BLASTULA PERIOD (2 1/4 - 5 1/4 h)

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.

We use the term blastula to refer to the period when the blastodisc begins to look ball-like, at the 128-cell stage, or 8th zygotic cell cycle, and until the time of onset of gastrulation, ca. cycle 14. Important processes occur during this blastula period; the embryo enters midblastula transition (MBT), the yolk syncytial layer (YSL) forms, and epiboly begins. Epiboly continues during the gastrulation period.

"Stereoblastula" would be more a appropriate term than blastula to describe the period, for it means no blastocoele is present, which is the case (Fig. 8). Only small irregular extracellular spaces exist between the deep cells of the blastodisc. The orientation of the cleavage planes is indeterminate, and they are much less regularly arranged than they were during the cleavage period. The daughter cells from these later cleavages slip out of what have been rather neat rows, in either side or top views, so that blastomere size is more useful than blastomere position in determining the stage during this period. The blastodisc looks slightly ellipsoidal in animal polar view (particularly during the early part of the period).

Early on, the cells of the blastula continue to divide synchronously, at the same rhythm as before (Fig. 5 and Fig. 9). More accurately, the cleavages during the early blastula period are "metasynchronous" because the mitoses do not all occur at quite the same time. Instead, a wave crosses the blastodisc at the end of each cell cycle. Cells near the animal pole enter the wave first, and the marginal cells enter the wave last. Generally the wave passes through the blastodisc obliquely, such that, from a face view, one can observe the wave pass across the field of cells along the long axis of the blastoderm, as it also passes from the animal pole to the margin.

Cell cycle lengthening marks the onset of the midblastula transition (MBT; Kane and Kimmel, 1993; Fig. 9A). Not all of the cycles begin to lengthen synchronously or to the same extent (Fig. 9B), and at any time point after MBT some cells are in interphase, and have nuclei easily visualized with Nomarski optics (Figs. 10A, D), while other cells are in mitosis (and have no nuclei; Figs. 10C, E): asynchrony among the blastomeres is thus immediately apparent from the morphology. The MBT begins during the tenth cell cycle (512-cell stage), two cycles earlier than in Xenopus (Newport and Kirshner, 1982a; 1982b), but otherwise the essentials of the MBT, its features and how its onset is controlled, appear the same in the two species (Kane and Kimmel, 1993). As interphases lengthen, cells become motile, and RNA synthesis increases over background levels.

Time lapse analysis reveals that after the onset of the MBT the deep cells are motile during their longer interphases (Kane and Kimmel, 1993). The deep cell movements appear to be unoriented, at least as seen from the surface.

The marginal tier of blastomeres in the early blastula have a unique fate. They lie against the yolk cell and remain cytoplasmically connected to it throughout cleavage. Beginning during cycle 10 (Kimmel and Law, 1985b), the marginal cells undergo a collapse, releasing their cytoplasm and nuclei together into the immediately adjoining cytoplasm of the yolk cell. Thus arises the yolk syncytial layer (YSL) a feature prominent with Nomarski optics, and important for staging (Fig. 10). After the YSL forms, the EVL cells that were in the second blastodisc tier now lie at the marginal position. They look like their former neighbors did, but with the important difference that these new marginal blastomeres are nonsyncytial: For the first time, none of the blastomeres have cytoplasmic bridges to other cells, except those present for a short time between newly divided sibling-cell pairs.

Although the YSL forms just at the time of the MBT, the regulatory control of these two events appears to be distinct; MBT alone depends on the nuclear-cytoplasmic ratio (Kane, 1991). The YSL nuclei continue to undergo mitotic divisions in the midblastula, but the nuclear divisions are unaccompanied by cytoplasmic ones, and the yolk cell remains uncleaved and syncytial. As in the blastodisc , the YSL division cycles lengthen during the midblastula period, but not as much, and the YSL nuclei continue to divide metasynchronously, not asynchronously. After about three cycles, and coinciding with the beginning of epiboly, the YSL divisions abruptly cease (Kane et al., 1992; see also Trinkaus, 1992). The YSL nuclei now begin to enlarge; possibly meaning that they are actively transcribing RNA.

