Axin2 Marks Quiescent Hair Follicle Bulge Stem Cells That Are Maintained by Autocrine Wnt/Β-Catenin Signaling

Axin2 marks quiescent hair follicle bulge stem cells that are maintained by autocrine Wnt/β-catenin signaling

Xinhong Lima,b,c,d,e,1, Si Hui Tana,c,f, Ka Lou Yua, Sophia Beng Hui Lima, and Roeland Nussec,d,1

aInstitute of Medical Biology, Agency for Science, Technology, and Research, Singapore 138648; bLee Kong Chian School of Medicine, Nanyang Technological University, Singapore 639798; cHoward Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305-5329; dDepartment of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305-5329; eProgram in Cancer and Stem Cell Biology, Duke- NUS Graduate Medical School, Singapore 169857; and fCancer Biology Program, Stanford University School of Medicine, Stanford, CA 94305-5329

Contributed by Roeland Nusse, January 28, 2016 (sent for review December 16, 2015; reviewed by Salvador Aznar-Benitah and Cheng-Ming Chuong) How stem cells maintain their identity and potency as tissues formation (9). Wnt signals are needed later during postnatal change during growth is not well understood. In mammalian hair, homeostasis as well, for the initiation of anagen in postnatal hair it is unclear how hair follicle stem cells can enter an extended (10). Therefore, in view of their well-established importance for period of quiescence during the resting phase but retain stem cell stem cell maintenance in multiple adult tissues, including the potential and be subsequently activated for growth. Here, we use skin (11), Wnts are candidate hair follicle stem cell (HFSC)- lineage tracing and gene expression mapping to show that the maintaining signals. However, Wnt signaling is generally be- Wnt target gene Axin2 is constantly expressed throughout the lieved to be inactive in the telogen bulge (8, 10, 12), which is hair cycle quiescent phase in outer bulge stem cells that produce thought to be quiescent. Wnt signaling becomes strongly elevated their own Wnt signals. Ablating Wnt signaling in the bulge cells when bulge cells are “activated” to undergo the transition from causes them to lose their stem cell potency to contribute to hair telogen to anagen (13, 14). During anagen, Wnt signaling has growth and undergo premature differentiation instead. Bulge cells been described to primarily specify differentiated cell fates in the express secreted Wnt inhibitors, including Dickkopf (Dkk) and se- anagen follicle (12, 15). As anagen proceeds and the follicle creted frizzled-related protein 1 (Sfrp1). However, the Dickkopf 3 enters catagen and telogen again, the bulge is thought to revert (Dkk3) protein becomes localized to the Wnt-inactive inner bulge to a Wnt-inhibited state (12, 13, 16, 17). that contains differentiated cells. We find that Axin2 expression Conversely, there is evidence for a functional requirement of remains confined to the outer bulge, whereas Dkk3 continues to Wnt/β-catenin signaling in the bulge other than initiating anagen be localized to the inner bulge during the hair cycle growth phase. and specifying differentiation during anagen. For instance, post- Our data suggest that autocrine Wnt signaling in the outer bulge natal deletion of β-catenin in outer bulge cells results in the loss of maintains stem cell potency throughout hair cycle quiescence and label-retention and HFSC markers, suggesting that β-catenin is growth, whereas paracrine Wnt inhibition of inner bulge cells required for maintenance of HFSC identity (10). reinforces differentiation. Here, beyond its role in hair differentiation and anagen initiation, we sought to determine whether Wnt/β-catenin signaling Wnt signaling | hair follicle | stem cells | homeostasis | regeneration is also involved in HFSC maintenance during telogen. We found that Axin2 expression persists in HFSCs in the outer bulge he hair follicle is a complex miniorgan that repeatedly cycles throughout telogen and anagen, suggesting that active Wnt Tthrough stages of rest (telogen), growth (anagen), and de- struction (catagen) throughout life (1). During anagen, growing Significance hair follicles emerge adjacent to the old telogen hair follicles that remain there throughout the cycle and create an epithelial pro- Hair follicle stem cells (HFSCs) remain quiescent for long pe- “ ” trusion known as the bulge. At the end of the hair cycle, in riods of time during the resting phase of the hair cycle. How catagen, cells from the follicle migrate along the retracting epi- they maintain their stemness and identity during quiescence thelial strand and join the two epithelial layers of the telogen while being responsive to growth-inducing cues remains poorly — — bulge the inner and outer bulge layers surrounding the club understood. Here, we identify Axin2 as a previously unidentified hair shaft (2). marker of HFSCs and use it to show that quiescent HFSCs un- Several studies have established that stem cells residing in the dergo and require active Wnt/β-catenin signaling. By mapping outer bulge are the source of the regenerative capacity of the Wnt and its inhibitors with high sensitivity, we show that HFSCs cycling hair follicle (3–5). During telogen, these stem cells are secrete their own self-renewing Wnt signals and inhibitors that thought to be generally quiescent (6). In response to signals from promote differentiation outside of the stem cell compartment. their microenvironment during anagen, the stem cells divide and Our findings suggest that careful modulation of Wnt signaling produce proliferative progeny that participate in the growth of may be important for the derivation and maintenance of HFSCs the new follicle (7). Some of these activated stem cells and their for alopecia treatment and drug screens. progeny are believed to migrate away from the bulge, but are subsequently able to rejoin it after anagen is complete (2, 5). Author contributions: X.L. and R.N. designed research; X.L., S.H.T., K.L.Y., and S.B.H.L. performed research; X.L. and S.H.T. contributed new reagents/analytic tools; X.L., S.H.T., Cells that return to the outer bulge take on a follicular stem cell K.L.Y., S.B.H.L., and R.N. analyzed data; and X.L., S.H.T., K.L.Y., and R.N. wrote the paper. identity, ready to divide and participate in the next hair cycle (2, Reviewers: S.A.-B., Institute for Research in Biomedicine, Barcelona; and C.-M.C., Univer- 8). Conversely, cells returning to the inner bulge do not divide sity of Southern California. and, instead, form an inner bulge niche of differentiated cells for The authors declare no conflict of interest. the outer bulge cells (2). Stem cells remain quiescent during Freely available online through the PNAS open access option. telogen for an extended period, and the identity of signals that 1To whom correspondence may be addressed. Email: [email protected] or maintain stem cell identity during this time are poorly understood. [email protected]. β In the hair, Wnt/ -catenin signaling is required right from the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. earliest stages of development, for the initiation of hair placode 1073/pnas.1601599113/-/DCSupplemental.

