Loss of a quiescent niche but not follicle stem cells in the absence of bone morphogenetic signaling

Krzysztof Kobielak, Nicole Stokes, June de la Cruz, Lisa Polak, and Elaine Fuchs*

Howard Hughes Medical Institute, Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York, NY 10021

Contributed by Elaine Fuchs, April 7, 2007 (sent for review March 8, 2007) During the hair cycle, follicle stem cells (SCs) residing in a special- These complexes translocate to the nucleus where they transac- ized niche called the ‘‘bulge’’ undergo bouts of quiescence and tivate their target (10). activation to cyclically regenerate new hairs. Developmental stud- When keratin 14 (K14)-driven Cre recombinase is used to ies have long implicated the canonical bone morphogenetic protein ablate Bmpr1a function in embryonic skin, the early stages (BMP) pathway in hair follicle (HF) determination and differentia- of HF occur, but matrix cells fail to differentiate tion, but how BMP signaling functions in the hair follicle SC niche (11, 12). Although these later differentiation steps rely on active remains unknown. Here, we use loss and gain of function studies BMP signaling, earlier steps in the lineage appear to require the to manipulate BMP signaling in the SC niche. We show that when impairment of the pathway. , an extracellular BMP the Bmpr1a gene is conditionally ablated, otherwise quiescent SCs inhibitor, is expressed by , where it induces follicle are activated to proliferate, causing an expansion of the niche and morphogenesis in the embryo and promotes new HF growth loss of slow-cycling cells. Surprisingly, follicle SCs are not lost, (anagen) postnatally (13, 14). Interestingly, once embryonic HFs however, but rather, they generate long-lived, tumor-like branches have initiated, they express BMP4, suggesting a negative feed- that express Sox4, Lhx2, and but fail to terminally back loop to prevent new HF initiation in the vicinity (9). differentiate to make hair. A key component of BMPR1A-deficient An early consequence of Noggin ablation in vivo is a loss of SCs is their elevated levels of both Lef1 and ␤-catenin, which form expression of Lef1, a DNA binding protein essential for efficient a bipartite transcription complex required for initiation of the hair HF morphogenesis (14, 15). Conversely, keratinocytes treated cycle. Although ␤-catenin can be stabilized by Wnt signaling, we with Noggin or deficient in BMP receptor 1A display nuclear show that BMPR1A deficiency enhances ␤-catenin stabilization in Lef1 in vitro (11, 13), and Lef1-positive matrix cells persist after the niche through a pathway involving PTEN inhibition and PI3K/ Bmpr1a is ablated in vivo (11, 12). Lef1 acts as a bipartite AKT activation. Conversely, sustained BMP signaling in the SC transcription factor in conjunction with stabilized ␤-catenin, niche blocks activation and promotes premature hair follicle dif- which is known to stimulate de novo HF morphogenesis and ferentiation. Together, these studies reveal the importance of increase follicle density when overexpressed (16). balancing BMP signaling in the SC niche. In adult follicle SCs, Smad1 is phosphorylated and BMP6 levels are elevated, suggesting that BMP signaling is active in the ␤-catenin ͉ BMPR1A ͉ hair follicle ͉ PTEN ͉ tumorigenesis bulge (5, 12) (see SI Fig. 6A, P19). Conversely, in the early hair germ that emerges from the activated bulge, nuclear P-Smad1 is dult stem cells (SCs) typically reside in specialized niches diminished (see SI Fig. 6A, P20) and remains low in the Awhere they receive microenvironmental cues that govern developing ORS and in the lower part of the matrix. The normal homeostasis and wound repair (1). Pulse–chase exper- strongest BMP signaling is in the cells that differentiate to iments with labeled nucleotides (2) or fluorescent histone pro- produce the IRS and hair shaft (11) (see SI Fig. 6A, P24). These teins (3) have designated the hair follicle (HF) bulge as the studies suggest a possible explanation for how bulge SCs might residence of skin’s slow cycling, label-retaining epithelial cells exist in a quiescent state and how this characteristic might (4). Clonal analyses reveal that bulge cells exhibit the properties change during subsequent lineage determination and differen- of self-renewing SCs in vitro, and when transplanted onto the tiation. backs of hairless (Nude) mice, they generate epidermis, HFs, and The possible role for BMPs in follicle SCs takes on greater sebaceous glands (5–7). interest given recent reports that conditional inactivation of Initiated during embryonic development, HFs continue to Bmpr1a leads to an increase in the number of stem and/or generate hair until approximately postnatal day 14 (P14), when progenitor cells in the hematopoietic