Fig. 5. Testing the predictions of the axin-degradation model of axis formation. (A) LRP6 is not a dorsally localized mRNA. Real-time RT-PCR analysis of groups of two wild-type embryos, and four dorsal- or ventral-half embryos at the 32-cell stage shows that endogenous LRP6 mRNA was not enriched dorsally, in comparison with Wnt11 mRNA. Data from two separate sets of embryos is shown. (B,C) Axin protein is less abundant dorsally than ventrally in wild-type 8-cell-stage embryos. (B) Total axin protein in western blots of ten dorsal- and ten ventral-half embryos at stage 4. Axin levels were reduced dorsally compared with ventrally. Blots were stripped and re-probed for ß-catenin levels. (C) Total axin protein levels in a series of sibling embryos from the 8- to the 64-cell stage. Only at the 8-cell stage was axin at a low level dorsally compared with ventrally. ß-catenin was enriched dorsally compared with ventrally throughout the 16- to 64-cell stage. (D) The dorsal enrichment of ß-catenin is lost in LRP6-depleted embryos. Two experiments showing western blots of ten dorsal or ten ventral halves of wild-type and LRP6-depleted embryos hemisected and frozen at the 32-cell stage, and probed with a ß-catenin polyclonal antibody. ß-catenin levels were enriched dorsally in control dorsal halves compared to ventral halves (32% more in experiment 2), and this difference was lost in LRP6-depleted embryos (dorsal halves have 8% less ß-catenin in experiment 2). (E) Embryos depleted of LRP6 and axin are less dorsalized than axin-depleted embryos. Real-time RT PCR analysis of the expression of Wnt target genes at the early gastrula stages in wild-type control embryos and embryos depleted of LRP6, axin or both LRP6 and axin. Axin depletion causes the upregulation, which is partially rescued by the depletion of LRP6, of Xnr3 and siamois.