Misu Regulates Stem Cell Fate Figure 5. Misu delays cell cycle entry of bulge stem cells at the onset of anagen. (A) Schematic representation of the first postnatal hair cycle after morphogenesis (M) including catagen (C), telogen (T) and anagen (A). Postnatal days (PD) are indicated on the right. Red marks the transition of telogen to anagen at P21. (B–I) Cell cycle profiles (B,D,F,H) and quantification (C,E,G,I) of whole epidermal preparations (B,C), Itga6high/ CD34+ve bulge stem cells (D,E), Itga6low/P-cadherinhigh hair germ cells (F,G) and Itga6high/CD34+ve bulge stem cells at P24 (H,I) using a minimum of three wild-type and three Misu 2/2 mice. Error bars represent SEM. doi:10.1371/journal.pgen.1002403.g005 in S- and G2/M-phase of the cell cycle was significantly reduced compared to wild-type cells at anagen initiation (Figure 5D, 5E). In contrast, the cell cycle profile of progenitor cells (Itga6low/P- cadherinhigh) in Misu 2/2 and wild-type epidermis was comparable (Figure 5F, 5G). At later stages, in anagen at P24, the percentage of Misu 2/2 bulge stem cell population dividing was comparable to that of wild-type controls (Figure 5H, 5I; Figure S6C). These data indicated that Misu was important to stimulate cell cycle entry of bulge stem cells at initiation of anagen and depletion of Misu might increase the quiescent phase of bulge stem cells. Deletion of Misu extends quiescence of bulge stem cells To test our hypothesis that lack of Misu resulted in increased number of quiescent bulge stem cells, we labelled Misu 2/2 and wild-type mice with BrdU, and detected label-retaining cells (LRC) after a chase period of four months (Figure 6A–6D). A long chase period allows detecting differences not only in the number of LRC but also in the intensity of the BrdU-label, which correlates with number of divisions. The number of LRC in Misu-depleted tail hair follicles was significantly increased in the outer follicles of triplets (Figure 6A–6C), which are known to go through the hair cycle concurrently, whereas the central follicle cycles asynchronously and usually has fewer LRC (data not shown) [25,26,27]. Additionally, the intensity of the BrdU-label was higher in Misu 2/2 skin suggesting that those cells divided slower than in their wt controls (Figure 6D). Flow cytometry for CD34 and Itga6 in anagen (P30), catagen (P40) and telogen (P49) confirmed that loss of Misu resulted in a two-fold increase of bulge stem cells only in telogen of the hair cycle (Figure 6E, 6F; Figure S7A). The number of hair germ cells in telogen (P49) was unchanged (Figure S7B). Strikingly, we found that the increase of bulge stem cells in Misu 2/2 epidermis was due to an enrichment of a distinct cell population with lower Itga6 expression compared to wild-type skin (Figure 6E; red line), which might represent those bulge cells that failed to enter the cell cycle at the initiation of anagen (Figure 5D). If our hypothesis was correct CD34+ve but Itga6low cells from Misu 2/2 mice should be more quiescent and therefore express higher levels of bulge stem cell markers than the comparable wild-type population. To test our hypothesis, we sorted bulge stem cells into two populations: L (CD34+ve/Itga6low) and H (CD34+ve/Itga6high) (Figure 6G). We then analysed both populations for expression of bulge markers in wild-type and Misu 2/2 epidermis (Figure 6H). We found that population L obtained from Misu 2/2 mice showed consistently higher expression of the stem cell markers FGF-18, Itga6, Sox9, CD34 and NFATc1 relative to wild-type mice (Figure 6H; Figure S8E). Importantly, population L did not show increased expression of the hair germ markers Wnt5a and Sox6, or Lgr5, a marker for cycling stem cells (Figure 6H). In contrast, population H from Misu 2/2 mice expressed similar levels of stem cell markers than wild-type mice, but showed an increase in the expression of hair germ markers. Thus, our data indicated that depletion of Misu resulted in an accumulation of quiescent CD34+ve/Itga6low stem cells in the bulge. To exclude the possibility that the increase in the quiescent stem cell population in Misu 2/2 mice was due to the general lack of Misu rather than being tissue-autonomous to skin, we generated a conditional knockout mouse model for Misu in the epidermis (Methods) (Figure S8A–S8C). Like in Misu 2/2 animals, also in mice with conditionally deleted Misu in the basal, undifferentiated layers of the epidermis (K14MisuD/D), a distinct cell population of stem cells with lower Itga6 expression accumulated in the bulge (Figure S8D; red line in right hand panel) and expressed higher levels of stem cell markers than the Cre-negative Misuf/f controls (Figure S8E). PLoS Genetics | www.plosgenetics.org 6 December 2011 | Volume 7 | Issue 12 | e1002403