Morpholinos for splice modificatio

Morpholinos for splice modification

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Hopx expression defines a subset of multipotent hair follicle stem cells and a progenitor population primed to give rise to K6+ niche cells
Norifumi Takeda, Rajan Jain, Matthew R. LeBoeuf, Arun Padmanabhan, Qiaohong Wang, Li Li, Min Min Lu, Sarah E. Millar, Jonathan A. Epstein

Summary

The mammalian hair follicle relies on adult resident stem cells and their progeny to fuel and maintain hair growth throughout the life of an organism. The cyclical and initially synchronous nature of hair growth makes the hair follicle an ideal system with which to define homeostatic mechanisms of an adult stem cell population. Recently, we demonstrated that Hopx is a specific marker of intestinal stem cells. Here, we show that Hopx specifically labels long-lived hair follicle stem cells residing in the telogen basal bulge. Hopx+ cells contribute to all lineages of the mature hair follicle and to the interfollicular epidermis upon epidermal wounding. Unexpectedly, our analysis identifies a previously unappreciated progenitor population that resides in the lower hair bulb of anagen-phase follicles and expresses Hopx. These cells co-express Lgr5, do not express Shh and escape catagen-induced apoptosis. They ultimately differentiate into the cytokeratin 6-positive (K6) inner bulge cells in telogen, which regulate the quiescence of adjacent hair follicle stem cells. Although previous studies have suggested that K6+ cells arise from Lgr5-expressing lower outer root sheath cells in anagen, our studies indicate an alternative origin, and a novel role for Hopx-expressing lower hair bulb progenitor cells in contributing to stem cell homeostasis.

INTRODUCTION

The hair follicle (HF) has emerged as an informative system in which to study adult stem cell biology because it is characterized by predictable and precisely choreographed phases of growth (anagen), regression (catagen) and rest (telogen; Fig. 1A). Hair growth is, in part, maintained by HF stem cells (SCs) located at the base of the hair follicle in telogen (Alonso and Fuchs, 2006; Fuchs, 2009; Li and Clevers, 2010). The HF stem cell compartment is primarily composed of three layers: (1) basal bulge (CD34+, integrin α6hi) cells; (2) suprabasal bulge (CD34+, integrin α6lo) cells; and (3) cytokeratin 6-positive (K6) innermost cells surrounding the hair shaft (Blanpain et al., 2004; Hsu et al., 2011; Fig. 1A). Populations of stem cells have been elucidated outside the bulge (MTS24+, Lrig1+, Lgr6+), although their contribution to hair follicle morphogenesis under physiological conditions appears limited (Nijhof et al., 2006; Jensen et al., 2009; Snippert et al., 2010). As telogen progresses, a rapidly cycling, biochemically distinct cohort of cells is established between the bulge and dermal papilla (DP), termed the secondary hair germ (HG). The secondary HG appears to house an activated subset of cells that provide the initial contributions to the growing hair follicle. These contributions combine with cells originating from the bulge during hair growth to migrate and proliferate during anagen, creating a linear trail of cells that will form the outer root sheath (ORS). The SC population of the outer bulge layer also includes quiescent cells that will contribute to subsequent rounds of hair follicle growth.

The growth phase (anagen) of the hair cycle is characterized by a stepwise differentiation of ORS cells into matrix progenitor cells of the hair bulb. As anagen progresses, matrix cells differentiate to produce the full complement of terminally differentiated cell lineages of the mature adult HF: the hair shaft, inner root sheath (IRS), and companion layer sandwiched between IRS and ORS (Fig. 1A). The growth phase is followed by catagen, which is characterized by involution of the lower half of the HF driven by widespread apoptosis (Botchkareva et al., 2006; Fig. 1A). A subset of matrix cells express sonic hedgehog (Shh) (St-Jacques et al., 1998; Kulessa et al., 2000; Blanpain et al., 2004), and initial studies posited that these cells survive catagen-induced apoptosis and contribute to the HG (Panteleyev et al., 2001). However, lineage studies in the adult do not support this hypothesis (Levy et al., 2005; Greco et al., 2009; White et al., 2011). The fate of Shh- matrix cells remains elusive, although it has been suggested that they may give rise to the companion layer (Winter et al., 1998).

Integrin α6hi cells, a slowly cycling population located in the basal layer, and CD34+, integrin α6lo cells in the suprabasal layer have long been thought to mark the hair follicle stem cells (Albert et al., 2001; Trempus et al., 2003; Blanpain et al., 2004). More recently, rapidly cycling Lgr5+ progenitor cells have been found to be in the secondary HG (Jaks et al., 2008). K6+ cells in the innermost layer of the bulge maintain the quiescence of the basal and suprabasal layer stem cells (Hsu et al., 2011). The K6+ layer is thought to share similarities with segments of the companion layer of anagen HFs (Higgins et al., 2009); both are adjacent to the ORS and express K6 (Winter et al., 1998; Wang et al., 2003; Hsu et al., 2011), plasminogen activator inhibitor 2 (PAI2) (Lavker et al., 1998), calretinin (Poblet et al., 2005) and S100 calcium-binding protein A6 (S100A6) (Ito and Kizawa, 2001). However, the lineage relationship between the K6+ inner bulge and companion layers remains a mystery.

