr E. Dursy. Lahr, 1866) on the primitive groove, shewing that it is a temporary structure, and not connected with the development of the neural canal, have in this country either been ignored or rejected. They are, nevertheless, perfectly accurate; and had Dursy made use of sections to support his statements I do not think they would so long have been denied. In Germany, it is true, Waldeyer has accepted them with a few modifications, but I have never seen them even alluded to in any English work. The observations which I have made corroborating Dr Dursy may, perhaps, under these circumstances be worth recording.

After about twelve hours of incubation the pellucid area of a hen's egg has become somewhat oval, with its longer axis at right angles to the long axis of the egg. Rather towards the hinder (narrower) end of this an opaque streak has appeared, with a somewhat lighter line in the centre. A section made at the time shews that the opaque streak is due partly to a thickening of the epiblast, but more especially to a large collection of the rounded mesoblast cells, which along this opaque line form a thick mass between the epiblast and the hypoblast. The mesoblast cells are in contact with both hypoblast and epiblast, and appear to be fused with the latter. The line of junction between them can, however, almost always be made out.

Soon after the formation of this primitive streak a groove is formed along its central line by a pushing inwards of the epiblast. The epiblast is not thinner where it lines the groove, but the mass of mesoblast below the groove is considerably thinner than at its two sides. This it is which produces the peculiar appearance of the primitive groove when the blastoderm is viewed by transmitted light as a transparent line in the middle of an opaque one.

This groove, as I said above, is placed at right angles to the long axis of the egg, and nearer the hind end, that is, the narrower end of the pellucid area. It was called the primitive groove by the early embryologists, and they supposed that the neural canal arose from the closure of its edges above. It is always easy to distinguish this groove, in transverse sections, by several well-marked characters. In the first place, the epiblast and mesoblast always appear more or less fused together underneath it; in the second place, the epiblast does not become thinner where it lines the groove; and in the third place, the mesoblast beneath it never shews any signs of being differentiated into any organ.

As Dursy has pointed out, there is frequently to be seen in fresh specimens, examined as transparent objects, a narrow opaque line running down the centre of this groove. I do not know what this line is caused by, as there does not appear to be any structural feature visible in sections to which it can correspond.

From the twelfth to the sixteenth hour the primitive groove grows rapidly, and by the sixteenth hour is both absolutely and considerably longer than it was at the twelfth hour, and also proportionately longer as compared with the length of the pellucid area.

There is a greater interval between its end and that of the pellucid area in front than behind.

At about the sixteenth hour, or a little later, a thickening of the mesoblast takes place in front of the primitive groove, forming an opaque streak, which in fresh specimens looks like a continuation from the anterior extremity of the primitive groove (vide Pl. 1, fig. 8). From hardened specimens, however, it is easy to see that the connection of this streak with the primitive groove is only an apparent one. Again, it is generally possible to see that in the central line of this streak there is a narrow groove. I do not feel certain that there is no period when this groove may not be present, but its very early appearance has not been recognized either by Dursy or by Waldeyer. Moreover, both these authors, as also His, seem to have mistaken the opaque streak spoken of above for the notochord. This, however, is not the case, and the notochord does not make its appearance till somewhat later. The mistake is of very minor importance, and probably arose in Dursy's case from his not sufficiently making use of sections. At about the time the streak in front of the primitive groove makes its appearance a semicircular fold begins to be formed near the anterior extremity of the pellucid area, against which the opaque streak, or as it had, perhaps, better be called, the medullary streak, ends abruptly.

This fold is the head fold, and the groove along the medullary streak is the medullary groove, which subsequently forms the cavity of the medullary or neural canal.

Everything which I have described above can without difficulty be made out from the examination of fresh and hardened specimens under the simple microscope; but sections bring out still more clearly these points, and also shew other features which could not have been brought to light without their aid. In Pl. 1, figs. 6 and 7, two sections of an embryo of about eighteen hours are shewn. The first of these passes through the medullary groove, and the second of them through the extreme anterior end of the primitive groove. The points of difference in the two sections are very obvious.

From fig. 6 it is clear that a groove has already been formed in the medullary streak, a fact which was not obvious in the fresh specimen. In the second place the mesoblast is thickened both under the groove and also more especially in the medullary folds at the sides of the groove; but shews hardly a sign of the differentiation of the notochord. So that it is clear that the medullary streak is not the notochord, as was thought to be the case by the authors above mentioned. In the third place there is no adhesion between the epiblast and the mesoblast. In all the sections I have cut through the medullary groove I have found this feature to be constant; while (for instance, as in Pl. 1, figs. 7, 9, 17) all sections through the primitive groove shew most clearly an adhesion between the epiblast and mesoblast. This fact is both strongly confirmatory of the separate origins of the medullary and primitive grooves, and is also important in itself, as leaving no loophole for supposing that in the region of embryo there is any separation of the cells from the epiblast to form the mesoblast.

By this time the primitive groove has attained its maximum growth, and from this time begins both absolutely to become smaller, and also gradually to be pushed more and more backwards by the growth of the medullary groove.

The specimen figured in Pl. 1, fig. 18, magnified about ten diameters, shews the appearance presented by an embryo of twenty-three hours. The medullary groove (mc) has become much wider and deeper than it was in the earlier stage; the medullary folds (A) are also broader and more conspicuous. The medullary groove widens very much posteriorly, and also the medullary folds separate far apart to enclose the anterior end of the primitive groove (pr).

All this can easily be seen with a simple microscope, but the sections taken from the specimen figured also fully bear out the interpretations given above, and at the same time shew that the notochord has at this age begun to appear. The sections marked 13-17 pass respectively through the lines with corresponding numbers in fig. 18. Section 1 (fig. 13) passes through the middle of the medullary canal.

In it the following points are to be noted. (1) That the epiblast becomes very much thinner where it lines the medullary canal (mc), a feature never found in the epiblast lining the primitive groove. (2) That the mesoblast is very much thickened to form the medullary folds at A, A, while there is no adherence between it and the epiblast, below the primitive groove. (3) The notochord (ch) has begun to be formed, though its separation from the rest of the mesoblast is not as yet very distinct[8].

