Fig. 7. Forced activation of epithelial β-catenin increases BMP signaling and compensates for the absence of Eda. (A-D) Immunohistological detection of phosphorylated Smad1/5/8 in E14 control (A,C) and Catnb^ex3K14/+ (B,D) skin, indicating stimulation of BMP pathway activity in the mutant skin. (E-H) Whole-mount in situ hybridization analysis of E14 embryos showed stimulated expression of Bmp2 (E,F) and Bmp4 (G,H) in Catnb^ex3K14/+ skin when compared with control. High magnifications of mutant wholemounts revealed prominent epithelial evaginations with Bmp2 and Bmp4 expression in the tips (inserts in F and H). (I-L) Vibratome sections of wholemounts (E-H) indicated more intense expression of Bmp2 and Bmp4 in Catnb^ex3K14/+ skin compared with controls (I-L). Ectopic expression of Bmp4 had been induced in mutant placode epithelium (L). (M) Quantitative RT-PCR showed 1.3-fold upregulation of Bmp2 and Bmp4 in E14.5 Catnb^ex3K14/+ skin compared with controls. No difference was seen in Bmp7 expression. Data are represented as mean±s.d. (N-Q) Expression of Sostdc1 in E13 and E14.5 Eda-deficient mice (Eda^-/-) and Eda^-/-; Catnb^ex3K14/+ mice. Sostdc1 was absent from E13 and E14.5 Eda^-/- skin (N,P) owing to lack of primary hair placodes. When Eda^-/- mutants were crossed to Catnb^ex3K14/+ mice, placode formation was rescued and the placode phenotypes at E13 and E14.5 were indistinguishable from the Catnb^ex3K14/+ mutant phenotype (O,Q). Broken lines indicate epithelial-mesenchymal borders. Scale bars: 200 µm in A; 100 µm in C and inset in F,I; 2 mm in E,N. (R) A model of the interactions between Wnts and BMPs in hair placode formation, suggesting roles for BMPs as reaction-diffusion inhibitors of placode patterning. Placodal cells display Wnt activity. Wnts induce the expression of their feedback inhibitors Dkk1 and Dkk4, as well as of Bmp2 and Bmp4, which in turn downregulate Wnt activation at least through inhibition of Lef1. In interplacode region BMPs induce the expression of Sostdc1 which may act by suppressing Wnt activity in interplacodal cells. Black lines indicate transcriptional regulation, and red lines show protein-protein interaction. Green area, placode; yellow area, condensed mesenchyme; white area, interfollicular epithelium.