Adult tissue-specific stem cells both self-renew and generate progeny that include components of their own niches and other functional derivatives (Morrison and Kimble, 2006; Hsu and Fuchs, 2012; Lander et al., 2012). The homeostatic mechanisms coordinating these activities are crucial to long-term regenerative potential, yet remain poorly understood. Defining lineage relationships and progression along alternative differentiation pathways is crucial for elucidation of this process. In many organs and tissues, the identities of various stem and niche cells remain controversial or unknown. In the intestinal crypt of adult mice, we recently demonstrated that Hopx (Chen et al., 2002; Shin et al., 2002) is strongly expressed by BrdU-retaining, +4 epithelial stem cells (Takeda et al., 2011). Interestingly, Hopx-expressing cells interconvert with the Lgr5-positive progenitor cell population found in the intestinal crypt base. Both Hopx and Lgr5 are expressed in the HF; hence, we sought to examine the lineage progression and relationship of these cell populations in the skin.

Here, we show that Hopx is expressed in the basal layer of the telogen hair follicle and its expression is more restricted than previously reported hair follicle stem cell markers. Lineage-tracing experiments using inducible Cre-Lox technologies demonstrate that Hopx+ cells give rise to all lineages of the hair follicle. Furthermore, we have identified a novel population of Hopx-expressing (Lgr5+, Shh-) lower hair bulb cells in the mature follicle that contribute to the K6+ inner bulge layer of the stem cell niche in telogen. The Hopx+ hair bulb progenitors are distinguished from Lgr5+ lower ORS cells as the lower ORS does not express Hopx. Rather, they are Shh- cells that escape catagen-induced apoptosis and give rise to K6+ cells in the subsequent telogen.

MATERIALS AND METHODS

Generation of Hopx3FlagGFP/+ mice

Hopx3FlagGFP/+ mice were generated by homologous recombination in embryonic stem cells targeting a cassette containing Exon3-3XFlag-Viral2A-eGFP-3UTR-Frt-PGK-neo-Frt to the third exon of Hopx. An AscI restriction enzyme site was introduced 70 bp upstream of exon 3 and sequential FseI, EcoRI restriction enzyme sites were introduced immediately following the 3′ UTR. The PGK-neo cassette was removed by breeding initial progeny to mice expressing ubiquitous FlpE recombinase (ACTFLPe) (Rodríguez et al., 2000).

Mice

HopxlacZ/+ (Chen et al., 2002), HopxERCre/+ (Takeda et al., 2011), Lgr5EGFP-ERCre/+ (Barker et al., 2007), ShhERCre/+ (Harfe et al., 2004), K14ERCre/+ (Vasioukhin et al., 1999), R26lacZ/+ (Soriano, 1999), R26mT-mG/+ (Muzumdar et al., 2007) and R26Tom/+ (Madisen et al., 2010) mice have been described previously. All mice were maintained on mixed genetic backgrounds. The University of Pennsylvania Institutional Animal Care and Use Committee approved all animal protocols.

Observation and description of HFs

The first two HF cycles are synchronized in mice for the first 60 days of life. Anagen and catagen are subdivided into 6 and 8 stages, respectively, based on a variety of morphological criteria (Müller-Röver et al., 2001). They are further organized as follows: middle anagen (anagen III-IV), late anagen (anagen V-VI), middle catagen (catagen V) and late catagen (catagen VII-VIII). Because hair cycles slightly vary among strains and sexes, stages of mouse ages were evaluated instead. Single hair follicles were dissected with fine forceps and visualized on an Olympus MVX10 fluorescence dissecting microscope.

Lineage-tracing experiments

Mice were injected intraperitoneally with tamoxifen (100 mg/kg body weight; Sigma) dissolved in corn oil, as a single dose or daily for 5 consecutive days, as indicated.

BrdU-labeling experiments

To mark label-retaining cells (LRCs), Hopx3FlagGFP/+ mice were injected intraperitoneally with BrdU (50 mg/kg body weight; Sigma) twice a day for 3 days at P3-5 (Braun et al., 2003; Jensen et al., 2009). Tissues were collected 7 weeks after BrdU administration, and then stained with an anti-BrdU monoclonal antibody (Rat, 1:20, Accurate).

β-Galactosidase staining

Tissues were fixed for 45 minute in ice-cold 2% paraformaldehyde (PFA) at 4°C, rinsed 3 times in PBS for 20 minutes at 4°C, and then incubated for 12-24 hours at 37°C in the staining solution [2 mM MgCl2, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 0.01% NP40, 0.01% sodium deoxycholate and 1 mg/ml X-gal in PBS]. The stained tissues were fixed in 2% PFA, ethanol dehydrated and sectioned using standard methods.

