FUSION OF EPIBLASTIC LAYERS IN RABBIT AND FROG. 165

On the Phenomenon of the Fusion of the Epiblastic Layers in the Rabbit and in the Frog.

By

Richard Assheton, M.A.

With Plate 18.

In my paper upon the early stages of the development of the rabbit I have given evidence in support of the views held by Balfour and Heape concerning the fate of the outer layer of epiblast over the embryonic disc of the rabbit embryo of the seventh day. The two layers of epiblast gradually fuse together, and cells from each take part in the formation of the permanent epiblast.

In the description I have given of the process, and in the attempt I have made to explain how the fusion is brought about, I have regarded the phenomenon as being entirely accidental and of no morphological importance.

It is, however, only right to point out that there is a fusion of two epiblastic layers in certain Amphibians which seems to have a deeper meaning, and to which in some way the condition in the rabbit may be comparable.

As far as I know, there is no account published of this phenomenon, and I am not aware that anyone else has noticed it. Therefore, it seems to me, a short account of the facts as they appear in young embryos of Rana temporaria may be of some interest.

For the purpose of following the fusion of the two epi

blast layers in the frog, it is best to examine the sections unstained.

In Rana temporaria the epiblast is from a very early period divided into two layers-an outer called the epidermic layer, and an inner called the nervous layer. If a section is taken, say, transversely though the neural plate of a tadpole about the time of the folding up of the neural folds, a section is obtained of which fig. 1 is a drawing.

The two layers are seen to be very sharply and distinctly divided, the outer or epidermic layer of epiblast (E. EP.) is a single cell in thickness. The cells are much more deeply pigmented than are the cells of the inner or nervous layer of epiblast (E. NE.) which forms a much thicker layer. The cells of this layer are very closely packed in the region of the neural plate through which this section is taken; so much so as to render it impossible at this stage, at any rate, to distinguish the boundary of the cells-if, indeed, there are any distinct boundaries. The nuclei are large, but are only seen with difficulty without staining. Some indication of the boundaries of the cells is to be seen in the slight increase of pigment along certain lines.

In fig. 2, which is taken at a slightly later stage, but before the neural folds have completely closed, the cells of the epidermic layer may be seen to have become much elongated, their inner borders being no longer truncated, but mostly pointed, and seem to be growing into the mass of nervous epiblast.

In fig. 3, which is from a section of a tadpole of about 3 mm. to 3 mm., in which the neural tube is now completely closed and separated off from the skin, the appearance of the darkly pigmented cells is very remarkable and instructive.

I can see no reason to doubt that the darkly pigmented cells of the epidermic epiblast, seen to be elongated in fig. 3, have by this time elongated and passed right through the mass of nervous cells (or nuclei) and spread out into fine filaments on the further side of the nervous layer. I cannot say for certain whether these fine filaments anastomose or not, but, however

that may be, they seem to form that which may, as a whole, be regarded as a reticulum, which is comparable to the myelospongium of His.

The outlines between the cells of the nervous layer are quite imperceptible in unstained specimens of this stage. The nuclei of the nervous layer are only seen with great difficulty.

The boundaries of the epidermic layer cells can in most cases be readily perceived, owing to the deeper pigmentation of the cells of this layer. The processes (PR.) are sharply and clearly defined, and are heavily loaded with pigment.

The bodies of the epidermic epiblast cells remain as yet lining the interior of the neural canal, although there is certainly a tendency for the nuclei in some of them to move more inwards. Also there seems to be a tendency for the epidermic cells to become pressed apart by the nervous cells. This is more marked at this stage in the spinal cord than in the brain, as may be seen in fig. 4.

I think we may take these appearances as a conclusive proof of the occurrence of an intimate fusion of the two epiblastic layers in the frog.

Observe the condition of the auditory vesicle in fig. 3. Here the walls of the vesicle are composed entirely of the lightcoloured elements. There is no trace of the dark, deeplypigmented strands such as are to be seen in the central nervous system. This is a further piece of evidence that the dark strands in fig. 3 are derived from the epidermic layer. For, as is well known, in the frog the nervous layer alone gives rise to the auditory vesicle-so it is a most significant fact that the dark strands should be entirely absent in this case.

After this stage (3—5 mm.) it is extremely difficult to trace the fate of the epidermic and nervous cells respectively. Up to now, the fact of the greater pigmentation of the former has rendered the inquiry easy.

The origin and meaning of the pigmentation is obscure, but its presence in the frog seems to be due to two causes, separate at any rate chronologically.

Firstly. Pigment is present in the unfertilised ovum as a

superficial layer covering the upper pole. Hence for a long time we find the superficial layer of cells after segmentation to be more deeply pigmented than the more internally situated segments.

Secondly. Pigment seems to be in some way connected with the actual protoplasmic activity, as it appears internally wherever division of cells takes place.

So also I believe the intensely black appearance of the processes of the epidermic epiblast which I have been describing is in some way connected with their intense activity just evinced by their growth inwards-which seems to be very rapid, and rather sudden.

When at a later period certain groups of nerve cells show a similar intense activity, there is a similar deposition of pigment in and around their processes, as, for instance, in the development of the ganglion habenula as shown in fig. 5. This pigment in each case becomes greatly lessened after the period of intense activity of growth has passed by.

The Question of Early Separation into Neuroblastic and Spongioblastic Elements.

Although the evidence to be drawn from the figures accompanying this paper is far from being conclusive, yet I think it tends very strongly towards the inference that the epidermic layer of epiblast in the frog gives rise to the spongioblastic elements, and the nervous layer to the neuroblasts.

His has shown how the spongioblastic network precedes the development of neuroblasts and nerve fibres and forms an irregular network with angular processes by no means unlike the dark strands in my fig. 3. These processes are not at all like the processes of nerve cells, which are always more tapering and less knotty. Further, at this stage there cannot be found any trace of definite nerve-fibres. These do not appear until later, till the tadpole has attained a length of about 6-6 mm.

I can only interpret these figures as showing the conversion of the epidermic layer of epiblast into a supporting