The YSL, an organ unique to teleosts, may be extraembryonic, making no direct contribution to the body of the embryo. At first the YSL has the form of a narrow ring around the blastodisc edge (Fig. 10), but soon (within two division cycles) it spreads underneath the blastodisc, forming a complete "internal" syncytium (the I-YSL), that persists throughout embryogenesis. In this position, between the embryonic cells and their yolk stores, the I-YSL might be presumed to be playing a nutritive role. Another portion of it, the E-YSL, is transiently "external" to the blastodisc edge during epiboly. Indeed, work with the teleost Fundulus shows that this region, the E-YSL, appears to be a major motor for epiboly (Trinkaus, 1984).

Epiboly, beginning in the late blastula (Solnica-Krezel and Driever, 1994), is the thinning and spreading of both the YSL and the blastodisc over the yolk cell, as you might model by pulling a knitted ski cap over your head. Eventually, at the end of the gastrula period, the yolk cell becomes engulfed completely. During the early stages of this morphogenetic movement the blastodisc thins considerably, changing from a high-piled cell mound (Fig. 8B) to a cup-shaped cell multilayer of nearly uniform thickness (Fig. 8F). This is accomplished by the streaming outwards, towards the surface, of the deepest blastomeres. As they move, they mix fairly indiscriminately among more superficial cells along their way (Wilson et al.,1993). Active cell repacking by these so-called "radial intercalations" (Keller, 1980) may be a part of the driving force of early epiboly. The intercalations do not drive deep cells into the EVL, which remains a compartmentalized monolayer (Kimmel et al., 1990b). Additionally, deep blastomeres at the margin mix together to a considerably lesser extent than do the central ones (Helde et al., 1994), where, as can be seen from Fig. 8, the pile is much thicker before epiboly begins. This relative lack of mixing among marginal blastomeres may have significance with respect to how pattern is established during early development. This is because the mesoderm will arise from these nonmixing marginal deep cells (see the fate map below, Fig. 14). Presuming that events specifying mesoderm begin to occur before epiboly, as they seem to do in Xenopus, then lack of mixing in the marginal deep cell population during epiboly could be important to maintain regions with different cellular "specifications" (Kimmel et al., 1991) or identities.

The yolk cell prominently changes shape at the same time that the radial intercalations occur. The I-YSL surface bulges or "domes" towards the animal pole, this change in shape being the clearest sign that epiboly is beginning (Fig. 8E), and serving to substantially increase the area of contact between the I-YSL and the blastodisc's inner surface. As the yolk cell domes, it occupies territory simultaneously vacated by the deep blastomeres during their radial intercalations; yolk cell doming could exert force that drives the deep cells outwards. Whatever its cause, epiboly clearly involves the blastodisc and yolk cell alike, and begins quite rapidly. As epiboly continues during the next several hours, the E-YSL continues to advance across the yolk ahead of the cells that are riding upon it. In Fundulus, at least, desmosomes serve to attach the E-YSL to the EVL margin (Betchaku and Trinkaus, 1978), and the spreading YSL appears to pull the EVL along behind it.

Epiboly appears to depend on functional microtubules (Strähle and Jesuthasan, 1993) and might be under control of early-acting zygotic genes (Kane, 1991). The earliest-expressed genes identified so far code for regionally localized putative transcription factors, and begin expression in the late blastula (e.g. the gene no tail, Schulte-Merker et al., 1992; goosecoid, Stachel et al., 1993; snail1, Thisse et al., 1993).

Changes also occur in the EVL during the blastula period. At the end of the cleavage period there are more EVL cells than deep cells, but with successive divisions the EVL cells become vastly outnumbered. The EVL flattens, its cells thinning and stretching markedly to eventually form a tightly sealed (Bennett and Trinkaus, 1970) epithelial monolayer that becomes increasingly difficult to see. By the late blastula period, EVL cell cycles are longer and less synchronous than deep cell cycles (Kane et al., 1992). At the same time the EVL cells become lineage-restricted; their cell divisions generate daughters that are always within the EVL (Kimmel et al., 1990b). However, their developmental potential does not seem to be restricted during the late blastula period; if they are transplanted singly among the deep cells, their descendants acquire new fates (Ho, 1992a).

Detailed description of Blastula stages.