Current Biology 19, R132–R142, February 10, 2009 ª2009 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2008.12.005 The Hair Follicle as a Dynamic Miniorgan Marlon R. Schneider,1,#,* Ruth Schmidt-Ullrich,2,# and Ralf Paus3,4 Hair is a primary characteristic of mammals, and exerts a wide range of functions including thermoregulation, physical protection, sensory activity, and social interactions. The hair shaft consists of terminally differentiated keratinocytes that are produced by the hair follicle. Hair follicle development takes place during fetal skin development and relies on tightly regulated ectodermal–mesodermal interactions. After birth, mature and actively growing hair follicles eventually become anchored in the subcutis, and periodically regenerate by spontaneously undergoing repetitive cycles of growth (anagen), apoptosis-driven regression (catagen), and relative quiescence (telogen). Our molecular understanding of hair follicle biology relies heavily on mouse mutants with abnormalities in hair structure, growth, and/or pigmentation. These mice have allowed novel insights into important general molecular and cellular processes beyond skin and hair biology, ranging from organ induction, morphogenesis and regeneration, to pigment and stem cell biology, cell proliferation, migration and apoptosis. In this review, we present basic concepts of hair follicle biology and summarize important recent advances in the field. Introduction Hair is composed of terminally differentiated, dead keratinocytes (trichocytes), which are compacted into a fibre of amazing tensile strength, the hair shaft. The presence of hair is characteristic for mammals, in which it exerts a wide range of tasks. These include physical protection, thermal insulation, camouflage, dispersion of sweat and sebum, sensory and tactile functions, and social interactions. In human society, hair is of enormous, psychosocial importance, and many human diseases are associated with hair loss or, less frequently, with overabundance of hair (Box 1). As inappropriate hair growth can cause considerable suffering in the affected individual, there is an ever-increasing demand for drugs that manipulate hair abundance and appearance [1–3]. Hair shafts are made by the hair follicle, a complex miniorgan of the skin, which constitutes the pilosebaceous unit together with its associated structures, the sebaceous gland, the apocrine gland and the arrector pili muscle (Figure 1 and Box 2). Hair follicle formation largely takes place during fetal and perinatal skin development. However, after skin wounding de novo hair follicle formation may also occur in adult mouse and rabbit skin [4], and can even be induced in adult human skin [5,6]. Hair follicle development 1Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany. 2Max-Delbrück-Center for Molecular Medicine, Dept. of Signal Transduction, Berlin-Buch, Germany. 3Dept. of Dermatology, University Hospital SchleswigHolstein, University of Lübeck, Lübeck, Germany. 4School of Translational Medicine, University of Manchester, Manchester, UK. #These authors contributed equally. *E-mail: schnder@lmb.uni-muenchen.de Review involves tightly coordinated prototypic ectodermal–mesodermal interactions [7,8]. Ectodermal hair follicle stem cells give rise to all epithelial components of the hair follicle, including the sebaceous gland and apocrine gland, while the mesoderm-derived cells will develop into the follicular dermal papilla and the connective tissue sheath. Instead, neural crest-derived melanocyte progenitors give rise to the hair follicle pigmentary unit [9,10]. The hair follicle undergoes cycles of growth (anagen), apoptosis-mediated regression (catagen) and relative quiescence (telogen) [2,11]. In each cycle, a new hair shaft is formed, and the old hair is eventually shed, mostly in an actively regulated process termed exogen. Generation of the new hair shaft depends on the activation of hair-specific epithelial stem cells, harboured in the bulge region of the hair follicle epithelium (Figure 1B, Box 2). Although the hair follicle is highly sensitive to numerous growth factors, cytokines, neuropeptides and hormones, which in part are produced by the hair follicle itself, hair follicle cycling as such is an autonomous phenomenon that is able to continue