| Establishment of dorsoventral polarity in Xenopus embryos requires activation of the canonical Wnt signal transduction pathway. Accumulated evidence has indicated that the key effector of canonical Wnt signaling, the beta-catenin transcriptional activator, is localized in nuclei of dorsally fated cells of the early embryo and is required for dorsal development. The importance of beta-catenin as a key element in body axis formation implies that factors which influence beta-catenin expression and activity play important roles during dorsal development. Our understanding, however, of the mechanism(s) that govern beta-catenin activity, for example, during embryonic development, is incomplete. Therefore, there is a need to identify factors that both inhibit and promote its activity. To this end, I have identified several novel proteins that interact with beta-catenin to modulate its transcriptional activity in Xenopus embryos.In a second set of experiments, I explored the role of a component of the beta-catenin transcriptional activation complex called B-cell lymphoma 9 (Bcl9), which is the orthologue to Legless (Lgs) of Drosophila and mammals. In Drosophila embryos, Lgs/Bcl9 was identified as a bridging protein between the downstream component, Pygopus, and beta-catenin. Furthermore, both Lgs/Bcl9 and Pygopus were demonstrated to be indispensable for beta-catenin-dependent embryonic patterning in Drosophila. Unlike Pygopus, however, the role of Lgs/Bcl9 in vertebrate development is unknown. I determined that like its fly counterpart, Xenopus Bcl9 (XBcl9) directly interacted in vitro, via conserved peptide sequences with the co-activator proteins, Pygopus and beta-catenin. Interestingly, XBcl9 preferentially accumulated in dorsal nuclei at a stage in development later than that reported for beta-catenin and just prior to Wnt target gene activation. Gain-of- function assays demonstrated that XBcl9 was dependent on Pygopus to ectopically promote beta-catenin target gene transcription, and that beta-catenin was dependent on its interaction with XBcl9 for dorsal axis formation. Additionally, loss-of-function assays determined that XBcl9 was required for body axis formation during Xenopus development. These results implied that the timing of XBcl9 nuclear localization may indicate an important step in dorsal cell fate determination.The role ofXBcl9 in axis formation suggested that its regulation is important for normal development. In my final set of experiments, I determined that XBcl9 is post-transcriptionally regulated in Xenopus embryos. The inhibition of XBc19 translation is dependent on a minimal 29nt element in the 5'UTR, proximal to the putative start of translation, and is well conserved in human Bc19. The minimal repression element is predicted to form a stable secondary structure, posing as a possible block to constitutive translation. Due to the dependence of beta-catenin on XBc19 for axis development in Xenopus embryos, these results suggest a novel mechanism regulating beta-catenin-dependent transcription.I first determined that the Xenopus Rel/NF-kappaB proteins, XRelA and XRel3, function as inhibitors of beta-catenin activity in embryos. Using gain-of-function assays, I found that both XRelA and XRel3 perturbed dorsal development by repressing the expression of multiple Xenopus nodal-related (Xnr) genes. Since dorsal development is a canonical Wnt-dependent process and the timing and level of Xnr expression is regulated by canonical Wnt signaling, I hypothesized that XRelA/XRel3 inhibits Canonical Wnt activity in embryos to regulate axis formation. Co-expression of either XRelA or XRel3 efficiently antagonized ectopic beta-catenin activity, as measured by their ability to prevent supernumerary axis formation in embryos injected at the 2-cell stage with beta-catenin and a constitutively active beta-catenin mutant. Furthermore, XRel3 directly interacted with beta-catenin, using in vitro co-immunoprecipitation assays. These results suggest a mechanism whereby Xenopus Rel proteins negatively regulate Xnr expression by inhibiting beta-catenin-dependent transcription thus controlling dorsal axis development. |
