Figure 2.

Rspo2 antagonizes Wnt signaling. (A) Scheme of the experiment. Four-cell embryos were injected animally into both dorsal blastomeres with the indicated constructs and cultured to stage 38. (B) Uninjected control embryo. (C) Headless embryo injected with Wnt8 DNA (50 pg). (D) Embryo injected with Rspo2 RNA (0.5 ng). (E) Embryo coexpressing Wnt8 DNA and Rspo2 mRNA. (F) Quantification of the data in (A–D) representative of 3 independent experiments. Numbers of embryos per group are shown above each bar. ****, p < 0.0001, Fisher's exact test. (G) Target gene expression in Wnt8 and Rspo2-stimulated ectoderm explants. Embryos were injected into four animal blastomeres with Wnt8 DNA (50 pg) and Rspo2 RNA (0.5 ng), as indicated, and ectoderm explants were prepared at stage 9 and cultured until stage 13. (H) Dorsal marginal zones (DMZ) were dissected at stage 10 from the control and Rspo2 RNA- or RMO^ATG-injected embryos and cultured until stage 12. (G,H) RT-qPCR analysis was carried out in triplicates for axin2 and cdx4, and normalized to eef1a1 levels. Means + /− s.d. are shown. Graphs are representative of three independent experiments. Statistical significance has been assessed by Student’s t test, *, p < 0.05; **, p < 0.01.