In fig. 14 the medullary groove has become wider and the medullary folds broader, the notochord has also become more expanded: the other features are the same as in section 1. In the third section (fig. 15) the notochord is still more expanded; the bottom of the now much expanded medullary groove has become raised to form the ridge which separates the medullary from the primitive groove. The medullary folds are also flatter and broader than in the previous section. Section 4 (fig. 16) passes through the anterior end of the primitive groove. Here the notochord is no longer visible, and the adherence between the mesoblast and epiblast below the primitive groove comes out in marked contrast with the entire separation of the two layers in the previous sections.

The medullary folds (A) are still visible outside the raised edges of the primitive groove, and are as distinctly as possible separate and independent formations, having no connection with the folds of the primitive groove. In the last section (fig. 17), which is taken some way behind section 4, no trace of the medullary folds is any longer to be seen, and the primitive groove has become deeper. This series of sections, taken in conjunction with the specimen figured in fig. 18, must remove all possible doubt as to the total and entire independence of the primitive and medullary grooves. They arise in different parts of the blastoderm; the one reaches its maximum growth before the other has commenced to be formed; and finally, they are distinguished by almost every possible feature by which two such grooves could be distinguished.

Soon after the formation of the notochord, the protovertebræ begin to be formed along the sides of the medullary groove (Pl. 1, fig. 19, pv). Each new protovertebra (of those which are formed from before backwards) arises just in front of the anterior end of the primitive groove. As growth continues, the primitive groove becomes pushed further and further back, and becomes less and less conspicuous, till at about thirty-six hours only a very small and curved remnant is to be seen behind the sinus rhomboidalis; but even up to the forty-ninth Dursy has been able to distinguish it at the hinder end of the embryo.

The primitive groove in the chick is, then, a structure which appears very early, and soon disappears without entering directly into the formation of any part of the future animal, and without, so far as I can see, any function whatever. It is clear, therefore, that the primitive groove must be the rudiment of some ancestral feature; but whether it is a rudiment of some structure which is to be found in reptiles, or whether of some earlier form, I am unable to decide. It is just possible that it is the last trace of that involution of the epiblast by which the hypoblast is formed in most of the lower animals. The fact that it is formed in the hinder part of the pellucid area perhaps tells slightly in favour of this hypothesis, since the point of involution of the epiblast not unfrequently corresponds with the position of the anus.

EXPLANATION OF PLATE 1, Figs. 6-8 and 13-19.

Figs. 6 and 7 are sections through an embryo rather earlier than the one drawn in fig. 8. Fig. 6 passes through the just commencing medullary groove (md), which appears in fresh specimens, as in fig. 8, merely as an opaque streak coming from the end of the primitive groove. The notochord is hardly differentiated, but the complete separation of mesoblast and hypoblast under the primitive groove is clearly shewn. Fig. 7 passes through the anterior end of the primitive groove (pr), and shews the fusion between the mesoblast and epiblast, which is always to be found under the primitive groove.

Fig. 8 is a view from above of a twenty hours' blastoderm, seen as a transparent object. Primitive groove (pr). Medullary groove (md), which passes off from the anterior end of the primitive groove, and is produced by the thickening of the mesoblast. Head fold (pf).

Figs. 13-17 are sections through the blastoderm, drawn in fig. 18 through the lines 1, 2, 3, 4, 5 respectively.

The first section (fig. 13) passes through the true medullary groove (mc); the two medullary folds (A, A) are seen on each side with the thickened mesoblast, and the mesoblast cells are beginning to form the notochord (nc) under the medullary groove. There is no adherence between the mesoblast cells and the epiblast under the medullary groove.

The second (fig. 14) section passes through the medullary groove where it has become wider. Medullary folds, A, A; notochord, ch.

In the third section (fig. 15) the notochord (ch) is broader, and the epiblast is raised in the centre, while the medullary folds are seen far apart at A.

In section fig. 16 the medullary folds (A) are still to be seen enclosing the anterior end of the primitive groove (pr). Where the primitive groove appears there is a fusion of the epiblast and mesoblast, and no appearance of the notochord.

In the last section, fig. 17, no trace is to be seen of the medullary folds.

Figs. 18 and 19 are magnified views of two hardened blastoderms. Fig. 18 is twenty-three hours old; fig. 19 twenty-five hours. They both shew how the medullary canal arises entirely independently of the primitive groove and in front of it, and also how the primitive groove gets pushed backwards by the growth of the medullary groove. pv, Protovertebræ; other references as above. Fig. 18 is the blastoderm from which sections figs. 13-17 were cut.

IV. The Development of the Blood-vessels of the Chick[9].

With Plate 2.

The development of the first blood-vessels of the yolk-sac of the chick has been investigated by a large number of observers, but with very discordant results. A good historical résumé of the subject will be found in a paper of Dr Klein (liii. Band der K. Akad. der Wissensch. Wien), its last investigator.

The subject is an important one in reference to the homologies of the blood-vascular system of the vertebrata. As I shall shew in the sequel (and on this point my observations agree with those of Dr Klein), the blood-vessels of the chick do not arise as spaces or channels between the cells of the mesoblast; on the contrary, they arise as a network formed by the united processes of mesoblast-cells, and it is through these processes, and not in the spaces between them, that the blood flows. It is, perhaps, doubtful whether a system of vessels arising in this way can be considered homologous with any vascular system which takes its origin from channels hollowed out in between the cells of the mesoblast.

My own researches chiefly refer to the development of the blood-vessels in the pellucid area. I have worked but very slightly at their development in the vascular area; but, as far as my observations go, they tend to prove that the mode of their origin is the same, both for the pellucid and the vascular area.

The method which I have principally pursued has been to examine the blastoderm from the under surface. It is very difficult to obtain exact notions of the mode of development of the blood-vessels by means of sections, though these come in as a valuable confirmation of the other method.

For the purpose of examination I have employed (1) fresh specimens; (2) specimens treated with spirit, and then mounted in glycerine; (3) specimens treated with chloride of gold for about half a minute, and then mounted in glycerine; and (4) specimens treated with osmic acid.

All these methods bring out the same appearances with varying clearness; but the successful preparations made by means of the gold chloride are the best, and bring out the appearances with the greatest distinctness.

The first traces of the blood-vessels which I have been able to distinguish in the pellucid area are to be seen at about the thirtieth hour or slightly earlier, at about the time when there are four to five protovertebræ on each side.

Fig. 1 shews the appearance at this time. Immediately above the hypoblast there are certain cells whose protoplasm sends out numerous processes. These processes vary considerably in thickness and size, and quickly come in contact with similar processes from other cells, and unite with them.

