CHAPTER III.

FORMATION OF THE LAYERS.

In the last chapter the blastoderm was left as a solid lens-shaped mass of cells, thicker at one end than at the other, its uppermost row of cells forming a distinct layer. There very soon appears in it a cavity, the well-known segmentation cavity, or cavity of von Baer, which arises as a small space in the midst of the blastoderm, near its non-embryonic end (Pl. 7, fig. 1.).

This condition of the segmentation cavity, though already[116] described, has nevertheless been met with in one case only. The circumstance of my having so rarely met with this condition is the more striking because I have cut sections of a considerable number of blastoderms in the hope of encountering specimens similar to the one figured, and it can only be explained on one of the two following hypotheses. Either the stage is very transitory, and has therefore escaped my notice except in the one instance; or else the cavity present in this instance is not the true segmentation cavity, but merely some abnormal structure. That this latter explanation is a possible one, appears from the fact that such cavities do at times occur in other parts of the blastoderm. Dr Schultz[117] does not mention having found any stage of this kind.

Footnote 116: _Qy. Journal of Microsc. Science_, Oct. 1874. [This Edition, No. V.]

Footnote 117: _Centr. f. Med. Wiss._ No. 38, 1875.

The position of the cavity in question, and its general appearance, incline me to the view that it is the segmentation cavity[118]. If this is the true view of its nature the fact should be noted that at first its floor is formed by the lower layer cells and not by the yolk, and that its roof is constituted by both the lower layer cells and the epiblast cells. The relations of the floor undergo considerable modifications in the course of development.

Footnote 118: Professor Bambeke ("Poissons Osseux," _Mém. Acad. Belgique_ 1875) describes a cavity in the blastoderm of Leuciscus rutilus, which he regards as the true segmentation cavity, but not as identical with the segmentation cavity of Osseous Fishes, usually so called. Its relations are the same as those of my segmentation cavity at this stage. This paper came into my hands at too late a period for me to be able to do more than refer to it in this place.

The other features of the blastoderm at this stage are very much those of the previous stage.

The embryonic swelling is very conspicuous. The cells of the blastoderm are still disposed in two layers: an upper one of slightly columnar cells one deep, which constitutes the epiblast, and a lower one consisting of the remaining cells of the blastoderm.

An average cell of the lower layer has a diameter of about 1/900 inch, but the cells at the periphery of the layer are in some cases considerably larger than the more central ones. All the cells of the blastoderm are still completely filled with yolk spherules. In the yolk outside the peculiar nuclei, before spoken of, are present in considerable numbers. They seem to have been mistaken by Dr Schultz[119] for cells: there can however be no question that they are true nuclei.

Footnote 119: _Loc. cit._

In the next stage the relations of the segmentation cavity undergo important modifications.

The cells which form its floor disappear almost completely from that position, and the floor becomes formed by the yolk.

The stage, during which the yolk serves as the floor of the segmentation cavity, extends over a considerable period of time, but during it I have been unable to detect any important change in the constitution of the blastoderm. It no doubt gradually extends over the yolk, but even this growth is not nearly so rapid as in the succeeding stage. Although therefore the stage I proceed to describe is of long continuance, a blastoderm at the beginning of it exhibits, both in its external and in its internal features, no important deviations from one at the end of it.

Viewed from the surface (Pl. 8, fig. A) the blastoderm at this stage appears slightly oval, but the departure from the circular form is not very considerable. The long axis of the oval corresponds with what eventually becomes the long axis of the embryo. From the yolk the blastoderm is still well distinguished by its darker colour; and it is surrounded by a concentric ring of light-coloured yolk, the outer border of which shades insensibly into the normal yolk.

At the embryonic portion of the blastoderm is a slight swelling, clearly shewn in Plate 8, fig. A, which can easily be detected in fresh and in hardened embryos. This swelling is to be looked upon as a local exaggeration of a slightly raised rim present around the whole circumference of the blastoderm. The roof of the segmentation cavity (fig. A, _s.c._) forms a second swelling; and in the fresh embryo this region appears of a darker colour than other parts of the blastoderm.

It is difficult to determine the exact shape of the blastoderm, on account of the traction exercised upon it in opening the egg; and no reliance can be placed on the forms assumed by hardened blastoderms. This remark also applies to the sections of blastoderms of this stage. There can be no doubt that the minor individual variations exhibited by almost every specimen are produced in the course of manipulations while the objects are fresh. These variations may affect even the relative length of a particular region and certainly the curvature of it. The roof of the segmentation cavity is especially apt to be raised into a dome-like form.

The main internal feature of this stage is the disappearance of the layer of cells which, during the first stage, formed the floor of the segmentation cavity. This disappearance is nevertheless not absolute, and it is doubtful whether there is any period in which the floor of the cavity is quite without cells.

Dr Schultz supposes[120] that the entire segmentation cavity is, in the living animal, filled with a number of loose cells. Though it is not in my power absolutely to deny this, the point being one which cannot be satisfactorily investigated in sections, yet no evidence has come under my notice which would lead to the conclusion that more cells are present in the segmentation cavity than are represented on Pl. 13, fig. 1, of my preliminary paper[121], an illustration which is repeated on Pl. 7, fig. 2.

Footnote 120: _Loc. cit._

Footnote 121: _Loc. cit._

The number of cells on the floor of the cavity differs considerably in different cases, but these cases come under the category of individual variations, and are not to be looked upon as indications of different states of development.

In many cases especially large cells are to be seen on the floor of the cavity (Pl. 7, fig. 2, _bd_). In my preliminary paper[122] the view was expressed that these are probably cells formed around the nuclei of the yolk. This view I am inclined to abandon, and to substitute for it the suggestion made by Dr Schultz, that they are remnants of the larger segmentation cells which were to be seen in the previous stages.

Footnote 122: _Qy. Journal of Micros. Science_, Oct. 1874. [This Edition, No. V.]

Plate 7, figs. 2, 3, 4 (all sections of this stage) shew the different appearances presented by the floor of the segmentation cavity. In only one of these sections are there any large number of cells upon the floor; and in no case have cells been observed imbedded in the yolk forming this floor, as described by Dr Schultz[123], but in all cases the cells simply rested upon it.

Footnote 123: _Loc. cit._ Probably Dr Schultz, here as in other cases, has mistaken nuclei for cells.

Passing from the segmentation cavity to the blastoderm itself, the first feature to be noticed is the more decided differentiation of the epiblast. This now forms a distinct layer composed of a single row of columnar cells. These are slightly more columnar in the region of the embryonic swelling than elsewhere, and become less elongated at the edge of the blastoderm. In my specimens this layer was never more than one cell deep, but Dr Schultz[124] states that, in the Elasmobranch embryos investigated by him, the epiblast was composed of more than a single row of cells.

Footnote 124: _Loc. cit._

Each epiblast cell is filled with yolk-spherules and contains a nucleus. Very frequently the nuclei in the layer are arranged in a regular row (vide Pl. 7, fig. 3). In the later blastoderms of this stage there is a tendency in the cells to assume a wedge-like form with their thin ends pointing alternately in opposite directions. This arrangement is, however, by no means strictly adhered to, and the regularity of it is exaggerated in Plate 7, fig. 4.

The nuclei of the epiblast cells have the same characters as those of the lower layer cells to be presently described, but their intimate structure can only be successfully studied in certain exceptionally favourable sections. In most cases the yolk-spherules around them render the finer details invisible.

There is at this stage no such obvious continuity as in the succeeding stage between the epiblast and the lower layer cells; and this statement holds good more especially with the best conserved specimens which have been hardened in osmic acid (Pl. 7, fig. 4). In these it is very easy to see that the epiblast simply thins out at the edge of the blastoderm without exhibiting the slightest tendency to become continuous with the lower layer cells[125].

Footnote 125: Prof. Haeckel ("Die Gastrula u. die Eifurchung d. Thiere," _Jenaische Zeitschrift_, Vol. IX.) has unfortunately copied a figure from my preliminary paper (_loc. cit._) (repeated now), which I had carefully avoided using for the purpose of describing the formation of the layers on account of the epiblast cells in the original having been much altered by the chromic acid, as a result of which the whole section gives a somewhat erroneous impression of the condition of the blastoderm at this stage. I take this opportunity of pointing out that the colouration employed by Professor Haeckel to distinguish the layers in this section is not founded on my statements, but is, on the contrary, in entire opposition to them. From the section as represented by Professor Haeckel it might be gathered that I considered the lower layer cells to be divided into two parts, one derived from the epiblast, while the other constituted the hypoblast. Not only is no such division present at this period, but no part of the lower layer cells, or the mesoblast cells into which they become converted, can in any sense whatever be said to be derived from the epiblast.

The lower layer cells form a mass rather than a layer, and constitute the whole of the blastoderm not included in the epiblast. The shape of this mass in a longitudinal section may be gathered from an examination of Plate 7, figs. 3 and 4.

It presents an especially thick portion forming the bulk of the embryonic swelling, and frequently contains one or two cavities, which from their constancy I regard as normal and not as artificial products.

In addition to the mass forming the embryonic swelling there is seen in sections another mass of lower layer cells at the opposite extremity of the blastoderm, connected with the former by a bridge of cells, which constitutes the roof of the segmentation cavity. The lower layer cells may thus be divided into three distinct parts:

(1) The embryo swelling.

(2) The thick rim of cells round the edge of the remainder of the blastoderm.