Histology

Back skins were fixed in 2% PFA, ethanol dehydrated, embedded in paraffin wax and sectioned in the sagittal plane. Antibodies used were: GFP (goat, 1:100, Abcam) (rabbit, 1:200, Molecular Probes), RFP (recognizes tdTomato) (rabbit, 1:50, Rockland), AE13 (mouse, 1:25, Abcam), cytokeratin 5 (goat, 1:20, Santa Cruz) (rabbit, 1:200, Abcam), cytokeratin 6 (mouse, prediluted, Abcam) (rabbit, 1:200, Abcam), K15 (mouse, 1:50, Vector), Sox9 (rabbit, 1:100, Santa Cruz), Gata3 (mouse, 1:25, Santa Cruz), CD34 (rat, 1:25, BD Pharmingen), Lrig1 (goat, 1:50, R&D Systems), P-cadherin (mouse, 1:20, R&D Systems), Shh (rabbit, 1:50, Santa Cruz) and Flag (mouse, 1:250, Sigma). TUNEL staining was performed using standard protocols (Roche). Tissue sections were incubated with terminal deoxynucleotidyl transferase (TdT) and fluorescein-dUTP at 37°C for 60 minutes. All immunohistochemistry was visualized on a Nikon Eclipse 80i fluorescence microscope, except the confocal images, which were evaluated on a Zeiss LSM 510 microscope. Radioactive in situ hybridization was performed using the previously described probe for Hopx (Chen et al., 2002). All images were analyzed using Adobe Photoshop (sizing, brightness or contrast adjustments, etc.). Brightness and contrast was adjusted linearly across the entirety of each image.

Wound healing assays

Mice that had been previously treated with tamoxifen at indicated days were anesthetized with ketamine and xylazine, and a 6 mm full-thickness excision of skin was made on the mid back (Ito et al., 2005). Skin samples were collected at indicated days after wounding and processed for β-galactosidase staining.

Keratinocyte isolation and FACS

Back skin keratinocytes were collected as previously described (Jensen et al., 2010). Briefly, mice were killed, dorsal hair was clipped and dorsal skin was taken and placed into Ca2+- and Mg2+-free Hank’s Balanced Salt Solution (HBSS; Invitrogen). Subcutaneous tissue and fat were removed by gentle scraping, and the skin was cut into 1 cm strips and incubated at 37°C in 0.25% trypsin solution in HBSS for 70 minutes. The epidermis was then scraped into S-minimal essential medium (SMEM; Invitrogen) supplemented with 5% FBS. After gentle pipetting, the suspension was passed through a 70 μm filter (BD Biosciences), and keratinocytes were counted. Single cells were analyzed using BD FACS Calibur (BD Biosciences). Cells were pre-incubated for 15 minutes with Fc Block (BD Pharmingen), and 7-amino-actinomycin D (7-AAD; BD Biosciences) was used to exclude dead cells. The following antibodies were used in a solution of 5% FBS in PBS for 30 minutes on ice: CD34-eFluor660 (1:100, eBioscience) and CD49f -PE (1:100, BD Pharmingen).

RESULTS

Hopx expression characterizes hair follicle stem cells

To identify Hopx-expressing cells in the hair follicle, we generated a novel Hopx allele in which the final exon of Hopx was appended to include a 3XFlag sequence followed by viral-2A-GFP (Hopx3FlagGFP/+; supplementary material Fig. S1A). GFP expression correlated with Hopx expression in the intestine and heart (Chen et al., 2002; Takeda et al., 2011) (supplementary material Fig. S1B-E). At postnatal day 21 (P21), the first adult mouse telogen, we detected strong Hopx expression in the basal layer of the hair follicle stem cell compartment, marked by cytokeratin 15 (K15; Krt15 - Mouse Genome Informatics) expression, and relatively weaker expression in the secondary HG, marked by P-Cadherin expression (Fig. 1B,C). Hopx+ cells in adult telogen HFs co-expressed CD34, but did not express K6, a marker of the innermost bulge cells, unlike many other known stem cell markers [e.g. Sox9 (Nowak et al., 2008), Lhx2 (Rhee et al., 2006), Tcf3 (Nguyen et al., 2006) and K15 (Lyle et al., 1998; Liu et al., 2003); Fig. 1D,E]. Hopx expression was more restricted to the bulge than that of CD34, and overlapped with, but was not identical to, K15 expression. In addition, Hopx+ cells retain BrdU 8 weeks after injection at P3-5, and hence we conclude they are LRCs (Fig. 1F). In contrast to Hopx, Lgr5 expression was localized to the lower basal layer and HG, as has been previously described (Jaks et al., 2008) (Fig. 1G). Very few Lrig1+ cells co-expressed Hopx (supplementary material Fig. S1F). FACS analysis during the second telogen (P55-P58) confirmed that Hopx-expressing cells are a subpopulation of CD34+, integrin α6hi and CD34+, integrin α6lo cells (data not shown).

Fig. 1.

Hopx specifically labels mouse hair follicle stem cells. (A) The hair cycle is characterized by successive phases of rest (telogen), growth (anagen) and regression (catagen). During anagen, Shh-expressing matrix progenitors in the lower hair bulb contribute to the IRS and hair shaft. During catagen, K6 protein-expressing cells surround the club hair. Some survive catagen-induced apoptosis and then home back to the innermost layer of the hair follicle. (B-E) Skin sections from Hopx3FlagGFP/+ mice from P21, the first mouse telogen. K15, bulge cells; PCad, secondary HG; CD34, basal bulge stem cells; K6, niche cells. (F) Double staining of Hopx3FlagGFP/+ skin for GFP and BrdU, demonstrating that LRCs labeled at P3-5 with BrdU express Hopx at P58. (G) GFP staining of Lgr5EGFP-ERCre/+ skin at P21. (H-L) GFP staining of HopxERCre/+;R26mT-mG/+ skin 3 months after a tamoxifen pulse at P50-54, with cell-specific markers as indicated. (M) β-galactosidase staining of HopxERCre/+;R26lacZ/+ skin 15 months after labeling. Arrowheads indicate double-positive cells. HG, secondary hair germ; DP, dermal papilla; ORS, outer root sheath; CL, companion layer; IRS, inner root sheath; ES, epithelial strand. Scale bars: 25 μm in B-L; 250 μm in M.

