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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.
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