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Fig. 5. XtSulf1 inhibits FGF signaling in Xenopus embryos and disrupts somitogenesis. (A–G) show the effects of over-expression of XtSulf1 on FGF activity, while (H–V) show the effects of XtSulf1 knock-down on FGF activity. (A) Western blotting shows the induction dpERK in stage 10 animal caps treated with FGF4 protein from stage 8 is inhibited in animal caps over-expressing XtSulf1. When the MAPK pathway is intracellularly activated by a drug inducible FGF receptor, XtSulf1 does not affect the induction of dpERK. In situ hybridisation shows that the FGF target genes MyoD (B–D) and Xbra (E–G) are down-regulated in embryos over-expressing XtSulf1. Panels B and E are uninjected stage 10 control embryos, panels C and F have been injected unilaterally at the 2-cell stage with 2 ng of XtSulf1 mRNA, panels D and G have been injected into all blastomeres at the 4-cell stage with a total of 4–8 ng of XtSulf1 mRNA. Western blotting to detect dpERK in X. tropicalis embryos at stage 14 in embryos where XtSulf1 has been knocked down shows elevated levels of dpERK; this effect is rescued by co-injecting mRNA coding for XtSulf1. In situ hybridisation shows that the FGF regulated gene Cdx4 (I–K) is up-regulated in neurula embryos where XtSulf has been knocked down. (I) uninjected control, (J) unilaterally injected at the 2-cell stage with AMO3, (K) injected into both blastomeres at the 2-cell stage. Panels L and M show the two sides of an embryos unilaterally injected with AMO3 and probed with MyoD. The uninjected side (L) shows normally segmented somites (arrowheads), while the side where XtSulf1 has been knocked down (M) continues to express MyoD, but does not segment to form somites. Whole mount immunostaining for dpERK (N–P) shows staining in the posterior mesoderm in stage 14 embryos unilaterally injected with a control morpholino (N). In embryos unilaterally injected with AMO3 (O and P) dpERK increases and extends anteriorly (shift is indicated by horizontal lines). In situ hybridisation for bowline and thylacine panels Q–V shows an anterior shift of the onset of somitogenesis where XtSulf1 is knocked down. (Q) Bowline expression in a stage 17 embryo unilaterally that was injected with a control morpholino at the 2-cell stage. (R and S) Bowline expression in stage 17 embryos that were unilaterally injected with AMO3 at the 2-cell stage. (T) Thylacine expression in a stage 17 embryo unilaterally that was injected with a control morpholino at the 2-cell stage. (U and V) Thylacine expression in stage 17 embryos that were unilaterally injected with AMO3 at the 2-cell stage. Arrow heads in panels Q–V indicate the most posterior, newly determined somitomere. |