E1498–E1505 | PNAS | Published online February 22, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1601599113 Downloaded by guest on September 27, 2021 signaling is a consistent feature of bulge stem cells. Furthermore, telogen (P49) and then tracked the fate of these cells and their PNAS PLUS these hair outer bulge stem cells produce autocrine Wnts and progeny for various lengths of time (Fig. 1C). Consistent with our paracrine-acting Wnt inhibitors that may specify the positional observations described above (Fig. 1 A and B), Axin2–CreERT2- identity of cells residing within the bulge niche. labeled cells in the bulge persisted from early to mid-telogen (Fig. 1D). As the hair follicles entered anagen, the labeled cells Results appeared to accumulate in the growing secondary hair germ and To determine whether Wnt/β-catenin signaling is active during migrate along the elongating outer root sheath and into the the telogen stage, we examined telogen follicles for the expres- developing matrix, where they lay adjacent to the dermal papilla sion of Axin2, a well-established Wnt/β-catenin target gene (18, and generated progeny spanning all of the layers of the anagen 19), using RNA in situ hybridization. We found that Axin2 was hair follicle (Fig. 1E). These cells were able to participate in expressed mostly in telogen outer bulge cells (Fig. 1A). Similarly, successive cycles of hair follicle growth up to a year after the when we examined telogen hair from Axin2–lacZ Wnt reporter initial labeling (Fig. 1F). We observed the same patterns of Axin2 mice, we observed clear reporter activity, specifically in the outer expression and long-term, self-renewing potential of outer bulge bulge (Fig. 1B). To determine whether Wnt signaling activity cells labeled during the first telogen (P21) occurring immediately varies during telogen, we looked at Axin2 mRNA expression after morphogenesis (Fig. S1 B–K), suggesting that Axin2- during early [postnatal day 43 (P43)], mid (P56), and late (P69) expressing outer bulge cells are present during successive telogen telogen using RNA in situ hybridization. We found that Axin2 cycles of hair growth. These data suggest that Axin2–CreERT2 mRNA is expressed in the bulge throughout telogen (Fig. S1A), labels long-lived HFSCs in the outer bulge. suggesting that Wnt signaling persists through all of the Hair bulge cells are thought to be quiescent during early sec- telogen phases. ond telogen (23). To determine whether Axin2-expressing bulge Because HFSCs are known to reside in the outer bulge (3, 5, cells were indeed quiescent cells, we performed cell-cycle analysis 20), we asked whether Axin2-expressing telogen bulge cells have on Axin2-expressing bulge cells harvested by flow cytometry from stem cell properties. We permanently labeled Axin2-expressing Axin2–lacZ reporter mice. Almost all of these cells exhibited a cells in the telogen bulge by exposing Axin2–CreERT2 Rosa26– G0/G1 nuclear profile (>90%; Fig. 1G), suggesting that Axin2- mTmGflox mice (21, 22) to tamoxifen (Tam) during early second expressing telogen bulge cells are quiescent.

AB C Axin2 mRNA Axin2-lacZ

Tam F injection E Chase D period

P49 P56 P88 P414

DE F G P56 P88 P88 P88 P414 P414 P414

Fig. 1. Axin2-expressing cells in the telogen bulge are quiescent HFSCs. (A and B) Axin2 mRNA (A) and Axin2–lacZ (B) expression in the early telogen hair bulge (P43). Dotted lines denote approximate outer boundary of the hair bulge. [Scale bars: 10 μm(Axin2 mRNA); 20 μm(Axin2lacZ).] (C) Schematic depicting

pulse-chase experiment to track the fate of Axin2+ cells and their progeny. (D–F) Histological sections of hair follicles from Axin2–CreERT2/Rosa26–mTmGflox BIOLOGY

mice chased from P49 for 1 wk (P56, mid-telogen; D), 39 d (P88, anagen; E), and 1 y (P414; F). Dotted lines denote approximate outer boundary of the hair DEVELOPMENTAL bulge. (Scale bars: 10 μm.) (G) Cell-cycle analysis of Axin2+ bulge cells by propidium iodide staining using flow cytometry.