(3) The cells which form the roof of the segmentation cavity.

These three parts form a continuous whole, but in addition to these there exist the previously mentioned cells, which rest on the floor of the segmentation cavity.

With the exception of these latter, the lower layer is composed of cells having a fairly uniform size, and exhibits no trace of a division into two layers.

The cells are for the most part irregularly polygonal from mutual pressure; and in their shape and arrangement, exhibit a marked contrast to the epiblast cells. A few of the lower layer cells, highly magnified, are represented in Pl. 7, fig. 2_a_. An average cell measures about 1/800 to 1/900 of an inch, but some of the larger ones on the floor attain to the 1/475 of an inch.

Owing to my having had the good fortune to prepare some especially favourable specimens of this stage, it has been possible for me to make accurate observations both upon the nuclei of the cells of the blastoderm, and upon the nuclei of the yolk.

The nuclei of the blastoderm cells, both of the epiblast and lower layer, have a uniform structure. Those of the lower layer cells are about 1/1600 of an inch in diameter. Roughly speaking each consists of a spherical mass of clear protoplasm refracting more highly than the protoplasm of its cell. The nucleus appears in sections to be divided by deeply stained lines into a number of separate areas, and in each of these a deeply stained granule is placed. In some cases two or more of such granules may be seen in a single area. The whole of the nucleus stains with the colouring reagents more deeply than the protoplasm of the cells; but this is especially the case with the granules and lines.

Though usually spherical the nuclei not infrequently have a somewhat lobate form.

Very similar to these nuclei are the nuclei of the yolk.

One of the most important differences between the two is that of size. The majority of the nuclei present in the yolk are as large or larger than an ordinary blastoderm cell; while many of them reach a size very much greater than this. The examples I have measured varied from 1/500 to 1/250 of an inch in diameter.

Though they are divided, like the nuclei of the blastoderm, with more or less distinctness into separate areas by a network of lines, their greater size frequently causes them to present an aspect somewhat different from the nuclei of the blastoderm. They are moreover much less regular in outline than these, and very many of them have lobate projections (Pl. 7, figs. 2_a_ and 2_c_ and 3), which vary from simple knobs to projections of such a size as to cause the nucleus to present an appearance of commencing constriction into halves. When there are several such projections the nucleus acquires a peculiar knobbed figure. With bodies of this form it becomes in many cases a matter of great difficulty to decide whether or no a particular series of knobs, which appear separate in one plane, are united in a lower plane, whether, in fact, there is present a single knobbed nucleus or a number of nuclei in close apposition. A nucleus in this condition is represented in Pl. 7, fig. 2_b_.

The existence of a protoplasmic network in the yolk has already been mentioned. This in favourable cases may be observed to be in special connection with the nuclei just described. Its meshes are finer in the vicinity of the nuclei, and its fibres in some cases almost appear to start from them (Pl. 7, fig. 12). For reasons which I am unable to explain the nuclei of the yolk and the surrounding meshwork present appearances which differ greatly according to the reagent employed. In most specimens hardened in osmic acid the protoplasm of the nuclei is apparently prolonged in the surrounding meshwork (Pl. 7, fig. 12). In other specimens hardened in osmic acid (Pl. 7, fig. 11), and in all hardened in chromic acid (Pl. 7, fig. 2_a_ and 2_c_), the appearances are far clearer than in the previous case, and the protoplasmic meshwork merely surrounds the nuclei, without shewing any signs of becoming continuous with them.

There is also around each nucleus a narrow space in which the spherules of the yolk are either much smaller than elsewhere or completely absent, vide Pl. 7, fig. 2_b_.

It has not been possible for me to satisfy myself as to the exact meaning of the lines dividing these nuclei into a number of distinct areas. My observations leave the question open as to whether they are to be looked upon as lines of division, or as protoplasmic lines such as have been described in nuclei by Flemming[126], Hertwig[127] and Van Beneden[128]. The latter view appears to me to be the more probable one.

Footnote 126: "Entwicklungsgeschichte der Najaden," _Sitz. d. k. Akad. Wien_, 1875.

Footnote 127: _Morphologische Jahrbuch_, Vol. 1. Heft 3.

Footnote 128: "Développement des Mammifères," _Bul. de l'Acad. de Belgique_, XL. No. 12, 1875.

Such are the chief structural features presented by these nuclei, which are present during the whole of the earlier periods of development and retain throughout the same appearance. There can be little doubt that their knobbed condition implies that they are undergoing a rapid division. The arguments for this view I have already insisted on, and, in spite of the observations of Dr Kleinenberg shewing that similar nuclei of Nephelis do not undergo division, the case for their doing so in the Elasmobranch eggs is to my mind a very strong one.

During this stage the distribution of these nuclei in the yolk becomes somewhat altered from that in the earlier stages. Although the nuclei are still scattered generally throughout the finer yolk-matter around the blastoderm, yet they are especially aggregated at one or two points. In the first place a special collection of them may be noticed immediately below the floor of the segmentation cavity. They here form a distinct row or even layer. If the presence of this layer is coupled with the fact that at this period cells are beginning to appear on the floor of the segmentation cavity, a strong argument is obtained for the supposition that around these nuclei cells are being produced, which pass into the blastoderm to form the floor. Of the actual formation of cells at _this_ period I have not been able to obtain any satisfactory example, so that it remains a matter of deduction rather than of direct observation.

Another special aggregation of nuclei is generally present at the periphery of the blastoderm, and the same amount of doubt hangs over the fate of these as over that of the previously mentioned nuclei.

The next stage is the most important in the whole history of the formation of the layers. Not only does it serve to shew, that the process by which the layers are formed in Elasmobranchii can easily be derived from a simple gastrula type like that of Amphioxus, but it also serves as the key by which other meroblastic types of development may be explained. At the very commencement of this stage the embryonic swelling becomes more conspicuously visible than it was. It now projects above the level of the yolk in the form of a rim. At one point, which eventually forms the termination of the axis of the embryo, this projection is at its greatest; while on either side of this it gradually diminishes and finally vanishes. This projection I propose calling, as in my preliminary paper[129], the embryonic rim.

Footnote 129: _Qy. Journal Microsc. Science_, Oct. 1874. [This Edition, No. V.]

The segmentation cavity can still be seen from the surface, and a marked increase in the size of the blastoderm may be noticed. During the stage last described, the growth was but very slight; hence the rather sudden and rapid growth which now takes place becomes striking.

Longitudinal sections at this stage, as at the earlier stages, are the most instructive. Such a section on the same scale as Pl. 7, fig. 4, is represented in Pl. 7, fig. 5. It passes parallel to the long axis of the embryo, through the point of greatest development of the embryonic ring.

The three fresh features of the most striking kind are (1) the complete envelopment of the segmentation cavity within the lower layer cells, (2) the formation of the embryonic rim, (3) the increase in distance between the posterior end of the blastoderm and the segmentation cavity. The segmentation cavity has by no means relatively increased in size. The roof has precisely its earlier constitution, being composed of an internal lining of lower layer cells and an external one of epiblast. The thin lining of lower layer cells is, in the course of mounting the sections, very apt to fall off; but I am absolutely satisfied that it is never absent.

The floor of the cavity has undergone an important change, being now formed by a layer of cells instead of by the yolk. A precisely similar but more partial change in the constitution of the floor takes place in Osseous Fishes[130].

Footnote 130: Götte, "Der Keim d. Forelleneies," _Arch. f. Mikr. Anat._ Vol. IX.; Haeckel, "Die Gastrula u. die Eifurchung d. Thiere," _Jenaische Zeitschrift_, Bd. IX.

The mode in which the floor is formed is a question of some importance. The nuclei, which during the last stage formed a row beneath it, probably, as previously pointed out, take some share in its formation. An additional argument to those already brought forward in favour of this view may be derived from the fact that during this stage such a row of nuclei is no longer present.

This argument may be stated as follows:

Before the floor of cells for the segmentation cavity is formed a number of nuclei are present in a suitable situation to supply the cells for the floor; as soon as the floor of cells makes its appearance these nuclei are no longer to be seen. From this it may be concluded that their disappearance arises from their having become the nuclei of the cells which form the floor.

It appears to me most probable that there is a growth inwards from the whole peripheral wall of the cavity, and that this ingrowth, as well as the cells derived from the yolk, assist in forming the floor of the cavity. In Osseous Fish there appears to be no doubt that the floor is largely formed by an ingrowth of this kind.

A great increase is observable in the distance between the posterior end of the segmentation cavity and the edge of the blastoderm. This is due to the rapid growth of the latter combined with the stationary condition of the former. The growth of the blastoderm at this period is not uniform, but is more rapid in the non-embryonic than in the embryonic parts.

The main features of the epiblast remain the same as during the last stages. It is still composed of a very distinct layer one cell deep. Over the segmentation cavity, and over the whole embryonic end of the blastoderm, the cells are very thin, columnar, and, roughly speaking, wedge-shaped with the thin ends pointing alternately in different directions. For this reason, the nuclei form two rows; but both the rows are situated near the upper surface of the layer (vide Pl. 7, fig. 5). Towards the posterior end of the blastoderm the cells are flatter and broader; and the layer terminates at the non-embryonic end of the blastoderm without exhibiting the slightest tendency to become continuous with the lower layer cells. At the embryonic end of the blastoderm the relations of the epiblast and lower layer cells are very different. At this part, throughout the whole extent of the embryonic rim, the epiblast is reflected and becomes continuous with the lower layer cells.

