Fig. 4. Neurosphere cultures from Gli2 null brains. (A-D) Phase contrast images of representative cortical nsps cultured from wild-type (A,B) and Gli2^-/- (C,D) animals at E18.5. (E) RT-PCR analyses of cortical nsps. Note the loss of Gli1 expression, the shift in the Gli2 mutant allele band (arrows), the reduced Gli3 expression, and the induction of Ihh and to a lesser extent Dhh in Gli2^-/- cells. Hprt is shown as a control. (F-I) Expression of nestin in precursors of Gli2^-/- nsps (F), of TuJ1 in neurons (G), of GFAP in astrocytes (H) and of O4 in oligodendrocytes (I) after differentiation. Nuclei were counterstained with DAPI. (J-M) Quantification of nsp size at E15.5 (J) and E18.5 (K). An average of 20 nsps from two independent experiments is shown (E15.5, P<0.05; E18.5, P<0.001). Given their rarity at E18.5, single Gli2^-/- nsps were measured. (L,M) Quantification of nsps obtained in cloning assays. Three independent experiments are shown for E15.5 (P<0.001) and one for E18.5. Scale bar: 75 µm for A-D; 10 µm for F-I.