Lim et al. PNAS | Published online February 22, 2016 | E1499 Downloaded by guest on September 27, 2021 ABCD Axin2 mRNA Axin2 mRNA het cKO

het cKO

Fig. 2. Bulge cells functionally require β-catenin for Axin2 expression and to maintain hair-forming potency. (A and B) Representative images of RNA in situ Δ + Δ hybridization for Axin2 gene expression as performed on Axin2–CreERT2/β-catenin ex2-6-fl/ (het; A) and mutant Axin2–CreERT2/β-catenin ex2-6-fl/fl (cKO; B) hair follicles. Dotted lines denote approximate outer boundary of the hair bulge. (Scale bars: 20 μm.) (C and D) Representative images of eosin-stained sections of control Axin2–CreERT2/β-cateninΔex2-6-fl/+ (het; C) and mutant Axin2–CreERT2/β-cateninΔex2-6-fl/fl (cKO; D) dorsal skins. (Scale bars: 20 μm.)

To test whether Axin2 is indeed a Wnt/β-catenin signaling bulge HFSCs themselves are a source of Wnt signals in the target gene in the hair bulge, we conditionally inactivated the telogen bulge. β-catenin gene in Axin2-expressing cells during telogen by crossing How is Wnt gene expression in the bulge regulated? When we Δ Axin2–CreERT2 mice with β-cat ex2-6 floxed mice. Axin2 looked at Wnt gene expression using RNA in situ hybridization Δ mRNA expression that was present in heterozygous Axin2– in Axin2–CreERT2/β-cat ex2-6-fl/fl mutant bulges, we found a loss Δ + CreERT2/β-cat ex2-6-fl/ control hair bulge cells was abrogated in of Wnt4 mRNA expression compared with Axin2–CreERT2/ Δ Δ + Axin2–CreERT2/β-cat ex2-6-fl/fl mutant bulge cells (Fig. 2 A and β-cat ex2-6-fl/ control bulges (Fig. 3D). This is similar to other B) cells. This result was consistent with the observation that reports where β-catenin deletion in keratin-15–expressing bulge Axin2 expression is also greatly decreased when β-catenin is cells also resulted in a great reduction in Wnt7b expression (Fig. deleted in Keratin-15–expressing bulge cells (Fig. S2B)(14). S2B) (14). Together, these data suggest that production of at Thus, Axin2 expression in the outer bulge cells is dependent least some Wnts is regulated by and requires autocrine Wnt/ on the presence of β-catenin. Furthermore, other Wnt target β-catenin signaling in the outer telogen bulge. genes like Lgr5 are also expressed in the outer bulge (Fig. S2A) Is Wnt secretion required for hair growth? We observed that (5) and similarly require β-catenin for their expression (Fig. S2B) Wntless, a Wnt cargo-binding protein required for Wnt secretion, (14). The expression of multiple Wnt target genes suggests that is expressed in bulge cells (Fig. 4A), consistent with previous Wnt/β-catenin signaling is active in the telogen bulge and that reports (25, 26). Wntless is also expressed during hair de- Axin2 expression is a reliable indicator of Wnt signaling activity in velopment (27), and our lineage tracing experiments show hair follicles. that embryonic Axin2-expressing cells become HFSCs (Fig. The ability to produce differentiated cells is a defining prop- S1L). Conditional knockout of Wntless from this stage in mutant erty of stem cells. We next asked whether Axin2-expressing HFSCs Axin2–rtTA/tetO–Cre/Wntlessfl/fl mice resulted in much less hair require Wnt/β-catenin signaling to function as stem cells by pro- and thickened epidermis in several regions compared with their ducing differentiated hair lineages during the anagen stage of heterozygous littermate controls (Fig. 4 B and C), suggesting that Δ + hair growth. In heterozygous Axin2–CreERT2/β-cat ex2-6-fl/ con- Wntless is required by Axin2-expressing cells for hair growth. This trol littermates, hair follicles entered anagen normally (Fig. 2C). In finding is consistent with other studies where hair is lost and Δ contrast, Axin2–CreERT2/β-cat ex2-6-fl/fl mutant hair follicles did not remaining hairs are arrested in telogen when Wntless is deleted grow, and most exhibited an abnormal telogen-like morphology in the entire epidermis (25–27) and also specifically in the hair (Fig. 2D). This result resembles the arrested hair growth seen in bulge (24, 25). Together, these data and the literature suggest that animals where β-catenin is mutated in all outer bulge cells (10, 24). autocrine Wnt signaling is required by HFSCs for hair growth The loss of hair follicle growth suggests that Axin2-expressing bulge and maintenance. HFSCs require β-catenin to maintain their stem cell potency/state. To determine whether Wnt production and β-catenin signaling To determine the source of Wnt signals for these Wnt-depen- change as the hair enters the anagen growth phase, we examined dent HFSCs, we screened for the expression of Wnt genes at follicles at the telogen-to-anagen transition and found that various stages of the hair cycle using RNA in situ hybridization Axin2, Wnt1, Wnt4, and Wnt7b continued to be expressed in the (Fig. 3 A–C). We found that Wnt1, Wnt4, and Wnt7b were ex- bulge (Fig. 3C). However, the expression of several Wnts, in- pressed in the bulge throughout telogen. The highest levels of cluding Wnt6, Wnt7b, Wnt10a, and Wnt10b, became up-regulated expression appeared to be in outer bulge cells. Consistent with in cells at the boundary of the secondary hair germ and the our Axin2 mRNA expression data, these Wnts remain expressed dermal papilla (Fig. S3C), consistent with the strong expression throughout telogen. In comparison, we did not detect any Wnt5a of Axin2 in the secondary hair germ and previous reports (7, 28). expression in the bulge throughout telogen, whereas other Wnts, This result suggests that specific Wnts may be involved in the including Wnt3, Wnt6, Wnt10a, and Wnt10b, are expressed, but at epithelial–mesenchymal cross-talk in secondary hair germ cells much lower levels (Fig. S3). These data suggest that the outer needed to initiate anagen, as has previously been proposed (28).

