Misu Regulates Stem Cell Fate Figure 4. Misu is expressed in bulge stem cells. (A,B) Co-localisation of LacZ and keratin 15 (K15) (A) and P-cadherin (PCad) (B) in Misu +/2 hair follicles. Arrows in (A) indicate K15/LacZ double positive cells. (C–E) Expression of Misu protein (green) in bulge (arrows) and hair germ (HG; arrowheads) at telogen (C), onset of anagen (D) and anagen (E). Bulge cells are labelled for K15 (red) and nuclei are counter stained with DAPI (blue) (C–E). (F–K) Percentage of FDG+ve sorted epidermal cells in the bulge (Itga6high/CD34+ve) (F–H) and the hair germ (Itga6low/P-cadherinhigh) (I–K) at P21 in Misu +/2 mice. (F,I) Cells were first gated by side scatter (SSLin) versus high expression of CD34 (F) or P-cadherin (I). The cells were further gated for Itga6high/FDG+ve (G,H) and Itga6low/FDG+ve (J,K). The percentage of Itga6high/CD34+ve/FDG+ve and Itga6low/P-cadherinhigh/FDG+ve cells is marked by the red squares (G,H,J,K). Wild-type cells were negative for FDG (G,J). Errors represent SEM (n = 3). (L–P) QPCR using FDG+ve and FDG2ve epidermal cells sorted from Misu +/2 mice at P21 as indicated in Figure S6 for Misu (L), CD34 (M), NFATc1 (N), Lgr5 (O), and Lef1 (P). Error bars represent SEM (n = 3). Scale bars: 50 mm (A–E). doi:10.1371/journal.pgen.1002403.g004 To further confirm co-expression of Misu with stem cells markers, we isolated FDG+ve and FDG2ve keratinocytes from Misu +/2 mice at P21 (Figure S5A, S5B). QPCR analysis demonstrated that the stem cell markers CD34, NFATc1, and Lgr5 were enriched in Misu-expressing cells (FDG+ve) (Figure 4L–4O). Both populations expressed Itga6 at similar levels (Figure S5C) but FDG+ve cells were also enriched for the hair germ markers Lef1, Wnt5a, and Sox6 (Figure 4P; Figure S5D, S5E). Expression of differentiation markers was comparable or decreased compared to FDG2ve cells (Figure S5F, S5G). We concluded that Misu was expressed in both bulge stem cells and cells of the hair germ at initiation of anagen. Lack of Misu delays cell cycle entry of bulge stem cells at telogen–anagen transition To test whether Misu might induce bulge stem cells to enter cell cycle at the transition of telogen to anagen, we FACS-sorted bulge stem cells, based on high expression of CD34 and Itga6 (Figure S6A) and early progenitor cells of the hair germ, based on high expression of P-cadherin and low expression of Itga6 (Figure S6B) at the onset of anagen at P21 in wild-type and Misu 2/2 mice. We then determined the cell cycle profile for all sorted populations (Figure 5A–5I). We did not observe any difference in cell cycle profile when the whole epidermal population was analyzed, and as expected most of the cells were in G1 (Figure 5B, 5C). However, the percentage of Misu 2/2 bulge stem cells (Itga6high/CD34+ve) PLoS Genetics | www.plosgenetics.org 5 December 2011 | Volume 7 | Issue 12 | e1002403