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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.
Fig. 1. Differential expression patterns of the bowline and ledgerline genes during Xenopus somitogenesis. (A,B) Expression patterns of bowline and ledgerline in the Xenopus PSM. Longitudi- nal serial sections of a stage 20 embryo were stained for bowline (A) and ledgerline (B), and the results are shown as a pseudocolor display. The white arrowheads in (A) and (B) represent the regions in which bowline and ledgerline, respectively, areexpressed. (C)Mergedimageof(A,B). The white arrowheads in (C) represent the regions in which the expression patterns of bowline and ledgerline overlap. The sec- tions are orientated so that the anterior part is to the left. (D,E) Whole-mount in situ hybridization for bowline (D) and ledgerline (E). The expression of bowline is restricted to the somitomeres. Interest- ingly, ledgerline is expressed in the newly formed somites, as well as in the somitomeres. The black bar indicates ledgerline expression in the newly formed somites. (F) Schematic diagram of the bowline and ledgerline expression patterns. Bowline is expressed in the anterior halves of the S-I to S-III somitomeres (left side). Ledgerline is expressed in the newly formed somites, in addition to the anterior halves of the S-0 to S-II somitomeres (right side). The dorsal view is shown, with
anterior being towards the top. The nomenclature used for the somitomeres follows that proposed earlier (Pourquie and Tam, 2001).
Fig. 2. Tbx6 is required for bowline and ledgerline expression during Xenopus development. (A) The ex- pression of bowline and ledgerline is induced by the synergistic effects of Tbx6, mespb, and E47. RT-PCR analysis shows that overexpression of the mixture of Tbx6, mespb, and E47 mRNA species induces ectopic bowline and ledgerline expression in gastrula-stage Xeno- pus embryos. ODC (ornithine decarboxylase) was used as an internal control. (B-E) Expression of bowline mRNA and ledgerline mRNA in Xtbx6 exint-MO-injected embryos. All the embryos were fixed at stage 19 for whole-mount in situ hybridization to detect the expression of bowline (B,C) and ledgerline (D,E). Magenta-Gal staining indicates the injected side. (B,D) A representative embryo injected with 50 ng Xtbx6 exint-MO. Inhibition of the splicing of Tbx6 mRNA results in loss of expression of bowline and ledgerline. The white arrowheads in (B) and (D) indicate reduced bowline and ledgerline expression, respectively. (C,E) Representative embryos injected with 50 ng Xtbx6 exint-MO and 2 pg or 10 pg Xenopus Tbx6 mRNA, respectively. The white arrowheads in (C,E) indicate rescued bowline and ledgerline expression, respectively.
Fig. 4. Knockdown of Ledgerline causes abnormal expression of mespb and Tbx6 during Xenopus somitogenesis. (A-D) Whole-mount in situ hybridization for mespb (A,B) and Tbx6 (C,D). Embryos at the 4-cell stage were injected with Control-MO (A,C) or Ledgerline-MO (B,D). In the embryos injected with Ledgerline-MO, the regions of mespb and Tbx6 expression were expanded anteriorly (white bar in B and D, respectively).
Fig. 4. Knockdown of Ledgerline causes abnormal expression of mespb and Tbx6 during Xenopus somitogenesis.
(E,F) Whole- mount in situ hybridization for mespb. Embryos were treated with 500 nM trichostatin A (TSA) or DMSO for 4 h. Anteriorly expanded mespb expression was induced by TSA (white bars in
F). Dorsal views are shown with the anterior towards the top.
Fig. 6. Expression profile of Ripply3 during X. laevis and mouse development. (A-C) Whole-mount in situ hybridization for xRipply3. (A) At the late neurula stage, xRipply3 is expressed in the region near the heartmesoderm. (B) At the tailbud stage, xRipply3 expression is restricted to the pharyngeal region. Panel (C) is a magnified view of (B). Lateral views are shown with the anterior towards the left. The arrowheads in (A) and (C) indicate xRipply3 expression. (D) Tailbud-stage embryos stained with the xRipply3 probe were sliced frontally. (E,F) Whole-mount in situ hybridization for murine Ripply3. (E) Murine Ripply3 expres- sion is observed in the pharyngeal region. Panel (F) is a magni- fied view of (E). The arrowheads in (F) indicate murine Ripply3 expression.
Fig. 7. xRipply3 suppresses the transcriptional ac- tivity of Tbx1. (A) Schematic diagram of pGL4-Tbox, which contains tandem T-box binding sites. (B,C) Luciferase assays for the pGL4-Tbox construct. (B) Tbx1 dose-dependently increases the luciferase activ- ity of pGL4-Tbox. COS7 cells were transfected with the pGL4-Tbox (100 ng) and the Tbx1 expression vector (50-200 ng). (C) xRipply3 represses the tran- scriptional activity of Tbx1 in the pGL4-Tbox construct. COS7 cells were transfected with pGL4-Tbox (100 ng) and a combination of the indicated expression vectors, Tbx1 (50 ng) and xRipply3 (25-100 ng). Error bars represent the SEM of three independent experiments. (D) Interaction between the xRipply3 and Tbx1 proteins in Xenopus embryos. Protein extracts of Xenopus embryos co-injected with myc-Tbx1 mRNA and HA-xRipply3 mRNA (500 pg each) were subjected to co-immunoprecipitation with the anti-myc antibody. (E) Interaction between the xRipply3 and Grg4 proteins in Xenopus embryos. Protein extracts of Xenopus embryos co-injected with myc-Grg4 mRNA and HA-xRipply3 mRNA (500 pg each) were subjected to co-immunoprecipitation with the anti-myc antibody. The asterisks indicate non-specific bands. (F-H) Frontal sections of Xenopus embryos were stained for xRipply3 (F), Fgf8 (G), and Tbx1 (H) transcripts. The regions of xRipply3 and Fgf8 expression differ from those of Tbx1.
ripply3 (ripply transcriptional repressor 3) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 20, lateral view, anteriorleft.
ripply3 (ripply transcriptional repressor 3) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 33 and 34, lateral view, anteriorleft.
ripply3 (ripply transcriptional repressor 3) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 33 and 34, lateral view, anteriorleft.
At NF stage 28, ripply3 (ripply transcriptional repressor 3) gene expression is restricted to the pharyngeal pouches, shown here in frontal section, through the pharyngeal region.
