Divergent functions of two ancientHydra Brachyuryparalogues suggest specific roles for their C-terminal domains in tissue fate induction

Abstract

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.

Early evolution of the T-box transcription factor family

“…We used Xenopus as a model system, as it has previously been used to characterize T-box genes from early-branching metazoans (4,7,18). Xenopus embryos injected with an mRNA encoding a dominant negative form of Brachyury (XBra_En) show defective gastrulation and impairment of muscle development (27).…”

“…Finally, ref. 18 proposed that a conserved motif in the C-terminal activation domain (called the R1 domain), which is present in Bilateria and Cnidaria, is responsible for Brachyury specificity. However, this domain is again not present in M. leidyi or S. ciliatum Brachyury homologs.…”

“…To obtain a glimpse into the functional conservation of the earliest Brachyury genes, we perform heterologous expression experiments of the Brachyury homologs from C. owczarzaki, Sycon ciliatum (Calcarea, Porifera), and Nematostella vectensis (Anthozoa, Cnidaria) in Xenopus laevis, a well-established model system for studying Brachyury (4,7,18). Our data show that C. owczarzaki Brachyury (CoBra) can partially rescue Xenopus laevis embryos injected with a dominant negative XBra construct.…”

An organizing region in metamorphosing hydrozoan planula larvae - stimulation of axis formation in both larval and in adult tissue

“…The T-Box transcription factor brachyury is involved in mesoderm differentiation (Smith 1999). A brachyury ortholog (like Wnt: Hobmayer et al 2000) is expressed in Hydra in the hypostome and in buds at an early stage in the region of the future hypostomes (Technau and Bode 1999;Bielen et al 2007). Its Hydractinia echinata ortholog, Hech-Bra, was isolated and identified by Kroiher (1999) and found to be expressed at the posterior tip of Hydractinia larvae.…”

A small molecule screen identifies a novel compound that induces a homeotic transformation inHydra

“…To distinguish among these possibilities, we used genes whose expression patterns define tissue identity along the oral/aboral axis. These include: (1) a marker of the organizer, the Hydra Wnt3 ortholog (HyWnt3), which is expressed at the tip of the hypostome (Hobmayer et al, 2000); (2) a marker of hypostome, the Hydra T-box gene HyBra2, which is expressed throughout the hypostome, but not in the tentacle zone (Bielen et al, 2007); (3) a marker of tentacles, HyAlx, an aristalessrelated homeobox gene that is expressed in rings at the bases of the tentacles (Smith et al, 2000); (4) a marker for tentacle zone, Hym-301 (Hobmayer et al, 2000;Smith et al, 2000;Takahashi et al, 2005); and (5) a marker for body column tissue, a gene for a novel secreted protein (GenBank Accession Number XP_002165635) (Hwang et al, 2007). This gene was described as being expressed in body column endodermal epithelial cells by Hwang et al (Hwang et al, 2007), but we have found that it is expressed in gland cells in the body column (Fig.…”