Many adult regenerative cells divide infrequently but have high proliferative capacity. We developed a strategy to fluorescently label slow-cycling cells in a cell type-specific fashion. We used this method to purify the label-retaining cells (LRCs) that mark the skin stem cell (SC) niche. We found that these cells rarely divide within their niche but change properties abruptly when stimulated to exit. We determined their transcriptional profile, which, when compared to progeny and other SCs, defines the niche. Many of the >100 messenger RNAs preferentially expressed in the niche encode surface receptors and secreted proteins, enabling LRCs to signal and respond to their environment.Epidermis and its appendages undergo continuous renewal and maintain reservoirs of multipotent SCs whose descendants are organized spatially and temporally. The epidermal basal layer (BL) contains putative SCs in addition to the transiently amplifying (TA) cells, which give rise to terminally differentiating suprabasal layers (1-3). The BL and the hair follicle outer root sheath (ORS) are contiguous and biochemically similar ( fig. S1A). In the hair bulb, the dermal papilla (DP) maintains contact with matrix TA cells until they differentiate to form the inner root sheath (IRS) and hair shaft. Follicles periodically undergo cycles of growth (anagen), destruction (catagen), and rest (telogen). The zone between noncycling and cycling segments is a SC niche, the ORS "bulge" (4,5).Multipotent epithelial SCs with high proliferative potential reside in the bulge (6,7). The bulge contains the majority of infrequently cycling, label-retaining cells (LRCs), which can respond to anagen DP signals to regenerate the follicle. After wounding or transplantation, bulge cells give rise to epidermis, follicles, and sebaceous glands. Additionally, when dissected from rat whiskers and cultured, bulge cells yield more colonies than other follicle segments (7). It is not known what features define this specialized SC niche, what its interactions with bulgeLRCs are, and whether all LRCs are SCs. To begin to address these issues, we devised a strategy based on the prediction that bulge SCs are uniquely both slow-cycling and active for a keratinocyte-specific promoter. With this strategy, we purify and characterize bulge LRCs and related keratinocyte progeny in the BL and ORS. Analyses of their transcriptional profiles reveal the skin LRC mRNAs; some of these mRNAs are found in SCs of other tissues, whereas others specify the unique environment of the skin SC niche.To mark infrequently cycling cells of adult skin epithelium, we engineered transgenic mice to express histone H2B-green fluorescent protein (GFP) (8) controlled by a tetracyclineresponsive regulatory element (TRE). A tightly regulated TRE-mCMV-H2B-GFP founder animal was crossed with mice harboring a keratin 5 (K5) promoter-driven tet repressor-VP16

SUMMARY Hair follicles (HFs) undergo cyclic bouts of degeneration, rest, and regeneration. During rest (telogen), the hair germ (HG) appears as a small cell cluster between the slow-cycling bulge and dermal papilla (DP). Here we show that HG cells are derived from bulge stem cells (SCs) but become responsive quicker to DP-promoting signals. In vitro, HG cells also proliferate sooner but display shorter-lived potential than bulge cells. Molecularly, they more closely resemble activated bulge rather than transit-amplifying (matrix) cells. Transcriptional profiling reveals precocious activity of both HG and DP in late telogen, accompanied by Wnt signaling in HG and elevated FGFs and BMP inhibitors in DP. FGFs and BMP inhibitors participate with Wnts in exerting selective and potent stimuli to the HG both in vivo and in vitro. Our findings suggest a model where HG cells fuel initial steps in hair regeneration, while the bulge is the engine maintaining the process.

SummaryStem cell niches in mammalian tissues are often heterogeneous and compartmentalized, however whether distinct niche locations determine different stem cell fates remains unclear. To test this hypothesis, we utilized the mouse hair follicle niche and devised a novel approach by combining intravital microscopy with genetic lineage tracing to re-visit the same stem cell lineages, from their exact place of origin, throughout regeneration in live mice. Using this method, we show directly that the position of a stem cell within the hair follicle niche can predict whether it is likely to remain uncommitted, generate precursors or commit to a differentiated fate. Furthermore, using laser ablation we demonstrate that hair follicle stem cells are dispensable for regeneration and that epithelial cells, which do not normally participate in hair growth, re-populate the lost stem cell compartment and sustain hair regeneration. This study provides a general paradigm for nicheinduced fate determination in adult tissues.