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Fig. 2. Precursor proliferation and neurosphere-forming cells in Shh null brains. (A) Morphology of wild-type (left, dorsal view) and Shh^-/- (right, side view) E18.5 dissected brains. Anterior is to the top. (B-E) Comparison of wild-type (B,D) and Shh^-/- (C,E) cortical nsp, obtained at E15.5 (B,C) or E18.5 (D,E). (F,G) BrdU incorporation assay on E18.5 attached nsps. (H-K) Differentiation of Nestin⁺ Shh^-/- nsps (H) into Tuj1⁺ neurons (I), GFAP⁺ astrocytes (J) and O4⁺ oligodendrocytes (K). (L) RT-PCR analyses of E18.5 wild-type and Shh null nsp cultures. (M) Quantification of wild-type versus Shh^-/- E15.5 and E18.5 nsp size in clonal dilution assays (E15.5 wild type, n=12; Shh null, n=11; E18.5 wild type, n=11; Shh null, n=14; P<0.001 for E15.5; P<0.05 for E18.5). (N) Quantification of the number of BrdU⁺ cells (after 1 week in culture for both E15.5 and E18.5) after a 7 hour pulse in wild-type versus Shh^-/- nsp (E15.5, P=0.006; E18.5, P<0.001). (O) Quantification of E15.5 and E18.5 wild-type versus Shh^-/- nsp number (P<0.001 for both). Scale bar in E: 620 µm for A; 90 µm for B,C,E; 70 µm for D; 45 µm for F,G; 15 µm for H-K.

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