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Fig. 5. Antisense morpholino oligos against Xhes6 block XmyoD expression in lateral and ventral mesoderm. (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. |
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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. |
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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. |
