Bowline, which is a member of the Xenopus Bowline/Ripply family of proteins, represses the transcription of somitogenesis-related genes before somite segmentation, which makes Bowline indispensable for somitogenesis. Although there are three bowline/Ripply family genes in each vertebrate species, it is not known whether the Bowline/Ripply family proteins share a common role in development. To elucidate their developmental roles, we examined the expression patterns and functions of the Xenopus Bowline/Ripply family proteins Bowline, Ledgerline, and a novel member of this protein family, xRipply3. We found that the expression patterns of bowline and ledgerline overlapped in the presomitic mesoderm (PSM), whereas ledgerline was additionally expressed in the newly formed somites. In addition, we isolated xRipply3, which is expressed in the pharyngeal region. Co-immunoprecipitation assays revealed that Ledgerline and xRipply3 interacted with T-box proteins and the transcriptional co-repressor Groucho/TLE. In luciferase assays, xRipply3 weakly suppressed the transcriptional activity of Tbx1, while Ledgerline strongly suppressed that of Tbx6. In line with the repressive role of Ledgerline, knockdown of Ledgerline resulted in enlargement of expression regions of the somitogenesis-related-genes mespb and Tbx6. Inhibition of histone deacetylase activity increased the expression of mespb, as seen in the Bowline and Ledgerline knockdown experiments. These results suggest that the Groucho-HDAC complex is required for the repressive activity of Bowline/Ripply family proteins during Xenopus somitogenesis. We conclude that although the Xenopus Bowline/Ripply family proteins Bowline, Ledgerline and xRipply3 are expressed differentially, they all act as negative regulators of T-box proteins.
We previously reported that Tbx6, a T-box transcription factor, is required for the differentiation of ventral body wall muscle and for segment formation and somitic muscle differentiation. Here, we show that Tbx6 is also involved, at later stages, in cartilage differentiation from the cranial neural crest and head muscle development. In Tbx6 knockdown embryos, the cranial neural crest was shown to be correctly induced at the border of the neural plate and migrated in a slightly delayed manner, but finally reached positions in the pharyngeal arches nearly similar to those in the normal embryos as revealed by in situ hybridization and the neural crest-transplantation experiments. However, the neural crest cells failed to maintain Sox9 expression. Tbx6 knockdown also reduced the expression of Tbx1, another T-box gene expressed in more anterior paraxial structures. Tbx1 knockdown caused phenotypes milder but similar to those of Tbx6 morphants, including reduced formation of head muscles and cartilages, and attenuated Sox9 expression. Furthermore, the phenotypes caused by Tbx6 knockdown were partially rescued by Tbx1 plasmid injection. These results suggest that Tbx6 is involved in the cranial cartilage and head muscle development by regulating anterior paraxial genes such as Tbx1 and Sox9.
T-box transcription factor tbx6 and basic-helix-loop-helix transcription factor pMesogenin1 are reported to be involved in paraxial mesodermal differentiation. To clarify the relationship between these genes in Xenopus laevis, we isolated pMesogenin2, which showed high homology with pMesogenin1. Both pMesogenin1 and 2 appeared to be transcriptional activators and were induced by a hormone-inducible version of Xtbx6 without secondary protein synthesis in animal cap assays. The pMesogenin2 promoter contained three potential T-box binding sites with which Xtbx6 protein was shown to interact, and a reporter gene construct containing these sites was activated by Xtbx6. Xtbx6 knockdown reduced pMesogenin1 and 2 expressions, but not vice versa. Xtbx6 and pMesogenin1 and 2 knockdowns caused similar phenotypic abnormalities including somite malformation and ventral body wall muscle hypoplasia, suggesting that Xtbx6 is a direct regulator of pMesogenin1 and 2, which are both involved in somitogenesis and myogenesis including that of body wall muscle in Xenopus laevis. Developmental Dynamics 237: 3749 -3761, 2008.
T-box proteins are important transcriptional regulators in animal development. We searched the Xenopus laevis expressed sequence tag (EST) database using zebrafish tbx24 (Ztbx24) as a query and found a sequence. We then obtained corresponding clones from a neurula cDNA library. This novel gene has a T-box showing 53% homology with Xenopus laevis tbx6 (Xtbx6) and 51% with Ztbx24 at the amino acid level and is relatively close to Xtbx6 indicated by alignment analysis. In situ hybridization showed that it is expressed in the paraxial mesoderm in the caudal region in a very similar manner to Xtbx6, with a slight difference in that the former is emphasized more dorsally and has a more restricted distribution along the antero-posterior axis. From these results we named this gene Xtbx6r (tbx6-related). Xtbx6r or Xtbx6r-En R , when overexpressed in animal caps, induced anterior neural markers but not mesodermal markers. In contrast, Xtbx6r-VP16, Xtbx6 and Ztbx24 induced various mesodermal markers. These results indicate that Xtbx6r is a transcriptional repressor and has activity different from that of Xtbx6 or Ztbx24. Xtbx6r induced Otx2, XAG and Pax6 in animal caps. This activity differed from that of Xbra-En R or Xtbx6-En R , suggesting that some differences in biological activity exist among the tested repressor-type T-box genes. Depletion of Xtbx6r by antisense morpholino oligo produced curved embryos, but did not affect expressions of MyoD, Myf5, XWnt8 or thylacine2, nor inhibited muscle differentiation or segmentation. The results of knockdown and overexpression experiments suggest that Xtbx6r is involved in some morphogenesis in the paraxial mesoderm.
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