ZFIN Gastrula Period Stages

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 Gastrula Period

50%-epiboly stage (5 1/4 h): Epiboly displaces the blastoderm margin to 50% of the distance between the animal and vegetal pole. The margin remains at about this same position for about an hour (Fig. 12), the duration of this stage and the next two. Hence, it is important to distinguish these early gastrula stages by examining the thickness at and along the marginal region of the blastoderm. At this 50%-epiboly stage radial intercalations have produced a blastoderm that is very uniform in thickness (Fig. 11A). In particular, an animal polar view with a dissecting microscope will reveal that the blastoderm rim is not yet distinctively thickened. Many DEL cells are in late cycle 14.

Germ-ring stage (5 2/3 h): The germ ring forms as a thickened annulus at the blastoderm margin, as one can discern in an animal-polar view (Fig. 1 and Fig. 11C; arrow). The germ ring appears nearly uniform in structure around the entire circumference of the blastoderm. Nomarski inspection, or sections of fixed embryos, reveal that within the germ ring the blastoderm consists of two layers, in addition to the EVL. The epiblast is uppermost, and is about three cells thick. The hypoblast lies beneath the epiblast, directly adjacent to the YSL. It is only about one to two cells thick. The yolk cell remains about half covered by the blastoderm (i.e epiboly remains at 50%). Many DEL cells are completing cycle 14.

Shield stage (6 h): An animal polar view most easily reveals the embryonic shield, as well as the germ ring, if one is using a dissecting microscope (arrows in Fig. 1 and Fig. 11E). A side view reveals that both the epiblast and hypoblast are locally thickened at the shield (Fig. 11D). Epiboly remains at 50% until late shield stage, when the yolk cell can be judged to be more than half-covered by the blastoderm. Many DEL cells are beginning cycle 15.

Because the embryonic shield marks the eventual dorsal side of the embryo, and because cells at the animal pole will develop into head structures, one can reliably determine both the AP and DV axes for the first time. From this stage onwards, because we can distinguish dorsal from ventral views, we use "side view" specifically with reference to the definitive embryonic axes.

75%-epiboly stage (8 h): Between about 60%- and 100%-epiboly we estimate the extent of epiboly for the staging index, and determine the standard developmental age from the relationship in Fig. 12. As epiboly continues the shape of the embryo itself becomes more along the animal-vegetal (approximately the AP) axis (Fig. 11F-J).

The embryonic shield becomes less distinctive, as compared to shield stage, as its cells repack to elongate the shield along the AP axis and narrow it mediolaterally. A ventral view reveals that the anterior axial hypoblast that will develop as the prechordal plate reaches the animal pole (Fig. 11G; arrow). A side view shows that the blastoderm is thinner than elsewhere on the ventral side, above the margin (Fig. 11H; arrow). This region is the evacuation zone; cells leave it by both epiboly and convergence (Ballard, 1981). In dorsal view, the boundaries in the hypoblast between the axial chorda mesoderm and paraxial segmental plate mesoderm become visible (Fig. 11I; arrows).

90%-epiboly stage (9 h): The bit of uncovered yolk cell protruding from the neighborhood of the vegetal pole may now be considered a yolk plug (Fig. 11I-K). The dorsal side of the blastoderm is very distinctively thicker than the ventral side (Fig 11J). Careful inspection with the dissecting microscope reveals Brachet's cleft between epiblast and hypoblast layers, particularly near, not just at, the dorsal midline. Cells have left the ventral region by epiboly and by convergence towards the dorsal side, leaving behind an enlarged cell-depleted evacuation zone (located approximately at 10:00 o'clock in the side view shown in Fig. 11J). The dorsal epiblast thickens anteriorly to form the neural plate (at 1:00 o'clock in the same panel), representing the brain anlagen. The prechordal plate extends just past the animal pole by 90%-epiboly Fig. 11K). Many DEL cells are in cycle 16, and the earliest postmitotic cells are present, including cells that will form the notochord, axial somite-derived muscles, and specific neurons in the hindbrain (Mendelson, 1986).

Bud stage (10 h): Epiboly comes to a close as the blastoderm completely covers the yolk plug, defining 100%-epiboly. Convergence and extension movements have spread the blastoderm across the yolk cell faster on the dorsal side than on the ventral side, and because of this asymmetry, the point at which the yolk plug disappears is not at the vegetal pole of the yolk cell, but somewhat ventral to it.

Just dorsal to the site of yolk plug closure, and usually within 10-15 min of the closure, the posterior or caudal end of the embryonic axis develops a distinct swelling, the tail bud, for which we name the stage (Fig. 11L, arrowhead). The region of cells may also contribute to posterior trunk, but the contribution is not a dramatic one, whereas a labeled cell in the tail bud at 10 h invariably will contribute progeny to the tail.

Along the dorsal side, anterior to the tail bud, the neural plate is now thickened along the entire embryonic axis; its more posterior cells will contribute to trunk spinal cord. The thickening is most prominent near the animal pole in the prospective head region, where the head will form, and here the surface of the plate has a shallow midsagittal groove. Prechordal plate hypoblast accumulating deep to the anterior-most neural plate now forms a prominent bulge, or polster ("pillow"; arrow in Fig. 11L) of postmitotic hatching gland cells, and possibly also giving rise to other fates. The polster, along with median epiblast cells of the neural groove begin to indent the yolk cell's surface in the embryo's midline, as can be seen by looking along the embryonic axis.