NSF Award
0131755
0131755<br/>Green<br/><br/>A central question in developmental biology is how the body axes are established in the vertebrate embryo. Previous studies in Xenopus have shown that the protein kinase GSK3b plays a pivotal role in establishing dorsoventral asymmetry. However, it was believed that asymmetry in GSK3b specific activity, rather than an asymmetry of GSK3b protein abundance per se, provided spatial orientation for the embryo. In preliminary studies, Dr. Green and colleagues have shown that dorsoventral regulation of GSK3b involves an actual depletion of GSK3b, perhaps through differential stability of the protein. The research proposed here builds upon this preliminary observation. The broad goal is to define the GSK3b regulatory mechanisms that establish the dorsoventral axis. The first aim is to characterize the mechanism of GSK3b protein depletion. The stability of purified recombinant GSK3b will be measured in early embryos. Ubiquitination/proteasome inhibitors will be tested for their ability to prevent GSK3b depletion. The second aim is to clarify the role of Wnt signaling in endogenous dorsoventral regulation of GSK3b. GSK3b abundance in embryos depleted for Wnt receptors will be measured and maternal Wnts will be compared with zygotic Wnts (that are known to reduce GSK3b specific activity only) for their ability to deplete GSK3b. To identify a possible temporal shift in GSK3b regulation from depletion to specific activity reduction, earlier and later stage unperturbed embryos will be compared. The third aim is to examine the role of GSK3b localization in its regulation. GSK3b protein depletion is tightly localized to the cell cortex, and has been shown to be involved in Wnt-dependent recruitment of the Wnt transduction protein Axin to the Wnt transmembrane receptor LRP5. The hypothesis that membrane-associated GSK3b forms a highly Wnt- and GBP-sensitive pool will be tested by examining GSK3b abundance and activity in cortical membrane fractions of normal and manipulated embryos. These projects will together provide a more precise molecular picture of GSK3b regulation in early Xenopus development.