To track the fate of telogen Hopx+ cells, we crossed HopxERCre/+ (Takeda et al., 2011) mice with R26lacZ/+ (Soriano, 1999) and R26mT-mG/+ (Muzumdar et al., 2007) indicator mice, in which β-galactosidase (β-gal) and membrane-bound GFP can be induced by Cre-mediated recombination, respectively. We injected mice with tamoxifen to activate Cre recombinase during the second synchronized telogen (P50-54). Examination at P55 revealed Hopx-derived cells in the basal layer and HG, consistent with the expression pattern of Hopx3FlagGFP/+ mice (supplementary material Fig. S1G). All HF compartments of the mature anagen stage follicle, including the ORS (K5+), companion layer (K6+), IRS (Gata3+), hair shaft (AE13+) and matrix contained derivatives of Hopx-expressing cells (Fig. 1H-K). Fated cells were also found in the subsequent telogen bulge and HG (Fig. 1L). Fifteen months after tamoxifen induction, Hopx derivatives were still present in all cell types of the mature follicle, suggesting that Hopx+ cells in adult telogen undergo self-renewal (Fig. 1M). The inefficiencies of tamoxifen-inducible Cre-Lox technologies probably account for, at least in part, the lack of labeling in every hair follicle. No ectopic reporter activity was detected in uninduced control mice (HopxERCre/+;R26mT-mG/+, data not shown).

Hair follicle SCs have been shown to contribute to the interfollicular epidermis during wound healing (Ito et al., 2005; Cotsarelis, 2006; Snippert et al., 2010; Brownell et al., 2011). We pulsed HopxERCre/+;R26lacZ/+ mice with tamoxifen during the second telogen phase (P50-54), and then created a full-thickness wound 48 hours later (P56). Two weeks after injury, streams of β-gal+ Hopx derivatives were seen within the wound in a radial pattern extending from hair follicles on the edge of the wound towards the wound center (Fig. 2A). Histological analysis revealed that Hopx derivatives contributed to the re-epithelialized skin (Fig. 2B). Nine weeks after wounding, clones of Hopx-derived cells were found within the newly formed cytokeratin 5 (K5)-positive epidermal cells and more differentiated suprabasal and granular layer cells (Fig. 2C-E). We could not detect β-gal expression in any uninjured areas of epidermis or ectopic reporter activity (data not shown). Taken together, these data suggest that Hopx+ cells of the telogen hair follicle can self-renew, give rise to all lineages of the hair follicle during subsequent cycles of hair growth, and contribute to the interfollicular epidermis upon injury. Hence, Hopx is a marker of resident adult HF stem cells.

Fig. 2.

Hopx+ cells contribute to regenerating epidermis. (A-E) β-galactosidase staining of wounds and regenerated epidermis in HopxERCre/+;R26lacZ/+ mice induced during the secondary telogen, 2 weeks (A,B) and 9 weeks (C-E) after a full-thickness skin wounding. (E) Double staining of β-galactosidase and K5, demonstrating a K5+ basal layer of scar tissue is derived from Hopx+ cells. Scale bars: 25 μm in B,D,E; 2 mm in A,C.

Hopx-expressing adult HF SCs derive from a Hopx-expressing precursor

The precise cellular origin and timing of hair follicle stem cell specification remain poorly defined. We sought to determine whether Hopx is expressed during postnatal hair follicle maturation and stem cell specification. We could not detect Hopx expression at P0 in the developing bulge, whereas Sox9+ cells were found in the ORS and primitive bulge (Fig. 3A). In immature P5 anagen stage follicles (anagen III) (Müller-Röver et al., 2001), Hopx expression was present in the K15+ upper ORS cells, but absent in the lower ORS (Fig. 3B,C). Hopx was expressed in the hair shaft, matrix, IRS and companion layer (Fig. 3D-G). Lineage-tracing experiments using HopxERCre/+;R26Tom/+ (Madisen et al., 2010) mice pulsed with a single dose of tamoxifen at P5 demonstrated that the anagen follicle at P85 and the telogen bulge was labeled (Fig. 3H,I). This result indicates that during early hair follicle maturation, Hopx-expression marks cells that are destined to give rise to all lineages of the adult hair follicle.

Fig. 3.

Lineage tracing of Hopx-expressing precursors. (A-G) GFP staining of Hopx3FlagGFP/+ skin at P0 (A) and P5 (B-G), the morphological middle anagen, with cell-specific markers. (H,I) TdTomato staining of HopxERCre/+;R26Tom/+ skin 80 days after labeling at P5. Arrowhead indicates a double-positive cell. Scale bars: 25 μm.

Hopx+ cells in the mature anagen lower hair bulb escape catagen apoptosis and give rise to K6+ niche cells in the subsequent telogen

Unlike several other HFSC markers [e.g. Sox9 (Nowak et al., 2008), Lhx2 (Rhee et al., 2006), Tcf3 (Nguyen et al., 2006) and K15 (Lyle et al., 1998; Liu et al., 2003)], Hopx expression in the bulge of telogen follicles was restricted to the outer stem cell compartment and not present in the K6+ innermost layer. It has been postulated that K6+ inner cells arise from Lgr5+ lower ORS cells in anagen (Hsu et al., 2011). Therefore, we investigated the Hopx+ population of cells in late anagen and their derivatives.

