Fig. 3. LRP6 is downstream of Wnt11 and upstream of ß-catenin in the axis-forming pathway. (A) The ventralized Wnt11-depletion phenotype at late neurula stage (top row; 9/9 cases ventralized), was rescued by 75 pg Wnt11 mRNA (second row; 0/8 cases ventralized), and by 75 pg LRP6 mRNA (third row; 0/12 cases ventralized), injected 24 hours before oocyte maturation. Additionally, 300 pg of LRP6 mRNA also rescued Wnt11-depleted embryos, but caused a dorsalized phenotype (fourth row; 0/8 cases ventralized). (B) The expression of Wnt target genes at the early gastrula stage (stage 10) in sibling embryos to those shown in A, assayed by real-time RT-PCR. LRP6 mRNA rescued the expression of siamois and Xnr3 in Wnt11-depleted embryos. (C) The ventralized LRP6-depletion phenotype at tailbud stage (red embryos; 9/9 cases ventralized) was not rescued by the injection of 100 pg Wnt11 mRNA 24 hours prior to maturation (bottom row; 6/6 cases ventralized). (D) The expression of Wnt target genes at the late blastula stage (stage 9.5) and early gastrula stages (stage 10) in embryos that were siblings to those shown in C, assayed by real-time RT-PCR. Wnt11 was not able to rescue the expression of Wnt target genes in LRP6-depleted embryos. (E) The ventralized LRP6-depletion phenotype (7/7 cases ventralized) shown at tailbud stage was rescued by the injection of 50 pg ß-catenin mRNA into one dorsal cell at the 4-cell stage (6/20 cases ventralized). (F) The expression of Wnt target genes at the early gastrula stages (stage 10) in embryos that were siblings to those shown in C, assayed by real-time RT-PCR. ß-catenin rescued the expression of Wnt target genes in LRP6-depleted embryos. (G) Lrp6-N-Myc interacts with Wnt11-HA in co-immunoprecipitation assays. Lrp6-N-Myc (1.5 ng) and Wnt11-HA (500 pg) were injected into two different blastomeres at the 4- to 8-cell stage. Embryos were harvested at stage 10.5. Panel on the right is a western blot of the embryo lysates, blotted with both anti-myc and anti-HA antibody. Panel on the left shows the result of the lysates immunoprecipitated with anti-HA antibody and blotted with anti-myc antibody. IgG band is indicated on the bottom of the left panel.