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Major developmental events in early Xenopus embryogenesis coincide with changes in the length and composition of the cell cycle. These changes are mediated in part through the regulation of CyclinB/Cdc2 and they occur at the first mitotic cell cycle, the mid-blastula transition (MBT) and at gastrulation. In this report, we investigate the contribution of maternal Wee1, a kinase inhibitor of CyclinB/Cdc2, to these crucial developmental transitions. By depleting Wee1 protein levels using antisense morpholino oligonucleotides, we show that Wee1 regulates M-phase entry and Cdc2 tyrosine phosphorylation in early gastrula embryos. Moreover, we find that Wee1 is required for key morphogenetic movements involved in gastrulation, but is not needed for the induction of zygotic transcription. In addition, Wee1 is positively regulated by tyrosine autophosphorylation in early gastrula embryos and this upregulation of Wee1 activity is required for normal gastrulation. We also show that overexpression of Cdc25C, a phosphatase that activates the CyclinB/Cdc2 complex, induces gastrulation defects that can be rescued by Wee1, providing additional evidence that cell cycle inhibition is crucial for the gastrulation process. Together, these findings further elucidate the developmental function of Wee1 and demonstrate the importance of cell cycle regulation in vertebrate morphogenesis.
Fig. 3. Wee1 depletion inhibits morphogenesis but not zygotic gene expression. (A) Animal cap explants prepared from uninjected embryos and embryos injected with MO-Control, MO-Wee1 or MO-Wee1+WT Wee1 RNA were left untreated or were treated with activin and cultured until stage 22-23. (B) RNA was isolated from animal cap explants prepared as in A and from stage 10.5 whole embryos (WE) that were either uninjected or had been injected with Control-MO or MO-Wee1 at the two-cell stage. Expression of brachyury, goosecoid and chordin was examined by RT-PCR analysis. cDNA levels were normalized to EF-1α, and a sample lacking reverse transcriptase (–RT) was also included. (C) Two-cell embryos were injected with MO-control, MO-Wee1 or MO-Wee1 + WT RNA. β-Gal RNA was injected into the B1 blastomeres at the 32-cell stage and β-gal activity visualized at stage 11.5-12. The B1 clone forms a narrow midline band extending between the blastopore (bottom) and animal hemisphere (top) in uninjected (n=12) and MO-Control injected embryos (n=23), while the B1 progeny form a broad band across the dorsal equator in MO-Wee1 embryos (MO-Wee1; n=36). this defect is significantly reversed by co-injection of WT Wee1 RNA (MO-Wee1+WT RNA; n=16). (D) The embryos shown in C were bisected through the area of β-gal staining. In uninjected embryos, the labeled cells extend from the animal hemisphere (top) to the dorsal blastopore lip (dbl). In Wee1-depleted embryos (MO-Wee1), no epibolic spread towards the vegetal pole (bottom) or involution occurs. However, some of the inner vegetal cells have moved upwards along the inner surface of the blastocoel roof (b.c.; arrow heads). (E) Expression of Xbrachyury (upper two panels, MO-Wee1, n=67) and chordin (MO-Wee1, n=54, lower two panels) was determined by in situ histochemistry (blue staining).
Fig. 1. Wee1-depletion using antisense morpholino oligonucleotides (MO). (A) MO-Wee1 was injected into both cells of a two-cell embryo. Uninjected and MO-Wee1-injected embryos were collected at various stages and lysates examined by immunoblot analysis using anti-Wee1 antibodies (* indicates a nonspecific band). (B) Two-cell embryos were injected with MO-Control, MO-Wee1 or co-injected with MO-Wee1 and WT Wee1 RNA (MO-Wee1+RNA). Embryos were collected at stage 10 and lysates examined as in A. (C) Embryos injected as in B were collected at stage 8, 10 or 12. Lysates were examined by immunoblot analysis using anti-phospho-Cdc2 or anti-Cdc2 antibodies. (D) The mitotic nuclei of injected stage 11 embryos were visualized by whole-mount immunostaining using phospho-histone H3 (αPH3). Mitotic index (n=10-12 embryos): MO-control, 8.9% (4739 nuclei); uninjected, 9.9% (1221 nuclei); MO-Wee1, 24.5% (4734 nuclei); MO-Wee1+RNA, 11.4% (2736 nuclei).
Fig. 7. Cdc25C overexpression disrupts gastrulation. (A,B) The two dorsal blastomeres of four-cell embryos were injected with β-gal RNA (4 ng) or co-injected with β-gal (100 pg) and His-Cdc25C (3 ng) and embryos were scored for gastrulation defects at stage 11.5-12. Number of embryos examined: Cdc25C (n=141), β-Gal (n=162). (A, lower panels) Stage 10.5-11 embryo lysates were examined by immunoblot analysis using anti-His-epitope, Cdc2 and phospho-Cdc2 antibodies. (C) Following injection as in A, mitotic nuclei of stage 11 embryos were visualized. Mitotic index (n=10-12 embryos): β-gal, 8.3% (6928 nuclei), Cdc25C, 28.1% (5550 nuclei). (D,E) Embryos were injected with 4 ng of Cdc25C RNA and either 0, 0.2 or 0.5 ng of Wee1 RNA. Number of embryos examined: Cdc25C + 0 ng Wee1 (n=74), Cdc25C + 0.2 ng Wee1 (n=100) and Cdc25C + 0.5 ng Wee1 (n=86). Note that increased expression of Wee1 counteracts the defects induced by Cdc25C overexpression.
