Misu Regulates Stem Cell Fate Figure 6. Deletion of Misu increases dormancy of bulge stem cell. (A,B) Whole mount staining of wild-type and Misu 2/2 tail skin for LRC (green), keratin 14 (K14) (red) and DAPI (blue) after 4 months chase period. (C) Automated quantification of LRC in tail outer hair follicles in (A,B). (D) Frequency distributions of the intensity of the BrdU-label in LRC in (A,B). Error bars represent SEM (n = 10) from at least 3 independent experiments. (E,F) Flow cytometry for Itga6 and CD34 in epidermis in telogen (P49) (E) and catagen (P40) (F). The percentage of double positive cells in each group is shown 6 SEM (n = 3). The red lines in (E) indicate a cell population that increases in Misu 2/2 skin in telogen. (G,H) Epidermal cells sorted for CD34+ve and low (L) and high (H) levels of Itga6 were subjected to QPCR for bulge and hair germ markers as indicated (G). RNA levels were measured relative to GAPDH and presented as fold enrichment in Misu 2/2 mice versus wild-type controls. Error bars represent SEM (n = 3). (I) BrdU labelling and chasing regime to measure migration of LCR from bulge to hair germ at telogen at P47 (arrow). (J,K) Detection of LRC (green), Ki67 (red) and DAPI (blue) in high and low part of the bulge (straight line) and hair germ (HG) in hair follicles, indicated by dotted line, from wild-type (wt) (J) and Misu 2/2 mice (K). (L) Automated quantification of LRC in the whole hair follicle (total), high and low bulge region and the hair germ. Error bars indicate SEM (n = 5). (M) Percentages of hair follicles without LRC in the lower bulge region. Scale bars: 250 mm (A,B); 50 mm (J,K). doi:10.1371/journal.pgen.1002403.g006 Deletion of Misu delays exit of stem cells from the bulge In vivo lineage tracing experiments suggest that a sub-population of bulge cells migrate into the hair germ in telogen to then undergo cell division at the onset of anagen [4,28]. We speculated that stem cells in the bulge of Misu 2/2 mice might fail to commit in the bulge and do not migrate into the hair germ. To determine whether lack of Misu affected stem cell migration into the hair germ, we labelled Misu 2/2 new born mice and control littermates with BrdU at late morphogenesis and chased them for one hair cycle until the second postnatal telogen (P47) We then detected quiescent bulge stem cells using an antibody to BrdU and compared the number and location of label-retaining cells (LRC) in the upper (high) and lower bulge region and the hair germ (Figure 6I–6M; Figure S9A–S9F) [4]. Wild-type and Misu 2/2 epidermal cells incorporated BrdU at the same rates (Figure S9A, S9B). After the chase period at P47, we found a significant higher number of fully labelled LRC in the high bulge area of Misu 2/2 epidermis (Figure 6J–6L). PLoS Genetics | www.plosgenetics.org 7 December 2011 | Volume 7 | Issue 12 | e1002403