lineage (17) and intestinal they enter a destructive phase, known as catagen (8, 9). During epithelium (18). That said, BMPs don’t always function to inhibit catagen, the lower two-thirds of the HF degenerates until the specialized dermal papilla (DP) cells at the base of the follicle come to rest below the bulge. In the resting (telogen) phase of Author contributions: K.K. and E.F. designed research; K.K., N.S., J.d.l.C., and L.P. performed research; K.K. contributed new reagents/analytic tools; K.K. analyzed data; and K.K. and the hair cycle, the bulge exists in a quiescent state in contact with E.F. wrote the paper. the DP. The first telogen is brief, lasting only a few days before The authors declare no conflict of interest. a new growth (anagen) phase begins, but the second resting phase lasts 3–4 weeks. With the end of telogen and the onset of Freely available online through the PNAS open access option. Abbreviations: HF, hair follicle; SC, stem cell; DP, dermal papilla; ORS, outer root sheath; IRS, the anagen, bulge SCs become activated to form the outer root inner root sheath; K14, keratin 14; Pn, postnatal day n; TM, tamoxifen; CON, control; MKs,

sheath (ORS) and matrix cells that fuel HF regeneration [see cultured primary keratinocytes; BMP, bone morphogenetic protein; cKOTM, tamoxifen CELL BIOLOGY supporting information (SI) Fig. 5]. Maintaining their associa- inducible conditional knockout; CONTM, control after tamoxifen treatment; Bmpr1A-CA, tion with the DP throughout anagen, matrix cells proliferate and constitutively active BMP receptor 1A; Dox, doxycycline; dTgDox, doxycycline inducible double transgenic for K14-rtTA and TRE-BmpR1A-CA-HA;CONDox, control after doxycy- differentiate upward to produce the hair shaft and its channel, cline treatment. the inner root sheath (IRS). *To whom correspondence should be addressed at: Howard Hughes Medical Institute, Although the mechanisms controlling the hair cycle are still Department of Mammalian Cell Biology and Development, The Rockefeller University, unfolding, bone morphogenetic protein (BMP) signaling is likely 1230 York Avenue, Box 300, New York, NY 10021. E-mail: [email protected]. to be involved. Interaction with exogenous BMPs stimulates the This article contains supporting information online at www.pnas.org/cgi/content/full/ transmembrane receptor BMPR1A to phosphorylate Smad1, 0703004104/DC1. Smad5, and Smad8, which then heterodimerize with Smad4. © 2007 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703004104 PNAS ͉ June 12, 2007 ͉ vol. 104 ͉ no. 24 ͉ 10063–10068 Downloaded by guest on September 28, 2021 SC self-renewal. BMPs can act to sustain self-renewal of both murine and human embryonic SCs (19, 20), and in the fly embryo, dpp (vertebrate BMP2/4 homolog) defines the niche that maintains anterior germline SCs (21). Here, we use an inducible conditional targeting strategy to uncover roles for BMP signaling in balancing quiescence and activation of hair follicle SCs in adult mice. We find that inhibition of BMP signaling in follicle SCs is essential for promoting the transition between quiescent bulge SCs to pro- liferating progeny. We further show that in the absence of BMPR1A function, premature anagen occurs, and although niche characteristics are perturbed, the SCs are not lost. Rather, both Lef1 and ␤-catenin are up-regulated in the SC niche, associated with signs of inactivation of PTEN and activation of the PI3K/AKT pathway in the Bmpr1a tamoxifen inducible conditional knockout (cKOTM) niche. These findings provide a mechanistic link for the convergence of BMP and Wnt pathways in follicle SC activation. Results BMP Signaling Is Necessary for Maintaining Quiescence of the SC Niche. Conditional knockout mice targeted by K14-Cre for Bmpr1a gene ablation die shortly after birth (11, 12). To evaluate how ablation of Bmpr1a affects postnatal homeostasis of the HF SC niche, we mated Bmpr1a fl/fl floxed mice (22) and inducible K14-CreTM mice (23). Topical application of tamoxifen (TM) was then used to control the inactivation of BMP signaling. Offspring from the matings of Bmpr1a(fl/ϩ)/K14-CreTM and Bmpr1a(fl/ϩ) mice yielded litters of the expected numbers, genotype, and Mendelian ratios. Before TM treatment, Bmpr1a(fl/fl)/K14-CreTM were indistinguishable from control (CON) animals, which included wild type (WT), Bmpr1a(fl/ϩ); Bmpr1a( fl/fl); Bmpr1a( fl/ϩ)/K14-CreTM, and Bmpr1a(ϩ/ϩ)/ K14-CreTM. At P44, HFs were just entering the extended second telogen, and both Bmpr1a(fl/fl)/K14-CreTM and Bmpr1a(fl/fl) HFs exhibited a resting-phase morphology (Fig. 1A and SI Fig. 6B). We shaved and applied TM topically at this time, and within 16d the Bmpr1a gene was efficiently targeted as judged by genotyping (SI Fig. 6C), anti-P-Smad-1 