Figure 2

Xfz4S acts synergistically with canonical Wnt ligands in activating the Wnt/β-catenin target gene Xnr3 and inhibits non-canonical Wnt ligands. (A) Experimental scheme. Embryos were injected at 4 cell stage into the animal blastomeres with synthetic mRNA for Xwnt-3a (0.5 pg/embryo), Xwnt-4 (150 pg/embryo), Xwnt-5a (50 pg/embryo), Xwnt-8 (0.5 pg/embryo), Xwnt-8b (15 pg/embryo) or Xwnt-11 (50 pg/embryo), either alone or in combination with 500 pg Xfz4S. Animal caps were dissected out at stage 8 – 9, grown until stage 10.5 at which expression of Xnr3 was analyzed by RT-PCR. (B) Xwnt-3a, -8, -8b induce Xnr3 expression only in combination with Xfz4S. Xwnt-5a, -4 or -11 do not synergize with Xfz4S in inducing Xnr3. (C) Xfz4S inhibits Xnr3 expression induced by coinjection of Hfz5 and non-canonical Wnt-5a class ligands. Wnt-5a class ligands such as Wnt-4 (150 pg/embryo), -5a (50 pg/embryo) or -11 (50 pg/embryo) when injected in combination with 250 pg Hfz5, Xnr3 expression was induced. Induction of Xnr3 expression by these Wnt ligands in combination with Hfz5 was inhibited by coinjection of 500 pg Xfz4S mRNA. (D) Coimmunoprecipitation of Xfz4S and Wnt-5a. Myc-tagged Xfz4S (500 pg/embryo) and flag-tagged Wnt-5a (500 pg/embryo) were injected into Xenopus embryos at 2–4 cell stage. Myc-tagged Xfz4S coimmunoprecipitates with flag-tagged Wnt-5a indicating that they interact. A part of the embryo extract was incubated with mouse IgG, which serves as a control against non-specific binding of proteins. Total embryo extract (EE) shows the expression of Xfz4S-myc and Wnt5a-flag constructs.