E1500 | www.pnas.org/cgi/doi/10.1073/pnas.1601599113 Lim et al. Downloaded by guest on September 27, 2021 A PNAS PLUS Negative Control Wnt5a mRNA Wnt1 mRNA Wnt4 mRNA Wnt7b mRNA

P43 P43 P43 P43 P43 B Negative Wnt5a mRNA Wnt1 mRNA Wnt4 mRNA Wnt7b mRNA Control

P57 P57 P57 P57 P57 C D Negative Control Wnt5a mRNA Wnt1 mRNA Wnt4 mRNA Wnt7b mRNA Wnt4 mRNA

P69 P69 P69 P69 P69 het cKO

Fig. 3. Bulge cells express Wnts throughout telogen. (A–C) Representative images of RNA in situ hybridization for Wnt gene expression as performed on hair follicles during early (P43; A), mid (P57; B), and late (P69; C) second telogen. (D) Representative images of RNA in situ hybridization for Wnt4 gene expression Δ + Δ as performed on Axin2–CreERT2/β-catenin ex2-6-fl/ (het) and mutant Axin2–CreERT2/β-catenin ex2-6-fl/fl (cKO) hair follicle bulges. Dotted lines denote approximate outer boundary of the hair bulge. (Scale bars: 20 μm.)

We next asked whether the bulge becomes Wnt-inhibited expression of Wnt4 and Wnt7b in the anagen bulge (Fig. 5 D and during anagen, as is widely believed (12, 13, 16, 17). Contrary to F). This finding is in contrast to other Wnt genes like Wnt5a, expectations, we observed that Axin2 mRNA and the Axin2–lacZ Wnt6, Wnt10a, and Wnt10b that are expressed elsewhere in the reporter gene continued to be expressed in the anagen bulge anagen follicle (Fig. S4C), but not in the anagen bulge (Fig. 5E). (Fig. 5 A–C and F). At the same time, we found the persistent By double-labeling RNA in situ hybridization, we found that

AB C Wls mRNA Wls-fl/+ Wls-fl/fl Wls-fl/+

Wls-fl/fl

n = 4 n = 4

Fig. 4. Axin2-expressing cells require Wnt secretion for hair growth. (A) Representative image of RNA in situ hybridization for Wntless gene expression as BIOLOGY μ

performed on telogen hair follicles. Dotted line denotes approximate outer boundary of the hair bulge. (Scale bar: 20 m.) (B and C) Representative images of DEVELOPMENTAL the whole body (B) or H&E-stained sections of the skins (C)ofAxin2–rtTA/tetO–Cre/Wls fl/+ or fl/fl mice. (Scale bars: 1 cm.)

Lim et al. PNAS | Published online February 22, 2016 | E1501 Downloaded by guest on September 27, 2021 A BC Axin2 mRNA Axin2-lacZ Anagen Bulge

inner bulge outer

outer root sheath

D E Negative control Wnt4 mRNA Wnt7b mRNA Wnt5a mRNA Wnt6 mRNA Wnt10a mRNA Wnt10b mRNA

F Negative Control Wnt4 mRNA Wnt7b mRNA Negative Control Axin2 mRNA Axin2 mRNA

Fig. 5. Anagen hair bulge cells express Axin2 and Wnts. (A) Schematic depicting cellular organization of the anagen follicle bulge. (B–E) Representative images of Axin2–lacZ (B) and RNA in situ hybridization (C)forAxin2 and Wnt gene expression (D and E) as performed on anagen hair follicles. Dotted lines denote approximate outer boundary of the hair bulge. Dotted lines denote approximate outer boundary of the hair bulge. [Scale bars: 20 μm(Axin2–lacZ image); 10 μm (RNA in situ hybridization images).] (F) Representative images of double-labeling RNA in situ hybridization in anagen hair follicles for Axin2 (red spots) and Wnt4 or Wnt7b (turquoise spots). Arrows indicate bulge cells expressing both Axin2 and Wnts. Boxes show magnified view of individual bulge cells expressing both Axin2 and Wnts. Dotted lines denote approximate outer boundary of the hair bulge. (Scale bars: 20 μm.)

