ABSTRACT 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,

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institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS TRACING THE ORIGIN OF HAIR FOLLICLE STEM CELLS Article 09 June 2021 DISTINCT

TYPES OF STEM CELL DIVISIONS DETERMINE ORGAN REGENERATION AND AGING IN HAIR FOLLICLES Article 11 February 2021 DEDIFFERENTIATION MAINTAINS MELANOCYTE STEM CELLS IN A DYNAMIC NICHE Article

Open access 19 April 2023 REFERENCES * Scadden, D. T. The stem-cell niche as an entity of action. _Nature_ 441, 1075–1079 (2006) Article  CAS  ADS  Google Scholar  * Spradling, A. C. et al.

Stem cells and their niches: integrated units that maintain _Drosophila_ tissues. _Cold Spring Harb. Symp. Quant. Biol._ 73, 49–57 (2008) Article  CAS  Google Scholar  * Fuchs, E. The

tortoise and the hair: slow-cycling cells in the stem cell race. _Cell_ 137, 811–819 (2009) Article  CAS  Google Scholar  * Li, L. & Clevers, H. Coexistence of quiescent and active adult

stem cells in mammals. _Science_ 327, 542–545 (2010) Article  CAS  ADS  Google Scholar  * Greco, V. & Guo, S. Compartmentalized organization: a common and required feature of stem cell

niches? _Development_ 137, 1586–1594 (2010) Article  CAS  Google Scholar  * Copley, M. R., Beer, P. A. & Eaves, C. J. Hematopoietic stem cell heterogeneity takes center stage. _Cell Stem

Cell_ 10, 690–697 (2012) Article  CAS  Google Scholar  * Wilson, A. et al. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. _Cell_

135, 1118–1129 (2008) Article  CAS  Google Scholar  * Sato, T. et al. Interferon regulatory factor-2 protects quiescent hematopoietic stem cells from type I interferon-dependent exhaustion.

_Nature Med._ 15, 696–700 (2009) Article  CAS  Google Scholar  * Kopp, H.-G., Avecilla, S. T., Hooper, A. T. & Rafii, S. The bone marrow vascular niche: home of HSC differentiation and

mobilization. _Physiology (Bethesda)_ 20, 349–356 (2005) CAS  Google Scholar  * Celso, C. L. et al. Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche.

_Nature_ 457, 92–96 (2009) Article  ADS  Google Scholar  * Xie, Y. et al. Detection of functional haematopoietic stem cell niche using real-time imaging. _Nature_ 457, 97–101 (2009) Article

  CAS  ADS  Google Scholar  * Ding, L. & Morrison, S. J. Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches. _Nature_ 495, 231–235 (2013) Article

  CAS  ADS  Google Scholar  * Greenbaum, A. et al. CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance. _Nature_ 495, 227–230 (2013) Article  CAS 

ADS  Google Scholar  * Sangiorgi, E. & Capecchi, M. R. _Bmi1_ is expressed _in vivo_ in intestinal stem cells. _Nature Genet._ 40, 915–920 (2008) Article  CAS  Google Scholar  * Takeda,

N. et al. Interconversion between intestinal stem cell populations in distinct niches. _Science_ 334, 1420–1424 (2011) Article  CAS  ADS  Google Scholar  * Barker, N. et al. Identification

of stem cells in small intestine and colon by marker gene Lgr5. _Nature_ 449, 1003–1007 (2007) Article  CAS  ADS  Google Scholar  * Buczacki, S. J. A. et al. Intestinal label-retaining cells

are secretory precursors expressing Lgr5. _Nature_ 495, 65–69 (2013) Article  CAS  ADS  Google Scholar  * Lopez-Garcia, C., Klein, A. M., Simons, B. D. & Winton, D. J. Intestinal stem

cell replacement follows a pattern of neutral drift. _Science_ 330, 822–825 (2010) Article  CAS  ADS  Google Scholar  * Snippert, H. J. et al. Intestinal crypt homeostasis results from

neutral competition between symmetrically dividing Lgr5 stem cells. _Cell_ 143, 134–144 (2010) Article  CAS  Google Scholar  * Cotsarelis, G., Sun, T. T. & Lavker, R. M. Label-retaining

cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. _Cell_ 61, 1329–1337 (1990) Article  CAS  Google Scholar  *

