Fig. 6. Rspo3 is required for Wnt/β-catenin mediated induction of Vegf. (A) Vegfr2 expression. (B) Vegfa expression. (C) Tpbpa expression in spongiotrophoblast layer. (D) Csh1 (placental lactogen 1) expression in giant cell border. (A-D) Representative samples from 20 wild-type and 20 knockout embryos are shown as indicated. (E) Immunofluorescence staining of β-catenin (green) and nuclei (Hoechst, blue) in the allantois (E8.5). Reduced β-catenin staining occurs in Rspo3^-/- allantois. (F) BATGAL reporter activity in wild-type and Rspo3^-/- chorionic plates. Representative samples from 20 wild-type and 20 knockout embryos are shown. (G) Quantification of lacZ staining in the allantois (all) (E8.5), somites (s) (E8.5) and chorionic plate (cp) (E9.5) and β-catenin staining in the allantois (all) (E8.5). The number of positive cells in wild-type embryos was set to 100% (see Materials and methods). Abbreviations: all, allantois; ch, chorion; gc, giant cells; sp, spongiotrophoblast; la, labyrinth; cp, chorionic plate.