Homologues of the T-box gene Brachyury play important roles in mesoderm differentiation and other aspects of early development in all bilaterians. In the diploblast Hydra, the Brachyuryhomologue HyBra1 acts early in the formation of the hypostome, the location of the organiser in adult Hydra. We now report the isolation and characterisation of a second Brachyury gene, HyBra2. Sequence analysis suggests that HyBra1 and HyBra2 are paralogues, resulting from an ancient lineage-specific gene duplication. We show that both paralogues acquired novel functions, both at the level of their cis-regulation as well as through significant divergence of the coding sequence. Both genes are expressed in the hypostome, but HyBra1 is predominantly endodermal, whereas HyBra2 transcripts are found primarily in the ectoderm. During bud formation, both genes are activated before any sign of evagination, suggesting an early role in head formation. During regeneration, HyBra1 is an immediate-early response gene and is insensitive to protein synthesis inhibition, whereas the onset of expression of HyBra2 is delayed and requires protein synthesis. The functional consequence of HyBra1/2 protein divergence on cell fate decisions was tested in Xenopus. HyBra1 induces mesoderm, like vertebrate Brachyury proteins. By contrast, HyBra2 shows a strong cement-gland and neural-inducing activity. Domain-swapping experiments show that the C-terminal domain of HyBra2 is responsible for this specific phenotype. Our data support the concept of sub- and neofunctionalisation upon gene duplication and show that divergence of cis-regulation and coding sequence in paralogues can lead to dramatic changes in structure and function.
Orthologues of Brachyury, a subfamily of T-box transcription factors, specify distinct cell types in different metazoan phyla, suggesting that the function of these genes has changed through the course of evolution. To investigate this evolutionary process, we have compared the activities of Brachyury orthologues from all major phyla in a single cellular context, the pluripotent Xenopus laevis animal cap. In this assay, an ancestral function is revealed: most orthologues, including the Hydra protein, mimic the action of endogenous Xenopus Brachyury, in that they induce mesoderm but not endoderm. Orthologues from Drosophila and ascidians, however, display an additional derived property, represented in our assay by the induction of endoderm. Misexpression of chimeric versions of Brachyury reveals that the C-terminal half of the protein is important for the strength of the induced response but not for its specificity. In contrast, amino acids located within the T-domain and in a short N-terminal peptide are involved in restricting the activity of Brachyury proteins to induction of mesoderm and not endoderm. Possession of this N-terminal motif is correlated with early circumblastoporal expression of Brachyury orthologues. We propose that restriction of Brachyury activity by this motif plays a conserved role in the control of Bilaterian gastrulation.
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