Tumbar, T. et al. Defining the epithelial stem cell niche in skin. _Science_ 303, 359–363 (2004) Article  CAS  ADS  Google Scholar  * Greco, V. et al. A two-step mechanism for stem cell

activation during hair regeneration. _Cell Stem Cell_ 4, 155–169 (2009) Article  CAS  Google Scholar  * Ito, M., Kizawa, K., Hamada, K. & Cotsarelis, G. Hair follicle stem cells in the

lower bulge form the secondary germ, a biochemically distinct but functionally equivalent progenitor cell population, at the termination of catagen. _Differentiation_ 72, 548–557 (2004)

Article  Google Scholar  * Rompolas, P. et al. Live imaging of stem cell and progeny behaviour in physiological hair-follicle regeneration. _Nature_ 487, 496–499 (2012) Article  CAS  ADS 

Google Scholar  * Trempus, C. S. et al. Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34. _J. Invest. Dermatol._ 120, 501–511 (2003)

CAS  PubMed  Google Scholar  * Blanpain, C., Lowry, W. E., Geoghegan, A., Polak, L. & Fuchs, E. Self-renewal, multipotency, and the existence of two cell populations within an epithelial

stem cell niche. _Cell_ 118, 635–648 (2004) Article  CAS  Google Scholar  * Claudinot, S., Nicolas, M., Oshima, H., Rochat, A. & Barrandon, Y. Long-term renewal of hair follicles from

clonogenic multipotent stem cells. _Proc. Natl Acad. Sci. USA_ 102, 14677–14682 (2005) Article  CAS  ADS  Google Scholar  * Liu, Y., Lyle, S., Yang, Z. & Cotsarelis, G. Keratin 15

promoter targets putative epithelial stem cells in the hair follicle bulge. _J. Invest. Dermatol._ 121, 963–968 (2003) Article  CAS  Google Scholar  * Ito, M. et al. Stem cells in the hair

follicle bulge contribute to wound repair but not to homeostasis of the epidermis. _Nature Med._ 11, 1351–1354 (2005) Article  CAS  Google Scholar  * Morris, R. J. et al. Capturing and

profiling adult hair follicle stem cells. _Nature Biotechnol._ 22, 411–417 (2004) Article  CAS  Google Scholar  * Zhang, Y. V., Cheong, J., Ciapurin, N., McDermitt, D. J. & Tumbar, T.

Distinct self-renewal and differentiation phases in the niche of infrequently dividing hair follicle stem cells. _Cell Stem Cell_ 5, 267–278 (2009) Article  CAS  Google Scholar  *

Müller-Röver, S. et al. A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. _J. Invest. Dermatol._ 117, 3–15 (2001) Article  Google

Scholar  * Jahoda, C. A., Horne, K. A. & Oliver, R. F. Induction of hair growth by implantation of cultured dermal papilla cells. _Nature_ 311, 560–562 (1984) Article  CAS  ADS  Google

Scholar  * Kaufman, C. K. et al. GATA-3: an unexpected regulator of cell lineage determination in skin. _Genes Dev._ 17, 2108–2122 (2003) Article  CAS  Google Scholar  * Legué, E. &

Nicolas, J.-F. Hair follicle renewal: organization of stem cells in the matrix and the role of stereotyped lineages and behaviors. _Development_ 132, 4143–4154 (2005) Article  Google Scholar

  * Hsu, Y.-C., Pasolli, H. A. & Fuchs, E. Dynamics between stem cells, niche, and progeny in the hair follicle. _Cell_ 144, 92–105 (2011) Article  CAS  Google Scholar  * Sequeira, I.

& Nicolas, J.-F. Redefining the structure of the hair follicle by 3D clonal analysis. _Development_ 139, 3741–3751 (2012) Article  CAS  Google Scholar  * Means, A. L., Xu, Y., Zhao, A.,

Ray, K. C. & Gu, G. A. CK19CreERT knockin mouse line allows for conditional DNA recombination in epithelial cells in multiple endodermal organs. _Genesis_ 46, 318–323 (2008) Article  CAS

  Google Scholar  * Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. _Nature Neurosci._ 13, 133–140 (2010) Article  CAS 

Google Scholar  * Youssef, K. K. et al. Identification of the cell lineage at the origin of basal cell carcinoma. _Nature Cell Biol._ 12, 299–305 (2010) Article  CAS  Google Scholar  * Jaks,

V. et al. Lgr5 marks cycling, yet long-lived, hair follicle stem cells. _Nature Genet._ 40, 1291–1299 (2008) Article  CAS  Google Scholar  * Chi, W., Wu, E. & Morgan, B. A. Dermal

papilla cell number specifies hair size, shape and cycling and its reduction causes follicular decline. _Development_ 140, 1676–1683 (2013) Article  CAS  Google Scholar  * Van Keymeulen, A.