Hopx was expressed in the matrix, companion layer, IRS and hair shaft of the mature anagen HF (anagen V-VI) (Müller-Röver et al., 2001; Fig. 4A-D). Hopx was expressed symmetrically and broadly in the anagen hair bulb, whereas Shh expression was more restricted and asymmetric (supplementary material Fig. S2). Hopx was also expressed in the Shh-/non-ORS lower hair bulb cells (Fig. 4A, red arrowheads). The K5-expressing lower ORS was devoid of Hopx expression, but K15+ upper ORS cells expressed Hopx (Fig. 4E,F). By middle catagen (catagen V) (Müller-Röver et al., 2001), low levels of Hopx were detected in the K6+ cells surrounding the club hair; however, expression in this region was extinguished by late catagen (catagen VII-VIII; Fig. 4G,H) (Müller-Röver et al., 2001).

Fig. 4.

Hopx+ cells in the lower hair bulb give rise to K6+ cells in telogen. (A) Hopx is expressed in both Shh+ and Shh- cells (white and red arrowheads, respectively). (B-E) Hopx is also expressed in the companion layer (B, arrowheads), IRS (C) and hair shaft (D), but is not expressed in the lower ORS (E) at late anagen, P14. (F) Hopx is expressed in K15+ bulge cells (F, arrowhead). (G,H) In middle catagen, Hopx is expressed in K6+ cells surrounding the club hair (G, arrowhead), but the expression there is absent in late catagen (H). (I-M) HopxERCre/+;R26Tom/+ mice were pulsed with tamoxifen once at P14. Samples were harvested during anagen (P15; I,J), middle catagen (P17, K), late catagen (P19, L) and telogen (P21, M). The IRS, matrix, companion layer and lower hair bulb cells are labeled in anagen (I,J). (I) Whole-mount image and the green background is a pseudocolored bright-field image. (K,L) Cells surrounding the club hair are labeled in catagen, whereas the epithelial strand is not derived from Hopx+ cells. (M) Derivatives were found mostly in the innermost layer, whereas isolated cells were found in the outer bulge layer in telogen (white arrowhead). (N-Q) Hopx derivatives were found in the K6+ layer surrounding the club hair in middle and late catagen. The outermost Hopx-derived cells surrounding the club hair in middle catagen as well as the Hopx derivatives in late catagen were TUNEL negative. (R) Hopx-derived cells are found in the basal bulge (red arrowhead) but mostly in the innermost layer of cells (white arrowheads) in the following telogen. Scale bars: 25 μm.

Pulse labeling of HopxERCre/+;R26Tom/+ mice with a single dose of tamoxifen in late anagen (P14) allowed for the identification of Hopx derivatives. As expected, 1 day after a single pulse of tamoxifen, tdTomato-positive cells were identified in the lower hair bulb, matrix, companion layer, IRS and upper ORS (Fig. 4I,J). We did not detect labeled cells in the lower ORS (Fig. 4I). During catagen, derivatives were found in the layers surrounding the club hair, but not in the epithelial strand or lower ORS (Fig. 4K,L). The lack of derivatives in the epithelial strand is consistent with the absence of Hopx expression in the lower ORS (Hsu et al., 2011). Examination of HFs in the subsequent telogen stage revealed numerous fated cells in the innermost layer with rare derivatives in the outer layer (Fig. 4M, white arrowhead). A subset of Hopx-fate mapped cells in middle catagen surrounding the newly formed club hair expressed K6 (Fig. 4N). The outermost layer of K6+ cells surrounding the hair shaft, derived from Hopx+ precursors, was resistant to apoptosis (TUNEL-negative, Fig. 4O). In late catagen, a single layer of tdTomato+, K6+, TUNEL- cells surrounded the newly formed club hair (Fig. 4P,Q). During the following telogen (P21), tdTomato+ cells were found in the K6+ innermost layer (Fig. 4R, white arrowheads), as well as in the outer layer (Fig. 4R, red arrowhead). Similar results were obtained with lineage-tracing experiments conducted with adult HopxERCre/+;R26Tom/+ mice (tamoxifen pulse P35-36, sacrificed at P50; data not shown). It is unlikely that the late anagen bulge cells are responsible for the contributions to the K6+ innermost layer in telogen, as they have been shown to give rise to only the outer bulge and HG in the subsequent telogen (Ito et al., 2004; Greco et al., 2009; Hsu et al., 2011).