The lower layer cells form, for the most part, a uniform stratum in which no distinction into mesoblast and hypoblast is to be seen.

Both the lower layer cells and the epiblast cells are still filled with yolk-spherules.

The structures at the embryonic rim, and the changes which are there taking place, unquestionably form the chief features of interest at this stage.

The general relations of these parts are very fairly shewn in Pl. 7, fig. 5, which represents a section passing through the median line of the embryonic region. They are however more accurately represented in Pl. 7, fig. 5_a_, taken from the same embryo, but in a lateral part of the embryonic rim; or in Pl. 7, fig. 6, from a slightly older embryo. In all of these figures the epiblast cells are reflected at the edge of the embryonic rim, and become perfectly continuous with the hypoblast cells. A few of the cells, immediately beyond the line of this reflection, precisely resemble in character the typical epiblast cells; but the remainder exhibit a gradual transition into typical lower layer cells. Adjoining these transitional cells, or partly enclosed in the corner formed between them and the epiblast, are a few unaltered lower layer cells (_m_), which at this stage are not distinctly separated from the transitional cells. The transitional cells form the commencement of the hypoblast (_hy_); and the cells (_m_) between them and the epiblast form the commencement of the mesoblast. The gradual conversion of lower layer cells into columnar hypoblast cells, is a very clear and observable phenomenon in the best specimens. Where the embryonic rim projects most, a larger number of cells have assumed a columnar form. Where it projects less clearly, a smaller number have done so. But in all cases there may be observed a series of gradations between the columnar cells and the typical rounded lower layer cells[131].

Footnote 131: When writing my earlier paper I did not feel so confident about the mode of formation of the hypoblast as I now do, and even doubted the possibility of determining it from sections. The facts now brought forward are I hope sufficient to remove all scepticism on this point.

In the last described embryo, although the embryonic rim had attained to a considerable development, no trace of the medullary groove had made its appearance. In an embryo in the next stage of which I propose describing sections, this structure has become visible.

A surface view of a blastoderm of this age, with the embryo, is represented on Pl. 8, fig. B; and I shall, for the sake of convenience, in future speak of embryos of this age as belonging to period B.

The blastoderm is nearly circular. The embryonic rim is represented by a darker shading at the edge. At one point in this rim may be seen the embryo, consisting of a somewhat raised area with an axial groove (_mg_). The head end of the embryo is that which points towards the centre of the blastoderm, and its free peripheral extremity is at the edge of the blastoderm.

A longitudinal section of an embryo of the same age as the one figured[132] is represented on Pl. 7, fig. 7. The general growth has been very considerable, though as before explained, it is mainly confined to that part of the blastoderm where the embryonic rim is absent.

Footnote 132: Owing to the small size of the plates this section has been drawn on a considerably smaller scale than that represented in fig. 5.

A fresh feature of great importance is the complete disappearance of the segmentation cavity, the place which was previously occupied by it being now filled up by an irregular network of cells. There can be little question that the obliteration of the segmentation cavity is in part due to the entrance into the blastoderm of fresh cells formed around the nuclei of the yolk. The formation of these is now taking place with great rapidity and can be very easily followed.

Since the segmentation cavity ceases to play any further part in the history of the blastoderm, it will be well shortly to review the main points in its history.

Its earliest appearance is involved in some obscurity, though it probably arises as a simple cavity in the midst of the lower layer cells (Pl. 7, fig. 1). In its second phase the floor ceases to be formed of lower layer cells, and the place of these is taken by the yolk, on which however a few scattered cells still remain (Pl. 7, figs. 2, 3, 4). During the third period of its history, a distinct cellular floor is again formed for it, so that it comes a second time into the same relations with the blastoderm as at its earliest appearance. The floor of cells which it receives is in part due to a growth inwards from the periphery of the blastoderm, and in part to the formation of fresh cells from the yolk. Coincidently with the commencing differentiation of hypoblast and mesoblast the segmentation cavity grows smaller and vanishes.

One of the most important features of the segmentation cavity in the Elasmobranchii which I have studied, is the fact that throughout its whole existence its roof is formed of _lower layer cells_. There is not the smallest question that the segmentation cavity of these fishes is the homologue of that of Amphioxus, Batrachians, etc., yet in the case of all of these animals, the roof of the segmentation cavity is formed of epiblast only. How comes it then to be formed of lower layer cells in Elasmobranchii?

To this question an answer was attempted in my paper, "Upon the Early Stages of the Development of Vertebrates[133]." It was there pointed out, that as the food material in the ovum increases, the bulk of the lower layer cells necessarily also increases; since these, as far as the blastoderm is concerned, are the chief recipients of food material. This causes the lower layer cells to encroach upon the segmentation cavity, and to close it in not only on the sides, but also above; from the same cause it results that the lower layer cells assume, from the first, a position around the spot where the future alimentary cavity will be formed, and that this cavity becomes formed by a simple split in the midst of the lower layer cells, and not by an involution.

Footnote 133: _Quart. Journ. of Microscop. Science_, July, 1875. [This Edition, No. VI.]

All the most recent observations[134] on Osseous Fishes tend to shew that in them, the roof of the segmentation cavity is formed alone of epiblast; but on account of the great difficulty which is experienced in distinguishing the layers in the blastoderms of these animals, I still hesitate to accept as conclusive the testimony on this point.

Footnote 134: Oellacher, _Zeit. f. Wiss. Zoologie_, Bd. XXIII. Götte, _Archiv f. Mikr. Anat._ Vol. IX. Haeckel, _loc. cit._

In the formation a second time of a cellular floor for the segmentation cavity in the third stage, the Elasmobranch embryo seems to resemble that of the Osseous Fish[135]. Upon this feature great stress is laid both by Dr Götte[136] and Prof. Haeckel[137]: but I am unable to agree with the interpretation of it offered by them. Both Dr Götte and Prof. Haeckel regard the formation of this floor as part of an involution to which the lower layer cells owe their origin, and consider the involution an equivalent to the alimentary involution of Batrachians, Amphioxus, &c. To this question I hope to return, but it may be pointed out that my observations prove that this view can only be true in a very modified sense; since the invagination by which hypoblast and alimentary canal are formed in Amphioxus is represented in Elasmobranchii by a structure quite separate from the ingrowth of cells to form the floor of the segmentation cavity.

Footnote 135: This floor appears in most Osseous Fish to be only partially formed. Vide Götte, _loc. cit._

Footnote 136: _Loc. cit._

Footnote 137: _Loc. cit._

The eventual _obliteration_ of the segmentation cavity by cells derived from the yolk is to be regarded as an inherited remnant of the involution by which this obliteration was primitively effected. The passage upwards of cells from the yolk, may possibly be a real survival of the tendency of the hypoblast cells to grow inwards during the process of involution.

The last feature of the segmentation cavity which deserves notice is its excentric position. It is from the first situated in much closer proximity to the non-embryonic than to the embryonic end of the blastoderm. This peculiarity in position is also characteristic of the segmentation cavity of Osseous Fishes, as is shewn by the concordant observations of Oellacher[138] and Götte[139]. Its meaning becomes at once intelligible by referring to the diagrams in my paper[140] on the Early Stages in the Development of Vertebrates. It in fact arises from the asymmetrical character of the primitive alimentary involution in all anamniotic vertebrates with the exception of Amphioxus.

Footnote 138: _Loc. cit._

Footnote 139: _Loc. cit._

Footnote 140: _Loc. cit._

Leaving the segmentation cavity I pass on to the other features of my sections.

There is still to be seen a considerable aggregation of cells at the non-embryonic end of the blastoderm. The position of this, and its relations with the portion of the blastoderm which at an earlier period contained the segmentation cavity, indicate that the growth of the blastoderm is not confined to its edge, but that it proceeds at all points causing the peripheral parts to glide over the yolk.

The main features of the cells of this blastoderm are the same as they were in the one last described. In the non-embryonic region the epiblast has thinned out, and is composed of a single row of cells, which, in the succeeding stages, become much flattened.

The lower layer cells over the greater part of their extent, have not undergone any histological changes of importance. Amongst them may frequently be seen a few exceptionally large cells, which without doubt have been derived directly from the yolk.

The embryonic rim is now a far more considerable structure than it was. Vide Pl. 7, fig. 7. Its elongation is mainly effected by the continuous conversion of rounded lower layer cells into columnar hypoblast cells at its central or anterior extremity.

This conversion of the lower layer cells into hypoblast cells is still easy to follow, and in every section cells intermediate between the two are to be seen. The nature of the changes which are taking place requires for its elucidation transverse as well as longitudinal sections. Transverse sections of a slightly older embryo than B are represented on Pl. 7, fig. 8_a_, 8_b_ and 8_c_.

Of these sections _a_ is the most peripheral or posterior, and _c_ the most central or anterior. By a combination of transverse and longitudinal sections, and by an inspection of a surface view, it is rendered clear that, though the embryonic rim is a far more considerable structure in the region of the embryo than elsewhere (compare fig. 6 and fig. 7 and 7_a_), yet that this gain in size is not produced by an outgrowth of the embryo beyond the rest of the germ, but by the conversion of the lower layer cells into hypoblast having been carried far further towards the centre of the germ in the axial line than in the lateral regions of the rim.