Axin2-expressing bulge cells also expressed Wnt4 and Wnt7b (Fig. phase (Fig. 6B). This finding is consistent with other studies of 5F). These data suggest that the anagen bulge is not Wnt- gene expression in isolated mouse bulge cells (3, 16) and is also inhibited and that Wnt production and β-catenin signaling persist similar to human hair follicle bulges, where both Dkk3 and in the anagen bulge. In addition, we found Axin2 (Fig. S4A) and SFRP1 are expressed in the outer bulge, but are either at very distinct Wnt gene expression patterns (Fig. S4 B and C)in low levels or not present in the inner bulge (Fig. 6C)(29). multiple differentiated lineages of the anagen hair follicle, con- When we performed antibody staining for the Dkk3 protein in sistent with previous reports (28). Other Wnts like Wnt7a and the telogen bulge, however, we found that the Dkk3 protein is Wnt16 were present either at very low levels or were not expressed localizedtotheinnerbulge(Fig.6D). This distribution of Dkk3 (Fig. S4D). Specific Wnts may thus play distinct functional roles in mRNA and protein in the bulge persists even through anagen the different compartments of the anagen hair follicle. (Fig. 6E), suggesting that HFSC stemness is restricted to the Our data show that Wnt signaling, as reported by Axin2 ex- outer bulge by constant inhibition of Wnt signaling in the inner pression, is restricted to the outer bulge (Fig. 1 A and B). If Wnt bulge cells. signals are available in the bulge, then why do inner bulge cells not also respond to Wnt and proliferate? In seeking to address Discussion this question, we found evidence for active Wnt inhibition of Our data lead us to propose a model in which Wnt/β-catenin the inner bulge. Using RNA in situ hybridization, we saw that signaling is a constant and persistent feature of the HFSC niche several secreted Wnt inhibitors, including Dkks and Sfrp1, are (Fig. 7). HFSCs located in the outer bulge are Wnt-dependent expressed in the outer bulge during telogen (Fig. 6A). Expres- and produce Wnt signals that act locally, while simultaneously sion of some of these Wnt inhibitors persists into the anagen inhibiting Wnt/β-catenin signaling in the inner follicle bulge by

E1502 | www.pnas.org/cgi/doi/10.1073/pnas.1601599113 Lim et al. Downloaded by guest on September 27, 2021 secreting Wnt inhibitors like Dkks and Sfrp1 (Fig. 7). This Wnt signaling, which in turn promotes the transition from telogen PNAS PLUS autocrine Wnt activation and paracrine Wnt inhibition may help to anagen (23, 32). to maintain the discrete identity of cells located in each bulge Our finding of Axin2–lacZ Wnt reporter gene expression in layer, even as cells traffic dynamically to and from the bulge the telogen follicle bulge contrasts with the lack of activity in niche (2) throughout multiple hair cycles. Such a model predicts other TCF-element-based Wnt reporter constructs such as that loss of Wnt/β-catenin signaling in the outer bulge would result TOPGAL (13). The more limited expression pattern of TOPGAL in disrupted compartmentalization of the inner vs. outer bulge. in hair follicles may result from endogenous Wnt-responsive Consistent with this prediction, we find that Keratin-6, which is cis-regulatory elements being absent in the TOPGAL reporter typically enriched in the inner bulge and is considered to be a construct (33). Consistent with this hypothesis, Axin2–lacZ is marker of that compartment (Fig. S2 C and D) (2), becomes more expressed widely in the anagen follicle in domains of Wnt mRNA strongly expressed in outer bulge cells upon deletion of β-catenin expression, whereas TOPGAL is not. That Axin2 is a more in Axin2-expressing outer bulge cells (Fig. S2 B and E) (14). sensitive and comprehensive Wnt reporter than TOPGAL is also If HFSCs generate their own source of Wnt, and Wnt pro- observed in other stem cell niches such as the intestine, where duction is regulated in an autocrine manner, what keeps hair Axin2 expression marks stem cells (22), even though TOPGAL follicles in telogen and prevents them from undergoing autocrine expression is absent. The expression of other Wnt target genes amplification of Wnt signaling to initiate anagen? A likely such as Lgr5 in the bulge further supports the notion that Wnt/ candidate is BMP signaling, which has been shown to inhibit Wnt β-catenin signaling is active in the bulge, and that Axin2 is a signaling and hair growth (30). It is thought that the reciprocal reliable, sensitive, and comprehensive reporter of Wnt/β-catenin interplay between BMP and Wnt signaling controls HFSC ac- signaling in the hair follicle. tivity and fate decisions and that BMP signaling suppresses Wnt Whether or not Wnt/β-catenin signaling is required for stem production (31). We found some evidence that Wnt signaling cell maintenance has been a matter of debate. Previous studies controls BMP pathway component expression. After deletion of showed that deletion of β-catenin in the bulge results in loss of β-catenin in Axin2-expressing bulge cells, we observed slightly hair stem cell identity, as determined by loss of both proliferation increased expression of several BMP antagonists, including Noggin, and expression of outer bulge cell markers like CD34 (10). More Bambi, and Gremlin1 (Fig. S2B), suggesting that autocrine Wnt recently, however, others have shown that CD34 and Keratin-15 signaling suppresses BMP antagonist expression. This finding leads continue to be expressed even after β-catenin deletion and have us to speculate that a low level of autocrine Wnt signaling in HFSCs proposed instead that Wnt/β-catenin signaling is not required for reduces BMP inhibition and thus permits BMP signaling, creating a maintenance of stem cell identity but rather to induce anagen feedback inhibition loop that moderates Wnt signaling levels and (14, 24). However, it is unclear that the expression of these genes prevents autocrine entry into anagen. As environmental BMP levels is a reliable indicator of stem cell identity or function. Indeed, are reduced, autocrine amplification could then lead to elevated when CD34 is knocked out, hair follicles continue to grow, suggesting

