Regulation and functional aspects of Xenopus Brachyury.

Lerchner, Walter (1999). Regulation and functional aspects of Xenopus Brachyury. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000ff54

Abstract

Formation of the mesoderm is one of the earliest patterning events in the development of the embryo. Brachyury is restricted to the mesoderm and has become one of the most widely used markers for mesoderm formation. The Xenopus homologue Xbra has been shown to be both necessary and sufficient for formation of mesoderm.

Employing a nuclear transplantation technique to create transgenic Xenopus embryos I studied spatial and temporal regulation of a reporter driven by Xbra genomic sequences. The proximal 5’ flanking region of the Xbra promoter was sufficient to confine expression of a reporter to the mesoderm during gastrulation. Deletion analysis and point mutations in putative transcription factor binding sites identified two repressor modules, which are necessary to restrict expression to the marginal zone during gastrulation. This part of the project suggests that the Xbra gene is not confined to the mesoderm by specific activation, but rather by repression in tissues where its activity is not required.

To learn more about the functional aspects of the Brachyury protein I created a series of constructs encoding Xbra protein with N-terminal and C-terminal deletions. Nuclear localisation of the protein requires two regions not related to known nuclear localisation sequences. Experiments investigating the dimérisation properties of the protein confirmed that the full-length Xbra protein, binds a palindromic consensus sequence as dimer. However, there was no evidence for Xbra dimers forming without binding to DNA. Finally, I investigated the possibility that the MAP kinase pathway regulates Xbra on a post-translational level. The experiments showed that Xbra is phosphoiylated by MAP kinase in vitro in the linker domain, but not in the DNA binding domain. These results will form the basis for a more detailed study investigating the post-translational regulation of Xbra. The possibilities for future experiments will be discussed in this context.