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Fig. 8. Comparative analysis of szl and other secreted Wnt inhibitors. (A-C) Increasing amounts of RNA encoding Myc-tagged versions of Xenopus Szl, Frzb-1 (Frzb), Crescent (Cres) and Dikkopf1 (Dkk1) were injected in the marginal zone of four-cell stage embryos. (A) Expression of the erythroid markers xGata1 and αT3 globin was analyzed by RT-PCR at stage 33/34. EF1α was used as an internal control. (B) Expression of the injected Myc-tagged proteins was analyzed by immunoblotting at stage 11 on embryos from the same round of injections. The same blot was probed for actin as a loading control. (C) At the same time, morphology of the embryos and shape of the VBI were visualized by whole-mount in situ hybridization for embryonic αT3 globin. (D) Activity of the injected proteins as inhibitors of the canonical Wnt pathway was assessed in a Luciferase reporter assay. Embryos were injected in the animal pole with 200 pg of pTOP-FLASH, 4 pg of xWnt8 RNA, and increasing amounts of RNA encoding Myc-tagged Wnt inhibitors. Relative LUC activity is normalized to activation of the reporter by xWnt8 alone, and amounts of injected sFRPs are plotted on a logarithmic scale. For each RNA dilution, injected proteins were expressed at comparable levels; i.e. Frzb-MT RNA was injected at a higher concentration to compensate for poor translation. |