A B C Dkk3 mRNA Dkk4 mRNA Sfrp1 mRNA Dkk3 mRNA Sfrp1 mRNA

telogen telogen telogen anagen anagen D E Dkk3 Axin2-CreERT2 Dkk3 Axin2-CreERT2 mRNA Dkk3 protein mRNA Dkk3 protein telogen Dkk3 Axin2 anagen Dkk3 Axin2

Fig. 6. Outer bulge cells express secreted Wnt inhibitors. (A and B) Representative images of RNA in situ hybridization for Dkk3, Dkk4, and Sfrp1 during telogen (A) and anagen (B). Dotted lines denote approximate outer boundary of the hair bulge. (Scale bars: 20 μm.) (C) Dkk3 and Sfrp1 expression in laser- capture microdissected primary outer vs. inner bulge cells from human hair follicles (error bars, SD). Expression values are from Gene Expression Omnibus accession no. GSE3419. (D and E) Representative images of Dkk3 immunostaining in telogen (D) and anagen (E) bulges of Axin2–CreERT2/Rosa26–mTmGflox

mice. Representative images of RNA in situ hybridization for Dkk3 in telogen and anagen bulges are shown for comparison. Dotted lines denote approximate BIOLOGY μ μ

outer boundary of the hair bulge. [Scale bars: 20 m (telogen Dkk3 RNA in situ hybridization image); 10 m (anagen Dkk3 RNA in situ hybridization and Dkk3 DEVELOPMENTAL immunostaining images).]

Lim et al. PNAS | Published online February 22, 2016 | E1503 Downloaded by guest on September 27, 2021 A B C Telogen Telogen-Anagen Anagen transition

Wnt1, Wnt1, Wnt4, Wnt4, Wnt4, stem Wnt7b Wnt7b Wnt7b cell maintenance Dkk3 Dkk3 Dkk3 Dkk4 Dkk4 Wnt6, Wnt7b, Dkk4 Sfrp1 Sfrp1 Wnt10a,Wnt10b Sfrp1

Wnt-active (Axin2+) Hair germ-dermal paplla boundary = Wnt5a Wnt-inhibited (Dkk protein+) = Wnt4, hair Wnt6, lineage Wnt7b specification Wnt10a, Wnt10b

Fig. 7. Model: Dual roles for Wnt/β-catenin signaling in HFSC maintenance and lineage determination. (A) During telogen, Wnt-responding stem cells in the outer bulge produce autocrine Wnt1, Wnt4,andWnt7b, and paracrine-acting secreted Wnt inhibitors like Dkk3, Dkk4, and Sfrp1. This expression pattern persists throughout the hair cycle, potentially to maintain the niche signaling microenvironment. (B) At the telogen–anagen transition, Wnt6, Wnt10a,and Wnt10b become strongly expressed in cells at the boundary of the hair germ and dermal papilla. (C) These and other Wnts continue to be expressed in various hair layers proximal to the dermal papilla during anagen and are likely involved in hair lineage specification.

that it is not functionally required for hair stem cell function (34). specified below. Axin2–CreERT2/Rosa26–mTmGflox mice received a single Additionally, Keratin-15 expression persists in follicles of balding dose of Tam at P21 or 49 (P49), totaling 1 mg per 25 g of body weight.

