Moreover, the number of hair follicles without any LRC in the lower bulge or hair germ was three-fold higher in Misu 2/2 skin than wild-type littermates (Figure 6M). Accordingly, we also found that the intensity of the BrdU-label increased in the higher bulge and decreased in the lower bulge and hair germ when Misu was depleted (Figure S9C–S9F). Importantly, at this stage of the hair cycle the majority of bulge cells are yet not dividing, as shown by labelling for Ki67 (Figure 6J, 6K), excluding the possibility that Misu 2/2 cells diluted the label by cell divisions. Our data showed that the increased quiescence of bulge stem cells in Misu 2/2 skin correlated with an accumulation of LRC in the upper part of the bulge, indicating that Misu 2/2 bulge stem cells are delayed in generating committed progenitor cells of the hair germ. Deletion of Misu increases stem cell self-renewal and causes aberrant hair cycling To test whether Misu-deletion led to increased self-renewal capacity of stem cells in vitro, we measured the colony forming efficiency (CFE) of sorted Itga6high/CD34+ve bulge stem cells (Figure 7A). Out of all cell populations derived from skin, epidermal stem cells exhibit the highest CFE [29]. When seeded in clonal density, keratinocytes derived from Misu 2/2 mice formed more colonies than wild-type littermates (Figure 7A), indicating that Misu 2/2 cells have a higher self-renewal capacity than wild-type cells. Similarly, unsorted keratinocytes obtained from Misu 2/2 epidermis showed higher CFE than control keratinocytes (Figure 7B, 7C). Although expression of Misu was undetectable in adult IFE (Figure 2N), cultured keratinocytes obtained from back skin expressed high levels of Misu RNA and protein (Figure 7D; data not shown). Our data indicated so far, that loss of Misu increased accumulation of bulge stem cells in their niche at late telogen leading to an increased self-renewing but quiescent stem cell population. Therfore, we speculated that Misu 2/2 hair follicles should be delayed in entering anagen. Indeed, depletion of Misu led to a delay of entry into the first and second synchronized hair cycle in males (Figure 7E–7L; Figure S10B, S10C). Compared to males, females exhibit a delayed hair cycle progression of around 2 days, yet even in Misu 2/2 females the percentage of hair follicles in the first anagen at P25 was lower than in their wild-type controls (Figure S10A; Table S1). Similarly, male mice with conditionally deleted Misu in the epidermis (K14MisuD/D), displayed a delay in entering the first adult hair cycle compared to controls (Misuf/f) (Figure 7M; Figure S10F–S10K). Later entry into anagen in Misu-depleted skin resulted in delayed differentiation of matrix cells (Figure 7N–7U). The number of Lef1-postive cells, marking lineage committed hair follicle cells, was lower in matrix cells of Misu 2/2 mice (Figure 7N, 7O; arrows) [30]. Accordingly, expression of Dlx3, Gata3 and BMP signalling, as determined by staining for phosphorylated Smad1/5/8, were absent in Misu 2/2 anagen hair follicles (Figure 7P–7U; arrows). Once Misu 2/2 mice entered anagen, the hair follicles were morphologically indistin- guishable from those of wild-type mice (Figure S10F, S10E, S10L– S10S). Our data suggested that Misu plays a role in accurately timing lineage commitment of hair follicle progenitor cells. Discussion The RNA methyltransferase Misu balances self-renewal and differentiation Here we show through generating general and skin-specific loss- of-function mouse models that the RNA methyltransferase Misu Misu Regulates Stem Cell Fate (NSun2) defines expanding, committed progenitor populations in mammalian skin. Expression of Misu is absent in adult mouse interfollicular epidermis and the quiescent phases of the hair cycle (catagen and telogen). As the hair follicle enters its growing phase (anagen), Misu is expressed in the bulge and hair germ, both of which contain multipotent stem cells [3,31,32]. Cells in the hair germ give rise to transit amplifying (TA) cells in the hair matrix [4,5]. Matrix cells, which collectively show the highest expression of Misu of all cell types, subsequently differentiate into all hair lineages [3]. Uniquely at the transition of telogen to anagen, Misu is co- expressed with markers for both quiescent (CD34+ve/Itga6high) and cycling (Lgr5+ve) stem cells from the bulge and the hair germ respectively. However, in contrast to those stem cell populations, Misu- expressing cells exhibit reduced self-renewal capacity. Thus, although Misu could reversibly commit stem cells to differentiate [33], we clearly show that stem and committed progenitor cells co- exist within the bulge, the hair follicle stem cell niche. Our findings now raise the question of how only a few selected stem cells are activated during each hair cycle. Misu is required for cellular division of bulge cells only at the telogen-to-anagen transition, indicating that its function is temporarily and spatially controlled in a strict manner. One key pathway that drives epidermal stem cells from telogen into anagen and specifies hair follicle lineages is the canonical Wnt pathway. Activation of Wnt signaling by transient expression of N- terminally truncated b-catenin in the epidermis is sufficient to induce ectopic hair follicle formation [34,35,36,37,38]. Converse- ly, when the pathway is inhibited by b-catenin ablation or expressing of a DNLef1 mutant, hair follicle formation is impaired [30,39,40,41]. The identification of Misu as a direct downstream target of Lef1 further supports Misu as a key component during lineage commitment of bulge stem cells at the initiation of anagen. Thus, our data support a model in which stem and committed progenitors are distinct populations within the hair follicle that can be distinguished by their expression of Misu. 5 m C RNA methylation and commitment to differentiate Misu belongs to a large family of highly conserved methyltrans- ferases, modifying cytosine-5 in RNA (RNA:m5C-MTase) [16]. Misu/NSun2 is the human orthologue of S. cerevisiae Trm4, both of which have substrate specificity towards tRNA [12,13,19]. Like for human and yeast NSun2, we confirm pre-tRNALeu (CAA) as a direct target substrate for mouse Misu in vivo. Although post- transcriptional methylation of tRNA at cytosine-5 is one of the most frequently encountered modifications, Dnmt2 and Misu are as yet the only identified tRNA:m5C MTases [13,17,18,42]. Although it has been recently shown that m5C methylation protects tRNA from cleavage and degradation, the biological function this may mediate remains unclear [17,43]. Similar to the depletion of Misu in mice, loss of Dnmt2 in zebrafish also results in reduced body size and impaired differentiation of specific tissues [44]. It remains, however, unclear why the RNA-methyltransferase activity of both proteins is critical in maintaining tissue homeostasis [44]. One possible mechanism of Misu’s function is that methylated tRNA species could be directly involved in regulating stem cell differentiation. An alternative and intriguing possibility could be that methylation regulates the cleavage of tRNAs or intron-splincing of pre-tRNAs to generate products with microRNA-like features, which may offer an additional mechanism of post-transcriptional control [17,45]. Recent RNA deep-sequencing data identified a new set of small RNAs derived from tRNAs, including intron sequences, which are PLoS Genetics | www.plosgenetics.org 8 December 2011 | Volume 7 | Issue 12 | e1002403