I have convinced myself, by the use of the hot stage, that these processes continually undergo alteration, sometimes uniting with other processes, sometimes becoming either more elongated and narrower or broader and shorter. In this way a network of somewhat granular protoplasm is formed with nuclei at the points from which the processes start.

From the first a difference may be observed in the character of this network in different parts of the pellucid area. In the anterior part the processes are less numerous and thicker, the nuclei fewer, and the meshes larger; while in the posterior part the processes are generally very numerous, and at first thin, the meshes small, and the nuclei more frequent. As soon as this network commences to be formed the nuclei begin to divide. I have watched this take place with the hot stage. It begins by the elongation of the nucleus and division of the nucleolus, the parts of which soon come to occupy the two ends of the nucleus. The nucleus becomes still longer and then narrows in the centre and divides. By this means the nuclei become much more numerous, and are found in almost all the larger processes. Whether they are carried out into the processes by the movement of the surrounding protoplasm, or whether they move through the protoplasm, I have been unable to determine; the former view, however, seems to be the most probable.

It is possible that some nuclei arise spontaneously in the protoplasm, but I am much more inclined to think that they are all formed by the division of pre-existing nuclei—a view favoured by the number of nuclei which are seen to possess two nucleoli. Coincidently with the formation of the new nuclei the protoplasm of the processes, as well as that surrounding the nuclei at the starting-points of the processes, begins to increase in quantity.

At these points the nuclei also increase more rapidly than elsewhere, but at first the resulting nuclei seem to be all of the same kind.

In the anterior part of the pellucid area (fig. 4) the increase in the number of nuclei and in the amount of protoplasm at the starting-points of the protoplasm is not very great, but in the posterior part the increase in the amount of the protoplasm at these points is very marked, and coincidently the increase in number of the nuclei is also great. This is shewn in figs. 2 and 3. These are both taken from the tail end of an embryo of about thirty-three hours, with seven or eight protovertebræ. Fig. 3 shews the processes beginning to increase in thickness, and also the protoplasm at the starting-points increasing in quantity; at the same time the nuclei at these points are beginning to become more numerous. Fig. 3 is taken from a slightly higher level, i.e. slightly nearer the epiblast. In it the protoplasm is seen to have increased still more in quantity, and to be filled with nuclei. These nuclei have begun to be slightly coloured, and one of them is seen to possess two nucleoli.

Very soon after this a change in the nuclei begins to be observed, more especially in the hinder part of the embryo. While before this time they were generally elongated, some of them now become more nearly circular. In addition to this, they begin to have a yellowish tinge, and the nuclei, when treated with gold (for in the fresh condition it is not easy to see them distinctly), have a more jagged and irregular appearance than the nucleoli of the other nuclei.

This change takes place especially at the starting-points of the processes, so that the appearance presented (fig. 5) is that of spherical masses of yellowish nuclei connected with other similar spherical masses by protoplasmic processes, in which nuclei of the original type are seen imbedded. These masses are surrounded by a thin layer of protoplasm, at the edge of which a normal nucleus may here and there be detected, as at fig. 5, a and a´, the latter possessing two nucleoli. Some of these processes are still very delicate, and it is exceedingly probable that they undergo further changes of position before the final capillary system is formed.

These differentiated nuclei are the first stage in the formation of the blood-corpuscles. From their mode of formation it is clear that the blood-corpuscles of the Sauropsida are to be looked upon as nuclei containing nucleoli, rather than as cells containing nuclei; indeed, they seem to be merely ordinary nuclei with red colouring matter.

This would make them truly instead of only functionally homologous with the red corpuscles of the Mammalia, and would well agree with the fact that the red corpuscles of Mammalia, in their embryonic condition, possess what have previously been called nuclei, but which might perhaps more properly be called nucleoli.

In the anterior part of the blastoderm the processes, as I have stated, are longer and thinner, and the spaces enclosed between them are larger. This is clearly brought out in Pl. 2, fig. 4. But, besides these large spaces, there are other smaller spaces, such as that at v. It is, on account of the transparency of the protoplasm, very difficult to decide whether these are vacuoles or simply spaces enclosed by the processes, but I am inclined to think that they are merely spaces. The difficulty of exactly determining this point is increased by the presence of numerous white-yolk spherules in the hypoblast above, which considerably obscure the view. At about the same time that the blood-corpuscles appear in the posterior end of the pellucid area, or frequently a little later, they begin to be formed in the anterior part also. The masses of them are, however, far smaller and far fewer than in the posterior part of the embryo. It is at the tail end of the pellucid area that the chief formation of blood-corpuscles takes place.

The part of the pellucid area intermediate in position between the anterior and posterior ends of the embryo is likewise intermediate as regards the number of corpuscles formed and the size of the spaces between the processes; the spaces being here larger than at the posterior extremity, but smaller than the spaces in front. Close to the sides of the embryo the spaces are, however, smaller than in any other part of the pellucid area. It is, however, in this part that the first formation of blood-corpuscles takes place, and that the first complete capillaries are formed.

We have then somewhat round protoplasmic masses filled with blood-corpuscles and connected by means of processes, a few of which may begin to contain blood-corpuscles, but the majority of which only contain ordinary nuclei. The next changes to be noticed take place in the nuclei which were not converted into blood-corpuscles, but which were to be seen in the protoplasm surrounding the corpuscles. They become more numerous and smaller, and, uniting with the protoplasm in which they were imbedded, become converted into flat cells (spindle-shaped in section), and in a short time form an entire investment for the masses of blood-corpuscles. The same change also occurs in the protoplasmic processes which connect the masses of corpuscles. In the case of those processes which contain no corpuscles the greater part of their protoplasm seems to be converted into the protoplasm of the spindle-shaped cells. The nuclei arrange themselves so as completely to surround the exterior of the protoplasmic processes. In this way each process becomes converted into a hollow tube, completely closed in by cells formed from the investment of the original nuclei by the protoplasm which previously formed the solid processes. The remainder of the protoplasm probably becomes fluid, and afterwards forms the plasma in which the corpuscles float. While these changes are taking place the formation of the blood-corpuscles does not stand still, and by the time a system of vessels, enclosed by cellular walls, is formed out of the protoplasmic network, a large number of the connecting processes in this network have become filled with blood-corpuscles. The appearances presented by the network at a slightly later stage than this is shewn in Pl. 2, fig. 6, but in this figure all the processes are seen to be filled with blood-corpuscles.

