Fig. 3. Smad3 activity reduces expression of progenitor proteins and promotes neuronal differentiation. (A,B) Forced expression of Smad3 reduces expression of progenitor markers Id1 and Id2. (C,D) Forced expression of Smad3 induces neural differentiation markers, 12 hours after electroporation, transfected cells upregulate the expression of NeuroM (C) and Tuj1 (D). (E,F) Smad3-3S/D mutant version mimics the induction of neural differentiation markers. (G) Electroporation of Smad3 shRNA efficiently reduces Smad3 endogenous expression. (H-J) Endogenous Smad3 activity is required for neuronal differentiation. 12 hours after electroporation of Smad3 shRNA, expression of Id1 (H) and Id2 (I) are ectopically activated, and Tuj1 expression is reduced (J). (K-P) 24 hours after transfection of either Smad3 (K) or TßR-I (L) most cells have upregulated the expression of the cyclin-dependent kinase inhibitor p27^kip1 (M,N), and the pan-neuronal marker Tuj-1 (O,P). (Q) Quantitative analysis shows an increase in expression of p27^kip1 after Smad3 or TßR-I transfection compared with the non-electroporated control side. Histograms show data points as mean values ± s.d. (n>3 embryos, six sections). (R) 36 hours after transfection of Smad3 shRNA reduces the expression of neurogenic markers p27^kip1 and Tuj-1, compared with control embryo transfected with the empty pSUPER vector. Histograms show data points as mean values ± s.d. (n>3 embryos).