immunofluorescence (SI Fig. 1. Postnatal ablation of Bmpr1a affects homeostasis of the SC niche in adult hair follicles. (A) Chart illustrating the temporal progression through the Fig. 6D), and in situ hybridization with a cRNA probe to the first and second postnatal hair cycles and the regiment for administering TM deleted exon 2 (SI Fig. 6E). and BrdU. (B) Phenotypes of CONTM and Bmpr1a cKOTM mice, after shaving TM Bmpr1a(fl/fl) mice carrying the TM-activated K14-Cre back skins during telogen, targeting Bmpr1a and monitoring after initiation transgene (cKOTM) failed to regrow hair. Despite the lack of of the next hair cycle. cKOTM mice did not regrow hair. (C and D) Skin sections visible hair, their HFs were in anagen by P59 (Fig. 1C, arrow- stained with H&E or with antibodies that are color-coded according to the heads). By contrast, CON follicles after tamoxifen treatment fluorescent tags of the secondary antibodies. Brackets denote bulge (Bu), and (CONTM) remained in telogen for Ͼ2 additional weeks (P75), arrowheads denote follicle cells outside the niche. Note that the CD34 mono- after which they entered anagen and regrew their hair (Fig. 1B). clonal antibody gives high dermal background in older animals; the hair shafts Before TM, both cKO and WT bulge cells consisted of an ␣6 typically display autofluorescence. FACS analyses are of SC populations sorted ␣ by ␣6-integrin and high CD34. R3, basal SCs; R2, i.e., the large box including R3, integrin-rich basal layer and an 6-low suprabasal layer, both of total bulge SCs. Note that both CD34-positive bulge pools are diminished 2 which were CD34-positive (5) (SI Fig. 6F). Following TM, weeks after Bmpr1a targeting in cKOTM skin. (E and F) Skin sections as in C and TM however, cKO follicles displayed Ϸ9–10ϫ fewer CD34(ϩ) D, but in this case, depicting analysis of bulge SCs after pulse–chase and pulse bulge cells by fluorescence-activated cell sorting (FACS) than experiments with BrdU (see A for time schedule). FACS analyses are based on CONTM follicles (Fig. 1D). In addition, when mice were pulsed ␣6-integrin and incorporation of BrdU during a 4-h pulse. Cells that incorpo- with BrdU 2ϫ/d for 3d in the first postnatal anagen (P23–25), rated BrdU above limits of detection by immunofluorescence microscopy are treated with TM from d44 to d55 and examined at 5 weeks after boxed as R5. Note that the pool of BrdU-incorporating cells is substantially TM administering BrdU (see Fig. 1A), label-retaining cells were increased in cKO skin. prevalent in CONTM but not in cKOTM bulges (2, 4) (Fig. 1E). The absence of CD34-positive, bulge label-retaining cells upon the inactivation of BMP signaling could be reflective of an telogen-to-anagen transition, and labeled cells were now de- activated follicle SC niche. To test this possibility, we adminis- tected in the developing hair germs (SI Fig. 6H). By contrast, P77 TM tered 4-h pulses of BrdU and processed the skins immediately cKO HFs had progressed well beyond anagen initiation. thereafter (see Fig. 1A). At P57 and P59, CONTM follicles were Developing HFs appeared morphologically enlarged, mis- in telogen and only an occasional bulge cell incorporated BrdU shapen, and hyperproliferative (SI Fig. 6H, arrowheads). during each 4-h pulse. By contrast, cKOTM follicles displayed numerous BrdU-labeled bulge cells (Fig. 1F, P57, and SI Fig. 6G, Expansion of Markers of Bulge and Proliferating Progeny Following P59, arrowheads). Quantification by FACS revealed Ͼ4ϫ more Ablation of Bmpr1a. To determine the extent to which SC prop- BrdU-labeled cells within the cKOTM versus CONTM skin at this erties are lost upon Bmpr1a targeting, we next examined the stage (Fig. 1F). At P77, CONTM HFs had just passed the expression of DNA binding Sox9 and Lhx2, which are

10064 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703004104 Kobielak et al. Downloaded by guest on September 28, 2021 hybridizations were markedly expanded in the cKOTM counter- parts. Sox4 was atypically detected in both cKOTM bulge and progeny (Fig. 2C and SI Fig. 7C). Shh positive cells were prevalent throughout the lower cKOTM follicle (Fig. 2D and SI Fig. 7D). Several months after TM treatment, each cKOTM HF ap- peared as a branch of amorphous tumor-like masses of cells consisting largely of the same two populations of cells seen at P77 (Fig. 2 E and F). Multiple branches of these masses formed over time. The P-cadherin and Shh-positive edges of the tumor masses remained proliferative many months after Bmpr1a targeting, and pulse–chase experiments showed no traces of BrdU label reten- tion (Fig. 2 G and H). Additionally, cells from these structures were able to replenish the epidermis when scraped or enzymat- ically removed (Fig. 2I and SI Fig. 8 B and A, respectively).

