Fig. 7. FoxD3 is necessary and sufficient for Nodal expression. At the early gastrula stage, Xnr1 (A) and Xnr2 (B) are expressed in two distinct domains: strong expression in the dorsal marginal zone and punctate expression throughout the vegetal pole. In the experiment shown, vegetal expression is more apparent for Xnr2. For FoxD3 gain-of-function, 200 pg of FoxD3 RNA was injected into the marginal region of two blastomeres at the four-cell stage and the expression of Xnr1 (C) and Xnr2 (D) was examined by in situ hybridization at the early gastrula stage (stage 10.25). Ectopic expression of Xnr1 and Xnr2 is indicated with brackets. For FoxD3 loss-of-function, 0.5 ng of VP16-FoxD3 (E,F) or 25 ng of FoxD3MO (G,H) was injected into each blastomere at the four-cell stage and the expression of Xnr1 and Xnr2 was examined. As a negative control, 25 ng of mismatch MO (I,J) was injected. The results shown are representative of three independent experiments (n=20-25 embryos per sample in each experiment). Vegetal views with dorsal side up are shown. (K) At the one-cell stage, the animal pole was injected with FoxD3 RNA (300 pg) and animal explants prepared at the blastula stage (stage 9) were analyzed by RT-PCR at the early gastrula stage (stage 10.25) for the expression of Brachyury (Xbra), Xnr1, Xnr2, Xnr4 and Derriere (Der). PCR controls are as described in Fig. 2. (L) Lysates of FoxD3- or Xnr1-expressing animal explants were examined for the presence of phospho-Smad2 protein by western blotting with a phospho-specific anti-Smad2 antibody. Stripped blots were analyzed for total Smad2/3 proteins as a loading control.