Previous studies have suggested that K6+ innermost telogen cells derive from lower ORS cells expressing Lgr5 and K14 in late anagen (Hsu et al., 2011). However, as noted above, Hopx is not expressed in the lower ORS in mature anagen. Therefore, we re-examined the fate of late anagen Lgr5+ cells using Lgr5EGFP-ERCre/+;R26Tom/+ mice (Barker et al., 2007) and an identical labeling protocol, as described for the analysis of Hopx derivatives (single pulse of tamoxifen at late anagen, P13; Fig. 5A). Consistent with previous reports (Hsu et al., 2011), we identified tdTomato+ derivatives in middle catagen in the outermost K6+ layer surrounding the club hair that were TUNEL negative (Fig. 5B,C). In contrast to Hopx lineage tracing, Lgr5 derivatives were also found in the ORS and epithelial strand (consistent with expression in the ORS), and, as expected, the derivatives were TUNEL positive (Fig. 5C). During late catagen, K6+ Lgr5-derived cells were resistant to apoptosis (Fig. 5D,E). In telogen, K6+ cells derived from Lgr5-expressing precursors were clearly evident, as previously reported (Hsu et al., 2011), as were Lgr5-derived lower basal layer and HG cells (Fig. 5F). Similar results were obtained when lineage-tracing experiments were conducted with K14ERCre/+;R26Tom/+ mice (Vasioukhin et al., 1999) (supplementary material Fig. S3). Taken together, these data suggest two possible interpretations for the source of K6+ innermost telogen cells. First, they may arise from at least two distinct sources in late anagen (either Lgr5+ or Hopx+ cells). Alternatively, there may be a population of cells that express both Lgr5 and Hopx during late anagen and give rise to K6+ niche cells in the subsequent telogen. Therefore, we investigated the relative expression of these factors in late anagen HFs, with particular attention to the ORS and the inner matrix where Shh is also expressed.

Fig. 5.

Lgr5 expression in the lower hair bulb and contribution to the telogen bulge. (A-F) Lgr5EGFP-ERCre/+;R26Tom/+ were pulsed with tamoxifen once at P13 and sacrificed at anagen (P15; A), middle catagen (P17; B,C), late catagen (P19; D,E) and the ensuing telogen (P21; F). (A) Cells were labeled in the lower hair bulb and ORS in late anagen. (B-E) In catagen, K6+, Lgr5-derived cells surrounding the club hair were TUNEL resistant (in contrast to derivatives in the epithelial strand). (F) In telogen, derivatives were found in the all layers of the bulge and the secondary HG. (G,H) Lgr5 expression was found in the ORS (K5+) and not in the companion layer (K6+) in late anagen, P14. Inset in H shows high magnification of companion layer adjacent to ORS. (I-K) Lgr5 is expressed in non-ORS, Shh- cells in the lower hair bulb (white arrowheads, I) and in the outermost K6+ layer surrounding the club hair in middle catagen (P17, J), whereas expression is absent from late catagen in K6+ cells (P19, K). (L) Lgr5 is expressed in the secondary HG and lower basal bulge in telogen, P21. (M) Hopx3FlagGFP/+;Lgr5EGFP-ERCre/+;R26Tom/+ were pulsed with one dose of tamoxifen at P14, sacrificed at P15. TdTomato+ cells were found in the lower hair bulb (white arrowheads), as well as the ORS (red arrowheads). White arrowheads demonstrate examples of a Flag+, tdTomato+ cell in the lower hair bulb. (N) Hopx3FlagGFP/+;Lgr5EGFP-ERCre/+;R26Tom/+ were pulsed with one dose of tamoxifen at P17 and sacrificed at P18, middle catagen. TdTomato+ cells were found in the epithelial strand (red arrowhead), whereas Flag+, tdTomato+ cells were found in the outermost layer of cells surrounding the club hair (white arrowheads). Confocal microscopy was used to image M and N. Scale bars: 25 μm; 10 μm in H (inset).

In accordance with previous reports (Jaks et al., 2008; Hsu et al., 2011), Lgr5-expressing cells were present in the ORS (K5+) and not in the companion layer (K6+) in late anagen (Fig. 5G,H). Surprisingly, however, we also identified Lgr5 expression in Shh-, lower hair bulb cells medial to the ORS (Fig. 5I). In middle catagen, the outermost K6+ layer of cells surrounding the club hair was Lgr5+ (Fig. 5J). However, by late catagen Lgr5 expression was present only in the ORS and epithelial strand (Fig. 5K). Lgr5+ cells in late catagen fate-mapped to the HG and basal layer of the stem cell compartment, but did not give rise to the K6+ innermost layer in telogen (supplementary material Fig. S4A). In telogen, Lgr5 was not expressed in the K6+ layer (Fig. 5L and supplementary material Fig. S4B).

To determine whether Lgr5 and Hopx are co-expressed in the lower hair bulb, we pulsed Hopx3FlagGFP/+;Lgr5EGFP-ERCre/+;R26Tom/+ mice with a single dose of tamoxifen at mature anagen and sacrificed the animals 24 hours later. (We chose this approach because, in our hands, available antibodies to Hopx, Lgr5 and Cre are unreliable in the skin, and available targeted alleles of both genes express GFP.) TdTomato expression was present in the ORS (Fig. 5M, red arrowheads) and closely resembled Lgr5 expression (supplementary material Fig. S4C,D), suggesting that most or all of the tdTomato+ cells were actively expressing Lgr5. Flag immunohistochemistry was specific to Hopx-expressing cells in the IRS and matrix (Fig. 5M). In the lower hair bulb, Flag and tdTomato double-positive cells were identified but not in the middle hair follicle (Fig. 5M; supplementary material Fig. S4E). We repeated this experiment in middle catagen; Hopx3FlagGFP/+;Lgr5EGFP-ERCre/+;R26Tom/+ mice were pulsed once with tamoxifen at P17 and sacrificed at P18. Our model predicts that double-positive cells will be found in the K6+ outermost layer surrounding the hair shaft in middle catagen, cells that are destined to become the K6+ niche cells in telogen. Indeed, we found multiple tdTomato cells co-expressing Hopx in the outermost layer of cells surrounding the club hair (Fig. 5N, white arrowheads). Taken together, these data suggest that Lgr5+, Hopx+ cells found in the lower hair bulb give rise to cells in catagen that are destined for the K6+ innermost layer in telogen.