Lef-1/␤-Catenin Stabilization and PI3 Kinase/AKT Pathway Activation Upon Bmpr1a Targeting. ␤-Catenin stabilization promotes the transition from quiescent bulge cells to proliferating Sox4- positive progeny (26). As expected, nuclear Lef1 and ␤-catenin were detected in the hair germs of P77 CONTM HFs but not in the bulge (SI Fig. 9 A and B). By contrast, nuclear Lef1 and ␤-catenin were expressed not only in precocious hair germs of P59 cKOTM HFs but also in the bulge (Fig. 3 A and B, respectively). By P77, most cells within down-growing masses were positive for these markers (SI Fig. 9 A and B). Most pathways that stabilize ␤-catenin inhibit GSK3␤ kinase, which phosphorylates and targets ␤-catenin for turnover (28). In CONTM HFs, GSK3␤ was not phosphorylated (active) at Ser-9 during telogen (Fig. 3C) but becomes phosphorylated as anagen ensues (SI Fig. 9C). In contrast, P59 and P77 cKOTM follicles showed expanded anti-P-GSK3␤ labeling, corresponding to the anti-␤-catenin pattern (Fig. 3C Right and SI Fig. 9C Right). These Fig. 2. Expansion of the proliferating populations and their ability to repair findings were consistent with a role for GSK3␤ inactivation in epidermal wounds when BMP signaling is ablated in hair follicles postnatally. generating the ␤-catenin-rich tumor masses when Bmpr1a was cKOTM and CONTM mice were targeted for Bmpr1a ablation at P44 and ␮ targeted. analyzed at P77 (A–D) or P90-P230 (E and F). Frozen skin sections (10 m) were ␤ subjected to indirect immunofluorescence for Sox9, P-cadherin, and Lhx2 (A, GSK3 -Ser-9 is an established target for AKT phosphoryla- B, and E), or to in situ hybridizations (ISH) with digoxygenin cRNA probes tion, and given the link between BMP signaling and PTEN specific for Sox4 and Shh (C, D, and F). Note that Sox9hiLhx2hi- and P-cadher- activation, we tested whether PTEN might be altered in inhiLhx2(ϩ)-positive cells mark two distinct populations emerging from the BMPR1A-deficient follicle SCs. From telogen to anagen in bulge lacking BMPR1A. These populations developed into tumor masses over CONTM (P77, 2nd cycle) or WT (P21 and P23, 1st cycle) HFs, time. (G) A 4-h pulse at P120 shows that the tumor borders are highly antibodies against phosphorylated (inactive) PTEN labeled proliferative. (H) Ablation was performed at P44, and a 3-day pulse was weakly in developing hair germs (SI Fig. 9D). No signal was administered at P67. Skin was analyzed after a 3-week chase. Note the absence detected in telogen CONTM (P59) or WT (P19) HFs (Fig. 3D Left of label-retaining cells in tumor masses. (I) Wound healing. cKOTM mice were mated to Rosa26-floxed-lacZ mice; after Bmpr1a ablation (LacZϩ), skins were and SI Fig. 9D Left). By contrast, anti-P-PTEN staining was TM scraped to remove the epidermis, and wound repair was monitored for the prevalent throughout the bulge and emerging progeny of cKO periods indicated. Note the reepithelialization (arrows) by BMPR1A-deficient HFs (Fig. 3D Right and SI Fig. 9D Right). follicle cells. Arrowhead denotes border between scab/blood clot and the AKT’s targets include Ser-552 of ␤-catenin, a mark which epidermis, dotted lines denote dermo–epithelial borders, and brackets de- facilitates 14–3-3␨ association and nuclear translocation (29). In note bulge (Bu). hg, hair germ; SG, sebaceous gland. cKOTM follicles, anti-14–3-3␨ staining was intense in developing tumor-like masses. Consistent with a role for AKT/14–3-3␨ in SC activation, staining was strong in the hair germs of cKO and essential for bulge SC maintenance (24, 25). Antibodies against CON HFs (Fig. 3E and SI Fig. 9E). Immunoblot analyses further TM each marker labeled the cKO bulge but at reduced intensity suggested that without BMPR1A, PTEN remained inactive and TM TM over CON (SI Fig. 7 A and B). For both cKO at P59 and PI3K/AKT/GSK3␤ pathway was active (Fig. 3F). TM CON at P77 follicles, Sox9 and Lhx2 levels were high in the Finally, we subjected cultured WT and KO keratinocytes ϩ P-cadherin( ) hair germs emerging after anagen induction (Fig. (MK) to assays with the TOPFlash reporter gene, active when 2 A and B and SI Fig. 7 A and B). By P77, labeling Sox9 and Lhx2 ␤-catenin and Lef1 are stabilized (30). WT MK required Wnt3a TM in cKO follicles was concentrated in the midzone below the and Noggin for optimal activity, and although Wnt3a was CELL BIOLOGY cKOTM bulge, while Sox9 was absent in the P-cadherin(ϩ) cells permissive for TOPFlash, expression was inhibited by BMP6 encased by the DP (Fig 2 A and B). In their patterns of (Fig. 3G). In all cases, expression depended on the Lef1 en- BrdU-incorporation and biochemical markers (24–26), these hancer element, as judged by assays with the mutated FOPFlash. two cell populations bore a resemblance to the proliferative cells In low serum-containing medium, TOPFlash was higher in KO of the lower ORS and matrix, respectively. than WT MK. Noggin elevated WT but not KO levels, suggesting In normal HFs, the Sox4 and Sonic hedgehog (Shh) genes are that low levels of endogenous BMPs accounted, in part, for these not expressed in the bulge but are coexpressed in the developing differences (13) (Fig. 3H). Differences in PI3K signaling were hair germ (26, 27). While Sox4 and Shh in situ hybridizations of also involved, given that Ly294002, a chemical inhibitor of PI3K, P59 and P77 CONTM follicles gave the expected patterns, markedly reduced TOPFlash activity and minimized the differ-

Kobielak et al. PNAS ͉ June 12, 2007 ͉ vol. 104 ͉ no. 24 ͉ 10065 Downloaded by guest on September 28, 2021 Fig. 3. Precocious and sustained activation of Lef1/␤-catenin and P-PTEN/PI3K/AKT signaling in hair follicles lacking BMPR1A. (A–E) cKOTM and CONTM mice were targeted for Bmpr1a ablation at P44 and analyzed at P59. Frozen skin sections (10 ␮m) were subjected to indirect immunofluorescence or immunohistochemistry by using the antibodies indicated in the lower right of each frame. Color coding is according to the fluorescent tags of the secondary antibodies. Arrowheads denote aberrant expression. (F) Immunoblot analyses of protein extracts isolated from P59 skin preparations as described (26). Antibodies used are indicated at the right. (G and H) TOPFlash assays. MKs were transfected with vector encoding the reporter gene TOPFlash or the control FOPFlash containing mutations in the multimerized Lef1 DNA binding sites. MKs were then treated with the recombinant factors or with the PI3K inhibitor Ly as indicated, and luciferase assays were conducted as described (13). Experiments were conducted in triplicate. Lam5, laminin5; ␤-cat, ␤-catenin; ␤4, ␤4-integrin; hg, hair germs; DP, dermal papilla; Nogg, Noggin; Wnt3, Wnt3a.

