Misu Regulates Stem Cell Fate Figure 2. Expression of Misu during embryogenesis, skin morphogenesis, and in adult epidermis. (A–F) LacZ staining (blue) in wild-type (wt) and Misu (+/2) embryos at E3.5 (A), E6.5 (B,B9), E9.5 (C), E13.5 (D) and E14.5 (E,F). HB: hindbrain; FB: forebrain; Br: branchial arch; H: heart; LB: limbs; YS: yolk sac. (B9) shows a section from the embryo whole mount in (B). (D,E) Arrows show LacZ staining in whiskers. Arrowheads indicate Misu expression in the eye. (G–I) LacZ staining of Misu (+/2) interfollicular epidermis at E15.5 (G), E16.5 (H) and E18.5 (I). (J,K) Misu-expression in the interfollicular epidermis after birth at P1 (J), P4 (K). (L,M) LacZ staining of hair follicles at P1 (L) and P4 (M). Dotted lines delineate the basement membrane in (J,K) and hair follicles in (L,M). Arrows in (J,K) indicate suprabasal layers of the interfollicular epidermis. Arrowhead in (M) indicate Misu- expression in the bulb of growing hair follicles. (N–R) Expression of Misu in adult skin is absent in telogen (N), low expressed in the bulge (Bu; arrows) and hair germ (HG; arrowhead) at early anagen (O) and high expressed in anagen hair follicles (P,Q). (R) mRNA levels of endogenous Misu during morphogenesis (M), catagen (C), telogen (T) and anagen (A). The grey box indicates anagen. Error bars indicate SEM (n = 2). Sections are counterstained with Eosin in (G–I, L, N–Q). Scale bars: 50 mm (G–Q). doi:10.1371/journal.pgen.1002403.g002 sebaceous glands [7]. Since a role for c-Myc in regulating bulge stem cells has yet not been identified and c-Myc expression levels remained unchanged during the hair cycle (Figure 3A), we asked whether expression of Misu might also be regulated by hair- specific transcription factors. We examined the mouse Misu proximal promoter and detected a putative Lef1-binding motif (Figure 3H). As described for Misu, also expression of Lef1 increased when hair follicles entered anagen (Figure 2R; Figure 3B, 3C) and expression of Misu and Lef1 overlapped in hair follicles at both early and late stages of anagen (Figure 3F, 3G). To validate Misu as a target gene of Lef1, we performed chromatin immunoprecipitation (ChIP) in mouse epidermis in anagen using an antibody for Lef1 (Figure 3I). We detected Lef1- binding to the promoters of Misu and Msx2, a known target gene of Lef1 [23]. We further confirmed Misu as a transcriptional target of the Lef1/b-catenin complex by luciferase assays using the Misu promoter (pMisu) and full-length Lef1 or DN63Lef1 (DLef1), a mutant construct lacking the b-catenin binding motif. The assays were performed in presence or as an additional negative control absence of b-Catenin (Figure 3J). The Lef7 synthetic promoter (pLef7) served as positive control (Figure 3J). Luciferase activity using the Misu promoter increased around two-fold when the Lef1/b-catenin complex was present com- pared to the controls (Figure 3J). Finally, we confirmed in vivo that Misu RNA levels decreased when DLef1 was over-expressed in the mouse epidermis (K14DLef1) (Figure 3K). We concluded that expression of Misu is up-regulated by Lef1 when hair follicles enter anagen. PLoS Genetics | www.plosgenetics.org 3 December 2011 | Volume 7 | Issue 12 | e1002403