Stem-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, here we use the mouse hair follicle niche and combine 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 model for niche-induced fate determination in adult tissues.
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We are grateful to S. King, S. Guo and A. Horwich for critical feedback on the manuscript. We thank E. Fuchs for the K14-H2BGFP, Lef1-RFP and pTRE-H2BGFP mice, A. Glick for the K5-tTA mice and G. Gu for the K19-CreER mice. We thank D. Gonzalez and A. Haberman for technical support with intravital microscopy. P.R. is a New York Stem Cell Foundation–Druckenmiller Fellow. This work was supported by The New York Stem Cell Foundation and by grants to V.G. from the American Cancer Society (RGS-12-059-01-DCC) and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (1RO1AR063663-01).
The authors declare no competing financial interests.
Extended data figures and tables
a, Scheme of a mouse hair follicle in quiescence. Different cell populations reside in defined anatomical compartments. b, c, In homeostasis the hair follicle undergoes repeated cycles of regeneration. b, Hair growth is fuelled by stem cells in the niche that proliferate and differentiate to form the seven concentric layers of the mature hair shaft and inner root sheath (IRS), whereas a basal epithelial layer called the outer root sheath (ORS) surrounds the entire structure. Notice that the seven inner layers expand from the matrix, at the interphase with the mesenchymal dermal papilla, where they are generated, towards the surface of the skin, whereas the ORS has a different mode of growth and expands in the opposite direction. c, A complete hair cycle alternates between phases of rest (telogen), growth (anagen) and regression (catagen).
a, Single hair follicle stem-cell labelling is achieved using a combination of either K19-CreER/Rosa-stop-tdTomato or Lgr5-CreER /Rosa-stop-tdTomato alleles and administration of a single low dose of tamoxifen to achieve a low frequency of Cre-mediated loxP recombination and mosaic expression of the fluorescent tdTomato reporter within the stem-cell niche. b, The lineage of single labelled stem cells is traced in vivo during hair growth. c, Using two-photon laser scanning microscopy we can re-visit the same hair follicles, non-invasively in live mice at different stages of hair regeneration. Each panel depicts low (top) and high (bottom) magnification images of live hair follicles captured in first telogen, second anagen and second telogen, respectively.
a, Panels depicting the green (left) and red (middle) channel as well as a composite image (right) of a group of follicles in rest phase (telogen). K14-H2BGFP marks all the epithelial cells in the skin including the hair follicles. Lef1-RFP marks mesenchymal cells in the dermis including the dermal papilla at the bottom of the hair follicles. The tdTomato-Cre reporter (K19- or Lgr5-driven) displays mosaic expression in the stem-cell niche after administering a low dose of tamoxifen. Notice that the fluorescent intensity of the tdTomato is several-fold higher and easily distinguishable from RFP in the red channel. b, Individual channels and composite images of a group of follicles three (P23) and five (P25) days after administering a low dose of tamoxifen show a non-leaky expression of the Cre reporter (tdTomato) and a quiescent niche between these time points. Scale bar, 100 μm.
a, Single optical sections (top) or 3D volume renderings (bottom) of the outer (ORS; left) or inner (right) hair follicle cell layers as seen in vivo using a K14-driven H2B–GFP fluorescent reporter. b, Single optical sections showing cells marked with the tdTomato-Cre reporter (in addition to K14-driven H2B–GFP) in the outer (ORS; left) and inner (right) hair follicle layers. Notice the differences in morphology between cells located in different layers within the hair follicle.
Examples of in vivo lineage tracing of bulge cells in rest and growth phases of a full hair cycle. Arrows point to the location of the original cell and that of its progeny occupying different positions in the niche after a full hair cycle. Scale bar, 50 μm.
Extended Data Figure 6 A single hair germ cell generates a spatially restricted differentiated lineage.
In vivo lineage tracing of a single cell located in the hair germ in rest and growth phases over a full hair cycle. In advanced hair growth (anagen) an IRS differentiated lineage can be visualized and it is restricted to one side of the follicle as the original founder cell. Scale bar, 50 μm.
Examples of in vivo lineage tracing of a single bulge cell in rest and growth phases over two consecutive hair cycles. Arrows point to the location of the stem cell that remains uncommitted in the niche during both hair cycles. Scale bar, 50 μm.
Examples of in vivo lineage tracing of a single bulge cell lineage in rest and growth phases over two consecutive hair cycles. A bulge cell positioned in the lower bulge undergoes limited and more extended amplification in the ORS over two consecutive hair cycles. After regression of the follicle at the end of the second hair cycle some surviving ORS clones form part of the hair germ before the third hair cycle begins. Arrows depict the clonal expansion and contraction of the bulge stem-cell lineage during the two hair cycles. Scale bar, 50 μm.
Extended Data Figure 9 Model for determining stem-cell fate based on the spatial organization of the hair follicle niche in homeostasis and after injury.
In homeostasis, a stem cell located in the upper bulge does not commit to a specific fate and is likely to remain quiescent, self-renew or become lost. By contrast, a cell positioned in the lower bulge is more likely to become activated and undergo limited amplification as part of the ORS, while still remaining relatively undifferentiated. ORS cells that survive the regression phase in one hair cycle can be situated in the niche compartment that becomes the new hair germ. Once positioned within the hair germ a cell will commit towards a differentiation pathway generating the cell types necessary to support hair growth. Loss of a stem-cell pool due to injury can induce an epithelial cell to enter the niche and contribute to its recovery. However, once a cell enters the niche it is subject to the same inputs as previous resident cells and as a result it will acquire a hair fate and actively contribute to hair regeneration.
Serial optical sections of a mouse hair follicle captured in vivo by multiphoton microscopy using a K14H2BGFP reporter. Also see Extended Data Figure 4. (MOV 1687 kb)
3D volume rendering of the tdTomato Cre reporter showing the spatial distribution of ORS clones during hair growth. Also see Figure 2a. (MOV 2440 kb)
Time-lapse recording of a hair follicle in growth (Anagen IIIa) as seen using a K14H2BGFP reporter. Notice the spatially restricted mode of proliferation and migration. Also see Fig. 2c-f. (MOV 814 kb)
Time-lapse recording of a hair follicle in growth (Anagen IV) as seen using a K14H2BGFP reporter. Notice the spatially restricted mode of proliferation and migration. (MOV 117 kb)
Time-lapse recording of a hair follicle in growth (Anagen IIIa) using a K14H2BGFP reporter, showing an example of ORS tdTomato+ clones being separated due to active cell migration. (MOV 905 kb)
Time-lapse recording of a regions above the hair follicles that previously had their bulges ablated, as seen using the K14H2BGFP reporter. (MOV 570 kb)
Time-lapse recording of a live hair follicle 24 hours after bulge ablation, as seen using the K14H2BGFP and Lef1RFP reporters. (MOV 1632 kb)
Serial optical sections of mouse hair follicles, captured by multiphoton microscopy in vivo using K14CreER/tdTomato, in addition to K14H2BGFP reporters. Notice the biased expression of the Cre reporter toward epithelial layers above the hair follicle niche. Also see Fig. 4a. (MOV 906 kb)
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Rompolas, P., Mesa, K. & Greco, V. Spatial organization within a niche as a determinant of stem-cell fate. Nature 502, 513–518 (2013). https://doi.org/10.1038/nature12602
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