ences between WT and KO MK. Noggin did not override the keratinocytes within the P35 dTg cyst-like structures, but P35 inhibitory effects of PI3K, consistent with the notion that CONDox HFs displayed BrdU-labeled cells throughout the ORS inhibition of BMP signaling operates through PI3K activation. and matrix (Fig. 4 E and EЈ). However, Wnt3A enhanced TOPFlash activity in both WT and As expected from prior studies, GATA-3 (33) and Lef1 (34) KO MK, and this observation was not markedly repressed by differentially marked the IRS and hair shaft precursor cells of PI3K inhibition (Fig. 3H). In summary, TOPFlash activity was CONDox HFs (Fig. 4F). In dTgDox skin, however, GATA-3 and optimal when WNT signaling was activated and BMPR1A Lef1 were precociously expressed in the follicle cysts that activity was impaired. WNT signaling and PI3K signaling were emanated from the bulge (Fig. 4F). In some cases, the same cells both able to stabilize ␤-catenin, but they appeared to do so by showed coexpression (Fig. 4F Right Inset). Cyst cells were also parallel, and at least partially independent, pathways. immunolabeled with monoclonal antibodies specific for the IRS protein trichohyalin (AE15) and hair keratins (AE13) (Fig. 4G Inducible Activation of BMPR1A Results in Premature Differentiation and SI 10D). dTgDox cysts did not label with antibodies against of Follicle SC Progeny. To further explore the role of BMP epidermal differentiation proteins, including keratin 10 (K10) signaling in follicle SC behavior, we engineered mice harboring and filaggrin (Fig. 4H and SI Fig. 10E). Thus, although dTgDox TRE-BmpR1A-CA-HA (BmpR1A-CA, constitutively active cysts did not exhibit the organized architecture typical of WT BMP receptor 1A), a tetracycline (Dox) regulatable transgene HFs, they displayed features of follicle, rather than epidermal, encoding a constitutively active and epitope-tagged form of differentiation. BMPR1A (31). Mice were mated to be doubly transgenic (dTg) While CD34 marked the bulge SC niche in P30 dTgDox HFs, for TRE-BmpR1A-CA-HA and K14-rtTA, encoding a Dox- its expression was reduced compared with P30 CONDox HFs, and inducible transactivator (32) (Fig. 4A and SI Fig. 10A). Before by P35, it was barely detectable (Fig. 4 I and IЈ; see also SI Fig. Dox administration, single transgenic CON and dTg mice were 10C). Similar results were obtained with Sox9 antibodies (SI indistinguishable. Fig.10 F and FЈ). No traces of proliferative bulge progenitors, At P19, during the short telogen of the first hair cycle, marked by Sox 4 and Shh, were seen (SI Fig. 10 G, GЈ, H, and posterior back skins were shaved and Dox was administered. HЈ, respectively). These collective results were consistent with CON mice, after doxycycline treatment (CONDox), developed a the view that follicle SCs may still be triggered to execute an HF new hair coat, reflecting the normal transition into the second fate, but when they do, they terminally differentiate in the hair cycle. By contrast, doxycycline inducible double transgenic presence of constitutive BMPR1A activity. for K14-rtTA and TRE-BmpR1A-CA-HA (dTgDox) mice dis- played defects in hair coat regeneration (Fig. 4B). Immunoflu- Discussion orescence confirmed efficient transgene induction (Fig. 4C), and Fine-Tuning BMP Signaling to Balance Quiescence, Proliferation, and reflective of BMPR1A-CA activity, anti-phospho-Smad1 stain- Differentiation of Follicle SCs. Follicle SCs display signs of activated ing was prevalent, with the exception of what appeared to be TGF␤ and BMP signaling in vivo, and in vitro, ligands specific for keratinized follicular masses (SI Fig. 10B). each pathway cause proliferating keratinocytes to transiently BmpR1A-CA expression severely perturbed the hair cycle. By withdraw from the cell cycle (3, 5, 6, 10). Thus, it was surprising P35, overall bulge morphology still appeared to be intact in dTg that ablation of BMP signaling alone was sufficient to disrupt the follicles, but a large keratinized cyst had developed beneath the quiescent state of the hair follicle SC niche. In the absence of bulge, where a mature HF was seen in WT counterparts (Fig. 4 BMPR1A, quiescent SCs were precociously activated to enter D and DЈ). BrdU labeling confirmed the paucity of proliferating the proliferative phases associated with the new hair cycle,

10066 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703004104 Kobielak et al. Downloaded by guest on September 28, 2021 nuclear anti-P-SMAD-1 within WT SCs is lower than that detected in the precursor cells of the hair shaft and IRS. Thus, the level of canonical BMP signaling in the SC niche may need to be sufficiently high to maintain quiescence, and yet not so high that terminal differentiation is triggered. We posit that the hair cycle may be activated when signaling to the SC niche dips below this threshold level, and when signaling rises above the threshold, proliferating progeny execute their terminal differentiation programs. BMP6 and BMP4 seem to play autocrine and paracrine roles, respectively, in niche quiescence (5, 35). Conversely, BMP antagonists, including Noggin, , and ectodysplasin, are made by the DP and, hence, are likely to play paracrine roles in SC activation (14, 36). LacZ knockin mice suggest that for at least one BMP inhibitor, namely Noggin, expression varies with the hair cycle, such that Noggin activity in the SC niche rises at the onset of anagen and wanes during telogen (35). Future studies will address how these competing patterns of BMPs and BMP inhibitors are temporally regulated to govern the hair cycle.

