Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Identification of hair shaft progenitors that create a niche for hair pigmentation Chung-Ping Liao,1 Reid C. Booker,1 Sean J. Morrison,2,3,4,5,6 and Lu Q. Le1,4,5 1Department of Dermatology, 2Department of Pediatrics, 3Children’s Research Institute, 4Simmons Comprehensive Cancer Center, 5Hamon Center for Regenerative Science and Medicine, 6Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA Hair differentiates from follicle stem cells through progenitor cells in the matrix. In contrast to stem cells in the bulge, the identities of the progenitors and the mechanisms by which they regulate hair shaft components are poorly understood. Hair is also pigmented by melanocytes in the follicle. However, the niche that regulates follicular melanocytes is not well characterized. Here, we report the identification of hair shaft progenitors in the matrix that are differentiated from follicular epithelial cells expressing transcription factor KROX20. Depletion of Krox20 lin- eage cells results in arrest of hair growth, confirming the critical role of KROX20+ cells as antecedents of structural cells found in hair. Expression of stem cell factor (SCF) by these cells is necessary for the maintenance of differen- tiated melanocytes and for hair pigmentation. Our findings reveal the identities of hair matrix progenitors that regulate hair growth and pigmentation, partly by creating an SCF-dependent niche for follicular melanocytes. [Keywords: stem cell factor (SCF); hair pigmentation; hair shaft progenitor cell; hair follicle stem cell; hair matrix; KROX20] Supplemental material is available for this article. Received March 10, 2017; revised version accepted April 13, 2017. Hair is a highly keratinized tissue differentiated from hair SCF and its receptor, KIT, for hair pigmentation has follicle (HF) stem cells, which are located primarily in a been supported by the absence of hair pigment in several discrete reservoir called the bulge (Cotsarelis et al. 1990; SCF- and KIT-deprived animals (Copeland et al. 1990; Blanpain et al. 2004; Tumbar et al. 2004). The cycle of Reith et al. 1990; Kunisada et al. 1998b; Botchkareva hair growth and involution occurs in three phases: anagen et al. 2001; Nishimura et al. 2002). It is apparent that me- (growth), catagen (regression), and telogen (resting) (Blan- lanocytes are the relevant target cells in this type of hypo- pain and Fuchs 2009; Shimomura and Christiano 2010; pigmentation, as they are the melanin-producing cells as Plikus et al. 2012; Solanas and Benitah 2013; Watt 2014). well as the predominant KIT-expressing cell in the HF. Hair is commonly pigmented because of the presence However, the sources of SCF that support melanocytic ac- of melanin in hair shafts, which in turn is synthesized tivity in the HF have not been fully characterized, al- by follicular melanocytes and then transferred to the though SCF expression by cells in the dermal papilla has neighboring hair progenitors that give rise to hair shafts been suggested to promote melanocyte stem cell differen- (Nishimura et al. 2002, 2005; Lin and Fisher 2007). De- tiation (Chang et al. 2013). spite recent advances in our understanding of HF stem The HF matrix is composed of epithelial cells surround- cell biology, the mechanisms that regulate hair pigmenta- ing the dermal papilla. The HF matrix cells proliferate and tion remain poorly understood, especially with respect to differentiate into the structures of the hair shaft and inner the cellular interactions between hair progenitor cells and root sheath (Driskell et al. 2011). There are also melano- melanocytes that take place in the HF matrix. cytes residing in the same niche to provide melanin to Stem cell factor (SCF, also known as KIT ligand or Steel hair shaft progenitor cells for hair pigmentation. A domi- factor) is a growth factor that regulates multiple physio- nant population of matrix cells is transit-amplifying cells. logical homeostatic events, including the maintenance They proliferate rapidly for several divisions and then pro- of hematopoietic stem cells (Ding et al. 2012), mast cells gress upward to become committed progenitors that dif- (Lennartsson and Ronnstrand 2012), and melanocytes ferentiate into central hair shafts (the medulla, cortex, (Lennartsson and Ronnstrand 2012). The critical role of and cuticle), its surrounding channels or the inner root sheaths (the cuticle, Henle, and Huxley layers) that guide Corresponding author: [email protected] Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/gad.298703.117. Free- © 2017 Liao et al. This article, published in Genes & Development,is ly available online through the Genes & Development Open Access available under a Creative Commons License (Attribution 4.0 Internation- option. al), as described at http://creativecommons.org/licenses/by/4.0/. GENES & DEVELOPMENT 31:1–13 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/17; www.genesdev.org 