Ito M et al. (2007), Xenopus Suppressor of Hairless 2 is involved in...

XB-ART-35025

Biochem Biophys Res Commun 2007 Feb 16;3533:644-9. doi: 10.1016/j.bbrc.2006.11.151.

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Xenopus Suppressor of Hairless 2 is involved in the cell fate decision during gastrulation through the transcriptional regulation of Xoct25/91.

Ito M , Nishitani E , Kinoshita T .

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We have previously indicated that Xenopus Suppressor of Hairless 2, XSu(H)2, is involved in the morphogenesis of gastrula embryos in a different manner from the XESR-1-mediated Notch signaling pathway. To address the downstream factors of XSu(H)2, we investigated the effect of XSu(H)2 on XenopusPOU-V genes, Xoct25 and Xoct91. Knockdown of XSu(H)2 caused the downregulation of Xoct25, Xoct91 and Xbrachyury. Dominant-negative Xoct25 caused the delay of blastopore closure with a decrease of Xbrachyury expression. Overexpression of Xoct25 or Xoct91 could rescue the decrease of Xbrachyury expression caused by XSu(H)2 depletion. XSu(H)2EnR inhibited Xoct25 and Xoct91 expressions in CHX-treated animal caps. Promoter analysis of the Xoct25 showed that the upstream region of Xoct25 contains the prospective Su(H) binding motif, which is essential for the transcription of Xoct25. These results suggest that XSu(H)2 is engaged in the cell fate decision during gastrulation through the gene expression of the Xoct25/91-mediated pathway.

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Species referenced: Xenopus laevis
Genes referenced: gata1 gata2 hdac1 hes5 myc nodal2 notch1 pou5f3 pou5f3.2 rbpj sms tal1 tbxt
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	Fig. 1. XSu(H)2 depletion suppressed Xoct25 and Xoct91 expressions. (A–F) Effect of XSu(H)2 depletion on embryogenesis. Embryos were injected with control-MO (A,D), XSu(H)2-MO (B,E) or XSu(H)2-MO + XSu(H)2 (C,F) into the marginal zone of one blastomere (D–F) or both blastomeres (A–C) at the 2-cell stage. The morphological phenotype of the injected embryo was examined at stage 13 (A–C). The injected embryos were cultured until stage 10.25 and the expression of Xbrachyury (brown) was examined using WISH (D–F). Reaction products of b-galactosidase (blue) indicate the injection side (right side). (G,H) The gene expressions of Xoct25 and Xoct91 were suppressed by XSu(H)2-MO in vivo and in vitro. (G) Embryos were injected with control-MO or XSu(H)2-MO into animal-marginal zone of one blastomere at the 2-cell stage. Injected embryos were cultured until stage 10.25 and the expressions of Xoct25 and Xoct91 were examined using WISH (lateral view). (H) Control-MO or XSu(H)2-MO was injected into the animal pole of both blastomeres of 2-cell stage embryos together with or without Xnr2 mRNA. Animal caps were isolated at stage 8 and were cultured until stage 10 for quantitative RT-PCR.
	Fig. 2. Xoct25 and Xoct91 could rescue the decrease of Xbrachyury expression caused by XSu(H)2 depletion. (A) Phenotype of DN-Xoct25- and DN-Xoct91-injected embryos (vegetal view). Embryos were injected with 500 pg DN-Xoct25 mRNA or 1 ng DN-Xoct91 mRNA into the marginal zone of both blastomeres at the 2-cell stage, and morphological changes were analyzed at stage 13. (B) Effect of DN-Xoct25 and DNXoct91 on gene expression (vegetal view). Embryos were injected with 250 pg DN-Xoct25 mRNA or 500 pg DN-Xoct91 mRNA into marginal zone of one blastomere at the 2-cell stage. Injected embryos were cultured until stage 10.25, when the expression of Xbrachyury (brown) was examined using WISH. Reaction products of b-galactosidase (blue) indicate the injection side (right side). (C) Effect of Xoct25 and Xoct91 on Xbrachyury suppressed by XSu(H)2-MO. At 2-cell stage, 40 ng XSu(H)2-MO was injected into the marginal zone of both blastomeres together with several doses (125, 250 or 500 pg) of Xoct25 or Xoct91. Gene expression was examined by quantitative RT-PCR at stage 10.25.
	Fig. 3. XSu(H)2EnR inhibited the gene expressions of Xoct25 and Xoct91 under CHX treatment. Synthesized mRNAs of 2 ng XSu(H)2, XSu(H)2EnR or XSu(H)2VP16 were injected into the animal pole of 2-cell stage embryos. Animal caps dissected at stage 8 were cultured in medium supplemented with (B) or without CHX (A), and gene expression was examined at stage 10.25.
	Fig. 4. XSu(H)2 directly bound to the upstream region of Xoct25 regulated its transcription. (A) Comparison of upstream sequences of Xoct25 between Xenopus laevis and tropicalis. Gray boxes indicate the high homology of S1 and S2 in the SPS motif. (B) Schematic drawings of several deletion forms in Xoct25 reporter construct. The upstream region of Xoct25 ( 1042) truncated with S1 (DS1), S2 (DS2) or both (DS1/DS2) was fused to the luciferase reporter gene. Adenine at the first methionine is defined as +1. (C) Effect of SPS motif mutation on reporter activity. All values were standardized by calculating luciferase activity in the upstream of Xoct25 ( 1042) as 1. Experiments were carried out in triplicate. (D) Two nanograms MT-XSu(H)2 into both blastomeres of two-cell stage embryos, the injected embryos were cultured until stage 10.25 for ChIP assay. A sample prepared from embryos without injection was used as ChIP control. Anti-myc, precipitation using anti-myc antibody; no Ab, precipitation without antibody; input, cross-linked chromatin supernatant before immunoprecipitation.