The most anterior of the series of transverse sections (Pl. 7, fig. 8_c_) I have represented, is especially instructive with reference to this point. Though the embryonic rim is cut through at the sides of the section, yet in these parts the rim consists of hardly more than a continuity between epiblast and lower layer cells, and the lower layer cells shew no trace of a division into mesoblast and hypoblast. In the axis of the embryo, however, the columnar hypoblast is quite distinct; and on it a small cap of mesoblast is seen on each side of the medullary groove. Had the embryonic rim resulted from a projecting growth of the blastoderm, such a condition could not have existed. It might have been possible to find the hypoblast formed at the sides of the section and not at the centre; but the reverse, as in these sections, could not have occurred. Indeed it is scarcely necessary to have recourse to sections to prove that the growth of the embryonic rim is towards the centre of the blastoderm. The inspection of a surface view of a blastoderm at this period demonstrates it beyond a doubt (Pl. 8, fig. B). The embryo, close to which the embryonic rim is alone largely developed, does not project outwards beyond the edge of the germ, but inwards towards its centre.

The space between the embryonic rim and the yolk (Pl. 7, fig. 7, _al._) is the alimentary cavity. The roof of this is therefore primitively formed of hypoblast and the floor of yolk. The external opening of this space at the edge of the blastoderm is the exact morphological homologue of the anus of Rusconi, or blastopore of Amphioxus, the Amphibians, &c. The importance of the mode of growth in the embryonic rim depends upon the homology of the cavity between it and the yolk, with the alimentary cavity of Amphioxus and Amphibians. Since this homology exists, the direction of the growth of this cavity ought to be, as it in fact is, the same as in Amphioxus, etc., viz. towards the centre of the germ and original position of the segmentation cavity. Thus though a true invagination is not present as in the other cases, yet this is represented in Elasmobranchii by the continuous conversion of lower layer cells into hypoblast along a line leading towards the centre of the blastoderm.

In the parts of the rim adjoining the embryo, the lower layer cells, on becoming continuous with the epiblast cells, assume a columnar form. At the sides of the rim this is not strictly the case, and the lower layer cells retain their rounded form, though quite continuous with the epiblast cells. One curious feature of the layer of epiblast in these lateral parts of the rim is the great thickness it acquires before being reflected and becoming continuous with the hypoblast (Pl. 7, fig. 8_c_). In the vicinity of the point of reflection there is often a rather large formation of cells around the nuclei of the yolk. The cells formed here no doubt pass into the blastoderm, and become converted into columnar hypoblast cells. In some cases the formation of these cells is very rapid, and they produce quite a projection on the under side of the hypoblast. Such a case is represented in Pl. 7, fig. 8_b_, _n.al_. The cells constituting this mass eventually become converted into the lateral and ventral walls of the alimentary canal.

The formation of the mesoblast has progressed rapidly. While many of the lower layer cells become columnar and form the hypoblast, others, between these and the epiblast, remain spherical. The latter do not at once become separated as a layer distinct from the hypoblast, and, at first, are only to be distinguished from them through their different character, vide Plate 7, figs. 6 and 7. They nevertheless constitute the commencing mesoblast.

Thus much of the mode of formation of the mesoblast can be easily made out in longitudinal sections, but transverse sections throw still further light upon it.

From these it may at once be seen that the mesoblast is not formed in one continuous sheet, but as two lateral masses, one on each side of the axial line of the embryo[141]. In my preliminary account[142] it was stated that this was a condition of the mesoblast at a very early period, and that it was probably its condition from the beginning. Sections are now in my possession which satisfy me that, from the very first, the mesoblast arises as two distinct lateral masses, one on each side of the axial line.

Footnote 141: Professor Lieberkühn (_Gesellschaft zu Marburg_, Jan. 1876) finds in Mammalia a bilateral arrangement of the mesoblast, which he compares with that described by me in Elasmobranchii. In Mammalia, however, he finds the two masses of mesoblast connected by a very thin layer of cells, and is apparently of opinion that a similar thin layer exists in Elasmobranchii though overlooked by me. I can definitely state that, whatever may be the condition of the mesoblast in Mammalia, in Elasmobranchii at any rate no such layer exists.

Footnote 142: _Loc. cit._

In the embryo from which the sections Pl. 7, fig. 8_a_, 8_b_, 8_c_ were taken, the mesoblast had, in most parts, not yet become separated from the hypoblast. It still formed with this a continuous layer, though the mesoblast cells were distinguishable by their shape from the hypoblast. In only one section (_b_) was any part of the mesoblast quite separated from the hypoblast.

In the hindermost part of the embryo the mesoblast is at its maximum, and forms, on each side, a continuous sheet extending from the median line to the periphery (fig. 8_a_). The rounder form of the mesoblast cells renders the line of junction between the layer constituted by them and the hypoblast fairly distinct; but towards the periphery, where the hypoblast cells have the same rounded form as the mesoblast, the fusion between the two layers is nearly complete.

In an anterior section the mesoblast is only present as a cap on both sides of the medullary groove, and as a mass of cells at the periphery of the section (fig. 8_b_); but no continuous layer of it is present. In the foremost of the three sections (fig. 8_c_) the mesoblast can scarcely be said to have become in any way separated from the hypoblast except at the summit of the medullary folds (_m_).

From these and similar sections it may be certainly concluded, that the mesoblast becomes first separated from the hypoblast as a distinct layer in the posterior region of the embryo, and only at a later period in the region of the head.

In an embryo but slightly more developed than B, the formation of the layer is quite completed in the region of the embryo. To this embryo I now pass on.

In the non-embryonic parts of the blastoderm no fresh features of interest have appeared. It still consists of two layers. The epiblast is composed of flattened cells, and the lower layer of a network of more rounded cells, elongated in a lateral direction. The growth of the blastoderm has continued to be very rapid.

In the region of the embryo (Pl. 7, fig. 9) more important changes have occurred. The epiblast still remains as a single row of columnar cells. The hypoblast is no longer fused with the mesoblast, and forms a distinct dorsal wall for the alimentary cavity. Though along the axis of the embryo the hypoblast is composed of a single row of columnar cells, yet in the lateral part of the embryo its cells are less columnar and are one or two deep.

Owing to the manner in which the mesoblast became split off from the hypoblast, a continuity is maintained between the hypoblast and the lower layer cells of the blastoderm (Pl. 7, fig. 9), while the two plates of mesoblast are isolated and disconnected from any other masses of cells.

The alimentary cavity is best studied in transverse sections. (Vide Pl. 7, fig. 10_a_, 10_b_ and 10_c_, three sections from the same embryo.) It is closed in above and at the sides by the hypoblast, and below by the yolk. In its anterior part a floor is commencing to be formed by a growth of cells from the walls of the two sides. The cells for this growth are formed around the nuclei of the yolk; a feature which recalls the fact that in Amphibians the ventral wall of the alimentary cavity is similarly formed in part from the so-called yolk cells.

We left the mesoblast as two masses not completely separated from the hypoblast. During this stage the separation between the two becomes complete, and there are formed two great lateral plates of mesoblast cells, one on each side of the medullary groove. Each of these corresponds to a united vertebral and lateral plate of the higher Vertebrates. The plates are thickest in the middle and posterior regions (Pl. 7, fig. 10_a_ and 10_b_), but thin out and almost vanish in the region of the head. The longitudinal section of this stage represented in Pl. 7, fig. 9, passes through one of the lateral masses of mesoblast cells, and shews very distinctly its complete independence of all the other cells in the blastoderm.

From what has been stated with reference to the development of the mesoblast, it is clear that in Elasmobranchii this layer is derived from the same mass of cells as the hypoblast, and receives none of its elements from the epiblast. In connection with its development, as two independent lateral masses, I may observe, as I have previously done[143], that in this respect it bears a close resemblance to mesoblast in Euaxes, as described by Kowalevsky[144]. This resemblance is of some interest, as bearing on a probable Annelid origin of Vertebrata. Kowalevsky has also shewn[145] that the mesoblast in Ascidians is similarly formed as two independent masses, one on each side of the middle line.

Footnote 143: _Quart. Journ. of Microsc. Science_, Oct., 1874. [This Edition, No. V.]

Footnote 144: "Embryologische Studien an Würmern u. Arthropoden." _Mémoires de l'Acad. S. Pétersbourg._ Vol. XIV. 1873.

Footnote 145: _Archiv für Mikr. Anat._ Vol. VII.

It ought, however, to be pointed out that a similar bilateral origin of the mesoblast had been recently met with in Lymnæus by Carl Rabl[146]. A fact which somewhat diminishes the genealogical value of this feature in the mesoblast in Elasmobranchii.

Footnote 146: _Jenaische Zeitschrift_, Vol. IX. 1875. A bilateral development of mesoblast, according to Professor Haeckel (_loc. cit._), occurs in some Osseous Fish. Hensen, _Zeit. für Anat. u. Entw._ Vol. 1., has recently described the mesoblast in Mammalia as consisting of independent lateral masses.

During the course of this stage the spherules of food-yolk immediately beneath the embryo are used up very rapidly. As a result of this the protoplasmic network, so often spoken of, comes very plainly into view. Considerable areas may sometimes be seen without any yolk-spherule whatever.

