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First published online 30 November 2006
doi: 10.1242/dev.02702

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The TGFß intracellular effector Smad3 regulates neuronal differentiation and cell fate specification in the developing spinal cord

Instituto de Biología Molecular de Barcelona, CSIC, Parc Científic de Barcelona, C/Josep Samitier 1-5, Barcelona, Spain.

View larger version (118K): [in this window] [in a new window]   Fig. 1. Expression of TGFß2/Smad3 in the developing chick spinal cord. (A) At stage HH13, ActR-II is expressed throughout the DV axis. (B) TGFß2 is highly expressed in the notochord and ventral mesoderm at HH13, and weakly in the floor plate. (C) At stage HH18, TGFß2 is highly expressed in the notochord and the floor plate. (D) Expression of Smad3 in the opened neural plate of a stage 6 chick embryo. (E) Smad3 is expressed at rostral levels of the neural tube by stage 13, along the DV axis, excluding dorsal and ventral midline cells. (F,G) Smad3 expression at stage 18 predominates in two stripes encompassing dorsal and intermediate domains of the VZ. (F) A third ventral stripe is detected at brachial levels. (H) Summary of the pattern of Smad3 expression along the DV axis of the stage 18 chick neural tube. Panels on the right indicate progenitor domains. (I,J) From stage HH18-19, Smad3 is absent from Olig2-expressing pMN progenitors (I) and from postmitotic Isl1+ MNs (J). (K) Smad3 is co-expressed with Nkx2.2 within p3 progenitor domain. (L) Smad3 is absent from Foxa2-expressing floor plate cells. (M) Double-labelling Smad3 with anti-Lhx1/5 assigns Smad3-expressing domains to V1-dI6, dI4 and dI1-2. (N) Double-labelling Smad3 with anti-Pax2 assigns Smad3 expressing domains to V1-dI6 and dI4. (O) At stage 25, Smad3 expression is high at the lateral edge of the VZ at most levels of the DV axis except in the dorsal domain. Virtually all Smad3+ cells at the edge of the VZ co-express Pax2.

View larger version (91K): [in this window] [in a new window]   Fig. 2. Regionalized expression of Smad3 depends on patterning genes. (A,C,F,I,L) Comparative expression analysis of Smad3 and progenitor class I/II genes at rostral levels of the chick neural tube at stages 12-18. (B) Diagram showing the expression pattern of selected class I/II genes in ventral progenitor domains. (D,E) Misexpression of Nkx6.1 (D) leads to a decrease in Smad3 in a position-dependent manner (E). Smad3 is repressed from intermediate and dorsal domains, but not from the p3 domain, filled arrow in (E). (G,H) Misexpression of Olig2 (G) leads to a decrease in Smad3 (H), including the p3 domain, open arrow in (H). (J,K) Misexpression of Nkx2.2 (J) induces ectopic expression of Smad3 along the DV axis (K). (M,N) Misexpression of Irx3 (M) induces Smad3 expression within the pMN domain (N). (O) Summary of regulatory interactions between class I/II proteins that prevent the maintenance of Smad3 expression in the pMN domain.

View larger version (105K): [in this window] [in a new window]   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 p27kip1 (M,N), and the pan-neuronal marker Tuj-1 (O,P). (Q) Quantitative analysis shows an increase in expression of p27kip1 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 p27kip1 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).

View larger version (106K): [in this window] [in a new window]   Fig. 4. TGFß signaling through Smad3 promotes neurogenesis in the spinal cord. HH stage 14-16 embryos electroporated with pCIG-based vectors driving the expression of nuclear GFP as reported, were harvested 48 hours later. (A-D) Control embryos electroporated with the empty pCIG vector. (E-H) Electroporation of Smad3 causes mediolateral displacement of GFP-expressing cells by >55% (E), decreases BrdU incorporation by >80% (F), cell autonomously increases p27kip1 expression by >30% (G) and cell-autonomously induces Tuj1 by >65% (H) compared with the empty pCIG vector transfection. (I-L) Electroporation of TßR-I causes similar phenotype changes to Smad3 electroporation. (M-P) Electroporation of Smad3-3S/A reverts all aspects of the Smad3 phenotype; lateral GFP+ cells (M) are reduced by 40%, transfected cells incorporating BrdU (N) are increased four- to five-fold and transfected cells co-expressing p27kip1 (O) or Tuj1 (P) are reduced by more than 27% and 53% respectively. (Q-T) Percentages of transfected cells at lateral positions (Q), double labeled GFP/BrdU (R), co-expressing GFP/p27kip1 (S) and co-expressing GFP/Tuj1 (T). The medial (m) or lateral (l) position of transfected cells was defined by cell location in relation to p27kip1+ cells. Histograms show data points as mean values ± s.d. (n=6 embryos, >6 sections were assessed in each group of experiments) *P<0.05; **P<0.01; ***P<0.001.

