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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.
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