On Pl. 7, fig. 7_a_, and figs. 11 and 12, I have attempted to reproduce the various appearances presented by this network: and these figures give a better idea of it than any description. My observations tend to shew that it extends through the whole yolk, and serves to hold it together. It has not been possible for me to satisfy myself that it had any definite limits, but on the other hand, in many parts all my efforts to demonstrate its presence have failed. When the yolk-spherules are very thickly packed, it is difficult to make out for certain whether it is present or absent, and I have not succeeded in removing the yolk-spherules from the network in cases of this kind. In medium-sized ovarian eggs this network is very easily seen, and extends through the whole yolk. Part of such an egg is shewn in Pl. 7, fig. 14. In full-sized ovarian eggs, according to Schultz[147], it forms, as was mentioned in the first chapter, radiating striæ, extending from the centre to the periphery of the egg. When examined with the highest powers, the lines of this network appear to be composed of immeasurably small granules arranged in a linear direction. These granules are more distinct in chromic acid specimens than in those hardened in osmic acid, but are to be seen in both. There can be little doubt that these granules are imbedded in a thread or thin layer of protoplasm.

Footnote 147: _Archiv für Mikr. Anat._ Vol. XI.

I have already (p. 252) touched upon the relation of this network to the nuclei of the yolk[148].

Footnote 148: A protoplasmic network resembling in its essential features the one just described has been noticed by many observers in other ova. Fol has figured and described a network or sponge-like arrangement of the protoplasm in the eggs of Geryonia. (_Jenaische Zeitschrift_, Vol. VII.) Metschnikoff (_Zeitschrift f. Wiss. Zoologie_, 1874) has demonstrated its presence in the ova of many Siphonophoriæ and Medusæ. Flemming ("Entwicklungsgeschichte der Najaden," _Sitz. der k. Akad. Wien_, 1875) has found it in the ovarian ova of fresh-water mussels (Anodonta and Unio), but regards it as due to the action of reagents, since he fails to find it in the fresh condition. Amongst vertebrates it has been carefully described by Eimer (_Archiv für Mikr. Anat._, Vol. VIII.) in the ovarian ova of Reptiles. Eimer moreover finds that it is continuous with prolongations from cells of the epithelium of the follicle in which the ovum is contained. According to him remnants of this network are to be met with in the ripe ovum, but are no longer present in the ovum when taken from the oviduct.

During the stages which have just been described specially favourable views are frequently to be obtained of the formation of cells in the yolk and their entrance into the blastoderm. Two representations of these are given, in Pl. 7, fig. 7_a_, and fig. 13. In both of these distinctly circumscribed cells are to be seen in the yolk (_c_), and in all cases are situated near to the typical nuclei of the yolk. The cells in the yolk have such a relation to the surrounding parts, that it is quite certain that their presence is not due to artificial manipulation, and in some cases it is even difficult to decide whether or no a cell area is circumscribed round a nucleus (Pl. 7, fig. 13). Although it would be possible for cells in the living state to pass from the blastoderm into the yolk, yet the view that they have done so in the cases under consideration has not much to recommend it, if the following facts be taken into consideration. (1) That the cells in the yolk are frequently larger than those in the blastoderm. (2) That there are present a very large number of nuclei in the yolk which precisely resemble the nuclei of the cells under discussion. (3) That in some cases (Pl. 7, fig. 13) cells are seen indistinctly circumscribed as if in the act of being formed.

Between the blastoderm and the yolk may frequently be seen a membrane-like structure, which becomes stained with hæmatoxylin, osmic acid etc. It appears to be a layer of coagulated albumen and not a distinct membrane.

SUMMARY.

At the close of segmentation, the blastoderm forms a somewhat lens-shaped disc, thicker at one end than at the other; the thicker end being termed the embryonic end.

It is divided into two layers--an upper one, the epiblast, formed by a single row of columnar cells; and a lower one, consisting of the remaining cells of the blastoderm.

A cavity next appears in the lower layer cells, near the non-embryonic end of the blastoderm, but the cells soon disappear from the floor of this cavity which then comes to be constituted by yolk alone.

The epiblast in the next stage is reflected for a small arc at the embryonic end of the blastoderm, and becomes continuous with the lower layer cells; at the same time some of the lower layer cells of the embryonic end of the blastoderm assume a columnar form, and constitute the commencing hypoblast. The portion of the blastoderm, where epiblast and hypoblast are continuous, forms a projecting structure which I have called the embryonic rim. This rim increases rapidly by growing inwards more and more towards the centre of the blastoderm, through the continuous conversion of lower layer cells into columnar hypoblast.

While the embryonic rim is being formed, the segmentation cavity undergoes important changes. In the first place, it receives a floor of lower layer cells, partly from an ingrowth from the two sides, and partly from the formation of cells around the nuclei of the yolk.

Shortly after the floor of cells has appeared, the whole segmentation cavity becomes obliterated.

When the embryonic rim has attained to some importance, the position of the embryo becomes marked out by the appearance of the medullary groove at its most projecting part. The embryo extends from the edge of the blastoderm inwards towards the centre.

At about the time of the formation of the medullary groove, the mesoblast becomes definitely constituted. It arises as two independent plates, one on each side of the medullary groove, and is entirely derived from lower layer cells.

The two plates of mesoblast are at first unconnected with any other cells of the blastoderm, and, on their formation, the hypoblast remains in connection with all the remaining lower layer cells. Between the embryonic rim and the yolk is a cavity,--the primitive alimentary cavity. Its roof is formed of hypoblast, and its floor of yolk. Its external opening is homologous with the anus of Rusconi, of Amphioxus and the Amphibians. The ventral wall of the alimentary cavity is eventually derived from cells formed in the yolk around the nuclei which are there present.

* * * * *

Since the important researches of Gegenbaur[149] upon the meroblastic vertebrate eggs, it has been generally admitted that the ovum of every vertebrate, however complicated may be its apparent constitution, is nevertheless to be regarded as a simple cell. This view is, indeed, opposed by His[150] and to a very modified extent by Waldeyer[151], and has recently been attacked from an entirely new standpoint by Götte[152]; but, to my mind, the objections of these authors do not upset the well founded conclusions of previous observations.

Footnote 149: "Wirbelthiereier mit partieller Dottertheilung." Müller's _Arch._ 1861.

Footnote 150: _Erste Anlage des Wirbelthierleibes._

Footnote 151: _Eierstock u. Ei._

Footnote 152: _Entwicklungsgeschichte der Unke._ The important researches of Götte on the development of the ovum, though meriting the most careful attention, do not admit of discussion in this place.

As soon as the fact is recognised that both meroblastic and holoblastic eggs have the same fundamental constitution, the admission follows, naturally, though not necessarily, that the eggs belonging to these two classes differ solely in degree, not only as regards their constitution, but also as regards the manner in which they become respectively converted into the embryo. As might have been anticipated, this view has gained a wide acceptance.

Amongst the observations, which have given a strong objective support to this view, may be mentioned those of Professor Lankester upon the development of Cephalopoda[153], and of Dr Götte[154] upon the development of the Hen's egg. In Loligo Professor Lankester shewed that there appeared, in the part of the egg usually considered as food-yolk, a number of bodies, which eventually developed a nucleus and became cells, and that these cells entered into the blastoderm. These observations demonstrate that in the eggs of Loligo the so-called food-yolk is merely equivalent to a part of the egg which in other cases undergoes segmentation.

Footnote 153: _Annals and Magaz. of Natural History_, Vol. XI. 1873, p. 81.

Footnote 154: _Archiv f. Mikr. Anat._ Vol. X.

The observations of Dr Götte have a similar bearing. He made out that in the eggs of the Hen no sharp line is to be found separating the germinal disc from the yolk, and that, independently of the normal segmentation, a number of cells are derived from that part of the egg hitherto regarded as exclusively food-yolk. This view of the nature of the food-yolk was also advanced in my preliminary account of the development of Elasmobranchii[155], and it is now my intention to put forward the positive evidence in favour of this view, which is supplied from a knowledge of the phenomena of the development of the Elasmobranch ovum; and then to discuss how far the facts of the growth of the blastoderm in Elasmobranchii accord with the view that their large food-yolk is exactly equivalent to part of the ovum, which in Amphibians undergoes segmentation, rather than some fresh addition, which has no equivalent in the Amphibian or other holoblastic ovum.

Footnote 155: _Quart. Journ. of Micr. Science_, Oct. 1874.

Taking for granted that the ripe ovum is a single cell, the question arises whether in the case of meroblastic ova the cell is not constituted of two parts completely separated from one another.

Is the meroblastic ovum, before or after impregnation, composed of a germinal disc in which _all_ the protoplasm of the cell is aggregated, and of a food-yolk in which _no_ protoplasm is present? or is the protoplasm present _throughout_, being simply _more concentrated_ at the germinal pole than elsewhere? If the former alternative is accepted, we must suppose that the mass of food-yolk is a something added which is not present in holoblastic ova. If the latter alternative is accepted, it may then be maintained that holoblastic and meroblastic ova are constituted in the same way and differ only in the proportions of their constituents.

My own observations in conjunction with the specially interesting observations of Dr Schultz[156] justify the view which regards the protoplasm as present throughout the whole ovum, and not confined to the germinal disc. Our observations shew that a fine protoplasmic network, with ramifications extending throughout the whole yolk, is present both before and after impregnation.

Footnote 156: _Archiv f. Mikr. Anat._ Vol. XXI.

The presence of this network is, in itself, only sufficient to prove that the yolk _may_ be equivalent to part of a holoblastic ovum; to demonstrate that it is so requires something more, and this link in the chain of evidence is supplied by the nuclei of the yolk, which have been so often referred to.