BMP Inhibition and SC Activation. Several main features of follicle SCs, namely CD34 and label retention, were lost upon Bmpr1a targeting. Nevertheless, Sox9, Lhx2, Sox4, and Shh, which are characteristic of quiescent bulge cells and/or their early prolif- erating progeny, were expanded after inhibiting BMP signaling. Given that this expansion of early progenitors persists for more than a year after Bmpr1a ablation, it suggests that the activated SCs of BMPR1A-deficient follicles are not lost. The ability of follicles to repair epidermal wounds further suggests that multipotent SCs survive Bmpr1a ablation. Our findings argue that BMPR1A inhibition is essential for the maintenance of proliferating bulge progeny but that CD34 and label retention are not essential features of follicle SCs. Skin tumors arise when Bmpr1a is targeted by either K14- CreTM (12), Engrailed1-Cre (37), or Mx1-Cre (35). We observed that the numbers of branches of these proliferative tumor-like masses increased with age, and yet they did not progress to malignancy. This is consistent with what might be expected if the destructive phase (catagen) of the hair cycle does not occur properly in the absence of BMPR1A (12), but the follicles continue to undergo cycling. We also noted that during the window of time when it was still recognizable, the Bmpr1a-null bulge expanded in size and was no longer label-retaining. We posit that in the absence of BMPR1A, Fig. 4. Mice expressing a dominant active BMPR1A transgene display pre- slow cycling SCs convert to activated SCs, which retain their cocious hair follicle differentiation. Bitransgenic mice harboring a Dox- ability to self-renew and to commit to the early proliferative regulatable, superactive BMPR1A (dTg), and noninducible CON mice were phases of the HF lineage. As shown for embryonic skin, HF treated with Dox at P19, after shaving the lower back skin areas (brackets). differentiation beyond the Lef1-expressing matrix cell stage was Analyses of mice were performed at P35, following a 4-h pulse with BrdU. (A) blocked in the absence of BMPR1A (11, 12). Transgene schematics. (B) Phenotypic abnormalities in the regrowth of the hair coat of dTgDox-treated mice. (C–I) P35 skin sections subjected to either H&E staining or immunofluorescence with the antibodies indicated. Color- Mechanisms Involved in BMPR1A Signaling in the SC Niche. The coding is according to the fluorescent tag of the secondary antibodies. AE15 importance of ␤-catenin stabilization and activation of Lef/Tcf recognizes trichohyalin, an IRS marker. Note that following BMPR1A super- target genes in follicle formation is well established (15, 16, 38). activation, dTgDox hair follicles develop cysts of terminally differentiated cells This process involves signaling by ectodermal Wnts, which when located just below the bulge (Bu, bracket). Dotted lines demarcate dermo– blocked by overexpression of Dkk1, suppress follicle formation epidermal boundaries. Mx, matrix cells. Boxed areas are magnified in Insets. (39). The process also involves inhibition of BMP signaling, given that Lef1 and follicle formation are repressed in the absence of ␤ Noggin. Additionally, Lef1/ -catenin-mediated transcription is CELL BIOLOGY underscoring a role for BMP inhibition in governing the telogen lost in Noggin-null mutants, and is elevated in the presence of to anagen transition. excessive Noggin or in the absence of BMPR1A (11, 13, 14). Our loss-of-function studies also highlighted an essential role Although Wnt signaling and BMP inhibition synergize during for BMP signaling in maintaining the biochemical and behav- HF morphogenesis in the embryo, whether and how this synergy ioral features of the SC niche. Our gain-of-function studies operates in controlling adult follicle SC behavior is less clear. demonstrate that overactivation of BMPR1A signaling causes Bulge cells display frizzled receptors, and their precocious precocious terminal differentiation, revealing the importance of activation in response to excess stabilized ␤-catenin underscores fine-tuning the levels of BMP signaling in the hair follicle its importance in the telogen to anagen transition (26) and lineage. In this regard, it is interesting that the intensity of references therein. Thus, several lines of evidence suggest that

