DEV022202 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Detection of Dlx3 mRNA expression by in situ hybridization in cross-sections of vibrissae (E16.5) and sections of neonatal skin (P1). In situ hybridizations were performed with antisense and sense (data not shown) probes according to Morasso et al. (Morasso et al., 1999).

• Supplemental Figure S2 -

  Fig. S2. Aberrant hair growth and cycling in Cre-mediated conditional Dlx3 knockout mice. (A) The deparaffinized sections of P5 and P18 were stained with Hematoxylin and Eosin. (B) K15 expression (red) was detected by immunohistochemistry on skin sections of P1, P5, P9, P12, P15, P18, P20 and P25 wild-type and K14Cre;Dlx3^{Kin/f or f/f} littermates using anti-K15 antibody. (C) Skin sections at P18 were also stained with anti-K1, anti-K10 and anti-adipophilin (APDH) antibodies. White arrowheads indicate abnormal K1 and K10 expression in mutant hfs. Scale bars: 100 µm in A,B; 50 µm in C.

• Supplemental Figure S3 -

  Fig. S3. In vitro and in vivo Dlx3 binding assays to hair keratin (K32, K35 and K37) and Hoxc13 promoters. (A) Western blot analysis was performed on protein extracts of hf cells at P10 using anti-Dlx3, anti-K35 and anti-tubulin. (B) Electrophoretic mobility shift assays (EMSA) using recombinant Dlx3 protein. The optimal Dlx3 binding sequence was used as a probe, whereas putative Dlx3 binding sites present in the promoter of human hair keratins (K32, K35 and K37) and mouse Hoxc13 were used as unlabeled competitors. The optimal (Con) and non-specific (Un) binding sequences were used as controls for binding specificity. (C) Sequence and localization of putative Dlx3 binding sites present in the promoter of human hair keratins (K32, K35 and K37) and mouse Hoxc13. Binding sites for Hoxc13 are from Jave-Suarez et al. (Jave-Suarez et al., 2002). +, binding; −, non-binding; +/−, weak binding; ++, strong binding. Circles indicate sequences used in ChIP assays (see Fig. 6C). (D) EMSA and supershift assays using anti-Dlx3 antibody. The number 2, 6 and 7 fragments of the K35 promoter were labeled and tested for ability to bind endogenous Dlx3 present in nuclear extracts of mouse hf cells. Arrow indicates supershifted complex.

• Supplemental Figure S4 -

  Fig. S4. Long-term epidermal defects in Cre-mediated conditional Dlx3 knockout mice. Skin samples were obtained from wild-type and mutant (K14Cre;Dlx3^f/f) littermates at 13 and 26 weeks, sectioned and stained with Hematoxylin and Eosin. Scale bar: 50 µm.