Interestingly, our data suggest that the region of Hopx and Lgr5 co-expression in the lower hair bulb is immediately adjacent to, but non-overlapping with, Shh-expressing cells. Recent studies have shown that Shh+ cells in late anagen do not give rise to K6+ innermost cells in telogen (Greco et al., 2009; White et al., 2011), and our own fate-mapping studies using ShhERCre/+ (Harfe et al., 2004) mice confirmed this finding (supplementary material Fig. S5). Therefore, although we cannot rule out the possibility that multiple distinct populations of progenitor cells give rise to K6+ innermost cells or migration of Lgr5+ ORS cells to the lower hair bulb, the most parsimonious interpretation of these data is that Hopx+, Lgr5+, Shh- cells in the lower hair bulb give rise to these K6+ niche cells (Fig. 6A,B).

Fig. 6.

Contribution of anagen Hopx+ and Lgr5+ cells to the telogen bulge. Model depicting Hopx and Lgr5 expression (A) and their relative contributions to the hair follicle (B). (For clarity and comparison purposes, Hopx and Lgr5 expression and their relative contributions are depicted on opposite sides of the same hair follicle.) (A) Hopx is expressed in companion layer, IRS, matrix, lower hair bulb and basal bulge. It is absent from the lower ORS. Its expression is extinguished by late catagen and is limited to the basal bulge in telogen. Lgr5 is expressed in the ORS and lower hair bulb in late anagen and in the outermost K6+ layer in middle catagen. Its expression is limited to the ORS and epithelial strand in late catagen and to the secondary HG and lower basal bulge in telogen. (B) Hopx+ cells in late anagen will eventually give rise to the K6+ cells surrounding the club hair in catagen, which transition to the niche cells in telogen. Lgr5+ cells in late anagen will also give rise to the outermost K6+ layer surrounding the club hair in catagen and niche cells in telogen.

DISCUSSION

In this report, we demonstrate that Hopx is specifically expressed in the basal layer of the HFSC compartment of telogen phase hair follicles, and that Hopx-expressing cells can give rise to all the lineages of the hair follicle over multiple cycles of hair growth. Some Hopx+ cells are label retaining and contribute to regenerating epidermis after wounding. Therefore, these and other data indicate that Hopx expression identifies a HFSC population. Our studies demonstrate that Hopx+ cells at P5, during early follicle maturation, contribute to all differentiated lineages of the hair follicle in the adult. Sox9-expressing cells in the suprabasal layer of the hair germ during embryogenesis may also give rise to HFSCs, although lineage-tracing studies are necessary to confirm the contribution of these cells in the adult (Vidal et al., 2005; Nowak et al., 2008). Finally, our work also provides evidence for a previously unrecognized population of cells in the lower hair bulb (Hopx+, Lgr5+, Shh-) that can give rise to K6+ niche cells in telogen.

Hopx marks stem cell populations in multiple tissues

With the advent of inducible Cre-Lox technologies, the stem cell biology field has been able to identify a variety of markers for adult resident tissue-specific stem cell populations in many tissues (Simons and Clevers, 2011; Kretzschmar and Watt, 2012). Hopx appears to be one of only a few genes that identifies quiescent, self-renewing adult stem cell populations in multiple tissues. Hopx expression marks epithelial stem cells in the hair follicle and intestinal crypt (Takeda et al., 2011). Hopx is also expressed in neural progenitor cells of the adult brain (De Toni et al., 2008). Expression in the adult pancreas (Dorrell et al., 2011), lung (Yin et al., 2006), mammary gland and kidney (J.A.E., unpublished) has also been identified. Future investigations will address whether Hopx marks progenitor cells in these organs. Additional genes that mark multiple adult stem cell populations include Sox9 (intestine and skin) (Vidal et al., 2005; Nowak et al., 2008; Gracz et al., 2010; Furuyama et al., 2011), Bmi1 (pancreas and intestine) (Sangiorgi and Capecchi, 2008; Sangiorgi and Capecchi, 2009), Lrig1 (intestine and skin) (Jensen and Watt, 2006; Jensen et al., 2009; Powell et al., 2012; Wong et al., 2012) and Lgr5 (intestine and skin) (Barker et al., 2007; Jaks et al., 2008). This conservation of gene expression raises the intriguing possibility that there may be shared homeostatic mechanisms regulated by these genes among tissue-specific stem cell populations, which is beyond the scope of this manuscript and will be addressed in future studies.

