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This material is from the 4th edition of The Zebrafish Book. The 5th edition is available in print and within the ZFIN Protocol Wiki.

Stages During the Blastula Period

Modified from: Kimmel et al., 1995. Developmental Dynamics 203:253-310. Copyright © 1995 Wiley-Liss, Inc. Reprinted only by permission of Wiley-Liss, a subsidiary of John Wiley & Sons, Inc.

128-cell stage (2 1/4 h): The eighth cycle begins with 128 blastomeres arranged as a high mound of cells, a solid half ball perched on the yolk cell. In contrast to earlier cleavages, the cleavage furrows that bring the 64-cell stage to an end generally occur so irregularly that with few exceptions one cannot after this time deduce a blastomere's cellular ancestry from its position. One can, however, distinguish whether a view from the side corresponds to a view along the earlier odd numbered cleavage planes (the face view, described above) or the even numbered ones because of the oblong shape of the blastodisc, which remains as described for the 4-cell stage. As seen from a face view, the EVL cells line up in about five irregular tiers between the animal pole and the margin.

256-cell stage (2 1/2 h): A face view (Fig. 8A) at the end of the eighth set of cleavages reveals the EVL cells in about seven irregular tiers. The EVL cells thin out considerably during the interphase of this ninth cycle. The deep cells substantially outnumber those of the EVL. The ninth cleavage divisions are the last highly synchronous, or metasynchronous, ones to occur.

512-cell stage (2 3/4 h): Here begins midblastula transition; cell cycles lengthen gradually during the next several divisions (Fig. 9A). In face view, about nine somewhat irregular tiers of EVL blastomeres occur between the margin and animal pole. During the last part of this stage and particularly as they enter the tenth mitosis, the marginal blastomeres (the first-tier EVL cells) begin to lose their lower borders where they join the yolk cell (Fig. 10A, B). This morphogenetic change signals the beginning of formation of the YSL; the marginal cells specifically do not undergo cytokinesis following the tenth mitosis. This mitosis occurs with enough synchrony that one can still find a minute or so of time when all cells, including the yolk cell, are in mitosis (i.e., the cells have no interphase nuclei; Fig. 10C). The 512-cell stage is the last cycle when this is possible.

1k-cell stage (3 h): One can usually, but not always, see a YSL (Fig. 10D), irregular in form and containing a total of about 20 nuclei within a single ring around the blastodisc margin. There are fewer than 1024 blastomeres after division 10 because the first-tier (marginal) EVL cells from the previous stage joined together in the YSL. Moreover, because of the manner of YSL formation, the cells making up the first EVL tier at this stage are descendants of those that were in the second tier a stage earlier.

In some embryos, the YSL forms over the course of two cycles, sometimes beginning a stage earlier and other times a stage later than the 1-k stage.

About eleven tiers of cells occupy the EVL. The eleventh set of mitoses occurs as a discernable wave passing through the blastodisc, and is the last one to do so. During this wave, for the first time, many of the cells can be seen to be out of phase with their neighbors; some have and some don't have interphase nuclei.

High stage (3 1/3 h): This stage marks the end of the period during which the blastodisc perches "high" upon the yolk cell (Figs. 8B, C). There is still a pinched ring, a constriction, where the marginal cells meet the YSL. One distinguishes the high stage from earlier ones by the appearance and numbers of both blastodisc cells and YSL nuclei. In side view there are substantially more than 11 tiers of EVL cells visible between the margin and the animal pole. In any region of the blastodisc throughout this whole stage there are some cells in interphase and others in mitosis. Most blastodisc cells complete zygotic cell cycle 12 whereas a few complete cycle 13 during this stage.

The YSL still has the form of a thin ring, but its outer edge, i.e. the edge away from the blastodisc, is now much smoother in contour. The YSL nuclei reappear from their second division without cytokinesis (their eleventh zygotic division) generally all still external to the blastodisc margin, and more tightly packed together (Fig 10F).

Oblong stage (3 2/3 h): The animal-vegetal axis of the blastula shortens, with the blastodisc compressing down upon the yolk cell, as one could imagine to result from a uniform increase in tension at the surface. The constriction at the blastodisc margin that has been present since the elevation of non-yolky cytoplasm during the 1-cell stage diminishes (Fig. 8C) and then disappears. Eventually the blastula acquires a smoothly outlined ellipsoidal shape, as viewed from the side, and the stage is named for this oblong shape. The blastomeres are dividing highly asynchronously, many of them being in cycle 12 or 13. The EVL is by now extremely thin, and careful observation with Nomarski is needed to detect its presence.

The YSL is spreading deep underneath the blastodisc, and spreading away from it as well. During their interphases the YSL nuclei form several rows, some nuclei invariably in the I-YSL.

Sphere stage (4 h): Continued shortening along the animal-vegetal axis generates a late blastula of smooth and approximately spherical shape. The overall shape then changes little the next several hours, well into the period of gastrulation, but cell rearrangements that begin now seem to occur more rapidly than at any other time in development. One distinguishes this stage from the dome stage that comes next by the appearance, at a very deep plane of focus, of the face between the lower part of the blastodisc and the upper part of the yolk cell, the I-YSL. At sphere stage specifically this interface is flat, or nearly flat (Fig. 8D).

Many of the cells of the blastodisc are in cycle 13, and in the EVL this cycle is often extremely long, such that mitoses occur very rarely in the EVL. The E-YSL is often noticeably thinner than earlier, and most YSL nuclei are internal. The last of the YSL nuclear divisions occur. Frequently, dechorionated embryos begin to be oriented animal pole-upwards, rather than lying on their sides as earlier.

Dome stage (4 1/3 h): Deep to the blastodisc the I-YSL surface begins to dome towards the animal pole (Fig. 8E). This prominent and rapidly occurring change in the interface between the yolk cell and the blastodisc represents the first sure sign that epiboly is beginning. EVL cells are in a long cycle 13 and many deep cells enter cycle 14. The E-YSL begins to narrow (Solnica-Krezel and Driever, 1994). The YSL nuclei are postmitotic; i.e., in contrast to when they were in mitotic cycles just preceding dome stage, the nuclei are now always visible. They begin to enlarge, and one can see them quite easily.

30%-epiboly stage (4 2/3 h): Epiboly, including doming of the yolk cell, produces a blastoderm, as we may now call it, of nearly uniform thickness (Fig. 8F). It now consists of a highly flattened EVL monolayer (hard to see except by focusing exactly in the plane of the blastoderm surface with Nomarski optics), and a deep cell multilayer (DEL) about 4 cells thick.

The extent to which the blastoderm has spread over across the yolk cell provides an extremely useful staging index from this stage until epiboly ends. We define percent-epiboly to mean the fraction of the yolk cell that the blastoderm covers; percent-coverage would be a more precise term for what we mean to say, but percent-epiboly immediately focuses on the process and is in common usage. Hence, at 30%-epiboly the blastoderm margin is at 30% of the entire distance between the animal and vegetal poles, as one estimates along the animal-vegetal axis.

The blastoderm thickness is not exactly uniform in many embryos at this stage. Observing the marginal region from a side view, as the blastula is rotated about the animal-vegetal axis will reveal one region along the margin that is noticeably thinner and flatter than elsewhere. This particular region will become the dorsal side of the embryo (Schmitz and Campos-Ortega, 1994). We observe the asymmetry most easily using low-mahtmlication Nomarski optics. To distinguish the dorsal side with absolute confidence, however, one needs to await shield stage (6 h).


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