The Xenopus Bowline/Ripply family proteins negatively regulate the transcriptional activity of T-box transcription factors

Abstract

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.

RIPPLY3 is a retinoic acid-inducible repressor required for setting the borders of the pre-placodal ectoderm

“…Tbx1 is also expressed in the otic placode, whereas Ripply3 is expressed in the pronephros. Similar results were reported for Tbx1 (Ataliotis et al, 2005;Showell et al, 2006) and for Ripply3 (Hitachi et al, 2009). Quantitative real-time RT-PCR (QPCR) analysis showed that Ripply3 is expressed maternally.…”

“…Control embryos were injected with 20 ng standard control MO: CCT CTT ACC TCA GTT ACA ATT TAT A (GeneTools). The following plasmids were constructed by PCR amplification of the protein-coding regions of the indicated genes and cloning into the expression vector pCDG1: xRARa2.2 (Sharpe, 1992), xTbx1 (Ataliotis et al, 2005) and xRipply3 (Hitachi et al, 2009). xRipply3 WRPWrAAAA , xRipply3 FPVQrAAAA and Ripply3…”

In vitro organogenesis from undifferentiated cells in Xenopus

“…Through this screening process, several somitogenesis-related genes, such as paraxial protocadherin and mesogenin 1 (msgn1), as well as a novel gene bowline (Kim et al, 2000;Yoon et al, 2000;Kondow et al, 2006), were isolated. Two bowline-related genes, ledgerline and xRipply3, were also isolated from X. laevis (Chan et al, 2006;Hitachi et al, 2008). Counterparts have been identified in the zebrafish and mouse (Ripply1, 2, 3), human (DSCR6), and amphioxus (AmphiSom) (Shibuya et al, 2000;Kawamura et al, 2005;Li et al, 2006;Chan et al, 2007;Morimoto et al, 2007).…”

Ripply3, a Tbx1 repressor, is required for development of the pharyngeal apparatus and its derivatives in mice

“…To reveal the developmental role of another member of the Ripply family, Ripply3 [also known as Down syndrome critical region 6 (Dscr6)] (Kawamura et al, 2005;Hitachi et al, 2009;Shibuya et al, 2000), we examined its expression and function in the early mouse embryo. On finding that both Ripply3 and Tbx1 are expressed in the pharyngeal endoderm, we examined the role of Ripply3 by generating Ripply3-deficient mice and investigated its relationship with Tbx1.…”