patients, whereas the expression of another HFSC marker, CD200,is Δ + – Conditional Knockout of β-Catenin and Wntless. Axin2–CreERT2/β-catenin ex2-6-fl/ lost (35). As such, although CD34-andKeratin-15 expressing cells – β Δex2-6-fl/fl β – and mutant Axin2 CreERT2/ -catenin animals were injected with a single may persist in -catenin deleted bulges, it is unclear that they re- dose of Tam corresponding to 4 mg per 25 g of body weight at 3 wk of age. main as stem cells. Skins were harvested and processed for histology upon morbidity or death after Stem cells are functionally defined by their potency rather 10–11 d of chase. For Wntless ablation, mothers carrying Axin2–rtTA/tetO–Cre/ + than by marker expression (36–38), and a more reliable indicator Wls fl/ and mutant Axin2–rtTA/tetO–Cre/Wls fl/fl pups were injected with a single of HFSC identity is a test of its ability to contribute to hair follicle dose of Tam corresponding to 1 mg per 25 g of body weight at embryonic day growth. Consistent with our own data (Fig. 2), all of the published 14.5 (E14.5). Skins were harvested and processed for histology when pups were studies we know of involving conditional ablation of β-catenin in at postnatal age 1 mo. the bulge result in the hair follicle being arrested in telogen and no β X-Gal Staining. Tissues were harvested and fixed in 4% (vol/vol) para- longer growing. Isolated bulge cells from these -catenin mutants formaldehyde (PFA) for 1 h at 4 °C, washed in PBS and detergent rinse (PBS cannot form hair follicles, even when transplanted (14). We in- with 2mM MgCl , 0.01% sodium deoxycholate, and 0.02% Nonidet P-40 β 2 terpret these observations to mean that Wnt/ -catenin signaling detergent), and then stained in staining solution (PBS with 2 mM MgCl2, is required to maintain HFSC potency and function. 0.01% sodium deoxycholate, and 0.02% Nonidet P-40, 5 mM potassium Our findings complement recent work which suggests that ferricyanide, 5 mM potassium ferrocyanate, and 1 mg/mL X-gal) in the dark attenuated Wnt signaling is part of the process of defining em- at room temperature overnight. Tissues were then washed in PBS, postfixed bryonic progenitors that become adult HFSCs (39). This study in 4% PFA in PBS overnight at 4 °C, and processed for paraffin embedding and histology. showed that hair progenitors start off as Axin2-expressing cells in the placode, but gradually lose Axin2 expression as they commit Histology and Immunostaining. Animals were killed and skins were removed to becoming adult HFSCs during late embryogenesis. Our work from the back (dorsal) and the tail regions. For cryosections, tissues were fixed suggests that Axin2 becomes expressed again in the adult HFSC for 1–2 h in 4% PFA at 4 °C, washed in PBS, and incubated overnight in 30% compartment, once it is established. sucrose/PBS at 4 °C. Tissues were then embedded in OCT medium and stored HFSCs have been proposed to actively organize their micro- at −80 °C before cryosectioning. Sections of 5- to 6-μm thicknesses were cut environment (16, 17). Here, we provide evidence that HFSCs using a Leica CM3050S cryostat (Leica Microsystems). may influence the organization of their niche by acting as their For paraffin sections, tissues were fixed overnight in 4% PFA at 4 °C. Tissues own source of self-renewing Wnt signals, while simultaneously were washed in PBS, then dehydrated through an ethanol series into xylene, and embedded in paraffin wax. Sections of 5-μm thickness were cut by using promoting the differentiation of their progeny. a Leica RM2255 microtome (Leica Microsystems), and then rehydrated and Materials and Methods counterstained with Eosin where specified. All immunofluorescence staining was performed in the dark. Cryosections – Animals. Axin2 CreERT2 mice were described (22). Axin2-lacZ (40) were a gift were washed in PBS and incubated in blocking buffer (2% normal donkey from W. Birchmeier, Max-Delbrück-Centre for Molecular Medicine, Berlin. serum and 0.2% Triton X in PBS) for 1 h at room temperature. Primary an- – flox β Δex2-6-flox/flox Rosa26 mTmG (21) and -catenin (41) mice were obtained tibodies were diluted in blocking buffer and incubated with cryosections from The Jackson Laboratory. All alleles were heterozygous, except where overnight at 4 °C for 1 h at room temperature. Sections were then washed in stated. All experiments were approved by the Stanford University Animal PBS, and incubated with secondary antibody diluted in blocking solution for Care and Use Committee and the A*STAR Animal Care and Use Committee 1 h at room temperature. Sections were then washed in PBS and mounted in and were performed according to NIH and Singapore Bioethics Council Prolong Gold with DAPI mounting medium (Life Technologies). The fol- guidelines. lowing primary antibodies were used: anti-GFP (Abcam), anti-Dkk3 (R&D systems), and anti–Keratin-6 (Covance). The following secondary antibodies Labeling and Tracing Experiments. All mice received a single i.p. injection of were used: anti-chicken conjugated to Alexa Fluor 488 (Life Technologies, 5–20 mg/mL stock solutions of Tam (Sigma, T5648) dissolved in corn oil/10% Jackson ImmunoResearch) and anti-goat conjugated to Alexa Fluor 647 (vol/vol) ethanol, corresponding to specific doses per gram body weight as (Jackson ImmunoResearch).

