From the description given above it will be clear that in the chick the mesoblast has an independent origin; it can be said neither to originate from the epiblast nor from the hypoblast. It is formed coincidently with the latter out of apparently similar segmentation cells. The hypoblast, as has been long known, shews in the chick no trace of its primitive method of formation by involution, neither does the mesoblast shew any signs of its primitive mode of formation. In so excessively highly differentiated a type as birds we could hardly expect to find, and certainly do not find, any traces of the primitive origin of the mesoblast, either from the epiblast or hypoblast, or from both. In the chick the mesoblast cells are formed directly from the ultimate products of segmentation. From having a secondary origin in most invertebrates the mesoblast comes to have, in the chick, a primary origin from the segmentation spheres, precisely as we find to be the case with the nervous layer in osseous fishes. It is true we cannot tell which segmentation-cells will form the mesoblast, and which the hypoblast; but the mesoblast and hypoblast are formed at the same time, and both of them directly from segmentation spheres.
The process of formation of the mesoblast in Loligo, as observed by Mr Ray Lankester (Annals and Magazine of Natural History, February, 1873), is still more modified. Here the mesoblast arises independently of the blastoderm, and by a process of free cell-formation in the yolk round the edge of the blastoderm. If Oellacher's observations in reference to the origin of formative cells are correct, then the modes of origin of the mesoblast in Loligo and the chick would have nothing in common; but if the formative cells are in reality derived from the white yolk, and also are alone concerned in the formation of the mesoblast, then the modes of formation of the mesoblast in the chick would be substantially the same as that observed by Mr Ray Lankester in Loligo.
No very important changes take place in the actual forms of the cells during the next few hours. A kind of fusion takes place between the epiblast and the mesoblast along the line of the primitive streak forming the axis-string of His; but the line of junction between the layers is almost always more or less visible in sections. In any case it does not appear that there is any derivation of mesoblast cells from the epiblast; and since the fusion only takes place in the region of the primitive groove, and not in front, where the medullary groove arises (see succeeding paper), it cannot be considered of any importance in reference to the possible origin of the Wolffian duct, &c., from the epiblast (as mooted by Waldeyer, Eierstock und Ei, Leipzig, 1870). The primitive groove, as can be seen in sections, begins to appear very early, generally before the twelfth hour. The epiblast spreads rapidly over the white yolk, and the area pellucida also increases in size.
From the mesoblast forming at first only a small mass of cells, which lies below the primitive streak, it soon comes to be the most important layer of the blastoderm. Its growth is effected by means of the formative cells. These cells are generally not very numerous in an unincubated blastoderm, but rapidly increase in numbers, probably by division; at the same time they travel round the edge of, and in some cases through, the hypoblast, and then become converted in the manner described into mesoblast cells. They act as carriers of food from the white yolk to the mesoblast till, after the formation of the vascular area, they are no longer necessary. The numerous cases in which two nucleoli and even two nuclei can be seen in one cell prove that the mesoblast cells also increase by division.
The growth of the hypoblast takes place in a very different way. It occurs by a direct conversion, cell for cell, of the white yolk spheres into hypoblast cells. This interpretation of the appearances, which I will describe presently, was first suggested to me by Dr Foster, from an examination of some of my specimens of about thirty-six hours, prepared with silver nitrate. Where there is no folding at the junction between the pellucid and opaque areas, there seems to be a perfect continuity in the silver markings and a gradual transition in the cells, from what would be undoubtedly called white yolk spheres, to as undoubted hypoblast cells (vide Pl. 1, fig. 5). In passing from the opaque to the pellucid areas the number of white yolk spherules in each cell becomes less, but it is not till some way into the pellucid area that they quite cease to be present. I at first thought that this was merely due to the hypoblast cells feeding on the white yolk sphericles, but the perfect continuity of the cells, and the perfect gradation in passing from the white yolk cells to the hypoblast, proves that the other interpretation is the correct one, viz. that the white yolk spheres become directly converted into the hypoblast cells. This is well shewn in sections (vide Pl. 1, fig. 4) taken from embryos of all ages from the fifteenth to the thirty-sixth hour and onwards. But it is, perhaps, most easily seen in embryos of about twenty hours. In such an embryo there is a most perfect gradation: the cells of the hypoblast become, as they approach the edge of the pellucid area, broader, and are more and more filled with white yolk sphericles, till at the line of junction it is quite impossible to say whether a particular cell is a white-yolk cell (sphere) or a hypoblast cell. The white-yolk cells near the line of junction can frequently be seen to possess nuclei. At first the hypoblast appears to end abruptly against the white yolk; this state of things, however, soon ends, and there supervenes a complete and unbroken continuity between the hypoblast and the white yolk.
Of the mode of increase of the epiblast I have but little to say. The cells undoubtedly increase entirely by division, and the new material is most probably derived directly from the white yolk.
Up to the sixth hour the cells of the upper layer retain their early regular hexagonal pattern, but by the twelfth hour they have generally entirely lost this, and are irregularly shaped and very angular. The cells over the centre of the pellucid area remain the smallest up to the twenty-fifth hour or later, while those over the rest of the pellucid area are uniformly larger.
In the hypoblast the cells under the primitive groove, and on each side as far as the fold which marks off the exterior limit of the protovertebræ are at the eighteenth hour considerably smaller than any other cells of this layer.
In all the embryos between the eighteenth and twenty-third hour which I have examined for the purpose, I have found that at about two-thirds of the distance from the anterior end of the pellucid area, and just external to the side fold, there is a small space on each side in which the cells are considerably larger than anywhere else in the hypoblast. These larger cells, moreover, contain a greater number of highly refractive spherules than any other cells. It is not easy to understand why growth should have been less rapid here than elsewhere, as the position does not seem to correspond to any feature in the embryo. In some specimens the hypoblast cells at the extreme edge of the pellucid area are smaller than the cells immediately internal to them. At about the twenty-third hour these cells begin rapidly to lose the refractive spherules they contained in the earlier stages of incubation, and come to consist of a nucleus surrounded simply by granular protoplasm.
At about this period of incubation the formative cells are especially numerous at the periphery of the blastoderm, and, no doubt, become converted into the mass of mesoblast which is found at about the twenty-fifth hour in the region of the vascular area. Some of them are lobate, and appear as if they were undergoing division. At this time also the greatest number of formative cells are to be found at the bottom of the now large segmentation cavity.