Chapter III: Formation of the Layers (1)

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._