E1504 | www.pnas.org/cgi/doi/10.1073/pnas.1601599113 Lim et al. Downloaded by guest on September 27, 2021 RNA in Situ Hybridization. Tissues were harvested and fixed in 4% PFA for 24 h, 4 °C, washed with 10% chelexed FBS/DPBS, centrifuged at 300xg for 5–10 min PNAS PLUS and then dehydrated and embedded in paraffin. Tissues sections were cut at at 4 °C and resuspended into 1 mL of single cell suspension with ice-cold 10% 5-μm thickness, air-dried at room temperature, and processed for RNA in situ chelexed FBS/DPBS which was then incubated with primary antibodies either detection by using the RNAscope 2.0 FFPE Reagent Kit according to the coupled directly with a fluorochrome or indirectly in chelexed FBS-precoated manufacturer’s instructions (Advanced Cell Diagnostics; ref. 42). RNAscope propylene tubes on ice for 60 min in the dark, washed two times before in- probes used were as follows: Axin2 (NM_015732, region 330–1287), Wnt1 cubation with secondary antibodies in 10% chelexed FBS in DPBS for 20 min (NM_021279.4, region 1204–2325), Wnt3 (NM_009521.2, region 135–1577), on ice, washed and analyzed with flow cytometry. The following primary Wnt4 (NM_009523, region 2147–3150), Wnt5a (NM_009524, region 200–1431), antibodies were used in our FACS analysis: Alexa 700-conjugated anti-mouse Wnt6 (NM_009526, region 780–2026), Wnt7a (NM_009527.3, region 1811–3013), CD34 (Affymetrix), APC-conjugated anti-human/mouse CD49f (α6Integrin) Wnt7b (NM_009528, region 1597–2839), Wnt10a (NM_009518, region 479– (eBioscience), Pacific Blue anti-mouse Ly-6A/E (Sca-1) (Biolegend), Polyclonal 1948), Wnt10b (NM_011718, region 989–2133), Wnt16 (NM_053116.4, re- goat antibody against mouse LRIG1 (R&D systems). The following secondary gion 453–1635) Dkk3 (NM_015814, region 1513–2703), Dkk4 (NM_145592.2, antibodies, Donkey anti-goat IgG (H+L)-Phycoerythrin (R&D systems) was used. region 22–1140), and SFRP1 (NM_013834.3, region 1810–2779), which were Cell viability was checked by staining with Zombie-Aqua Fixable Viability kit detected using the Fast Red detection reagent. (Biolegend). Living cells were sorted positive for CD34 and α6 Integrin, and negative for Lrig1 and Sca1. This CD34+ α6Integrin+ bulge cell population was Microscope Image Acquisition and Quantification. All sections were imaged by further sorted into Axin2+ and Axin2− populations. using the Axioplan 2 and Imager.Z2 Microscopes, the AxioCam MRm (fluores- For detection of intracellular β-galactosidase activity of Axin2-expressing cence) and MRC5 (bright field) cameras, and using Axiovision AC software (Re- cells, a fluorescein di-β-D-galactopyranoside (FDG) staining kit (Thermo Fisher) lease 4.1, Carl Zeiss) or Zen software (Carl Zeiss). Image acquisitions were done at was used. Keratinocytes stained with the antibodies were loaded with FDG × × room temperature using 10 NA 0.3, X20 NA 0.5, X20 NA 0.8, and 40 NA 0.75 using hypotonic shock at 37 °C. Cell sorting was carried out using a FACSAria EC Plan-Neofluar objectives (Carl Zeiss). For some images, contrast, color, and cell sorter using unstained control, single color compensation controls. Axin2- dynamic range were globally adjusted in Adobe Photoshop (Adobe Systems). positive cells were defined by gating using fluorescence minus one without FDG. Axin2+ and Axin2− cell populations were fixed in ice-cold methanol for Cell Labeling and Flow Cytometry. For isolation of bulge cells, 6- to 8-wk-old 15 min, washed and centrifuged in DPBS. Cell cycle analysis was performed by Axin2–LacZ animals were killed, hair was removed, and dorsal skins were staining fixed cells with propidium iodide supplemented with RNase in the ’ harvested and washed in ice-cold calcium and magnesium-free Dulbecco s dark at room temperature and analyzed with a FACSAria cell sorter. PBS (DPBS; GE Healthcare). After removing fat and s.c. tissues using a scalpel, – the dorsal skins were then trypsinized in 0.25% Trypsin EDTA (Gibco) at ACKNOWLEDGMENTS. We thank Poh Hui Chia for cell quantification assis- 37 °C for 1 h. Epidermal cell suspension was neutralized with ice-cold 10% tance. This work was supported by the Howard Hughes Medical Institute; Cal- chelexed FBS (GE Healthcare) in DPBS and strained with 70 and 40 μMcell ifornia Institute of Regenerative Medicine Grant TR1-01249; and the Agency for strainers (Fisher Scientific). Cells were centrifuged at 300 × g for 30 min at Science, Technology, and Research in Singapore.

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Lim et al. PNAS | Published online February 22, 2016 | E1505 Downloaded by guest on September 27, 2021