This investment of the masses of corpuscles by a cellular wall occurs much earlier in some specimens than in others, both in relation to the time of incubation and to the completion of the network. It is generally completed in some parts by the time there are eight or nine protovertebræ, and is almost always formed over a great part of the pellucid area by the thirty-sixth hour. The formation of the corpuscles, as was pointed out above, occurs earliest in the central part of the hour-glass shaped pellucid area, and latest in its anterior part. In the hinder part of the pellucid area the processes, as well as their enlarged starting-points, become entirely filled with corpuscles; this, however, is by no means the case in its anterior part. Here, although the corpuscles are undoubtedly developed in parts as shewn in fig. 7, yet a large number of the processes are entirely without them. Their development, moreover, is in many cases very much later. When the development has reached the stage described, very little is required to complete the capillary system. There are always, of course, a certain number of the processes which end blindly, and others are late in their development, and are not by this time opened; but, as a general rule, when the cellular investment is formed for the masses of corpuscles, there is completed an open network of tubes with cellular walls, which are more or less filled with corpuscles. These become quickly driven into the opaque area in which at that time more corpuscles may almost always be seen than in the pellucid area.

By the formation of a network of this kind it is clear that there must result spaces enclosed between the walls of the capillaries; these spaces have under the microscope somewhat the appearance of being vesicles enclosed by walls formed of spindle-shaped cells. In reality they are only spaces enclosed at the sides, and, as a general rule, not above and below. They have been mistaken by some observers for vesicles in which the corpuscles were supposed to be developed, and to escape by the rupture of the walls into the capillary spaces between. This mistake has been clearly pointed out by Klein (loc. cit.).

At the time when these spaces are formed, and especially in the hinder two-thirds of the pellucid area, and in the layer of blood-vessels immediately above the hypoblast, a formation takes place which forms in appearance a secondary investment of the capillaries. Dr Klein was the first to give a correct account of this formation. It results from the cells of the mesoblast in the meshes of the capillary system. Certain of these cells become flattened, and send out fine protoplasmic processes. They arrange themselves so as completely to enclose the spaces between the capillaries, forming in this way vesicles.

Where seen on section (vide fig. 6) at the edge of the vesicles these cells lining the vesicles appear spindle-shaped, and look like a secondary investment of the capillaries. This investment is most noticeable in the hinder two-thirds of the pellucid area; but, though less conspicuous, there is a similar formation in its anterior third, where there would seem to be only veins present. Dr Klein (loc. cit., fig. 12) has also drawn this investment in the anterior third of the pellucid area. He has stated that the vessels in the mesoblast between the splanchnopleure and the somatopleure, and which are enclosed by prolongations from the former, do not possess this secondary investment; he has also stated that the same is true for the sinus terminalis; but I am rather doubtful whether the generalisation will hold, that veins and arteries can from the first be distinguished by the latter possessing this investment. I am also rather doubtful whether the spaces enclosed by the protoplasmic threads between the splanchnopleure and somatopleure are the centres of vessels at all, since I have never seen any blood-corpuscles in them.

It is not easy to learn from sections much about the first stages in the formation of the capillaries, and it is impossible to distinguish between a completely-formed vessel and a mere spherical space. The fine protoplasmic processes which connect the masses of corpuscles can rarely be seen in sections, except when they pass vertically, as they do occasionally (vide Pl. 2, fig. 9) in the opaque area, joining the somatopleure and the splanchnopleure. Dr Klein considers these latter processes to be the walls of the vessels, but they appear rather to be the processes which will eventually become new capillaries.

From sections, however, it is easy to see that the appearances of the capillaries in the vascular area are similar to the appearances in the pellucid area, from which it is fair to conclude that their mode of formation is the same in both. It is also easy to see that the first formation of vessels occurs in the splanchnopleure, and that even up to the forty-fifth hour but few or no vessels are found in the somatopleure. The mesoblast of the somatopleure is continued into the opaque area as a single layer of spindle-shaped cells.

Sections clearly shew in the case of most of the vessels that the secondary investment of Klein is present, even in the case of those vessels which lie immediately under the somatopleure.

In reference to the origin of particular vessels I have not much to say. Dr Klein's account of the origin of the sinus terminalis is quite correct. It arises by a number of the masses of blood-corpuscles, similar to those described above, becoming connected together by protoplasmic processes. The whole is subsequently converted into a continuous vessel in the usual way.

From the first the sinus terminalis possesses cellular walls, as is clear from its mode of origin. I am inclined to think that Klein is right in saying that the aortæ arise in a similar manner, but I have not worked out their mode of origin very fully.

It will be seen from the account given above that, in reference to the first stages in the development of the blood-vessels, my observations differ very considerably from those of Dr Klein; as to the later stages, however, we are in tolerable agreement. We are in agreement, moreover, as to the fundamental fact that the blood-vessels are formed by a number of cells becoming connected, and by a series of changes converted into a network of vessels, and that they are not in the first instance merely channels between the cells of the mesoblast.

By the forty-fifth hour colourless corpuscles are to be found in the blood whose exact origin I could not determine; probably they come from the walls of the capillaries.

In the vessels themselves the coloured corpuscles undergo increase by division, as has already been shewn by Remak. Corpuscles in the various stages of division may easily be found. They do not appear to show very active amœboid movements in the vessels, though their movements are sometimes very active when removed from the body.

To recapitulate—some of the cells of the mesoblast of the splanchnopleure send out processes, these processes unite with the processes from other cells, and in this way a network is formed. The nuclei of the original cells divide, and at the points from which the processes start their division is especially rapid. Some of them acquire especially at these points a red colour, and so become converted into blood-corpuscles; the others, together with part of the protoplasm in which they are imbedded, become converted into an endothelium both for the processes and the masses of corpuscles; the remaining protoplasm becomes fluid, and thus the original network of the cells becomes converted into a network of hollow vessels, filled with fluid, in which corpuscles float.