1 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Liao et al. hair shafts toward the skin surface, and the companion mostly absent from the proximal end (Fig. 1B). Fontana- layer (the innermost layer of the outer root sheath) Masson staining confirmed that this hypopigmentation (Hsu et al. 2011, 2014). In contrast to HF stem cells in was caused by the absence of melanin in the hair shaft cor- the bulge, the identities of the committed progenitor cells tex and medulla (the compartments housing the most that give rise directly to hair shafts (the medulla, cortex melanin in pigmented hair shaft) (Fig. 1C). We then deter- and cuticle) are currently not well characterized. In mined the time of onset of decreased pigmentation. The addition, the development of hair is also accompanied amount of pigment in individual hairs appeared normal by pigmentation that is contributed to the hair shafts at P9 (Fig. 1D), with the loss of hair pigmentation becom- by melanocytes. The reciprocal interactions between ing noticeable at P11; it progressed quickly after that (Fig. melanocytes and hair progenitor cells as well as the source 1D,E). Analysis of melanin density in the HF revealed sig- of SCF, which regulates hair pigmentation, are not well nificant reductions at P11 and P13 (P < 0.0001) (Fig. 1F), ex- characterized. plaining the early loss of hair pigmentation in Scfflox/gfp; KROX20 (also known as EGR2) is a zinc finger tran- Krox20Cre mice (Fig. 1B). Scfflox/gfp; Krox20Cre mice ex- scription factor that regulates hindbrain segmentation hibited progressive hair graying; their coat color under- (Schneider-Maunoury et al. 1993), Schwann cell mye- went a dynamic change from black to white within 9 lination (Topilko et al. 1994), and lymphocyte immune re- mo (Fig. 1A; Supplemental Fig. S1A). However, their new- sponses (Li et al. 2012). In addition, KROX20 is expressed ly synthesized hairs were already largely depigmented at in subpopulations of HF cells (Gambardella et al. 2000). P20 (Fig. 1B). These results suggested that the long period However, the role of the KROX20-expressing cells in of hair graying is due to the mixture of partly pigmented HF development is not known. We now report that the (old) and unpigmented (new) hair shafts (Supplemental transcription factor KROX20 identifies a sublineage of Fig. S1B,C) rather than the continuous production of HF epithelial cells toward the differentiation of hair shaft new gray hairs. To confirm this, gray Scfflox/gfp; Krox20Cre during HF morphogenesis. We observed that they are a mice were depilated at 2 mo to remove all hairs, including necessary source of SCF for the maintenance of mature old hairs, and stimulate new hair regeneration; as expect- melanocytes in the hair matrix to produce hair pigmen- ed, their new hairs showed a complete loss of pigmenta- tation, as evidenced by the complete loss of pigment tion (Fig. 1G). Most importantly, the fact that Scfflox/gfp; when Scf is deleted in Krox20 lineage cells. We also dem- Krox20Cre mice underwent a complete loss of hair pig- onstrate that deletion of Krox20 lineage cells in vivo mentation suggested that Krox20 lineage cells are the results in a complete arrest of new hair growth, demon- main source of SCF for follicular melanocytes to produce strating the critical role of Krox20 lineage cells in hair hair pigment. development. Hair pigmentation is dependent on SCF expression Results by epithelial keratinocytes In order to identify the cell type that produces SCF in the Mice lacking SCF in Krox20 lineage cells exhibit skin, thereby regulating hair pigmentation and being re- progressive hair graying sponsible for the hair hypopigmentation in Scfflox/gfp; While studying the role of mast cells and SCF during the Krox20Cre mice, we examined the hair color phenotype initiation of neurofibroma, a Schwann cell neoplasm, we of SCF ablation in different cell lineages in the skin by us- conditionally deleted Scf in neurofibroma neoplastic cells ing several cell type-specific Cre mouse lines, including using the Schwann cell lineage Krox20Cre mouse line. DhhCre (Schwann cell), PLPCreERT2 (Schwann cell), Tyr- Serendipitously, we found that the mice without SCF in CreERT2 (melanocyte), and K14Cre (keratinocyte). In addi- Krox20 lineage cells developed hair graying and, early in tion, CMVCreERT (all cells) was included as a control for life, lost all hair pigmentation. Invariably, all Scfflox/gfp; the inducible Cre systems. The R26-LacZ reporter was in- Krox20Cre mice (n = 20) displayed progressive hair gray- troduced into all of the above conditional knockouts to ing. The first round of hair graying occurred homoge- validate Cre expression specificity and efficiency. nously during postnatal days 30–40 (P30–P40). As the In mice lacking SCF in Schwann cells (Scfflox/gfp; mice aged, the hairs underwent further depigmentation DhhCre), we did not observe any hair color changes for in waves.