Morphogenesis during Xenopus gastrulation requires Wee1-mediated inhibition of cell proliferation.

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.

Altered expression of Chk1 disrupts cell cycle remodeling at the midblastula transition in Xenopus laevis embryos.

Studies in several model systems, including Xenopus laevis oocytes and embryos, have indicated that the checkpoint kinase, Chk1, is required for early development, even in the absence of damaged or unreplicated DNA. Chk1 is transiently activated at the midblastula transition (MBT) in Xenopus, a time when the cell cycle remodels from rapid embryonic cleavage cycles to longer, more regulated somatic cell cycles. To better understand the role of Chk1 in cell cycle remodeling, mRNA encoding Chk1 was microinjected into 1-cell stage embryos, and the effects on both the MBT and on the expression of several cell cycle regulators were examined. Zygotic transcription, a hallmark of the MBT that depends upon the nucleocytoplasmic (N/C) ratio, was blocked, as was degradation of maternal cyclin E, an event of the MBT that occurs independent of the N/C ratio. Levels of mitotic cyclins were elevated throughout early development, consistent with cell cycle arrest at G2/M. In these embryos, Cdc25A level was low, whereas Cdc25C level was not affected. Furthermore, the level of Wee1 increased at 6 hrs post-fertilization (pf), the time at which the MBT normally occurs, even though these embryos did not demonstrate any known markers of the MBT. These studies suggest that in addition to targeting Cdc25A for degradation, Chk1 may also function in cell cycle remodeling at the MBT by stabilizing Wee1 until it is replaced by the somatic Wee2 protein during gastrulation.

Tome-1, a trigger of mitotic entry, is degraded during G1 via the APC.

Entry into mitosis requires the activation of cdk1/cyclin B, while mitotic exit is achieved when the same kinase activity decreases, as cyclin B is degraded. Cyclin B proteolysis is mediated by the anaphase promoting complex, or APC, an E3 ligase that is active at anaphase in mitosis through G1. We have identified a G1 substrate of the APC that we have termed Tome-1, for trigger of mitotic entry. Tome-1 is a cytosolic protein required for proper activation of cdk1/cyclin B and mitotic entry. Tome-1 associates with Skp-1 and is required for degradation of the cdk1 inhibitory tyrosine kinase wee1; Tome-1 therefore appears to be acting as part of an SCF-type E3 for wee1. Degradation of Tome-1 during G1 allows for wee 1 accumulation during interphase, thereby providing a critical link between the APC and SCF pathways in regulation of cdk1/cyclin B activity and thus mitotic entry and exit.