In reference to the development of the heart, my observations are not quite complete. It is, however, easy to prove from sections (vide figs. 10 and 11, Pl. 2) that the cavity of the heart is produced by a splitting or absorption of central cells of the thickened mesoblast of the splanchnopleure, while its muscular walls are formed from the remaining cells of this thickened portion. It is produced in the following way:—When the hypoblast is folded in to form the alimentary canal the mesoblast of the splanchnopleure follows it closely, and where the splanchnopleure turns round to assume its normal direction (fig. 11) its mesoblast becomes thickened. This thickened mass of mesoblast is, as can easily be seen from figs. 10 and 11, Pl. 2, entirely distinct from the mesoblast which forms the outside walls of the alimentary canal. At the point where this thickening occurs an absorption takes place to form the cavity of the heart. The method in which the cavity is formed can easily be seen from figs. 10 and 11. It is in fig. 11 shewn as it takes place in the mesoblast on each side, the folds of the splanchnopleure not having united in the middle line; and hence a pair of cavities are formed, one on each side. It is, however, probable that, in the very first formation of the heart, the cavity is single, being formed after the two ends of the folded mesoblast have united (vide hz, fig. 10). In some cases the two folds of the mesoblast appear not at first to become completely joined in the middle line; in this case the cavity of the heart is still complete from side to side, but the mesoblast-cells which form its muscular walls are deficient above. By the process of absorption, as I said, a cavity is produced in the thickened part of the mesoblast of the splanchnopleure, a cavity which is single in front, but becomes divided further behind, where the folds of the mesoblast have not united, into two cavities, to form the origin of the omphalomeseraic veins. As the folding proceeds backwards the starting-point of the omphalomeseraic veins is also pushed backwards, and the cavities which were before separated become joined together. From its first formation the heart is lined internally by an endothelium; this is formed of flattened cells, spindle-shaped in section. The exact manner of the origin of this lining I have not been able to determine; it is, however, probable that some of the central mesoblast-cells are directly converted into the cells of the endothelium.

I have obtained no evidence enabling me to determine whether Dr Klein is correct in stating that the cells of the mesoblast in the interior of the heart become converted partly into blood-corpuscles and partly into a cellular lining forming the endothelium of the heart, in the same way that the blood-vessels in the rest of the blastoderm are formed. But I should be inclined to think that it is very probable—certainly more probable than that the cavity of the heart is formed by a process of splitting taking place. Where I have used the word absorption in speaking of the formation of the cavity of the heart, I must be understood as implying that certain of the interior cells become converted into the endothelium, while others either form the plasma or become blood-corpuscles.

The originally double formation of the hinder part of the heart probably explains Dr Afanassiev's statement (Bullétin de l'Académ. Impériale de St Pétersb., tom. xiii, pp. 321-335), that he finds the endothelium of the heart originally dividing its interior into two halves; for when the partition of the mesoblast which separated at first the two halves of the heart became absorbed, the endothelium lining of each of the originally separate vessels would remain complete, dividing the cavity of the heart into two parts. The partition in the central line is, however, soon absorbed.

The account given above chiefly differs from that of Remak by not supposing that the mesoblast-cells which form the heart are in any way split off from the wall of the alimentary canal.

There can be no doubt that His is wrong in supposing that the heart originates from the mesoblast of the splanchnopleure and somatopleure uniting to form its walls, thus leaving a cavity between them in the centre. The heart is undoubtedly formed out of the mesoblast of the splanchnopleure only.

Afanassiev's observations are nearer to the truth, but there are some points in which he has misinterpreted his sections.

Sections Pl. 2, figs. 10 and 11, explain what I have just said about the origin of the heart. Immediately around the notochord the mesoblast is not split, but a very little way outside it is seen to be split into two parts so and sp; the former of these follows the epiblast, and together with it forms the somatopleure, which has hardly begun to be folded at the line where the sections are taken. The latter (sp) forms with the hypoblast (hy) the splanchnopleure, and thus has become folded in to form the walls of the alimentary canal (d). In fig. 11 the folds have not united in the central line, but in fig. 10 they have so united. In fig. 11, where the mesoblast, still following the hypoblast, turns back to assume its normal direction, it is seen to be thickened and to have become split, so that a cavity (of) (of the omphalomeseraic vein) is formed in it on each side, lined by endothelium.

In the section immediately behind section fig. 11 the mesoblast was thickened, but had not become split.

In fig. 10 the hypoblast folds are seen to have united in the centre, so as to form a completely closed digestive canal (d); the folds of the mesoblast have also united, so that there is only a single cavity in the heart (hz), lined, as was the case with the omphalomeseraic veins, by endothelium.

In conclusion, I have to thank Dr Foster for his assistance and suggestions throughout the investigations which have formed the subject of these three short papers, and which were well carried on in the apartments used by him as a Physiological Laboratory.

EXPLANATION OF PLATE 2.

Fig. 1 is taken from the anterior part of the pellucid area of a thirty hours' chick, with four protovertebræ. At n is a nucleus with two nucleoli.

Figs. 2 and 3 are taken from the posterior end of the pellucid area of a chick with eight protovertebræ. In fig. 3 the nuclei are seen to have considerably increased in number at the points of starting of the protoplasmic processes. At n is seen a nucleus with two nucleoli.

Fig. 4 is taken from the anterior part of the pellucid area of an embryo of thirty-six hours. It shews the narrow processes characteristic of the anterior part of the pellucid area, and the fewer nuclei. Small spaces, which have the appearance of vacuoles, are shewn at v.

Fig. 5 is taken from the posterior part of the pellucid area of a thirty-six hours' embryo. It shews the nuclei, with somewhat irregular nucleoli, which have begun to acquire the red colour of blood-corpuscles; the protoplasmic processes containing the nuclei; the nuclei in the protoplasm surrounding the corpuscles, as shewn at a, a´.

Fig. 6 shews fully formed blood-vessels, in part filled with blood-corpuscles and in part empty. The walls of the capillaries, formed of cells, spindle-shaped in section, are shewn, and also the secondary investment of Klein at k, and at b is seen a narrow protoplasmic process filled with blood-corpuscles.

Fig. 7 is taken from the anterior part of the pellucid area of a thirty-six hours' embryo. It shews a collection of nuclei which are beginning to become blood-corpuscles.

Figs. 1-5 are drawn with an 1/8 object-glass. Fig. 6 is on a much smaller scale. Fig. 7 is intermediate.

Fig. 8.—A transverse section through the dorsal region of a forty-five hours' embryo; ao, aorta with a few blood-corpuscles. v, Blood-vessels, all of them being formed in the splanchnopleure, and all of them provided with the secondary investment of Klein; pe, pellucid area; op, opaque area.