& Blanpain, C. Tracing epithelial stem cells during development, homeostasis, and repair. _J. Cell Biol._ 197, 575–584 (2012) Article  CAS  Google Scholar  * Barroca, V. et al. Mouse

differentiating spermatogonia can generate germinal stem cells _in vivo_. _Nature Cell Biol._ 11, 190–196 (2009) Article  CAS  Google Scholar  * Nystul, T. & Spradling, A. An epithelial

niche in the _Drosophila_ ovary undergoes long-range stem cell replacement. _Cell Stem Cell_ 1, 277–285 (2007) Article  CAS  Google Scholar  * Plikus, M. V. et al. Epithelial stem cells and

implications for wound repair. _Semin. Cell Dev. Biol._ 23, 946–953 (2012) Article  CAS  Google Scholar  * Vasioukhin, V., Degenstein, L., Wise, B. & Fuchs, E. The magical touch: genome

targeting in epidermal stem cells induced by tamoxifen application to mouse skin. _Proc. Natl Acad. Sci. USA_ 96, 8551–8556 (1999) Article  CAS  ADS  Google Scholar  * Rendl, M., Lewis, L.

& Fuchs, E. Molecular dissection of mesenchymal-epithelial interactions in the hair follicle. _PLoS Biol._ 3, e331 (2005) Article  Google Scholar  * Diamond, I., Owolabi, T., Marco, M.,

Lam, C. & Glick, A. Conditional gene expression in the epidermis of transgenic mice using the tetracycline-regulated transactivators tTA and rTA linked to the keratin 5 promoter. _J.

Invest. Dermatol._ 115, 788–794 (2000) Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS 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). AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Department of Genetics, Department of Dermatology, Yale Stem Cell Center, Yale Cancer Center, Yale School of Medicine, New Haven, 06510, Connecticut, USA Panteleimon Rompolas,

 Kailin R. Mesa & Valentina Greco Authors * Panteleimon Rompolas View author publications You can also search for this author inPubMed Google Scholar * Kailin R. Mesa View author

publications You can also search for this author inPubMed Google Scholar * Valentina Greco View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS

P.R. and V.G. designed experiments and wrote the manuscript; P.R. performed the experiments and analysed the data; K.R.M. assisted with the revisions. CORRESPONDING AUTHOR Correspondence to

Valentina Greco. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. EXTENDED DATA FIGURES AND TABLES EXTENDED DATA FIGURE 1 HAIR FOLLICLE ANATOMY

AND PHYSIOLOGY. 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). EXTENDED DATA FIGURE 2 METHOD FOR SINGLE STEM-CELL LINEAGE TRACING

IN LIVE MICE. 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. EXTENDED DATA FIGURE 3 FLUORESCENT PROTEINS AND KINETICS OF THE INDUCIBLE TDTOMATO-CRE REPORTER. 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. EXTENDED DATA FIGURE 4 CLASSIFICATION OF HAIR FOLLICLE CELL TYPES _IN VIVO_. 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. EXTENDED DATA FIGURE 5 RELOCATION OF BULGE STEM CELLS AND PROGENY OVER A HAIR

CYCLE. 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. EXTENDED DATA FIGURE 7 LONG-TERM FATE OF BULGE STEM CELLS. 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. EXTENDED DATA FIGURE 8 ORIGIN OF THE HAIR GERM. 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. SUPPLEMENTARY INFORMATION SERIAL OPTICAL SECTIONS OF A LIVE HAIR FOLLICLE 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) ORS CLONAL DISTRIBUTION 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) I - ORS EXPANSION CAPTURED _IN VIVO_ 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) II - ORS EXPANSION CAPTURED _IN VIVO_ 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) ORS CLONE FRAGMENTATION

CAPTURED _IN VIVO_ 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) POPULATIONS ABOVE THE BULGE PROLIFERATE FOLLOWING NICHE ABLATION 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) NICHE RECOVERY AFTER ABLATION Time-lapse recording of a live hair follicle 24 hours after bulge ablation, as seen using the K14H2BGFP and

Lef1RFP reporters. (MOV 1632 kb) BIASED EXPRESSION OF K14CREER/TDTOMATO REPORTER 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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ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 09 July 2013 * Accepted: 23 August 2013 * Published: 06 October 2013 * Issue Date: 24 October 2013 * DOI:

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