Fig. 4. Wnt11-LRP6 signaling regulates axin levels in the early embryo. (A) Western blot of embryos derived from wild-type and LRP6-depleted oocytes (LRP6^-) that were frozen at the 8-cell (stage 4) and early blastula (stage 7) stages and probed with an affinity purified axin polyclonal antibody. Blots were stripped and re-probed for ß-catenin, and -tubulin antibody was used as a loading control. Axin levels were increased in LRP6-depleted embryos (arrow; increased by 47% compared with control). ß-catenin levels were decreased by LRP6 depletion (arrowhead; decreased by 17% compared with controls). (B) Western blot of embryos derived from wild-type and Wnt11-depleted oocytes (Wnt11^-) that were frozen at stage 4 and probed with an affinity purified axin polyclonal antibody. Blots were stripped and re-probed for ß-catenin. Axin levels were increased (arrow; 46% compared to control) in Wnt11-depleted embryos and ß-catenin levels were decreased (arrowhead; 23% compared with control). (C) A model of LRP6 function in the axis-forming pathway. (Ci) Sperm activates the cortical rotation movements of the first cell cycle, leading to a dorsal asymmetry of Wnt11 mRNA and protein, which causes increased Wnt11 signaling dorsally (purple arrows). (Cii) By the 8-cell stage, Wnt11 protein is secreted, mostly by dorsal vegetal cells, and binds to LRP6. Signaling is shown between a dorsal vegetal and animal cell, although LRP6 is likely to be present in both cells, and autocrine signaling may occur. In LRP6-depleted embryos, Wnt11 molecules are secreted dorsally but are unable to activate the pathway. (Ciii) An enlargement of the animal cells shown in Fig. 3Cii at the 8-cell stage. Association of Wnt11 with the extracellular domain of LRP6 causes the degradation of axin. ß-catenin enters nuclei and binds to Xenopus Tcf3 on the dorsal side. In LRP6-depleted embryos, Wnt11 molecules do not bind LRP6, so axin increases in concentration and ß-catenin is degraded to the same extent on dorsal and ventral sides, and does not enter nuclei.