Fig. 9.—Small portion of a section through the opaque area of a thirty-five hours' embryo, showing protoplasmic processes, with nuclei passing from the somatopleure to the splanchnopleure.

Fig. 10.—Section through the heart of a thirty-four hours' embryo. a. Alimentary canal; hb, hind brain; nc, notochord; e, epiblast; so, mesoblast of the somatopleure; sp, mesoblast of the splanchnopleure; hy, hypoblast; hz, cavity of the heart.

Fig. 11.—Section through the same embryo as fig. 10, and passing through the orifice of the omphalomeseraic vein. of, Omphalomeseraic vein; other references as above.

These two sections shew that the heart is entirely formed from the mesoblast of the splanchnopleure, and that it is formed by the splitting of that part of the mesoblast which has turned to assume its normal direction after being folded in to form the muscular wall of the alimentary canal. In fig. 11 the cavities so formed on each side have not yet united, but in fig. 10 they have united. When the folding becomes more complete the cavities (of, of) in fig. 11 will unite, and in this way the origin of the omphalomeseraic veins will be carried further backwards. In the section immediately behind section 11 the mesoblast had become thickened, but had not split.

V. A Preliminary Account of the Development of the Elasmobranch Fishes[10].

With Plates 3 and 4.

During the spring of the present year I was studying at the Zoological Station, founded by Dr Dohrn at Naples, and entirely through its agency was supplied with several hundred eggs of various species of Dog-fish (Selachii)—a far larger number than any naturalist has previously had an opportunity of studying. The majority of the eggs belonged to an oviparous species of Mustelus, but in addition to these I had a considerable number of eggs of two or three species of Scyllium, and some of the Torpedo. Moreover, since my return to England, Professor Huxley has most liberally given me several embryos of Scyllium stellare in a more advanced condition than I ever had at Naples, which have enabled me to fill up some lacunæ in my observations.

On many points my investigations are not yet finished, but I have already made out a number of facts which I venture to believe will add to our knowledge of vertebrate embryology; and since it is probable that some time will elapse before I am able to give a complete account of my investigations, I have thought it worth while preparing a preliminary paper in which I have briefly, but I hope in an intelligible manner, described some of the more interesting points in the development of the Elasmobranchii. The first-named species (Mustelus sp.?) was alone used for the early stages, for the later ones I have also employed the other species, whose eggs I have had; but as far as I have seen at present, the differences between the various species in early embryonic life are of no importance.

Without further preface I will pass on to my investigations.

The Egg-shell.

In the eggs of all the species of Dog-fishes which I have examined the yolk lies nearest that end of the quadrilateral shell which has the shortest pair of strings for attachment. This is probably due to the shape of the cavity of the shell, and is certainly not due to the presence of any structures similar to chalazæ.

The Yolk.

The yolk is not enclosed in any membrane comparable to the vitelline membrane of Birds, but lies freely in a viscid albumen which fills up the egg-capsule. It possesses considerable consistency, so that it can be removed into a basin, in spite of the absence of a vitelline membrane, without falling to pieces. This consistency is not merely a property of the yolk-sphere as a whole, but is shared by every individual part of it.

With the exception of some finely granular matter around the blastoderm, the yolk consists of rather small, elliptical, highly refracting bodies, whose shape is very characteristic and renders them easily recognizable. A number of striæ like those of muscle are generally visible on most of the spherules, which give them the appearance of being in the act of breaking up into a series of discs; but whether these striæ are normal, or produced by the action of water I have not determined.

Position of the Blastoderm.

The blastoderm is always situated, immediately after impregnation, near the pole of the yolk which lies close to the end of the egg-capsule. Its position varies a little in the different species and is not quite constant in different eggs of the same species. But this general situation is quite invariable. It is of about the same proportional size as the blastoderm of a bird.

Segmentation.

In a fresh specimen, in which segmentation has only just commenced, the blastoderm or germinal disc appears as a circular disc, distinctly marked off by a dark line from the rest of the yolk. This line, as is proved by sections, is the indication of a very shallow groove. The appearance of sharpness of distinction between the germ and the yolk is further intensified by their marked difference of colour, the germ itself being usually of a darker shade than the remainder of the yolk; while around its edge, and apparently sharply separated from it by the groove before mentioned, is a ring of a different shade which graduates at its outer border into the normal shade of the yolk.

These appearances are proved by transverse sections to be deceptive. There is no sharp line either at the sides or below separating the blastoderm from the yolk. In the passage between the fine granular matter of the germ to the coarser yolk-spheres every intermediate size of granule is present; and, though the space between the two is rather narrow, in no sense of the word can there be said to be any break or line between them.

This gradual passage stands in marked contrast with what we shall find to be the case at the close of the segmentation. In the youngest egg which I had, the germinal disc was already divided into four segments by two furrows at right angles. These furrows, however, did not reach its edge; and from my sections I have found that they were not cut off below by any horizontal furrow. So that the four segments were continuous below with the remainder of the germ without a break.

In the next youngest specimen which I had, there were already present eighteen segments, somewhat irregular in size, but which might roughly be divided into an outer ring of larger spheres, separated, as it were, by a circular furrow from an inner series of smaller segments. The furrows in this case reached quite to the edge of the germinal disc.

The remarks I made in reference to the earlier specimen about the separation of the germ from the yolk apply in every particular to the present one. The external limit of the blastoderm was not defined by a true furrow, and the segmentation furrows still ended below without meeting any horizontal furrows, so that the blastoderm was not yet separated by any line from the remainder of the yolk, and the segments of which it was composed were still only circumscribed upon five sides. In this particular the segmentation in these animals differs materially from that in the Bird, where the horizontal furrows appear very early.

In each segment a nucleus was generally to be seen in sections. I will, however, reserve my remarks upon the nature of the nuclei till I discuss the nuclei of the blastoderm as a whole.

For some little time the peripheral segments continue larger than the more central ones, but this difference of size becomes less and less marked, and before the segments have become too small to be seen with the simple microscope, their size appears to be uniform over the whole surface of the blastoderm.

