Xenbase Image ID: 1811

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. Image published in: Murai K et al. (2007) Copyright © 2007. Image reproduced with permission of the Publisher, Elsevier B. V. Gene Synonyms Species Stage(s) Tissue tbxt.S bra, brachyury, ntl, t, t-a, t-b, X-bra, Xbra, Xbrachyury X. laevis Throughout NF stage 10 mesoderm marginal zone ventral marginal zone ventro-lateral marginal zone dorso-lateral marginal zone myod1.S mf25, MyoD, myod1-a, myod1-b, myogenic factor 3, XMyoD, XtmyoD X. laevis Throughout NF stage 10 mesoderm ventro-lateral marginal zone ventral marginal zone dorso-lateral marginal zone Image source: Published Larger Image Printer Friendly View

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