Total DNA was isolated from embryos expressing Wee1 or luciferase, resolved by gel electrophoresis and stained with ethidium selleck Nutlin-3a bromide. Embryos expressing exogenous Wee1 contained less DNA throughout early development, compared to luciferase controls. However, the DNA content increased through the early development of embryos overexpresssing Wee1 indicative of continuing DNA replication. The rate of increase in DNA content was consistent with the lengthened cleavage cycles, suggesting that nuclear and cytoplasmic cycles were coordinated. To examine nuclear morphology, embryos were collected at several stages, sectioned and stained with DAPI. Repre sentative fields from embryos collected at Stage 9 are shown in Figure 1E. Embryos overexpressing Wee1 con tained interphase and mitotic nuclei with normal mor phology.
There was no loss of nuclei as seen in embryos overexpressing cyclin E, although there were fewer and larger cells in these embryos, consistent with the delayed cell cycles. These results indicate that the overex pression of Wee1 delays both nuclear and cleavage Inhibitors,Modulators,Libraries cycles, but these cycles appear to remain coupled. Overexpression of Wee1 triggers apoptosis after the MBT In previous studies, we showed that X. laevis embryos expressing exogenous checkpoint kinases, Chk1 or Chk2, exhibited a similar cell cycle delay and premature Cdk phosphorylation. Furthermore, embryos express Inhibitors,Modulators,Libraries ing exogenous Chk2 developed normally through Inhibitors,Modulators,Libraries neuru lation and beyond, and exogenous Chk2 protects embryos form apoptosis induced by ionizing radiation.
Inhibitors,Modulators,Libraries Embryos overexpressing Wee1 developed normally through the MBT until early gastrulation. However, during gastrulation, these embryos began to appear abnormal, exhibiting a loss of cellular attachment and overall organization. The gross Inhibitors,Modulators,Libraries morphol ogy of embryos expressing exogenous Wee1 appeared consistent with that of embryos that had undergone apop tosis in response to ionizing radiation, unreplicated DNA, or expression of dominant negative Chk1/Chk2. To determine whether the kinase activity of Wee1 was required for its disruption of development, embryos were microinjected with wild type or kinase dead Wee1 mRNA. The embryos expressing KD Wee1 developed normally through gastrulation, indicating that kinase activity is required for the effects of exogenous Wee1.
To determine whether overexpression of Wee1 induced apoptosis, embryos were assayed for caspase activity in embryo lysates by the cleavage of exogenous human inhibitor Volasertib PARP protein. Embryos expressing exogenous Wee1 were positive for caspase activity at gastrulation indicated by the presence of a cleaved PARP frag ment. These results were surprising since exogenous Chk1 and Chk2, lengthen cleavage cycles and promote phosphorylation to the same extent as Wee1, yet exogenous Chk1 and Chk2 kinases inhibit rather than promote apoptosis.