Misu Regulates Stem Cell Fate Figure 3. Misu is a down-stream target gene of the Lef1/b-Catenin complex in adult skin. (A–B) QPCR for c-Myc (A) and Lef1 (B) using total RNA isolated from back skin of wild-type mice during hair morphogenesis and the first hair cycle after birth. M; morphogenesis, C; catagen, T; telogen and A; anagen. The grey box marks anagen. Error bars represent SEM (n = 3). (C–E) Localisation of Lef1 protein (red) in the bulge (B, arrowheads) and hair germ (HG) at telogen (C), onset of anagen (D) and anagen (E). (F,G) Co-localisation of Misu (LacZ) and Lef1 (brown) in the lower bulge (B, arrowheads) and hair germ (HG) at the onset of anagen (F) and in the matrix later in anagen (G). DP indicates the dermal papilla. (H) Schematic overview of the Misu promoter indicating the putative Lef1-binding site, the TATA box and the transcriptional start site (TSS) for Misu. The red line indicates the PCR fragment amplified after ChIP. (I) Enrichment of promoters of Misu and as positive control Msx2 by ChIP using an antibody for Lef1. IgG and Gapdh served as negative controls. (J) Luciferase reporter assays using 2 kb of the mouse proximal promoter of Misu in Hela cells in the absence and presence of b-Catenin, Lef1 or DN63-Lef1. pLef7-fos-luc synthetic promoter was used as a positive control for Lef1/b-Catenin activity. Error bars represent SD (n = 3). (K) RNA levels of Misu in wild-type and K14-DLef1 skin. Nuclei are counterstained with DAPI (blue) (C–E). Scale bars: 20 mm (C–E). Scale bar: 25 mm (F,G). doi:10.1371/journal.pgen.1002403.g003 Misu is expressed in bulge stem cells and the hair germ at the initiation of anagen The complete lack of expression of Misu in adult skin in both the IFE and the bulge in either the catagen or telogen phase of the hair cycle excludes its expression in quiescent stem cells. However, Misu-expression, detected by LacZ staining, was up-regulated in the bulge region (arrows) and the hair germ (HG) as early as telogen to anagen transition (Figure 4A, 4B; Figure S3A, S3B). We confirmed expression of Misu protein in the bulge (arrows) and the hair germ (arrowheads) in anagen by immunoflourescence using an antibody against mouse Misu (Figure 4C–4E; Figure S3C). We next asked whether Misu-positive cells in the bulge were indeed stem cells. Hair follicle stem cells can be isolated by fluorescence activated cell sorting (FACS), based on high expression of CD34 and a6 integrin (Itga6) (Figure 4F–4H) [24]. Progenitor cells of the hair germ are characterized by high expression of P-cadherin and low expression of Itga6 (Figure 4I– 4K; Figure S4A–S4C) [5]. Both, stem and progenitor popula- tions were sorted from Misu +/2 mice at the onset of anagen (P21) and tested for expression of Misu based on b-galactosidase activity (LacZ) using fluorescein di-galactoside (FDG) (Figure 4F–4K). Around 12% of bulge stem cells (Itga6high/ CD34+ve) and 17% of progenitor cells in the hair germ (Itga6low/P-cadherinhigh) expressed Misu (Figure 4F–4K). No signal for FDG was detected in keratinocytes from wild-type mice (Figure 4G, 4J). PLoS Genetics | www.plosgenetics.org 4 December 2011 | Volume 7 | Issue 12 | e1002403