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The specification of mesoderm into distinct compartments sharing the same lineage restricted fates is a crucial step occurring during gastrulation, and is regulated by morphogenic signals such as the FGF/MAPK and activin pathways. One target of these pathways is the transcription factor XmyoD, which in early gastrulation is expressed in the lateral and ventral mesoderm. Expression of the hairy/enhancer of split transcription factor hes6, is also restricted to lateral and ventral mesoderm in gastrula stage Xenopus embryos, leading us to investigate whether it has a role in XmyoD regulation. In vivo, Xhes6 is required for FGF-mediated induction of XmyoD expression but not for induction of early mesoderm. The WRPW domain of Xhes6, which binds Groucho family transcriptional co-regulators, is essential for the XmyoD-inducing activity of Xhes6. Two Groucho proteins, Xgrg2 and Xgrg4, are expressed in lateral and ventral mesoderm, and inhibit expression of XmyoD. Xhes6 binds both Xgrg2 and Xgrg4 and relieves their inhibition of XmyoD expression. We also find that lowering Xhes6 expression levels blocks normal myogenic differentiation at tail bud stage. We conclude that Xhes6 is essential for XmyoD induction and acts by relieving Groucho-mediated repression of gene expression.
Fig. 1. Xhes6 is induced by mesoderm inducing signals and its expression is dependent on FGF-signaling. 5 pg of Xnr2 (C, D), 1 pg of eFGF (E, F) or 125 pg of XFD (G, H) mRNA was injected into a blastomere at 2-cell stage, along with β-galactosidase mRNA (red staining) and analyzed at gastrula stage for expression of Xhes6 (A, C, E and G) and XmyoD (B, D, F and H) by whole-mount in situ hybridisation. The side view of the area within the yellow box is shown on the right of each panel.
Fig. 2. Over-expression of Xhes6 increases XmyoD expression in vivo. (A) Constructs used. Xhes6 constructs were myc tagged at the N-terminus. In the DBM mutant, the basic amino acids in the DNA domain were mutated to acidic residues (underlined) whilst in the δWRPW mutant, the C-terminal WRPW protein–protein interaction motif was replaced by a stop codon (red triangle). (B) Western blotting with an anti-myc antibody to detect Xhes6 protein (upper panel) and an anti-α-tubulin antibody loading control (lower panel). (C–L) Effects of overexpression of wild-type and mutant Xhes6 500 pg of Xhes6, DBM or δWRPW mRNA was injected into marginal zone of a blastomere at 2-cell stage, along with β-galactosidase mRNA (red staining) and analyzed at gastrula stage for expression of XmyoD (C–F), Xbra (G, H), Xwnt8 (I, J) and Chordin (K, L) by whole-mount in situ hybridisation. The side view of the area within the yellow box is shown on the right of each panel.
Fig. 4. Xhes6 requires FGF signaling to induce XmyoD. (A–E) 500 pg of Xhes6 mRNA was injected alone or in combination with 125 pg of XFD mRNA into either the marginal zone of one blastomere at 2-cell stage, along with β-galactosidase mRNA (red staining). Gastrula stage embryos were analysed for XmyoD expression (A–D). (E) Summary of in situ hybridisation results.
Fig. 5. Antisense morpholino oligos against Xhes6 block XmyoD expression in lateral and ventralmesoderm. (A) The 5′ region of Xhes6 cDNA. The previously reported 5′ end of the transcript is indicated by the triangle; an additional 81 base pairs of sequence were identified by 5′RACE PCR. Two initiation codons are present (bold). The sequences targeted by Xhes6 antisense morpholino oligonucleotides, MO-1 and MO-2 are underlined in red. (B) Antisense morpholino oligonucleotide (MO) validation. Both cells of 2-cell stage embryos were injected with 1 ng Xhes6-HA mRNA together with either no MO (−), a standard control MO (CTL) or Xhes6 MO-1 in the amounts shown. Embryos were lysed at stage 11 and analyzed for Xhes6-HA protein expression by western blotting with an anti-HA antibody. The same membrane was also blotted with anti-α-tubulin antibody as a loading control. (C–L) Phenotype of Xhes6 MO. Whole-mount in situ hybridisations showing XmyoD in uninjected or MO-injected embryos. 40 ng of each indicated MO together with 0.25 ng of β-galactosidase mRNA, as a tracer, were injected into marginal zone of one blastomere of embryos at the 8-cell stage. Embryos were analyzed by whole-mount in situ hybridisation to detect mRNA encoding Xbra (C–G) or XmyoD (H–L) at gastrula stage; red colour indicates presence of β-galactosidase detected by salmon-gal, the yellow arrow indicates the targeted region. STD-CTL, standard control MO (D, I); 5-mis MO-2, a 5 base pair mismatched control version of Xhes6 MO-2 (E, J); Xhes6 MO-1 (F, K) and Xhes6 MO-2 (G, L) are directed against Xhes6 mRNA as shown in panel A. (M–R) Rescue of Xhes6 MO phenotype. 40 ng of MO-1 was injected with mRNA encoding Xhes6 (M, N), DBM (O, P) or MT-δWRPW (Q, R) together with β-galactosidase mRNA into the marginal zone of one blastomere of 8-cell stage embryos. Gastrula stage embryos were analysed by whole-mount in situ hybridisations for XmyoD; red colour indicates presence of β-galactosidase, the yellow box outlines the targeted regions shown enlarged in panels I, K and M.
Fig. 6. Xhes6 morpholino inhibits ectopic XmyoD expression by FGF-signaling. 1 pg of eFGF was injected alone or with 40 ng of STD CTL or Xhes6 in the animal pole of a blastomere at 2-cell stage, along with β-galactosidase mRNA (red staining) and analyzed at gastrula stage for Xbra (A–D) and XmyoD (E–H) expression by whole-mount in situ hybridisation. The side view of the area within the yellow box is shown on the right of each panel. (I) Summary of in situ hybridisation results.
Fig. 7. Xgrg2 and Xgrg4 inhibit XmyoD induction by MAPK signaling. Gastrula stage embryos were analysed for Xhes6 (A), Xgrg2 (B), Xgrg4 (C) and XmyoD (D–J) expression by whole-mount in situ hybridisation. (A–C) Uninjected embryos. (D–G) 1 ng flag-tagged Xgrg2, Xgrg4 or non-phosphorylatable mutant (Xgrg2AAA or Xgrg4AA) was injected with β-galactosidase mRNA (red staining) into marginal zone of a blastomere at 2-cell stage. (H–J) 1 ng of active MEK1 (MEK1S → E) mRNA was injected alone or with flag-tagged Xgrg2AAA or Xgrg4AA into a blastomere at the 2-cell stage.
