Fig. 2. SHH upregulates Gli1 transcription and promotes proliferation of vz cells in perinatal mouse neocortical explants. (A) RT-PCR analyses of gene expression in untreated control (con) or SHH-treated (+SHH) mouse E17.5 cerebral cortical explants and meninges (m). One-fifth the concentration of SHH (1 nM) induced a smaller increase in Gli1 expression (not shown). The expression of the housekeeping gene Hprt is used as internal quantitative control. Dorsal brain-derived SHH could affect adjacent cells of the meninges, which express Gli1. (B) RT-PCR analyses of gene expression in untreated control or SHH-treated mouse P3 cerebral cortical explants. At this time, the choroid plexus expresses low levels of both Gli1 and Shh (not shown). (C) Sketch of a lateral view of a 
P3 mouse brain showing the position of explanted tissue within the parietal neocortex. (D,E) In situ hybridization analyses on cryostat sections of P3 mouse cerebral cortex explants left untreated (D) or grown in the presence of SHH (E). Each panel shows a representative section. vs, ventricular surface. (F-J) Localization of BrdU-positive cells in the vz of a P3 mouse cortical explants left untreated (F), or treated with SHH (G), anti-SHH blocking monoclonal antibody (H), dideoxyforskolin (I) or forskolin (J). ps, pial surface; vs, ventricular surface. (K) Quantification of cell proliferation by SHH treatment or inhibition of SHH signaling in P3 mouse cortical explants. Numbers of cells±s.e.m. are given. n>10 sections of six independent explants counted for each condition. Ten independent explants were used for RT-PCR in two separate experiments, and four or five independent explants for in situ analyses.
