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Fig. S1. Smad3 activity represses expression of HD and bHLH progenitor proteins. To assess the effect of Smad3 activity on neural tube patterning, we misexpressed Smad3 and examined the resulting pattern of class II (Nkx6.1 and Olig2) and class I (Dbx1, Irx3, Pax6 and Pax7) progenitor proteins. (A,B) Ectopic expression of Smad3 is sufficient to efficiently repress Nkx6.1 (A) and Olig2 (B) from their endogenous ventral domains. (C) Repression of Olig2 expression from pMN progenitors is cell-autonomous. Since cross-repressive interactions between Olig2/Irx3, Nkx6.1/Dbx2 and Nkx6.2/Dbx1 contribute to sharpening pMN/p2, p2/p1 and p1/p0 domain boundaries, respectively, we next tested whether Smad3-mediated repression of pMN progenitor proteins was dependent on ectopic activation of their class I counterparts. (D,E) Forced expression of Smad3 does not expand the ventral boundaries of Dbx2 (D) or Irx3 (E), indicating that Smad3 repression of pMN determinants is not dependent on derepression of their counterpart class I genes. (F) Smad3-mediated repression of progenitor proteins does not result from increased apoptosis, as assessed by caspase3 immunostaining. (G-J) Overexpression of Smad3, monitored by GFP expression (green), causes cell-autonomous repression of Pax6 (J) and Pax7 (H) class I progenitor proteins, together with a moderate ventral expansion of Pax6 expression in the pMN domain (G,I) and dorsal expansion of Nkx2.2 in the pMN domain (I).
Fig. S2. Smad3 pseudo-phosphorylated and non-phosphorylable mutant versions activate and repress expression of a TGFβ-responsive luciferase reporter gene in vivo, respectively. (A) Schematic representation of the p3TP-Lux reporter construct. 3TP promoter contains three consecutive TPA (12-O-tetradecanoyl phorbol 12-myristete 13-acetate) response elements (TREs) and a portion of the plasminogen activator inhibitor-1 (PAI-1) promoter region. Constructs used for the transcriptional assay: wild-type Smad3, Smad3-3S/D and Smad3-3S/A mutant versions. Diagram shows MH1 (red) and MH2 (purple) domains separated by the linker region (light grey). Black box represents the phosphorylation motif at the C-terminal end of the protein; amino acid changes in phosphorylation-site mutants are indicated. (B) Transcriptional activity of the wild-type Smad3 and the pseudo-phosphorylated Smad3-3S/D mutant was tested by in ovo electroporation. We found a higher transcriptional activity for the Smad3-3S/D mutant version (∼40 fold increase in 3TP-Lux activity) compared with the wild-type Smad3, which in turn induced a ∼10 fold increase compared with the control pCIG transfection in the absence of ectopic activation of the TGFβ pathway (-TβR-I condition). TβR-I turns on TGFβ response pathways through its constitutively activated kinase activity, bypassing the need for ligand binding to receptor II. The Smad3-3S/A mutant abrogates transactivation of the 3TP-Lux reporter gene under these conditions (+TβR-I). Data are normalized to renilla-luciferase as a control for transfection efficiency. Luciferase activity is expressed as relative luminometer units (RLU) and shown as the mean ± s.d. (n=8-10 embryos were assessed in each group of experiments).**P<0.01, ***P<0.001; using the one-tailed Student’s t-test.
Fig. S3. Smad4/Smad3 promotes neuronal differentiation. Smad4 is a common cofactor for the transcriptional activity of all phospho-Smad proteins. We co-transfected HA-Smad4 together with Smad3 and analyzed changes in the expression of the neuronal marker Tuj1. (A-C) 6 hours after co-electroporation, a high proportion of cell expressed both DNAs. (D-F) Smad4 alone does not increase Tuj1 expression. Co-electroporation of Smad4 with Smad3 (G-I) or with Smad3-3S/D (J-L) increases Tuj-1 expression.
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