Shh induces tectal NSC proliferation.

<p>Nsp suspension cultures were immobilized in collagen type-I gels. Proliferation experiments were performed for 8 hours and included a BrdU (1 µg/ml) pulse for the last 2 hours in the presence of EGF and FGF-2 (used at 1 ng/ml or 10 ng/ml); combined with either Shh (3,3 µg/ml) or Cyc (5 or 10 µM). (<b>A</b>) Representative images of double immunostaining for BrdU and the RGC marker Blbp after indicated treatments. Bar, 20 µm. (<b>B</b>) Quantification of the percentage of positive cells for Blbp over total numbers of cells, in Z-stacks of confocal microscope. Anti-Shh antibody 5E1 affects both endogenous Shh source as well as recombinant Shh effect resulting in less number of Blbp positive cells. (<b>C</b>) Quantification of the effect of endogenous Shh on the number of Blbp positive cells shows significant inhibition after Cyc treatment. (<b>D</b>) Histogram showing proliferation of nsps after exposures to treatments as indicated. The anti-Shh antibody 5E1 reduces significantly number of BrdU positive cells in the Blbp positive cell population. (<b>E</b>) Quantification of positive cells for BrdU over Blbp positive cells in Z-stacks of confocal microscope treated with Cyc. *, p<0.05; **, p<0.01; ***, p<0.0001. W/O GF: without growth factors, E: EGF, F: FGF-2.