β-catenin in the DP regulates Agouti and Corin expression inversely. (A) Real-time PCR analysis of whole-skin preparations from P1–10 compares the RNA levels of Agouti between wild type (WT) and mutant (Mut) (mean ± SD). In both genotypes, Agouti expression declines dramatically after the peak to stable levels, but these levels are 10-fold higher in the mutant. (B) In situ hybridization readily detects Agouti transcripts (blue) in the DP of wild-type and mutant P4 skins. Saturated signals are obtained after 1 d of detection. In both genotypes, follicles with black pigment and no detectable Agouti transcript can be identified (arrowheads). (C) In situ hybridization for Agouti in wild-type and mutant P8 skins. After 6 d of detection, Agouti transcripts are observed only in the mutant. Insets in the upper left corners show higher magnification of hair bulbs to illustrate the presence and absence of Agouti transcript in the DP of mutant and wild type, respectively. Rare follicles with black pigment deposition and lack of Agouti expression are observed in the mutant (Lower Left Inset). (D) Real-time PCR analysis monitors the RNA levels of Corin and Prss12 from P1–10 in wild-type and mutant mice (mean ± SD). (E and F) Immunostaining of Corin in P8 wild-type (E) and mutant (F) mice. The same follicle is shown in the left and right images with YFP (green) marking the DP at Left and Corin staining (red) at Right. Blue labels nuclei.

β-catenin activity in the DP positively regulates a novel pathway that promotes eumelanogenesis by an Agouti- and Corin-independent mechanism. (A) Real-time PCR analysis compares eumelanogenic gene expression between mice lacking β-catenin in the DP (Mut) to littermate controls (WT) on an Agouti (A/a; yellow lines) or nonagouti (a/a; black lines) background (mean ± SEM). For statistical analysis, see Fig. S4. (B) Eumelanogenic gene expression in nonagouti mice homozygous or heterozygous for a mutant allele of Corin between P7–10, a period when robust changes in eumelanogenic gene expression are observed in nonagouti mice lacking β-catenin in the DP.

β-Catenin ablation in the DP results in reduced melanocyte activity. (A) Confocal images of P8 wild-type (Upper) and mutant (Lower) follicle immunostained for Tyrosinase (Left) and Mitf (Center). (B) Melanocyte number (mean ± SD) was scored by counting Mitf+ cells per follicle per section in P8 mice. Three hundred follicles from 3 mice per genotype were analyzed. Rd, reduction in perentage relative to wild type. Two-tailed unpaired Student's t test was used (**P < 0.0001). (C) Three dorsal skin biopsies of 12.6 mm² along the anterior-posterior axis from 11 wild-type and 8 mutant P8 mice were obtained by using skin-biopsy punches of 2-mm radius to prepare and measure total RNA yield (mean ± SD). Two-tailed unpaired Student's t test was used (**P < 0.0001). (D) Absorbance at 500 nm (A₅₀₀) was measured for total melanin extracted from 1 mg of hair (mean ± SD). Hair coat was harvested at P20 after the first hair cycle from 9 mice per genotype. Two-tailed unpaired Student's t test was used (**P < 0.0001).