These nuclei arise independently in the yolk, and become the nuclei of cells which enter the germ and the bodies of which are derived from the protoplasm of the yolk. Not only so, but the cells formed around these nuclei play the same part in the development of Elasmobranchii as do the largest so-called yolk cells in the development of Amphibians. Like the homologous cells in Amphibians, they mainly serve to form the ventral wall of the alimentary canal and the blood-corpuscles. The identity in the fate of the so-called yolk cells of Amphibians with the cells derived from the yolk in Elasmobranchii, must be considered as a proof of the homology of the yolk cells in the first case with the yolk in the second; the difference between the yolk in the two cases arising from the fact that in the Elasmobranch ovum the yolk-spherules bear a larger proportion to the protoplasm than they do in the Amphibian ovum. As I have suggested elsewhere[157], the segmentation or non-segmentation of a particular part of the ovum depends solely upon the proportion borne by the protoplasm to the yolk particles; so that, when the latter exceed the former in a certain fixed proportion, segmentation is no longer possible; and, as this limit is approached, segmentation becomes slower, and the resulting segments larger and larger.

Footnote 157: "Comparison," &c., _Quart. Journ. Micr. Science_, July, 1875. [This Edition, No. VI.]

The question how far the facts in the developmental history of the various vertebrate blastoderms accord with the view of the nature of the yolk just propounded is one of considerable interest. An answer to it has already been attempted from a general point of view in my paper[158] entitled 'The Comparison of the early stages of development in Vertebrates'; but the subject may be conveniently treated here in a special manner for Elasmobranch embryos.

Footnote 158: _Loc. cit._

In the woodcut, fig. 1, _A_, _B_, _C_[159], are represented three diagrammatic longitudinal sections of an Elasmobranch embryo. _A_ nearly corresponds with the longitudinal section represented on Pl. 7, fig. 4, and _B_ with Pl. 7, fig. 7. In Pl. 7, fig. 7, the segmentation cavity has however completely disappeared, while it is still represented as present in the diagram of the same period. If these diagrams, or better still, the woodcuts fig. 2 _A_, _B_, _C_ (which only differ from those of the Elasmobranch fish in the smaller amount of food-yolk), be compared with the corresponding ones of Bombinator, fig. 3, _A_, _B_, _C_, they will be found to be in fundamental agreement with them. First let fig. 1, _A_, or fig. 2, _A_, or Pl. 7, fig. 4, be compared with fig. 3, _A_. In all there is present a segmentation cavity situated not centrally but near the surface of the egg. The roof of the cavity is thin in all, being composed in the Amphibian of epiblast alone, and in the Elasmobranch of epiblast and _lower layer cells_. The floor of the cavity is, in all, formed of so-called yolk (vide Pl. 7, fig. 4), which in all forms the main mass of the egg. In the Amphibian the yolk is segmented, and, though it is not segmented in the Elasmobranch, it contains in compensation the nuclei so often mentioned. In all, the sides of the segmentation cavity are formed by lower layer cells. In the Amphibian the sides are enclosed by smaller cells (in the diagram) which correspond exactly in function and position with the lower layer cells of the Elasmobranch blastoderm.

Footnote 159: This figure, together with figs. 2 and 3, are reproduced from my paper upon the comparison of the early stages of development in vertebrates.

The relation of the yolk to the blastoderm in the Elasmobranch embryo at this stage of development very well suits the view of its homology with the large cells of the Amphibian ovum. The only essential difference between the two ova arises from the roof of the segmentation cavity being in the Elasmobranch embryo formed of lower layer cells, which are absent in the Amphibian embryo. This difference no doubt depends upon the greater quantity of yolk particles present in the Elasmobranch ovum. These increase the bulk of the lower layer cells, which are thus compelled to creep up the sides of the segmentation cavity till they close it in above.

In the next stage for the Elasmobranch, fig. 1 and 2 _B_ and Pl. 7, fig. 7, and for the Amphibian, fig. 3, _B_, the agreement between the two types is again very close. In both for a small portion (_x_) of the edge of the blastoderm the epiblast and hypoblast become continuous, while at all other parts the epiblast, accompanied by lower layer cells, grows round the yolk or round the large cells which correspond to it. The yolk cells of the Amphibian ovum form a comparatively small mass, and are therefore rapidly enveloped; while in the case of the Elasmobranch ovum, owing to the greater mass of the yolk, the same process occupies a long period. In both ova the portion of the blastoderm, where epiblast and hypoblast become continuous, forms the dorsal lip of an opening--the anus of Rusconi--which leads into the alimentary cavity. This cavity has the same relation in both ova. It is lined dorsally by lower layer cells, and ventrally by yolk or what corresponds with yolk; the ventral epithelium of the alimentary canal being in both cases eventually supplied by the yolk cells.

As in the earlier stage, so in the present one, the anatomical relations of the yolk to the blastoderm in the one case (Elasmobranch) are nearly identical with those of the yolk cells to the blastoderm in the other (Amphibian). The main features in which the two embryos differ, during the stage under consideration, arise from the same cause as the solitary point of difference during the preceding stage.

In Amphibians, the alimentary cavity is formed coincidently with a true ingrowth of cells from the point where epiblast and hypoblast become continuous, and from this ingrowth the dorsal wall of the alimentary cavity is formed. The same ingrowth causes the obliteration of the segmentation cavity.

In the Elasmobranchii, owing to the larger bulk of the lower layer cells caused by the food-yolk, these have been compelled to arrange themselves in their final position during segmentation, and no room is left for a true invagination; but instead of this there is formed a simple split between the blastoderm and the yolk. The homology of this with the primitive invagination is nevertheless proved by the survival of a number of features belonging to the ancestral condition in which a true invagination was present. Amongst the more important of these are the following:--(1) The continuity of epiblast and hypoblast at the dorsal lip of the anus of Rusconi. (2) The continuous conversion of indifferent lower layer cells into hypoblast, which gradually extends backwards towards the segmentation cavity, and exactly represents the course of the invagination whereby in Amphibians the dorsal wall of the alimentary cavity is formed. (3) The obliteration of the segmentation cavity during the period when the pseudo-invagination is occurring.

The asymmetry of the gastrula or pseudo-gastrula in Cyclostomes, Amphibians, Elasmobranchii and, I believe, Osseous Fishes, is to be explained by the form of the vertebrate body. In Amphioxus, where the small amount of food-yolk present is distributed uniformly, there is no reason why the invagination and resulting gastrula should not be symmetrical. In other vertebrates, where more food-yolk is present, the shape and structure of the body render it necessary for the food-yolk to be stored away on the ventral side of the alimentary canal. This, combined with the unsymmetrical position of the anus, which primitively corresponds in position with the blastopore or anus of Rusconi, causes the asymmetry of the gastrula invagination, since it is not possible for the part of the ovum which will become the ventral wall of the alimentary canal, and which is loaded with food-yolk, to be invaginated in the same fashion as the dorsal wall. From the asymmetry, so caused, follow a large number of features in vertebrate development, which have been worked out in some detail in my paper already quoted[160].

Footnote 160: _Quart. Journ. of Micr. Science_, July, 1875. [This Edition, No. VI.]

Prof. Haeckel, in a paper recently published[161], appears to imply that because I do not find absolute invagination in Elasmobranchii, I therefore look upon Elasmobranchii as militating against his Gastræa theory. I cannot help thinking that Prof. Haeckel must have somewhat misunderstood my meaning. The importance of the Gastræa theory has always appeared to me to consist not in the fact that an actual ingrowth of certain cells occurs--an ingrowth which might have many different meanings[162]--but in the fact that the types of early development of all animals can be easily derived from that of the typical gastrula. I am perfectly in accordance with Professor Haeckel in regarding the type of Elasmobranch development to be a simple derivative from that of the gastrula, although believing it to be without any true ingrowth or invagination of cells.

Footnote 161: "Die Gastrula u. Eifurchung d. Thiere," _Jenaische Zeitschrift_, Vol. IX.

Footnote 162: For instance, in Crustaceans it does not in some cases appear certain whether an invagination is the typical gastrula invagination, or only an invagination by which, at a period subsequent to the gastrula invagination, the hind gut is frequently formed.

Professor Haeckel[163] in the paper just referred to published his view upon the mutual relationships of the various vertebrate blastoderms. In this paper, which appeared but shortly after my own[164] on the same subject, he has put forward views which differ from mine in several important details. Some of these bear upon the nature of food-yolk; and it appears to me that Professor Haeckel's scheme of development is incompatible with the view that the food-yolk in meroblastic eggs is the homologue of part of the hypoblast of the holoblastic eggs.

Footnote 163: _Loc. cit._

Footnote 164: _Loc. cit._

The following is Professor Haeckel's own statement of the scheme or type, which he regards as characteristic of meroblastic eggs, pp. 98 and 99.

