Fig. 6. Verification of array data and identification of Sp5 as a direct β-catenin target capable of suppressing epidermal differentiation gene expression. (A) qRT-PCR assays for the indicated transcripts in epidermal extracts from control and activated β-catenin mutant E15.5 embryos. Average relative expression levels, measured in triplicate assays of two independent pairs of experimental and control samples, were normalized to levels of actin transcripts. (B,C) Immunohistochemistry for hair keratin KRT82 (brown) demonstrates its upregulation in activated mutant epithelium at E16.5. (D,E) Similar patterns of Topgal (whole-mount X-gal staining, blue) and Sp5 (whole-mount in situ hybridization, purple) expression (arrows) in wild-type primary hair follicle placodes at E16.0. The less-sensitive in situ hybridization assay does not detect Sp5 expression in all secondary hair follicles at this stage. D,E were photographed at the same magnification. (F-I) Expanded Sp5 expression in KRT14-Cre Ctnnb1^(Ex3)fl/+ mutant embryos at E14.5 (F,G; yellow arrows) and E15.5 (H,I; yellow arrows), and its ectopic expression in mutant cornea (white arrow). (J) qRT-PCR shows decreased expression of Sp5 in epidermal extracts of KRT5-rtTA tetO-Dkk1 E14.5 embryos doxycycline treated from E0.5 compared with control littermates. (K,L) Absence of placodal Sp5 expression in Dkk1-expressing E15.5 embryos (L) compared with littermate controls (K). (M) ChIP assay for β-catenin binding to a region of the Sp5 promoter containing multiple conserved LEF/TCF-binding sites. Chromatin prepared from E15.5 control and KRT14-Cre Ctnnb1^(Ex3)fl/+ epidermis was incubated with anti-β-catenin antibody (β-cat Ab) or control IgG. Semi-quantitative PCR detects β-catenin binding to the Sp5 promoter in control extracts and increased binding in KRT14-Cre Ctnnb1^(Ex3)fl/+ extracts. β-catenin does not bind to Gapdh sequences (bottom panel). (N) qRT-PCR detects expression of transfected mouse Sp5, decreased levels of endogenous KRT10 and involucrin, and increased expression of endogenous DLX3 in Sp5-transfected HaCAT cells compared with empty vector-transfected controls. Average relative expression levels measured in triplicate assays of three independent transfection experiments are shown, normalized to levels of actin transcripts.