View larger version (90K): [in this window] [in a new window]   Fig. 5. Smad3 activity inhibits MN differentiation. HH14-16 embryos transfected with Smad3 were analyzed 48 hours after electroporation for the expression of p27kip1, 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.

View larger version (95K): [in this window] [in a new window]   Fig. 6. Smad3 activity is both required and sufficient for the generation of ventral interneurons. (A-F) HH stage 14 embryos electroporated with Smad3 or TßR-I were analyzed 24 hours later for the expression of selective IN markers. Pax2+ (A-D) and Lhx1/5+ (E,F) were cell-autonomously induced in the electroporated side. (G) Quantitative analysis shows an increase in expression of Pax2+ and Lhx1/5+ cells after Smad3 or TßR-I transfection compared with the non-electroporated control side. (H) Quantitative analysis of p27kip1/Pax2 double labeled cells after electroporation of Smad3 shows a increased proportion of Pax2+cells. Histograms show data points as mean values ± s.d. (n=embryos, 6 sections). (I,J) 48 hours co-electroporation of pSUPER with TßR-I resulted in a dramatic increase in Pax2+ cells. (K,L) Co-electroporation of pSUPER-Smad3 shRNA with TßR-I resulted in the rescue of Pax2 ectopic expression. (M-R) HH stage 14-16 embryos electroporated with pSUPER-Smad3 shRNA were analyzed 48 hours later for the expression of selective LIM-HD factors. shRNA Smad3 and empty pSUPER vector were co-electroporated with pCIG (5:1) to use GFP as a reporter protein. 48 hours after electroporation (M,N) the expression of interneuron markers such as Pax2 (O,P) and Lhx1/5 (Q,R) is reduced. (S) Percentage of cells that express Pax2, and Lhx1/5 in control vs. electroporated sides. Pax2+ cells are reduced by 41.29%, Lhx1/5+ cells by 42.31% (n=5 embryos were assessed in each experiment). Histograms show data points as mean values ± s.e.m. Lhx1/5, P=0.00022; Pax2, P=0.000025; using the two-tailed Student's t-test.

View larger version (135K): [in this window] [in a new window]   Fig. 7. Smad3 activity promotes differentiation of V0-V2 and V3 interneurons at the expenses of MNs. HH stage 14-16 embryos electroporated with Smad3 were analyzed 48 hours later for the expression of selective LIM-HD factors. (A,C,E,G,I,K) Control embryos showing markers for specific neuronal subpopulations. Lhx3 expression identifies postmitotic pre-migratory MNs and V2 INs. (B,D,F,H,J,L) Forced expression of Smad3 results in the ectopic induction of V2 INs. Lhx3+; Isl1-V2 INs are dorsally and ventrally expanded (F). LMC, lateral MN column; MMC, medial MN column (E). Chx10-expressing V2 INs (I-L) are also dorsally and ventrally expanded. (M,O,Q,S,U)Control embryos showing markers for specific ventral IN populations. (N,P,R,T,V) Misexpression of Smad3 induces Pax2+, En1+ V1 and Evx1+ V0 INs. Ectopic expression of Pax2 (N), En1 (P) and Evx1 (R) is detected ventral to the p2/p1 domain boundary. (S-V) Misexpression of Smad3 induces few ectopic Nkx2.2+ cells (T) and a dorsal expansion of Sim1+ V3 INs (V). (W,X) Diagrammatic representation of ventral progenitor domains and ventral neuronal subtypes, generated in a normal spinal cord and after Smad3 misexpression.