Evolution of Brachyury proteins: identification of a novel regulatory domain conserved within Bilateria

Abstract

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.

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).…”

“…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.…”

“…This family is characterized by an evolutionary conserved DNA-binding domain of 180-200 amino acids, known as the T-box domain (1)(2)(3). Brachyury is the founding and best-characterized member of the T-box family, with well-established roles in blastopore specification, mesoderm differentiation and, in chordates, notochord formation (4)(5)(6). It has been hypothesized that the ancestral role of Brachyury was primarily that of blastopore determination and gastrulation (5,7).…”

T‐box genes in early embryogenesis

“…However, not all properties of Brachyury may be evolutionarily conserved. For example, Marcellini and coworkers have demonstrated recently that, while most orthologues of Brachyury can induce mesoderm, a second class of orthologues including those of Drosophila and ascidians can also induce endoderm (Marcellini et al 2003).…”

In silico and in vivo identification of the intermediate filament vimentin that is downregulated downstream of Brachyury during Xenopus embryogenesis

“…For example, Brachyury plays a role in gastrulation in the sea urchin, Xenopus, and the mouse as well as in the invagination of the stomodeum in the sea urchin and ctenophore (Conlon and Smith, 1999;Gross and McClay, 2001;Kwan and Kirschner, 2003;Wilson and Beddington, 1997;Wilson, 1995;Yamada et al, 2010). In addition, Brachyury orthologs derived from various animals mimic the action of Xenopus Brachyury (Xbra) in experiments wherein their synthetic RNAs are introduced in the animal caps of Xenopus laevis (Marcellini et al, 2003;Yamada et al, 2010). These findings suggest the possibility that the Brachyury-downstream mechanisms are conserved among the metazoans.…”