Jetzt folgt der höchst wichtige und interessante Vorgang, den ich als Einstülpung der Blastula auffasse und der zur Bildung der Gastrula führt (Fig. 63, 64)[165]. Es schlägt sich nämlich der verdickte Saum der Keimscheibe, der "Randwulst" oder das _Properistom_, nach innen um und eine dünne Zellenschicht wächst als directe Fortsetzung desselben, wie ein immer enger werdendes Diaphragma, in die Keimhöhle hinein. Diese Zellenschicht ist das entstehende Entoderm (Fig. 64 _i_, 74 _i_). Die Zellen, welche dieselbe zusammensetzen und aus dem innern Theile des Randwulstes hervorwachsen, sind viel grösser aber flacher als die Zellen der Keimhöhlendecke und zeigen ein dunkleres grobkörniges Protoplasma. Auf dem Boden der Keimhöhle, d. h. also auf der Eiweisskugel des Nahrungsdotters, liegen sie unmittelbar auf und rücken hier durch centripetale Wanderung gegen dessen Mitte vor, bis sie dieselbe zuletzt erreichen und nunmehr eine zusammenhängende einschichtige Zellenlage auf dem ganzen Keimhöhlenboden bilden. Diese ist die erste vollständige Anlage des Darmblatts, Entoderms oder "Hypoblasts", und von nun an können wir, im Gegensatz dazu den gesammten übrigen Theil des Blastoderms, nämlich die mehrschichtige Wand der Keimhöhlendecke als Hautblatt, Exoderm oder "Epiblast" bezeichnen. Der verdickte Randwulst (Fig. 64 _w_, 74 _w_), in welchem beide primäre Keimblätter in einander übergehen, besteht in seinem oberen und äusseren Theile aus Exodermzellen, in seinem unteren und inneren Theile aus Entodermzellen.

In diesem Stadium entspricht unser Fischkeim einer Amphiblastula, welche mitten in der Invagination begriffen ist, und bei welcher die entstehende Urdarmhöhle eine grosse Dotterkugel aufgenommen hat. Die Invagination wird nunmehr dadurch vervollständigt und die Gastrulabildung dadurch abgeschlossen, dass die Keimhöhle verschwindet. Das wachsende Entoderm, dem die Dotterkugel innig anhängt, wölbt sich in die letztere hinein und nähert sich so dem Exoderm. Die klare Flüssigkeit in der Keimhöhle wird resorbirt und schliesslich legt sich die obere convexe Fläche des Entoderms an die untere concave des Exoderms eng an: die Gastrula des discoblastischen Eies oder die "Discogastrula" ist fertig (Fig. 65, 76; Meridiandurchschnitt Fig. 66, 75).

Die Discogastrula unsers Knochenfisches in diesem Stadium der vollen Ausbildung stellt nunmehr eine kreisrunde Kappe dar, welche wie ein gefüttertes Mützchen fast die ganze obere Hemisphäre der hyalinen Dotterkugel eng anliegend bedeckt (Fig. 65). Der Ueberzug des Mützchens entspricht dem Exoderm (_e_), sein Futter dem Entoderm (_i_). Ersteres besteht aus drei Schichten von kleineren Zellen, letzteres aus einer einzigen Schicht von grösseren Zellen. Die Exodermzellen (Fig. 77) messen 0.006 - 0.009 Mm., und haben ein klares, sehr feinkörniges Protoplasma. Die Entodermzellen (Fig. 78) messen 0.02 - 0.03 Mm. und ihr Protoplasma ist mehr grobkörnig und trüber. Letztere bilden auch den grössten Theil des Randwulstes, den wir nunmehr als Urmundrand der Gastrula, als "_Properistoma_" oder auch als "RUSCONI'schen After" bezeichnen können. Der letztere umfasst die Dotterkugel, welche die ganze Urdarmhöhle ausfüllt und weit aus der dadurch verstopften Urmund-Oeffnung vorragt.

Footnote 165: The references in this quotation are to the figures in the original.

My objections to the view so lucidly explained in the passage just quoted, fall under two heads.

(1) That the facts of development of the meroblastic eggs of vertebrates, are not in accordance with the views here advanced.

(2) That even if these views be accepted as representing the actual facts of development, the explanation offered of these facts would not be satisfactory.

* * * * *

Professor Haeckel's views are absolutely incompatible with the facts of Elasmobranch development, if my investigations are correct.

The grounds of the incompatibility may be summed up under the following heads:

(1) In Elasmobranchii the hypoblast cells occupy, even before the close of segmentation, the position which, on Professor Haeckel's view, they ought only eventually to take up after being involuted from the whole periphery of the blastoderm.

(2) There is no sign at any period of an invagination of the periphery of the blastoderm, and the only structure (the embryonic rim) which could be mistaken for such an invagination is confined to a very limited arc.

(3) The growth of cells to form the floor of the segmentation cavity, which ought to be part of this general invagination from the periphery, is mainly due to a formation of cells from the yolk.

It is this ingrowth of cells for the floor of the segmentation cavity which, I am inclined to think, Professor Haeckel has mistaken for a general invagination in the Osseous Fish he has investigated.

(4) Professor Haeckel fails to give an account of the asymmetry of the blastoderm; an asymmetry which is unquestionably also present in the blastoderm of most Osseous Fishes, though not noticed by Professor Haeckel in the investigations recorded in his paper.

The facts of development of Osseous Fishes, upon which Professor Haeckel rests his views, are too much disputed, for their discussion in this place to be profitable[166]. The eggs of Osseous Fishes appear to me unsatisfactory objects for the study of this question, partly on account of all the cells of the blastoderm being so much alike, that it is a very difficult matter to distinguish between the various layers, and, partly, because there can be little question that the eggs of existing Osseous Fishes are very much modified, through having lost a great part of the food-yolk possessed by the eggs of their ancestors[167]. This disappearance of the food-yolk must, without doubt, have produced important changes in development, which would be especially marked in a pelagic egg, like that investigated by Professor Haeckel.

Footnote 166: A short statement by Kowalevsky on this subject in a note to his account of the development of Ascidians, would seem to indicate that the type of development of Osseous Fishes is precisely the same as that of Elasmobranchii. Kowalevsky says, _Arch. f. Mikr. Anat._ Vol. VII. p. 114, note 5, "According to my observations on Osseous Fishes the germinal wall consists of two layers, an upper and lower, which are continuous with one another at the border. From the upper one develops skin and nervous system, from the lower hypoblast and mesoblast." This statement, which leaves unanswered a number of important questions, is too short to serve as a basis for supporting my views, but so far as it goes its agreement with the facts of Elasmobranch development is undoubtedly striking.

Footnote 167: The eggs of the Osseous Fishes have, I believe, undergone changes of the same character, but not to the same extent, as those of Mammalia, which, according to the views expressed both by Professor Haeckel and myself, are degenerated from an ovum with a large food-yolk. The grounds on which I regard the eggs of Osseous Fishes as having undergone an analogous change, are too foreign to the subject to be stated here.

The Avian egg has been a still more disputed object than even the egg of the Osseous Fishes. The results of my own investigations on this subject do not accord with those of Dr Götte, or the views of Professor Haeckel[168].

Footnote 168: I find myself unable without figures to understand Dr Rauber's (_Centralblatt für Med. Wiss._ 1874, No. 50; 1875, Nos. 4 and 17) views with sufficient precision to accord to them either my assent or dissent. It is quite in accordance with the view propounded in my paper (_loc. cit._) to regard, with Dr Rauber and Professor Haeckel, the thickened edge of the blastoderm as the homologue of the lip of the blastopore in Amphioxus; though an invagination, in the manner imagined by Professor Haeckel, is no necessary consequence of this view. If Dr Rauber regards the _whole_ egg of the bird as the homologue of that of Amphioxus, and the inclosure of the yolk by the blastoderm as the equivalent to the process of invagination in Amphioxus, then his views are practically in accordance with my own.

Apart from disputed points of development, it appears to me that a comparative account of the development of the meroblastic vertebrate ova ought to take into consideration the essential differences which exist between the Avian and Piscian blastoderms, in that the embryo is situated in the centre of the blastoderm in the first case and at the edge in the second[169].

Footnote 169: I have suggested in a previous paper ("Comparison," &c., _Quart. Journal of Micr. Science_, July, 1875) that the position occupied by the embryo of Birds at the centre, and not at the periphery, of the blastoderm may be due to an abbreviation of the process by which the Elasmobranch embryos cease to be situated at the edge of the blastoderm (vide p. 296 and Pl. 9, fig. 1, 2). Assuming this to be the real explanation of the position of the embryo in Birds, I feel inclined to repeat a speculation which I made some time ago with reference to the primitive streak in Birds (_Quart. Journ. of Micr. Science_, 1873, p. 280). In Birds there is, as is well known, a structure called the primitive streak, which has been shewn by the observations of Dursy, corroborated by my observations (_loc. cit._), to be situated behind the medullary groove, and to take no part in the formation of the embryo. I further shewed that the peculiar fusion of epiblast and mesoblast, called by His the axis cord, was confined to this structure and did not occur in other parts of the blastoderm. Nearly similar results have been recently arrived at by Hensen with reference to the primitive streak in Mammals. The position of the primitive streak immediately behind the embryo suggests the speculation that it may represent the line along which the edges of the blastoderm coalesced, so as to give to the embryo the central position which it has in the blastoderms of Birds and Mammals, and that the peculiar fusion of epiblast and mesoblast at this point may represent the primitive continuity of epiblast and lower layer cells at the dorsal lip of the anus of Rusconi in Elasmobranchii. I put this speculation forward as a mere suggestion, in the hope of elucidating the peculiar structure of the primitive streak, which not improbably may be found to be the keystone to the nature of the blastoderm of the higher vertebrates.

This difference entails important modifications in development, and must necessarily affect the particular points under discussion. As a result of the different positions of the embryo in the two cases, there is present in Elasmobranchii and Osseous Fishes a true anus of Rusconi, or primitive opening into the alimentary canal, which is absent in Birds. Yet in neither Elasmobranchii[170] nor Osseous Fishes does the anus of Rusconi correspond in position with the point where the final closing in of the yolk takes place, but in them this point corresponds rather with the blastopore of Birds[171].

