Fig. 4. Pax6^-Crx⁺ RPCs do not complete the photoreceptor-specification program. (A-I) The expression pattern of factors involved in photoreceptor differentiation; Crx (A,D,G), Otx2 (B,E,H) and Trβ2 (C,F,I) in control (A-C), Pax6^lacZ/lacZ (D-F) and Pax6^flox/flox;a-Cre;Z/AP (G-I) E15 eyes. (J) The region of Pax6 inactivation was determined by detection of human alkaline phosphatase (hAP) expressed from the Z/AP reporter (J is adjacent to G-I). (K) Chromatin immunoprecipitation (ChIP) was conducted on chromatin from E13 eyes with Pax6 or rabbit IgG (IgG). PCR amplification was carried out with specific primers for detection of the Crx promoter. The Crx 3' UTR sequence was amplified as a control that does not bind Pax6 in vivo. The same pairs of primers were used for amplification of the chromatin samples prior to immunoprecipitation (input lane). When ChIP was conducted on limb tissue, where Pax6 is not expressed, no amplification of Crx promoter sequences was detected. co, cornea; le, lens; oc, optic cup; ov, optic vesicle; prp, photoreceptor layer; rpe, retinal pigmented epithelium. Scale bar: 100 µm.