even in isolated hair follicles in organ culture [2,11]. Spontaneous mouse mutants as well as genetically engineered mouse models have been extremely helpful in the molecular characterization of hair-follicle biology and pathology [12]. Furthermore, several genes responsible for defined hair defects have been identified in the spontaneous mutants, which include Ragged/Opossum (abnormal hair numbers), Waved 2 (abnormal morphogenesis), Nude and Balding (abnormal hair shaft structure) or Lethal spotting (abnormal hair pigmentation) (Table 1). In this review, we will emphasize recent insights into the molecular controls of murine hair follicle development and cycling, stem cell biology, and pigmentation. Functional Hair Anatomy The mature (anagen) hair follicle can be divided into a ‘permanent’ upper part, which does not cycle visibly, and a lower part, which is continuously remodelled in each hair cycle. The upper part of the hair follicle consists of the infundibulum, which is the opening of the hair canal to the skin surface, and the isthmus. The lower, cycling part represents the actual hair shaft factory, the anagen bulb (Figure 1A) [3]. The lower end of the infundibulum is marked by the insertion of the sebaceous gland duct — a constant trouble area in patients with acne. At the proximal end, the infundibulum joins the isthmus region of the outer root sheath, where the arrector pili muscle is inserted (Figure 1A). The lower isthmus also harbours epithelial and melanocytic hair follicle stem cells in the so-called bulge region. The bulge is the end of the permanent, non-cycling region. Bulge and anagen bulb, the bulbar end of the hair follicle, are separated by a long stretch of suprabulbar hair-follicle epithelium (Figure 1A,C). The anagen bulb contains the matrix keratinocytes and the hair follicle pigmentary unit. Activated matrix keratinocytes, which have migrated out of the bulge to colonize the matrix area, are rapidly proliferating cells and their number determines hair bulb size and hair shaft diameter. When matrix cells stop proliferating and differentiate, they give rise to the various cell lineages of the hair shaft and the inner root Review R133 Box 1. Frequent diseases of hair growth. Alopecia Abnormal hair loss; androgenetic alopecia is baldness caused by miniaturization of genetically predisposed follicles; alopecia areata (patchy hair loss) is thought to be caused by an autoimunne reaction to anagen follicles; scarring or cicatricial alopecia, caused by destruction of hair follicles after inflammation and other causes. Anagen Abrupt hair shedding caused by interruption of effluvium hair growth, for instance in patients undergoing chemotherapy. Hirsutism Excessive hair growth in androgen-dependent areas in women. Hypertrichosis Excessive hair growth beyond the normal pattern. Miniaturization Conversion of large terminal hairs into small vellus hairs. Telogen Poorly defined thinning of scalp hair, mostly effluvium associated with physical or psychological stress. sheath, while the outer root sheath is derived from separate progenitor cells (Figure 1C,D) [13,14]. While infundibulum, isthmus, bulge and hair bulb are all part of the hair follicle epithelium, i.e. of ectodermal origin, the dermal papilla is mesoderm-derived. The dermal papilla (Figure 1C,D), which consists of a small cluster of densely packed fibroblasts, dictates hair bulb size, hair shaft diameter and length, and anagen duration [2,11,14,15]. When looking at a cross-section of the hair follicle, its epithelium forms a cylinder with at least eight different concentric layers, each one expressing a distinct pattern of keratins [3]. Starting from the periphery, these layers include the outer root sheath, the companion layer, the inner root sheath, and finally the hair shaft (Figure 1D). In its bulge region, the outer root sheath contains the epithelial hair follicle stem cells [14]. The central part of the hair follicle epithelium holds the hair shaft (see below and Figure 1D). The entire hair follicle epithelium is surrounded by a mesoderm-derived connective tissue sheath (Figure 1C,D), a loose accumulation of collagen and stromal cells resting upon a basement membrane. The hair shaft is enwrapped by the cuticle, and further components are the cortex and the medulla (Figure 1D). In