Figure 3.

The effects of Rspo2 manipulation on Wnt reporter activity in transgenic embryos. (A) Experimental scheme. Xla.Tg(WntREs:dEGFP)^Vlemx embryos were injected into one dorsal blastomere with mRFP RNA (50 pg) with (C) or without (B) Rspo2 RNA (0.5 ng). GFP fluorescence of the injected embryos at stage 18 is shown. Embryo images are representative of 3 different experiments. Asterisk indicates the injected side of the embryo, brackets in C show the comparison of the injected and the control sides. (D,E) Rspo2 modulates Wnt reporter activation. (D) Rspo2 RNA (0.5 ng), RMO^ATG (10 ng) or RMO^SB (20 ng) were injected at two dorsal blastomeres at 4-cell stage. The embryos were lysed at stage 20 and immunoblotted with anti-GFP antibodies. (E) Four-cell stage embryos were injected animally with Wnt3a RNA (50 pg) and Rspo2 RNAs (0.5 ng) or RMO^ATG (10 ng). Ectoderm explants were dissected at stage 9 and cultured until stage 13, then lysed and immunoblotted with anti-GFP antibodies. Erk1 is a control for loading. In (E) two right lanes were run in the same gel but away from the left lanes (see Supplementary Fig. 5). Five embryos or 10 explants were pooled for each experimental condition in (D,E).