Shh induction of Hes1 requires Gli2. (a) Retinal explants were cultured from wild-type (Wt; n = 5), Gli1^−/− (n = 3), Gli2^−/− (n = 6), and Gli2^−/−Gli1^−/− (n = 3) mice with or without a Smo agonist at E18 for 3 d, then analyzed for Hes1 expression by RT-qPCR. Values represent fold mRNA induction in Smo agonist–treated explants relative to untreated explants. (b) RT-qPCR on acutely dissected retinas from E18 wild-type (n = 5) and Gli2^−/− (n = 5) animals. Values represent fold mRNA induction in Gli2^−/− retinas compared with the wild type. The black lines in the Western blot indicate that intervening lanes have been spliced out. (c) Western blot analysis of protein lysates from Myc-Gli2–transfected or control COS cells blotted with an anti-Gli2 antibody. The β-tubulin protein level was used as a loading control. (d) Schematic of the 10-kb region of the Hes1 promoter. The Gli2-binding sites are indicated with the mismatched nucleotides relative to the ideal Gli consensus sequence in small letters. ChIP reveals enrichment of Gli2 at sites −7,808 bp and −146 bp upstream of the transcription start site in the Hes1 promoter in retinal explants treated with a Smo agonist. No enrichment of Gli2 was detected at these sites in untreated retinal explants. Association of Gli2 at a region of the Hes1 promoter that does not contain a Gli consensus sequence was used as a negative control. (e) Western blot analysis for Gli2 on retinal explants treated with or without a Smo agonist (P0 + 3 DIV). The β-tubulin protein level was used as a loading control. (f and g) Retinal explants cultured with or without a Smo agonist for 3 DIV and subjected to ISH for Gli2. Bars, 100 μm. (h) Retinal explants (P0) were cultured with (n = 5) or without a Smo agonist (n = 4) for 6 h and analyzed for Hes1 and Gli1 expression by RT-qPCR. Values represent fold mRNA induction in Smo agonist–treated explants relative to untreated explants. Error bars represent SEM. *, P < 0.05; **, P < 0.001.