adult humans, there are two major hair types: heavily pigmented terminal hairs, found on the scalp, and vellus hairs, which are most abundant e.g. in facial and truncal skin [3]. Mice display at least 8 major hair types: pelage, or coat hairs, vibrissae or whiskers, cilia or eyelashes, tail hairs, ear hairs, and hairs around the feet, the genital and perianal area and the nipples [12]. Discerning these various hair types is very important when analysing mouse hair mutants, since distinct hair follicle populations show substantial differences in their molecular controls [7]. Hair Follicle Development and Its Molecular Control The key prerequisite for hair follicle development is the molecular communication between the epidermis and the underlying mesenchyme (Figure 2) [7,8,16]. The fundamentals of this reciprocal interaction are evolutionarily ancient and required for the development of all ectodermal appendages across species, including scales, feathers, hair, nails and teeth, as well as most exocrine glands [17,18]. The signals controlling epidermal–dermal communication include secreted molecules of the Wnt/wingless family, the hedgehog family, and members of the TGF-b/BMP (transforming growth factor-b/bone morphogenetic protein), FGF (fibroblast growth factor) and TNF (tumour necrosis factor) families [7,8,16]. Different combinations of these signals may dictate whether a tooth or a hair follicle will develop [7,8,16]. In mice, both Wnt and BMP are essential for the development of skin epithelium, which originates from the ectoderm Figure 1. Histomorphology of the hair follicle. (A) Sagittal section through a human scalp hair follicle (anagen VI) showing the permanent (infundibulum, isthmus) and anagenassociated (suprabulbar and bulbar area) components of the hair follicle. (B) High magnification image of the isthmus. The dashed square indicates the approximate location of the bulge. (C) High magnification image of the bulb. (D) Schematic drawing illustrating the concentric layers of the outer root sheath (ORS), inner root sheath (IRS) and shaft in the bulb. The inner root sheath is composed of four layers: Companion layer (CL), Henle’s layer, Huxley’s layer, and the inner root sheath cuticle. The companion layer cells are tightly bound to Henle’s layer, but not to the outer root sheath, thus allowing the companion layer to function as a slippage plane between the stationary outer root sheath and the upwards moving inner root sheath. Further inwards, the inner root sheath cuticle is composed of scales that interlock with the scales of the hair shaft cuticle, anchoring the shaft in the follicle and enabling both layers to jointly move during hair follicle growth. The hair shaft is wrapped by a protective layer of overlapping scales and in mice, but not in humans, shows in its centre regularrows of air spaces believed to play a role in thermal insulation (BM: basal membrane; APM: arrector pili muscle; CTS: connective tissue sheath; DP: dermal papilla; M: matrix; HS: hair shaft, IRS: inner root sheath; ORS: outer root sheath; SG: sebaceous gland). (Histology kindly provided by Katja Meyer.) Current Biology Vol 19 No 3 R134 Box 2. Glossary of hair follicle anatomy. Arrector pili muscle Bulb Bulge Club hair Dermal papilla Hair canal Hair germ Hair peg Hair shaft Infundibulum Inner root sheath Isthmus Outer root sheath Pelage hairs Sebaceous gland Tiny smooth muscle that connects the hair follicle with the dermis. When contracted the arrector pili causes the ‘raising’ of the hair. Thickening of the proximal end of the hair follicle. Contains rapidly proliferating, rather undifferentiated matrix cells (transient amplifying cells), melanocytes and outer root sheath cells. Convex protrusion of the outer root sheath in the most distal permanent portion of the hair follicle, just below the sebaceous gland and at the insertion site of the muscle arrector pili. Contains the hair follicle stem cells. Fully keratinized, dead hair (telogen follicle) formed during catagen and telogen. Mesodermal signaling center of the hair follicle consisting of closely packed specialized mesenchymal fibroblasts. Framed by the enlarged bulb matrix in anagen. Tubular connection between the epidermal surface and the most distal part of the inner root sheath. Contains the hair shaft. Also