Body axis formation in Xenopus laevis : positive and negative regulation of canonical Wnt-mediated transcription

Kennedy, Mark (2009) Body axis formation in Xenopus laevis : positive and negative regulation of canonical Wnt-mediated transcription. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

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 β-catenin transcriptional activator, is localized in nuclei of dorsally fated cells of the early embryo and is required for dorsal development. The importance of β-catenin as a key element in body axis formation implies that factors which influence β-catenin expression and activity play important roles during dorsal development. Our understanding, however, of the mechanism(s) that govern β-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 β-catenin to modulate its transcriptional activity in Xenopus embryos. -- I first determined that the Xenopus Rel/NF-κB proteins, XRelA and XRel3, function as inhibitors of β-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 β-catenin activity, as measured by their ability to prevent supernumerary axis formation in embryos injected at the 2-cell stage with β-catenin and a constitutively active β-catenin mutant. Furthermore, XRel3 directly interacted with β-catenin, using in vitro co-immunoprecipitation assays. These results suggest a mechanism whereby Xenopus Rel proteins negatively regulate Xnr expression by inhibiting β-catenin-dependent transcription thus controlling dorsal axis development. -- In a second set of experiments, I explored the role of a component of the β-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 β-catenin. Furthermore, both Lgs/Bcl9 and Pygopus were demonstrated to be indispensable for β-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 β-catenin. Interestingly, XBcl9 preferentially accumulated in dorsal nuclei at a stage in development later than that reported for β-catenin and just prior to Wnt target gene activation. Gain-of-function assays demonstrated that XBcl9 was dependent on Pygopus to ectopically promote β-catenin target gene transcription, and that β-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 of XBcl9 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 XBcl9 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 Bcl9. 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 β-catenin on XBcl9 for axis development in Xenopus embryos, these results suggest a novel mechanism regulating β-catenin-dependent transcription.

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