Wnt and Hedgehog signalings affect Hes4 expression in an opposite manner. (A–E):In situ hybridization analysis of Hes4 expression on retinal sections, following a 24-hour (from stage 25 to stage 35; A, C–E) or a 15-hour (from stage 25 to stage 32; B, C) treatment with the indicated drug. Shown beneath each retinal section is a higher magnification of the dorsal CMZ delineated with dotted lines. (C, E): Quantification of Hes4 staining area in the dorsal CMZ in each condition. (F): Quantitative polymerase chain reaction analysis of retinal Hes4 expression at stage 35 following 24 hours of Hedgehog or Wnt signaling inhibition. Ptc1 and CyclinD1 serve as controls of drug efficiency. Scale bar = 50 μm. Abbreviations: BIO, 6-bromoindirubin-3′-oxime; CMZ, ciliary marginal zone; CR, central retina; Cyclo, cyclopamine; IWR1, inhibitor of wnt response 1; L, lens; N, number of analyzed sections; Purm, purmorphamine.