Ryk promotes internalization of the Wnt11-induced Fz7 and Dsh aggregates in animal cap cells. (A–D) Four-cell stage embryos were injected in the animal pole with various mRNAs in combination as indicated (500 pg GFP Dsh, 500 pg myc Rab5, 1 ng Fz7, 1 ng myc Fz7, 1 ng CFP Fz7, 1 ng YFP Fz7, 500 pg GFP Rab5, 1 ng XRyk, 1 ng myc XRyk, and 1 ng Wnt11). Animal caps were dissected at stage 9–10 and then subjected to fluorescence analysis. In the presence of Ryk, the Wnt11-induced punctate aggregates of Dsh and Fz7 (A) overlapped with Rab 5 (B and C). (D) Fz7 colocalized with Ryk in both cytoplasmic (yellow arrows) and membrane (white arrows) puncta. Bars, 20 μm. (E and F) HEK293FT cells were transfected with various constructs, either alone or in combination as indicated. Cell lysates were immunoprecipitated with anti-Flag (E) or anti-GFP (F) antibodies and the immunocomplexes were blotted with specific antibodies. (E) Ryk bound to the noncanonical Wnt11 as well as Wnt5a. (F) Ryk and Ryk-ΔC, which lacks the cytoplasmic domain, interacted with Dsh. Arrows indicate a size of epitope-tagged Ryk or Ryk-ΔC. Numbers to the right of the gel blots indicate molecular mass standards in kD.