XB-IMG-24133
Xenbase Image ID: 24133
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Fig. 4. XtSulf1 inhibits BMP signaling in Xenopus embryos. (A–I) show the effects of over-expression of XtSulf1 on BMP4 activity, while (J–P) show the effects of XtSulf1 knock-down on BMP4 activity. (A) Western blotting to detect phospho-SMAD1/5/8 in stage 10 animal caps injected with mRNA coding for BMP4 or the constitutively active BMP receptor Alk3. Over-expression of XtSulf1 results in the inhibition of BMP4 activation of pSMAD1, while it has no effect on the pSMAD1 activation by Alk3. (B) A GST-BMP receptor fusion protein was used to pull down an HA-tagged BMP4 in whole embryos. In the presence of XtSulf1 less BMP4 is associated with the BMP receptor. (C) XtSulf1 over-expression in stage 18 animal caps results in low levels of NCAM expression as analysed by RNAase protection analysis. In situ hybridisation shows that the BMP target genes sizzled (D–F) and Msx1 (G–I) are down-regulated in embryos over-expressing XtSulf1. Panels D and G are uninjected stage 10 control embryos, panels E and H have been injected unilaterally at the 2-cell stage with 2 ng of XtSulf1 mRNA, and panels F and I have been injected into all blastomeres at the 4-cell stage with a total of 4 ng of XtSulf1 mRNA. (J) Western blotting to detect phospho-SMAD1/5/8 in X. tropicalis embryos at stage 14. Embryos where XtSulf1 has been knocked down show elevated levels of pSMAD1; this effect is rescued by co-injecting mRNA coding for XtSulf1. In situ hybridisation of X. tropicalis embryos at stage 16 shows that embryos unilaterally injected at the 2-cell stage with an AMO against XtSulf1 (L, N, P) have reduced levels of Otx2 (K and L) and Pax6 (M–P) which are known to be negatively regulated by BMP4 signaling. Image published in: Freeman SD et al. (2008) Copyright © 2008. Image reproduced with permission of the Publisher, Elsevier B. V.
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