Several markers have been identified to mark the skin stem cell compartment, including CD34 (Albert et al., 2001; Trempus et al., 2003; Blanpain et al., 2004), Sox9 (Nowak et al., 2008), Tcf3 (Nguyen et al., 2006), Lhx2 (Rhee et al., 2006), K15 (Lyle et al., 1998; Liu et al., 2003), Lrig1 (Jensen and Watt, 2006; Jensen et al., 2009), Lgr5 (Jensen et al., 2009) and Lgr6 (Snippert et al., 2010). Many of these have overlapping expression domains, but careful inspection identifies subtle differences. Hopx appears to be specific to the basal bulge layer and is absent from the K6+ layer in telogen, in contrast to Sox9, Lhx2, K15 and Tcf3, which mark both layers (Hsu et al., 2011). The secondary hair germ robustly expresses Lgr5, whereas Hopx may be weakly expressed there, although we cannot exclude the possibility of GFP perdurance. Therefore, combinations of markers may be useful to best identify subsets of stem cell populations and allow for the dissection of the heterogeneity of cells composing stem cell compartments in the skin. Ongoing studies are aimed at elucidating the function of Hopx in progenitor populations.

Hopx expression defines a previously unrecognized progenitor pool in anagen

Hopx is expressed in the companion layer as well as in a Shh-, lower hair bulb population of cells in anagen, which is marked by K6 mRNA expression (Winter et al., 1998). Recent studies have suggested the existence of a distinct population of cells in the lower hair bulb in position 4-5 that can give rise to the companion layer (Sequeira and Nicolas, 2012), but the additional functions and potential of these cells, especially during hair follicle cycling, is unknown. Our lineage-tracing studies demonstrate that a Hopx+, Shh- population of lower hair bulb cells in anagen gives rise to the K6+ innermost layer of the telogen bulge. Previous reports have shown that Lgr5-expressing cells in the lower ORS are one source of K6+ niche cells in telogen (Hsu et al., 2011), but Hopx is not expressed in the lower ORS. It is possible that two distinct progenitor populations can give rise to K6+ niche cells, but our data suggesting the existence of Lgr5+, Hopx+, Shh- lower hair bulb cells provides an alternative explanation that reconciles our findings with prior observations. We suggest that Hopx+, Lgr5+ cells in the lower hair bulb can give rise to K6+ niche cells. Alternatively, we cannot exclude the possibility that Lgr5+ lower ORS cells migrate into the lower hair bulb and subsequently activate Hopx expression. However, we believe this is less likely in light of recent data indicating that the ORS and the lower hair bulb represent distinct lineages (Sequeira and Nicolas, 2012). As the lower hair bulb cells transition/differentiate into K6-protein expressing cells surrounding the club hair during catagen, Hopx and Lgr5 expression is extinguished whereas Sox9, Lhx2 and Tcf3 expression commences (DasGupta and Fuchs, 1999; Vidal et al., 2005; Rhee et al., 2006; Hsu et al., 2011; supplementary material Fig. S6). These latter HFSC markers remain expressed into telogen as the innermost K6+ layer fully matures. It should also be noted that Shh expression marks the matrix, and lineage tracing of Hopx- or Shh-expressing cells demonstrates that they give rise to the IRS. A subset of Shh+ cells in anagen co-expresses Hopx (Fig. 4A, white arrowhead).

During anagen, the upper companion layer expresses K6 protein whereas the lower companion layer and hair bulb express K6 mRNA exclusively (Winter et al., 1998). In telogen, the niche cells express K6 protein. The shared expression of K6 by the companion layer and the niche has led to the investigation of potential similarities and differences among these populations of cells and raised the issue of whether there is a shared lineage relationship (Higgins et al., 2009). In fact, K6-expressing cells in telogen and anagen, as well as the cells surrounding the club hair in catagen, share a common molecular signature [expression of K6 (Winter et al., 1998; Wang et al., 2003; Hsu et al., 2011), PAI-2 (Lavker et al., 1998), calretinin (Poblet et al., 2005) and S100A6 (Ito and Kizawa, 2001)]. The companion layer is composed of thin long cells adjacent to the IRS, whereas the K6+ niche (and catagen K6+) cells are cuboidal (Hsu et al., 2011). Although they exhibit morphological differences, both layers remain adjacent to the ORS and appear to serve anchorage functions (Hsu et al., 2011; Chapalain et al., 2002). However, although the companion layer and K6+ niche cells share some gene expression characteristics, our data demonstrate that the K6 protein-expressing companion layer is not the source of the K6+ niche cells in telogen.

In summary, our data demonstrate that Hopx is a unique marker of multiple stem cell populations and marks a subset of known stem cell populations in the telogen hair follicle. Our data in anagen demonstrate the presence of a previously unappreciated progenitor population residing in the anagen lower hair bulb that expresses both Hopx and Lgr5, and gives rise to K6-expressing cells in telogen that are responsible for stem cell homeostasis, providing another example in which the progeny of a lineage cycles back to regulate its stem cell compartment.

Acknowledgments

We thank the Epstein laboratory for helpful discussions, the CDB Microscopy core for confocal imaging and the Penn SDRC Stem Cell Core for help with isolation of cells.

Footnotes

  • Funding

    This work was supported by the National Institutes of Health (NIH) [2T32 HL007843-16]; National Marfan Foundation (R.J.) and the NIH [R01 HL071546, U01 HL100405]; the Spain fund for Regenerative Medicine; and the W. W. Smith Endowed Chair (J.A.E.). Deposited in PMC for release after 12 months.

  • Competing interests statement

    The authors declare no competing financial interests.

  • Supplementary material

    Supplementary material available online at http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.093005/-/DC1

  • Accepted February 4, 2013.

References

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