The zebrafish midblastula transition

Donald A. Kane 0 1 Charles B. Kimmel 0 0 Institute of Neuroscience, University of Oregon , Eugene, Oregon 97403 , USA 1 Present address: Max Planck Institut fur Entwicklungsbiologie, Abteilung III , Spemannstrasse 35, 72076 Tubingen , Germany SUMMARY The zebrafish midblastula transition (MBT) begins at cycle 10. It is characterized by cell cycle lengthening, loss of cell synchrony, activation of transcription and appearance of cell motility. Superceding a 15 minute oscillator that controls the first nine cycles, the nucleocytoplasmic ratio appears to govern the MBT. This timing mechanism operates cell autonomously: clones of labeled cells initiate cell cycle lengthening independently of neighbors but dependent on immediate lineal ancestors. Unequal divisions, when they occur, produce asymmetThe early development of many animals is characterized by rapid and synchronous cleavages that quickly subdivide the zygote into a large population of blastomeres. This stage is followed by a midblastula transition (MBT) to a longer cell cycle that is accompanied by the loss of cell synchrony (Signoret and Lefresne, 1971; Gerhart, 1980), and activation of transcription and cell motility (Newport and Kirschner, 1982a), both characteristics essential for the ensuing process of gastrulation. The lengthening of the cell cycle is thought to be the primary cause of many of the associated behaviors at MBT. If inhibitors are used to stop the cell cycle early in Drosophila, then transcriptional activation occurs early (Edgar and Schubiger, 1986) and, in Xenopus, both onset of cell motility and activation of transcription occur early (Kimelman et al., 1987). MBT begins when the cleaving cells reach a particular nucleocytoplasmic ratio: MBT is late in haploid embryos (Signoret and Lefresne, 1973; Edgar and Schubiger, 1986) and is early in tetraploid or polyploid embryos (Newport and Kirschner, 1982a; Mita and Obata, 1984). MBT also occurs early when the nucleocytoplasmic ratio is increased by ligating a portion of the cytoplasm away from the early nuclei in the early fly blastula (Edgar and Schubiger, 1986), in starfish embryos (Mita and Obata, 1984) and in the newt (Kobayakawa and Kubota, 1981). Ligating frog embryos (Newport and Kirschner, 1982a), similar to the classic experiment by Spemann on Triturus (Spemann, 1938), suggest that neither absolute time nor counting ric cell cycle lengthening based on the volume of each daughter. During the several cycles after the MBT begins, cycle length is correlated with the reciprocal of the blastomere volume, suggesting a continuation of cell cycle regulation by the nucleocytoplasmic ratio during an interval that we term the MBT period. models of MBT timing are likely MBT-triggering mechanisms. The basis for the loss of synchrony at MBT is not understood. One idea, which we propose and examine here, is that individual cells or lineages of cells could start their MBT cell cycle lengthening based on their individual volumes, which would give each of them a unique nucleocytoplasmic ratio. The volume differences of individual cells could be by chance or design the result of unequal earlier cleavage divisions. This hypothesis is consistent with the mitotic behavior of invertebrate embryos which have early, extremely unequal cleavages, such as in sea urchin micromere formation (Okazaki, 1975), in Caenorhabditis Pblast lineage divisions (Sulston et al., 1983) and in leech teloblast divisions (Bissen and Weisblat, 1989). An alternative view, though not mutually exclusive, is that as cells become specified to different programs of development, these differences are subsequently mirrored in differential lengthening of the cell cycle. Evidence for this hypothesis comes from the study of mitotic domains in the fly embryo, where during cycle 14 the cell cycle of patches of cells correlates to cell fate (Foe, 1989) and mutations that change cell fate change cell cycle length (Arora and Nsslein-Volhard, 1992). MBT in teleosts is not well characterized. During cleavage cycles 1 to 7, divisions are stereotyped and highly synchronous (Oppenheimer, 1936, 1937; Hisaoka and Battle, 1958), each consecutive cycle having approximately the same length (Roosen-Runge, 1938; Marrable, 1959). At the blastula stage, beginning at cycle 8, the embryos show slight metasynchrony, with waves of mitosis emanating from the animal pole; during this time, Marrable (1965) reported a gradual increase in the cell cycle in the zebrafish, indicating possible MBT-like cell cycle lengthening. In studies on the loach, Misgurnus fossilis, Rott and Shevelava (1967) found a more defined cell cycle lengthening, which did not occur until cycle 11, and, in the same study, they found the equivalent cell cycle lengthening in haploid embryos occurred at cycle 12, indicating that onset of teleost cell cycle lengthening is timed by the nucleocytoplasmic ratio. Motility of cells in the early blastula stage has not been reported, but cells are motile by the late blastula stage, as established for Fundulus (Trinkaus and Erickson, 1983) and zebrafish (Thomas and Waterman, 1978). Therefore, although studies of teleosts are incomplete, many of the elements of a frog-like MBT seem to be present. In this paper, as a prelude to studies relating the blastula cell cycle to cell fate, we characterize the zebrafish MBT and examine the role that the nucleocytoplasmic ratio plays in cell cycle control during the MBT. We find that cell cycle lengthening and loss of cell synchrony begins at cycle 10, with a concomitant activation of transcription and motility. The timing of MBT onset appears to be under the control of the nucleocytoplasmic ratio. Afterwards, during the next several cycles, we find a correlation of the cell volume and hence nucleocytoplasmic ratio with cycle lengths, suggesting that the rapid loss of cell synchrony at MBT is based on unequal cell division. Finally, we examine a potential role for the nucleocytoplasmic ratio in cell cycle control during early development. MATERIALS AND METHODS Eggs were produced for experiments by either natural crosses or by in vitro fertilization (Streisinger et al., 1981). All embryos with experimental ploidys were fertilized in vitro with the following modifications: activated eggs were produced by activation with the addition of water without the addition of sperm and are equivalent to unfertilized eggs. Haploid embryos were produced by fertilization with sperm inactivated by a short exposure to ultraviolet irradiation. Tetraploid eggs were produced by fertilization with untreated sperm but then briefly exposed to heat to prevent the first mitotic division, a method used for the production of clonal diploids. After fertilization, embryos were maintained at 28.51C in E medium (13.7 mM NaCl, 0.5 mM KCl, 1.3 mM CaCl2, 1 mM MgSO4, 4.2 mM NaHCO3 and 0.07 mM sodium/potassium phosphate buffer, pH 7.2). From early cleavage until dye injections, embryos were time-lap (...truncated)