Footnote 170: Vide p. 296 and Plate 9, fig. 1 and 2, and Self, "Comparison," &c., _loc. cit._

Footnote 171: The relation of the anus of Rusconi and blastopore in Elasmobranchii was fully explained in the paper above quoted. It was there clearly shewn that neither the one nor the other exactly corresponds with the blastopore of Amphioxus, but that the two together do so. Professor Haeckel states that in the Osseous Fish investigated by him the anus of Rusconi and the blastopore coincide. This is not the case in the Salmon.

Owing also to the respective situations of the embryo in the blastoderm, the alimentary and neural canals communicate posteriorly in Elasmobranchii and Osseous Fishes, but _not_ in Birds. Of all these points Professor Haeckel makes no mention.

The support of his views which Prof. Haeckel attempts to gain from Götte's researches in Mammalia is completely cut away by the recent discoveries of Van Beneden[172] and Hensen[173].

Footnote 172: "Développement Embryonnaire des Mammifères," _Bulletin de l'Acad. r. d. Belgique_, 1875.

Footnote 173: _Loc. cit._

It thus appears that Professor Haeckel's views but ill accord with the facts of vertebrate development; but even if they were to do so completely it would not in my opinion be easy to give a rational explanation of them.

Professor Haeckel states that no sharp and fast line can be drawn between the types of 'unequal' and 'discoidal' segmentation[174]. In the cases of unequal segmentation he admits, as is certainly the case, that the larger yolk cells (hypoblast) are simply enclosed by a growth of the epiblast around them; which is to be looked on as a modification of the typical gastrula invagination, necessitated by the large size of the yolk cells (vide Professor Haeckel's paper, Taf. II. fig. 30). In these instances there is no commencement of an ingrowth in the _manner supposed for meroblastic ova_.

Footnote 174: For an explanation of these terms, vide Prof. Haeckel's original paper or the abstract in _Quart. Journ. of Micr. Science_ for January, 1876.

When the food-yolk becomes more bulky, and the hypoblast does not completely segment, it is not easy to understand why an ingrowth, which had no existence in the former case, should occur; nor where it is to come from. Such an ingrowth as is supposed to exist by Professor Haeckel would, in fact, break the continuity of development between meroblastic and holoblastic ova, and thus destroy one of the most important results of the Gastræa theory.

It is quite easy to suppose, as I have done, that in the cases of discoidal segmentation, the hypoblast (including the yolk) becomes enclosed by the epiblast in precisely the same manner as in the cases of unequal segmentation.

But even if Professor Haeckel supposes that in the unsegmented food-yolk a fresh element is added to the ovum, it remains quite unintelligible to me how an ingrowth of cells from a circumferential line, to form a layer which had no previous existence, can be equivalent to, or derived from, the invagination of a layer, which exists before the process of invagination begins, and which remains continuous throughout it.

If Professor Haeckel's views should eventually turn out to be in accordance with the facts of vertebrate development, it will, in my opinion, be very difficult to reduce them into conformity with the Gastræa theory.

Although some space has been devoted to an attempt to refute the views of Professor Haeckel on this question, I wish it to be clearly understood that my disagreement from his opinions concerns matters of detail only, and that I quite accept the Gastræa theory in its general bearings.

* * * * *

Observations upon the formation of the layers in Elasmobranchii have hitherto been very few in number. Those published in my preliminary account of these fishes are, I believe, the earliest[175].

Footnote 175: I omit all reference to a paper published in Russian by Prof. Kowalevsky. Being unable to translate it, and the illustrations being too meagre to be in themselves of much assistance, it has not been possible for me to make any use of it.

Since then there has been published a short notice on the subject by Dr Alex. Schultz[176]. His observations in the main accord with my own. He apparently speaks of the nuclei of the yolk as cells, and also of the epiblast being more than one cell deep. In Torpedo alone, amongst the genera investigated by me, is the layer of epiblast, at about the age of the last described embryo, composed of more than a single row of cells.

Footnote 176: _Centralblatt f. Med. Wiss._ No. 33, 1875.

EXPLANATION OF PLATE 7.

COMPLETE LIST OF REFERENCE LETTERS.

_c._ Cells formed in the yolk around the nuclei of the yolk. _ep._ Epiblast. _er._ Embryonic ring. _es._ Embryo swelling. _hy._ Hypoblast. _ll._ Lower layer cells. _ly._ Line separating the yolk from the blastoderm. _m._ Mesoblast. _mg._ Medullary groove. _n´._ Nuclei of yolk. _na._ Cells to form ventral wall of alimentary canal which have been derived from the yolk. _nal._ Cells formed around the nuclei of the yolk which have entered the hypoblast. _sc._ Segmentation cavity. _vp._ Combined lateral and vertebral plate of mesoblast.

Fig. 1. Longitudinal section of a blastoderm at the first appearance of the segmentation cavity.

Fig. 2. Longitudinal section through a blastoderm after the layer of cells has disappeared from the floor of the segmentation cavity. _bd._ Large cell resting on the yolk, probably remaining over from the later periods of segmentation. Magnified 60 diameters. (Hardened in chromic acid.)

The section is intended to illustrate the fact that the nuclei form a layer in the yolk under the floor of the segmentation cavity. The roof of the segmentation cavity is broken.

Fig. 2_a_. Portion of same blastoderm highly magnified, to shew the characters of the nuclei of the yolk _n´_ and the nuclei in the cells of the blastoderm.

Fig. 2_b_. Large knobbed nucleus from the same blastoderm, very highly magnified.

Fig. 2_c_. Nucleus of yolk from the same blastoderm.

Fig. 3. Longitudinal section of blastoderm of same stage as fig. 2. (Hardened in chromic acid.)

Fig. 4. Longitudinal section of blastoderm slightly older than fig. 2. Magnified 45 diameters. (Hardened in osmic acid.)

It illustrates (1) the characters of the epiblast; (2) the embryonic swelling; (3) the segmentation cavity.

Fig. 5. Longitudinal section through a blastoderm at the time of the first appearance of the embryonic rim, and before the formation of the medullary groove. Magnified 45 diameters.

Fig. 5_a_. Section through the periphery of the embryonic rim of the blastoderm of which fig. 5 represents a section.

Fig. 6. Section through the embryonic rim of a blastoderm somewhat younger than that represented on Pl. 8, fig. B.

Fig. 7. Section through the most projecting portion of the embryonic rim of a blastoderm of the same age as that represented on Pl. 8, fig. B. The section is drawn on a very considerably smaller scale than that on fig. 5. It is intended to illustrate the growth of the embryonic rim and the disappearance of the segmentation cavity.

Fig. 7_a_. Section through peripheral portion of the embryonic rim of the same blastoderm, highly magnified. It specially illustrates the formation of a cell (_c_) around a nucleus in the yolk. The nuclei of the blastoderm have been inaccurately rendered by the artist.

Figs. 8_a_, 8_b_, 8_c_. Three sections of the same embryo. Inserted mainly to illustrate the formation of the mesoblast as two independent lateral masses of cells; only half of each section is represented. 8_a_ is the most posterior of the three sections. In it the mesoblast forms a large mass on each side, imperfectly separated from the hypoblast. In 8_b_, from the anterior part of the embryo, the main mass of mesoblast is far smaller, and only forms a cap to the hypoblast at the highest point of the medullary fold. In 8_c_ a cap of mesoblast is present, similar to that in 8_b_, though much smaller. The sections of these embryos were somewhat oblique, and it has unfortunately happened that while in 8_a_ one side is represented, in 8_b_ and 8_c_ the other side is figured, had it not been for this the sections 8_b_ and 8_c_ would have been considerably longer than 8_a_.

Fig. 9. Longitudinal section of an embryo belonging to a slightly later stage than B.

This section passes through one of the medullary folds. It illustrates the continuity of the hypoblast with the remaining lower layer cells of the blastoderm.

Figs. 10_a_, 10_b_, 10_c_. Three sections of the same embryo belonging to a stage slightly later than B, Pl. 8. The space between the mesoblast and the hypoblast has been made considerably too great in the figures of the three sections.

10_a_. The most posterior of the three sections. It shews the posterior flatness of the medullary groove and the two isolated vertebral plates.

10_b_. This section is taken from the anterior part of the same embryo and shews the deep medullary groove and the commencing formation of the ventral wall of the alimentary canal from the nuclei of the yolk.

10_c_ shews the disappearance of the medullary groove and the thinning out of the mesoblast plates in the region of the head.

Fig. 11. Small portion of the blastoderm and the subjacent yolk of an embryo at the time of the first appearance of the medullary groove × 300. It shews two large nuclei of the yolk (_n_) and the protoplasmic network in the yolk between them; the network is seen to be closer round the nuclei than in the intervening space. There are no areas representing cells around the nuclei.

Fig. 12. Nucleus of the yolk in connection with the protoplasmic network hardened in osmic acid.

Fig. 13. Portion of posterior end of a blastoderm of stage B, shewing the formation of cells around the nuclei of the yolk.

Fig. 14. Section through part of a young Scyllium egg, about 1/15th of an inch in diameter.

_nl._ Protoplasmic network in yolk. _zp._ Zona pellucida. _ch._ Structureless chorion. _fep._ Follicular epithelium. _x._ Structureless membrane external to this.