Masazumi Tada

T‐targets: Clues to understanding the functions of T‐box proteins

Members of the T‐box gene family have been identified in both vertebrates and invertebrates, where they play key roles in the regulation of embryonic development, and particularly in morphogenesis and the assignment of cell fate. T‐box proteins act as transcription factors which regulate the expression of downstream effector genes. This review focuses on the identification of T‐box target genes and the basis of T‐box functional specificity.

A screen for targets of the Xenopus T-box gene Xbra.

Brachyury (T), a member of the T-box gene family, is essential for the formation of posterior mesoderm and notochord in vertebrate development. Expression of the Xenopus homologue of Brachyury, Xbra, causes ectopic ventral and lateral mesoderm formation in animal cap explants and co-expression of Xbra with Pintallavis, a forkhead/HNF3beta-related transcription factor, induces notochord. Although eFGF and the Bix genes are thought to be direct targets of Xbra, no other target genes have been identified. Here, we describe the use of hormone-inducible versions of Xbra and Pintallavis to construct cDNA libraries enriched for targets of these transcription factors. Five putative targets were isolated: Xwnt11, the homeobox gene Bix1, the zinc-finger transcription factor Xegr-1, a putative homologue of the antiproliferative gene BTG1 called Xbtg1, and BIG3/1A11, a gene of unknown function. Expression of Xegr-1 and Xbtg1 is controlled by Pintallavis alone as well as by a combination of Xbra and Pintallavis. Overexpression of Xbtg1 perturbed gastrulation and caused defects in posterior tissues and in notochord and muscle formation, a phenotype reminiscent of that observed with a dominant-negative version of Pintallavis called Pintallavis-En(R). The Brachyury-inducible genes we have isolated shed light on the mechanism of Brachyury function during mesoderm formation. Specification of mesodermal cells is regulated by targets including Bix1-4 and eFGF, while gastrulation movements and perhaps cell division are regulated by Xwnt11 and Xbtg1.

Dual specificity of activin type II receptor ActRIIb in dorso-ventral patterning during zebrafish embryogenesis.

Members of the transforming growth factor‐β (TGF‐β) superfamily are thought to regulate specification of a variety of tissue types in early embryogenesis. These effects are mediated through a cell surface receptor complex, consisting of two classes of ser/thr kinase receptor, type I and type II. In the present study, cDNA encoding zebrafish activin type II receptors, ActRIIa and ActRIIb was cloned and characterized. Overexpression of ActRIIb in zebrafish embryos caused dorsalization of embryos, as observed in activin‐overexpressing embryos. However, in blastula stage embryos, ActRIIb induced formation of both dorsal and ventro‐lateral mesoderm. It has been suggested that these inducing signals from ActRIIb are mediated through each specific type I receptor, TARAM‐A and BMPRIA, depending on activin and bone morphogenetic protein (BMP), respectively. In addition, it was shown that a kinase‐deleted form of ActRIIb (dnActRIIb) suppressed both activin‐ and BMP‐like signaling pathways. These results suggest that ActRIIb at least has dual roles in both activin and BMP signaling pathways during zebrafish embryogenesis.

Regulation of apoptosis in theXenopus embryo by Bix3.

Members of the Bix family of homeobox-containing genes are expressed in the vegetal hemisphere of the Xenopus embryo at the early gastrula stage. Misexpression of at least some of the family members causes activation of mesoderm- and endoderm-specific genes and it is known that some of the proteins, including Bix2 and Bix3, interact with Smad proteins via a motif that is also present in the related protein Mixer. In this paper we study the function of Bix3. Misexpression of Bix3, similar to misexpression of other members of the Bix family, causes the activation of a range of mesendodermal genes, but the spectrum of genes induced by Bix3 differs from that induced by Bix1. More significantly, we find that overexpression of Bix3 also causes apoptosis, as does depletion of Bix3 by use of antisense morpholino oligonucleotides. The ability of Bix3 to causes apoptosis is not associated with its ability to activate transcription and nor with its possession of a Smad interaction motif. Rather, Bix3 lacks a C-terminal motif, which, in Bix1, acts in cis to inhibit apoptosis. Mutation of this sequence in Bix1 causes the protein to acquire apoptosis-inducing activity.