In the blastoderms somewhat older than the one last described the segments have already become completely separate masses, and each of them already possesses a distinct nucleus. They form a layer one or two segments deep. The limits of the blastoderm are not, however, defined by the already completed segments, but outside these new segments continue to be formed around nuclei which appear in the yolk. At this stage there is, therefore, no line of demarcation between the germ and the yolk, but the yolk is being bored into, so to speak, by a continuous process of fresh segmentation.

The further segmentation of the already existing spheres, and the formation of new ones from the yolk below and to the sides, continues till the central cells acquire their final size, the peripheral ones being still large, and undefined towards the yolk. These also soon reach the final size, and the blastoderm then becomes rounded off towards the yolk and sharply separated from it.

The Nuclei of the Yolk.

Intimately connected with the segmentation is the appearance and history of a number of nuclei which arise in the yolk surrounding the blastoderm.

When the horizontal furrows appear which first separate the blastoderm from the yolk, the separation does not occur along the line of passage from the fine to the coarse yolk, but in the former at some distance from this line.

The blastoderm thus rests upon a mass of finely granular material, from which, however, it is sharply separated. At this time there appear in this finely granular material a number of nuclei of a rather peculiar character.

They vary immensely in size—from that of an ordinary nucleus to a size greater than the largest blastoderm-cell.

In Pl. 3, fig. 1, n, is shewn their distribution in this finely granular matter and their variation in size. But whatever may be their size, they always possess the same characteristic structure. This is shewn in Pl. 3, figs. 1 and 2, n.

They are rather irregular in shape, with a tendency when small to be roundish, and are divided by a number of lines into distinct areas, in each of which a nucleolus is to be seen. The lines dividing them into these areas have a tendency (in the smaller specimens) to radiate from the centre, as shewn in Pl. 3, fig. 1.

These nuclei colour red with hematoxylin and carmine and brown with osmic acid, while the nucleoli or granules contained in the areas also colour very intensely with all the three above-named reagents.

With such a peculiar structure, in favourable specimens these nuclei are very easily recognised, and their distribution can be determined without difficulty. They are not present alone in the finely granular yolk, but also in the coarsely granular yolk adjoining it. They form very often a special row, sometimes still more markedly than in Pl. 3, fig. 1, along the floor of the segmentation cavity. They are not, however, found alone in the yolk. All the blastoderm-cells in the earlier stages possess precisely similar nuclei! From the appearance of the first nucleus in a segmentation-sphere till a comparatively late period in development, every nucleus which can be distinctly seen is found to be of this character. In Pl. 3, fig. 2, this is very distinctly shewn.

(1) We have, then, nuclei of this very peculiar character scattered through the sub-germinal granular matter, and also universally present in the cells of the blastoderm. (2) These nuclei are distributed in a special manner under the floor of the segmentation cavity on which new cells are continually appearing. Putting these two facts together, there would be the strongest presumption that these nuclei do actually become the nuclei of cells which enter the blastoderm, and such is actually the case. In my account of the segmentation I have, indeed, already mentioned this, and I will return to it, but before doing so will enter more fully into the distribution of these nuclei in the yolk.

They appear in small numbers around the blastoderm at the close of segmentation, and round each one of them there may at this time be seen in osmic acid specimens, and with high powers, a fine network similar to but finer than that represented in Pl. 3, fig. 2. This network cannot, as a general rule, be traced far into the yolk, but in some exceptionally thin specimens it may be seen in any part of the fine granular yolk around the blastoderm, the meshes of the network being, however, considerably coarser between than around the nuclei. This network may be seen in the fine granular material around the germ till the latest period of which I have yet cut sections of the blastoderm. In the later specimens, indeed, it is very much more distinctly seen than in the earlier, owing to the fact that in parts of the blastoderm, especially under the embryo, the yolk-granules have disappeared partly or entirely, leaving only this fine network with the nuclei in it.

A specimen of this kind is represented in Pl. 3, fig. 2, where the meshes of the network are seen to be finer immediately around the nuclei, and coarser in the intervals. The specimen further shows in the clearest manner that this network is not divided into areas, each representing a cell and each containing a nucleus. I do not know to what extent this network extends into the yolk. I have never yet seen the limits of it, though it is very common to see the coarsest yolk-granules lying in its meshes. Some of these are shewn in Pl. 3, fig. 2, yk.

This network of lines[11] (probably bubbles) is characteristic of many cells, especially ova. We are, therefore, forced to believe that the fine granular and probably coarser granular yolk of this meroblastic egg consists of an active organized basis with passive yolk-spheres imbedded in it. The organized basis is especially concentrated at the germinal pole of the egg, but becomes less and less in quantity, as compared with the yolk-spheres, the further we depart from this.

Admitting, as I think it is necessary to do, the organized condition of the whole yolk-sphere, there are two possible views as to its nature. We may either take the view that it is one gigantic cell, the ovum, which has grown at the expense of the other cells of the egg-follicle, and that these cells in becoming absorbed have completely lost their individuality; or we may look upon the true formative yolk (as far as we can separate it from the remainder of the food-yolk) as the remains of one cell (the primitive ovum), and the remainder of the yolk as a body formed from the coalescence of the other cells of the egg-follicle, which is adherent to, but has not coalesced with, the primitive ovum, the cells in this case not having completely lost their individuality; and to these cells, the nuclei, I have found, must be supposed to belong.

The former view I think, for many reasons, the most probable. The share of these nuclei in the segmentation, and the presence of similar nuclei in the cells of the germ, both support it, and are at the same time difficulties in the way of the other view. Leaving this question which cannot be discussed fully in a preliminary paper like the present one, I will pass on to another important question, viz.:

How do these nuclei originate? Are they formed by the division of the pre-existing nuclei, or by an independent formation? It must be admitted that many specimens are strongly in favour of the view that they increase by division. In the first place, they are often seen two together; examples of this will be seen in Pl. 3, fig. 1. In the second place, I have found several specimens in which five or six appear close together, which look very much as if there had been an actual division into six nuclei. It is, however, possible in this case that the nuclei are really connected below and only appear separate, owing to the crenate form of the mass. Against this may be put the fact that the division of a nucleus is by no means so common as has been sometimes supposed, that in segmentation it has very rarely been observed that the nucleus of a sphere first divides[12], and that then segmentation takes place, but segmentation generally occurs and then a new nucleus arises in each of the newly formed spheres. Such nuclei as I have described are rare; they have, however, been observed in the egg of a Nephelis (one of the Leeches), and have in that case been said to divide. Dr Kleinenberg, however, by following a single egg through the whole course of its development, has satisfied himself that this is not the case, and that, further, these nuclei in Nephelis never form the nuclei of newly developing cells.