Kobielak et al. PNAS ͉ June 12, 2007 ͉ vol. 104 ͉ no. 24 ͉ 10067 Downloaded by guest on September 28, 2021 Wnt signaling is not the sole regulator of ␤-catenin stabilization Materials and Methods in adult HFs. Mice and BrdU labeling. Bmpr1a-floxed mice (22) were mated to ␤ Wnt signaling is thought to disrupt the Axin-GSK3- -catenin K14-CreER mice (23), and targeting was achieved by 2.5 mg/ complex, thereby inhibiting the phosphorylation and destruction ␤ ␤ mouse TM (Sigma–Aldrich, St. Louis, MO) applied daily for 11 d of -catenin by GSK3 kinase (40). However, conditional gene to back skins of shaved mice at P44, corresponding to the start targeting also suggests a role for the small GTPase Cdc42 and the of the second postnatal telogen. For pulse–chase experiments, PKC␨/Par3/Par6 polarity complex in regulating GSK3␤ activity BrdU (Sigma–Aldrich) was administered by either peritoneal in HFs (41). Our findings now link the inhibition of BMP ␮ signaling to ␤-catenin stabilization through the inactivation of injection of P23 mice (50 g/g/d) or by adding 0.8 mg/ml BrdU PTEN, leading to the activation of the PI3 kinase and Akt. Our to the drinking water from P23 to P25 (5). Mice were then chased findings are consistent with a role for Akt both in inactivating from day 23 to day 59. For pulse experiments, a 4-h BrdU pulse GSK3␤ through phosphorylation at Ser-9 and in directly phos- (50 ␮g/g) was administered peritoneally before analyses. phorylating ␤-catenin at Ser 552, leading to its enhanced asso- For gain-of-function studies, Fvb/n transgenic mice were made ciation with 14-3-3␨ and preferential nuclear location. to express BMPR1A-CA under the control of a Dox regulatory The first link between PTEN and BMPR1A emerged from element (TRE) and were mated to mice harboring the Dox- genetic studies revealing mutations in these genes as causes of sensitive transactivator, rTA2SM2-VP16 (rtTA), under the con- patients with Cowden syndrome, a hyperproliferative disorder of trol of the K14 promoter (32). Mice doubly transgenic for the colon. Subsequent studies revealed that BMP exposure TRE-BmpR1A-CA-HA and K14-rTA2SM2-VP16 (rtTA) were resulted in increased levels of the PTEN tumor suppressor (42). shaved and induced by oral administration of Dox at P19 PTEN, which acts by inhibiting PI3K-Akt, thereby antagonizes (telogen) of the first postnatal hair cycle. Wnt’s effects on ␤-catenin stabilization (18). This PI3K pathway appears to be activated in several tissues where Bmpr1a has been We thank Y. Mishina (National Institutes of Health) for floxed-Bmpr1a inactivated, including the intestine (18) and Mx1-positive skin mice; N. D. Rosenblum for plasmid BMPR1A-CA (Hospital for Sick tumors (35). Our findings now extend this pathway to the follicle Children, Toronto); our colleagues who provided reagents and are cited SC niche and provide a molecular explanation as to why PTEN in the text; the Rockefeller University core facility staff (Fred Quimby, inhibition and constitutive activation of AKT in skin lead to the Rockefeller University Laboratory Animal Research Center) for mouse induction of new hair in resting-phase follicles (43). Precisely husbandry; C. Blanpain and W. Lowry for discussions and advice; and how BMP signaling, Wnt signaling, and polarization/cytoskeletal other Fuchs laboratory members for their willingness to share ideas, cues are regulated to tip the intricate balance of ␤-catenin/Lef/ reagents, and protocols. This work was supported by National Institutes Tcf transcriptional activity in SC activation is an interesting of Health Grant R01 AR050452. E.F. is an Investigator of Howard question which merits further studies. Hughes Medical Institute.

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