de Almeida I , Rolo A , Batut J , Hill C , Stern CD , Linker C .

GM60156 NIGMS NIH HHS , R01 MH060156-01 NIMH NIH HHS, R01 MH060156-02 NIMH NIH HHS, R01 MH060156-03 NIMH NIH HHS, R01 MH060156-04 NIMH NIH HHS, R01 MH060156-05 NIMH NIH HHS, R01 MH060156-06 NIMH NIH HHS, Medical Research Council , G0400559 Medical Research Council , MRC_G0400559 Medical Research Council

	Fig. 1. Smad7 is not sufficient for neural induction in chick. Electroporation of Smad7 (A–C; 0/12), Cerberus (D–F; 0/4) or a combination of Smad7 + Smad6 + Noggin + Chordin + dnBMPR (0/9) does not induce either Brachyury (light blue in A, D and G) or Sox2 (dark blue in B, E and H; the same embryo to the left). Electroporated cells were recognised by GFP expression (C, F, I and I’ in the same embryo to the left). The plane of section is indicated by a black line.
	Fig. 2. Smad7 induces dorsal mesoderm in Xenopus ventral epidermis, and neural markers only indirectly. Injection of Smad7 (2 ng) into the A4 blastomere induces Sox3 (A–C), Sox2 (D–E), Chordin (H–J) and Brachyury (K–L), but not Six3 (F–G). (C and J) Histological sections through the levels indicated in B and I, respectively. A, D, F, H and K are dorsal views. B, E, G, I and L show ventral views of the embryo to their left.
	Fig. 3. Smad7 blocks Smad1 and Smad2 phosphorylation but induces neural markers only indirectly. (A and B) Inhibition of phospho-Smad1 and phospho-Smad2 by Smad7, tAlk4+Smad6 and CerS+Smad6, revealed by Western blot analysis of animal caps from injected embryos. Fldx-injection and whole embryos are included as controls. (B) Quantification of the level of phosphorylated Smad1 and Smad2 in the above experiment. (C) Effects of decreasing concentration of Smad7 on Sox3, Sox2 and Chordin induction after injection into the A4 blastomere. 2 ng: Sox3 92%, Sox2 81%, Chordin 100%; 1–0.5 ng: Sox3 89%, Sox2 80%, Chordin 100%; 100 pg: Sox3 66%, Sox2 36%, Chordin 91%; 50 pg: Sox3 33%, Sox2 14%, Chordin 88%; 10 pg: Sox3 0%, Sox2 0%, Chordin 72%. (D) The induction of Sox3 is not cell-autonomous to the progeny of the Smad7-injected A4 blastomere. Embryos were injected with either Rldx or Smad7+Fldx in the A4 blastomere at the 32-cell stage, and isotopic transplants performed at stage 8, replacing Rldx-labelled cells with the progeny of Smad7(2 ng)+Fldx-injected cells. Embryos were grown to stage 18 and stained for Sox3 and Fldx. (E) Section through the transplanted area showing expression of Sox3 in uninjected cells of the host (8/8).
	Fig. 4. Smad7 induces dorsal mesodermal and neural markers in Xenopus ventral epidermis at gastrula stages. Injection of Smad7 (2 ng) into the A4 blastomere induces Brachyury (A–B), Chordin (C–D), Sox3 (E–F) and Sox2 (G–H). A, C, E and G are shown in vegetal view, B, D, F and H in animal view.
	Fig. 5. Inhibition of TGFβ signalling by Smad7 has different consequences from its inhibition by Smad6 (or δSmad7) with CerS. Inhibition of the BMP and the Nodal/Activin pathway does not recapitulate the effect of Smad7 injection. Injection of Smad6+CerS into A4 does not induce Sox3 (A–B) or Sox2 (C–D), but does induce Chordin (E–F). Injection of δSmad7+CerS into A4 does not induce Sox3 (G–H) or Sox2 (I–J), but does induce Chordin (K–L). Smad7+CerS injection induces Sox3 (M–N), Sox2 (O–P) and Chordin (Q–R).
	Fig. 6. The non-cell-autonomous effects of Smad7 are not mediated by the Wnt/β-catenin or the FGF pathways. When co-injected with the Wnt/β-catenin inhibitor GSK3, Smad7 still induces Sox3 (A–B), Sox2 (C–D) and Chordin (E–F). The same result is obtained when Smad7-injected embryos are grown in the presence of the FGF inhibitor SU5402: Sox3 (G), Sox2 (H) and Chordin (I) are still induced. Note that the concentration of SU5402 used is sufficient to block gastrulation and endogenous expression of these markers.
	Supplementary Figure S2. δSmad7 acts like Smad6. (A-D) Injection of δSmad7 (together with 10 pg of TEV) and immediate activation by Dexamethasone in one blastomere at the 2-cell stage expands the neural plate, as seen by Sox3 (A, B) and Sox2 (C, D) expression. (E) Effects of decreasing concentrations of δSmad7 mRNA on the expansion of Sox3 expression, in a similar experiment. +DEX, injected embryos activated by Dexamethasone; −DEX, non-activated controls. This experiment shows that at 10 pg δSmad7 the effect is maximal.

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