Passing from the external features of this stage to those which are displayed by sections, the striking point to be noticed is the persisting continuity of the segments, marked out on the surface, with the floor of the germinal disc.
The furrows which are visible on the surface merely form a pattern, but do not isolate a series of distinct segments. They do not even extend to the limit of the finely granular matter of the germinal disc.
The section represented, Pl. 6, fig. 6, bears out the statements about the segments as seen on the surface. There are three smaller segments in the middle of the section, and two larger at the two ends. These latter are continuous with the coarser yolk-spheres surrounding the germinal disc and are not separated from them by a segmentation furrow.
In a slightly older embryo than the one figured I met with a few completely isolated segments at the surface. These segments were formed by the apparent bifurcation of furrows as they neared the surface of the germinal disc. The segments thus produced are triangular in form. They probably owe their origin to the meeting of two oblique furrows. The last-formed of these furrows apparently ceases to be prolonged after meeting the first-formed furrow. I have not in any case observed an example of two furrows crossing one another at this stage.
The furrows themselves for the most part are by no means simple slits with parallel sides. They exhibit a beaded structure, shewn imperfectly in Pl. 6, fig. 6, but better in Pl. 6, fig. 6a, which is executed on a larger scale. They present intervals of dilatations where the protoplasms of the segments on the two sides of the furrow are widely separated, alternating with intervals where the protoplasms of the two segments are almost in contact and are only separated from one another by a very narrow space.
A closer study of the germinal disc at this period shews that the cavities which cause the beaded structure of the furrows are not only present along the lines of the furrows but are also found scattered generally through the germinal disc, though far more thickly in the neighbourhood of the furrows. Their appearance is that of vacuoles, and with these they are probably to be compared. There can be little question that in the living germinal disc they are filled with fluid. In some cases, they are collected in very large numbers in the region of a furrow. Such a case as this is shewn in Pl. 6, fig. 6b. In numerous other cases they occur, roughly speaking, alternately on each side of a furrow. Some furrows, though not many, are entirely destitute of these structures. The character of their distribution renders it impossible to overlook the fact that these vacuole-like bodies have important relations with the formation of the segmentation furrows.
Lining the two sides of the segmentation furrows there is present in sections a layer which stains deeply with colouring reagents; and the surface of the blastoderm is stained in the same manner. In neither case is it permissible to suppose that any membrane-like structure is present. In many cases a similar very delicate, but deeply-stained line, invests the vacuolar cavities, but the fluid filling these remains quite unstained. When distinct segments are formed, each of these is surrounded by a similarly stained line.
The yolk-spherules are so numerous, and render even the thinnest section so opaque, that I have failed to make satisfactory observations on the behaviour of the nucleus. I find nuclei in many of the segments, though it is very difficult even to see them, and only in very favourable specimens can their structure be studied. In some cases, two of them lie one on each side of a furrow; and in one case at the extreme end of a furrow I could see two peculiar aggregations of yolk-spherules united by a band through which the furrow, had it been continued, would have passed. The connection (if any exists) between this appearance and the formation of the fresh nuclei in the segments, I have been unable to elucidate.
The peculiar appearances attending the formation of fresh nuclei in connection with cell-division, which have recently been described by so many observers, have hitherto escaped my observation at this stage of the segmentation, though I shall describe them in a later stage. A nucleus of this stage is shewn on Pl. 6, fig. 6c. It is lobate in form and is divided by lines into areas in each of which a deeply-stained granule is situated.
The succeeding stages of segmentation present from the surface no fresh features of great interest. The somewhat irregular (Pl. 6, figs. 4 and 5) circular line, which divides the peripheral larger from the central smaller segments, remains for a long time conspicuous. It appears to be the representative of the horizontal furrow which, in the Batrachian ovum, separates the smaller pigmented spheres from the larger spheres of the lower pole of the egg.