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Dev Biol
2008 Jan 15;3132:816-28. doi: 10.1016/j.ydbio.2007.10.015.
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Tbx6, Thylacine1, and E47 synergistically activate bowline expression in Xenopus somitogenesis.
Hitachi K
,
Kondow A
,
Danno H
,
Inui M
,
Uchiyama H
,
Asashima M
.
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T-box factor, Tbx6, is a prerequisite for somite segmentation in vertebrates. We recently identified a negative regulator of Tbx6, Bowline, which represses the expression of genes involved in somite segmentation by suppressing the transcriptional activity of Tbx6. According to this function, bowline gene expression is restricted to the most anterior presomitic mesoderm where the somite segmentation program terminates, although it remains unclear how bowline expression is activated. To address this, we investigated the cis-regulatory region of bowline. Measuring luciferase activity driven by the bowline promoter, we found that Tbx6, Thylacine1, and E47 synergistically activate bowline expression in vitro. We also found that Tbx6, Thylacine1, and E47 are spatiotemporally sufficient to induce bowline expression in Xenopus somitogenesis. Our findings indicated that besides being a negative regulator of Tbx6, bowline itself is also regulated by Tbx6, suggesting the negative feedback loop of Tbx6-Bowline in the termination step of somite segmentation.
Fig. 1. Isolation of a bowline upstream region, and reconstitution of bowline expression in transgenic embryos. (A) In situ hybridization for endogenous bowline. (B, C) In situ hybridization for EGFP reporter gene in transgenic embryos with δ3UTRGFP (B), or with FullGFP (C). (D–F) Magnified views of panels A (D), B (E), and C (F). (D′–F′) Schematic diagrams of gene expression in panels D (D′), E (E′), and F (F′). (G) In situ hybridization for endogenous Xhairy2a (h, brown). (H) Double in situ hybridization for Xhairy2a (h, brown) and bowline (b, blue) in wild-type embryos. (I) Double in situ hybridization for Xhairy2a (h, brown) and EGFP reporter gene (g, blue) in transgenic embryos with FullGFP. (G′–I′) Schematic diagrams of gene expression in panels G (G′), H (H′), and I (I′). Arrowheads indicate segmental patterns of expression. The bar indicates the broad expression pattern in transgenic embryos with δ3UTRGFP.
Fig. 7. Tbx6, Thy1, and E47 are spatiotemporally sufficient to induce bowline expression in Xenopus somitogenesis. (A) Schematic diagram of somites, somitomeres, and tailbud region of Xenopus neurula. Dorsal view is shown with anterior towards the top. The fictive expression shown in panel A is restricted to the anterior half of S-0, S-I, S-II, and S-III. The nomenclature of somites and somitomeres is based on the annotation of Pourquie and Tam (2001). (B) In situ hybridization for Tbx6 (blue). (C) In situ hybridization for Thy1 (arrowhead; brown). (D) Double in situ hybridization for Tbx6 (blue) and Thy1 (arrowhead; brown). (E) Double in situ hybridization for Tbx6 (blue) and bowline (arrowhead; brown). (B′–E′) Schematic diagrams of gene expression in panels B (B′), C (C′), D (D′), and E (E′). (F–K) In situ hybridization for bowline. Embryos were unilaterally injected with DNA for β-gal (100 pg; F), Tbx6 (50 pg; G), Thy1 (50 pg; H), a mixture of Tbx6 and Thy1 (50 pg each; I), and a mixture of Tbx6, Thy1, and E47 (33 pg each; J and K). The white bar indicates expanded bowline expression (G). The white arrowhead indicates disrupted bowline expression (H). The white brackets indicate ectopically induced bowline expression (I, J, and K). Injected sides are to the right and dorsal views are shown with anterior towards the top. (L) Diagram illustrating a stepwise regulatory model of somitogenesis involving Tbx6. (1) Tbx6 may participate in the segmentation clock system via the regulation of Notch ligand Dll1 (Notch signaling component) in mouse ([Hofmann et al., 2004] and [White and Chapman, 2005]). (2) Tbx6 may be involved in the formation of somite boundaries via regulation of Thy1/Mesp2 in mouse and X. laevis ([Kondow et al., 2007] and [Yasuhiko et al., 2006]). (3) We propose a novel participation of Tbx6 in termination of the somite segmentation program, whereby Tbx6 directly activates bowline expression together with Thy1/E47, and Bowline in turn suppresses the transcriptional activity of Tbx6 (Kondow et al., 2007). The actions represented by red arrows are based on our new findings.