I must leave it an open question, and indeed one which can hardly be solved from sections, whether these nuclei arise freely or increase by division, but I am inclined to believe that both processes may possibly take place. In any case their division does not appear to determine the segmentation or segregation of the protoplasm around them.

As was mentioned in my account of the segmentation, these nuclei first appear during that process, and become the nuclei of the freshly formed segmentation spheres. At the close of segmentation a few of them are still to be seen around the blastoderm, but they are not very numerous.

From this period they rapidly increase in number, up to the commencement of the formation of the embryo as a body distinct from the germ. Though before this period they probably become the nuclei of veritable cells which enter the germ, it is not till this period, when the growth of the blastoderm becomes very rapid and it commences to spread over the yolk, that these new cells are formed in large numbers. I have many specimens of this age which shew the formation of these new cells with great clearness. This is most distinctly to be seen immediately below the embryo, where the yolk-spherules are few in number. At the opposite end of the blastoderm I believe that more of these cells are formed, but, owing to the presence of numerous yolk-spherules, it is much more difficult to make certain of this.

As to the final destination of these cells, my observations are not yet completed. Probably a large number of them are concerned in the formation of the vascular system, but I will give reasons later on for believing that some of them are concerned in the formation of the walls of the digestive canal and of other parts.

I will conclude my account of these nuclei by briefly summarizing the points I have arrived at in reference to them.

A portion, or more probably the whole, of the yolk of the Dog-fish consists of organized material, in which nuclei appear and increase either by division or by a process of independent formation, and a great number of these subsequently become the nuclei of cells formed around them, frequently at a distance from the germ, which then travel up and enter it.

The formation of cells in the yolk, apart from the general process of segmentation, has been recognised by many observers. Kupffer (Archiv. für Micr. Anat., Bd. IV. 1868) and Owsjannikow (Entwicklung der Coregonus, Bulletin der Akad. St Petersburgh, Vol. XIX.) in osseous fishes[13], Ray Lankester (Annals and Mag. of Nat. Hist. Vol. IX. 1873, p. 81) in Cephalopoda, Götte (Archiv. für Micr. Anat. Vol. X.) in the chick, have all described a new formation of cells from the so-called food-yolk. The organized nature of the whole or part of this, previous to the formation of the cells from it, has not, however, as a rule, been distinctly recognised. In the majority of cases, as, for instance, in Loligo, the nucleus is not the first thing to be formed, but a plastide is first formed, in which a nucleus subsequently makes its appearance.

Formation of the Layers.

Leaving these nuclei, I will now pass on to the formation of the layers.

At the close of segmentation the surface of the blastoderm is composed of cells of a uniform size, which, however, are too small to be seen by the aid of the simple microscope.

The cells of this uppermost layer are somewhat columnar, and can be distinguished from the remainder of the cells of the blastoderm as a separate layer. This layer forms the epiblast; and the Dog-fish agree with Birds, Batrachians, and Osseous fish in the very early differentiation of it.

The remainder of the cells of the blastoderm form a mass, many cells deep, in which it is impossible as yet or till a very considerably later period to distinguish two layers. They may be called the lower layer cells. Some of them near the edge of this mass are still considerably larger than the rest, but they are, as a whole, of a fairly uniform size. Their nuclei are of the same character as the nuclei in the yolk.

There is one point to be noticed in the shape of the blastoderm as a whole. It is unsymmetrical, and a much larger number of its cells are found collected at one end than at the other. This absence of symmetry is found in all sections which are cut parallel to the long axis of the egg-capsule. The thicker end is the region where the embryo will subsequently appear.

This very early appearance of distinction in the blastoderm between the end at which the embryo will appear, and the non-embryonic end is important, especially as it shews the affinity of the modes of development of Osseous fishes and the Elasmobranchii. Oellacher (Zeitschrift für Wiss. Zoologie, Vol. XXXIII. 1873) has shewn, and, though differing from him on many other points, on this point Götte (Arch. für Micr. Anat. Vol. IX. 1873) agrees with him, that a similar absence of symmetry by which the embryonic end of the germ is marked off, occurs almost immediately after the end of segmentation in Osseous fishes. In the early stages of development there are a number of remarkable points of agreement between the Osseous fish and the Dog-fish, combined with a number of equally remarkable points of difference. Some of these I shall point out as I proceed with my description.

The embryonic end of the germ is always the one which points towards the pole of the yolk farthest removed from the egg-capsule.

The germ grows, but not very rapidly, and without otherwise undergoing any very appreciable change, for some time.

The growth at these early periods appears to be particularly slow, especially when compared with the rapid manner in which some of the later stages of the development are passed through.

The next important change which occurs is the formation of the so-called segmentation cavity.

This forms a very marked feature throughout the early stages. It appears, however, to have somewhat different relations to the blastoderm than the homologous structure in other vertebrates. In its earliest stage which I have observed, it appears as a small cavity in the centre of the lower layer cells. This grows rapidly, and its roof becomes composed of epiblast and only a thin lining of lower layer cells, while its floor is formed by the yolk (Pl. 3, fig. 3, sg). In the next and third stage (Pl. 3, fig. 4, sg) its floor is formed by a thin layer of cells, its roof remaining as before. It has, however, become a less conspicuous formation than it was; and in the last (fourth) stage in which it can be distinguished it is very inconspicuous, and almost filled up by cells.

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

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

Dog-fish resemble Osseous fish in the fact that their embryos are entirely formed from a portion of the germ which does not form part of the roof of the segmentation cavity, so that the cells forming the roof of the segmentation cavity take no share at any time in the formation of their embryos. They further agree with Osseous fish (always supposing that the descriptions of Oellacher, loc. cit., and Götte, Archiv. für Micr. Anat. Bd. IX. are correct) in the floor of the segmentation cavity being formed at one period by yolk. Together with these points of similarity there are some important differences.

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

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

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

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

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

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

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

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

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

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

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

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

(1) The increase in size of the blastoderm.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The explanation of this is, I believe, the keystone to the whole position. The vertebrates may be divided as to their early development into two classes, viz. those with holoblastic ova, in which the digestive canal is formed by an involution with the presence of an anus of Rusconi.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Development of the Embryo.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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