Fig. 5. Smad3 activity inhibits MN differentiation. HH14-16 embryos transfected with Smad3 were analyzed 48 hours after electroporation for the expression of p27^kip1, Tuj1 and MN markers. (A-D) At this stage, the total number of cells that exited the cell cycle in the non-electroporated control side and the electroporated side does not differ significantly (400 cells, n=6 embryos) (D). Immunohistochemical analysis of MN differentiation (MNR2+ cells) in embryos electroporated with pCIG empty vector (E), Smad3 (F) or Smad3-3S/D (G), and analyzed 24 hours (left panel) or 36 hours (right panel) after transfection. Overexpression of Smad3 decreases the number of MNR2+ cells from 56% at 24 h (F, left panel) to 90% at 36 h after transfection (F, right panel) compared with the pCIG empty vector. Forced expression of the Smad3-3S/D mutant version further reduces GFP+/MNR2+ cell numbers, 90% reduction at 24 h (G, left panel) to 96% at 36 h (G, right panel). (H) Quantitative data of transfected cells that co-express GFP/MNR2 within the domain of MN generation (n=6 embryos were assessed in each experiment). Histograms show data points as mean values ± s.d. ^**P<0.01; ^***P<0.001. (I-L) 48 hours after electroporation of Smad3, transfected cells (GFP+ cells) do not express Isl1 in a cell-autonomous way. (L) Quantitative data on Isl1-expressing cells, 48 hours after Smad3 electroporation. Isl1+ cells are reduced by 30% compared with the non-electroporated control side (n>6 embryos, at least four sections/embryo). Histograms show data points as mean values ± s.d. ^*P<0.05. (M) Diagrammatic representation of ventral progenitor domains and ventral neuronal subtypes, generated in a normal spinal cord and after Smad3 misexpression.