called ‘hair placode’ depending on the developmental stage. Bud-like thickening in the fetal epidermis consisting of elongated keratinocytes, which at the distal end are in touch with numerous aggregated specialized dermal fibroblasts, the dermal condensate. Column of keratinocytes growing into the dermis during embryonic hair follicle development (developmental stages 3-5). The concave proximal end starts to encase the dermal condensate, the future dermal papilla. The hair per se, composed of trichocytes, which are terminally differentiated hair follicle keratinocytes. It is composed of the medulla, the central part with loosely connected keratinized cells and large air spaces, and the cortex, which is the bulk of the hair shaft, consisting of keratinized cells, keratin filaments, and melanin granules in pigmented hairs. Most proximal part of the hair follicle relative to the epidermis, extending from the sebaceous duct to the epidermal surface. Includes the hair canal and the distal Outer root sheath. A multilayered, rigid tube composed of terminally differentiated hair follicle keratinocytes, surrounded by the outer root sheath. Middle part of the hair follicle extending from the sebaceous duct to the bulge. The outermost layer of the hair follicle. Merges proximally with the basal layer of the interfollicular epidermis and distally with the hair bulb. Pelage hair covers most of the body’s surface, and at the molecular level it is the most extensively studied hair type in mice. Divided into four types: the large primary monotrich or guard hairs, the secondary intermediate awl and auchene hairs, and the secondary downy zigzag hairs. Acinar gland composed of lipid-filled sebocytes, localized close to the insertion of the arrector pili muscle. Secretes sebum to the epidermal surface via a holocrine mechanism. Sebum helps making hair and skin waterproof. Together with the hair follicle and the arrector pili muscle it forms the pilosebaceous unit. whereas Wnt/b-catenin signalling alone decides on dermal fate around stage E10.5 [8,19]. Once mesenchymal cells from diverse origins populate the dermis, they start interacting with the overlying epidermis to eventually induce the growth of regularly interspaced hair placodes [7,19]. Specific cues from the dermis are thought to induce the overlying epidermal keratinocytes to assume an upright position and to start proliferating. This becomes apparent in the form of a small epithelial ingrowth into the dermis, called the hair placode (Figure 2). The molecular nature of the earliest hair follicle-inducing cue from the dermis remains unclear. Hair follicle induction results in hair placode formation, which is followed by hair follicle organogenesis and cytodifferentiation (or maturation), each phase being characterized by specific molecular interactions (Figure 2 and Table 2). These three developmental phases are subdivided into eight morphologically distinguishable developmental stages (Figure 2) [7,20]. Canonical Wnt/b-catenin signalling provides the master switch for hair follicle fate, as ectopic epithelial expression of the secreted Wnt inhibitor Dkk1 or lack of epidermal b-catenin expression results in absence of hair follicle Table 1. Classical mouse mutants with hair follicle defects and the characterization of their molecular defect. Mutant Hair follicle phenotype Gene affected References Ragged/Opossum Hypoplasia of selected pellage follicles; absence of auchene, and very few zigzag hairs Alopecia behind the ears and on the tail; lack of guard and zigzag hairs Premature protein truncation in Sox18 [98] Mutations in the Ectodysplasin-A1 gene (Eda-A1). Defective NF-kB signaling and disturbed formation of skin appendages Point mutation in the epidermal growth factor receptor gene resulting in a ‘dead’ kinase Mutations in the gene encoding Sgk3 [99] Insertion in the hairless gene; mutation of the human genes causes alopecia universalis Deletion of fibroblast growth factor 5 (FGF5) increases anagen duration and delays initiation of catagen Truncation of forkhead transcription factor Foxn1 Truncation of desmosome protein Desmoglein-3 [101] [47] [44] Tabby Waved 2 Wavy hairs, curly whiskers Fuzzy Sparse fur, abnormal hair morphology and acceleration of hair follicle cycling Initially normal hair growth. Total alopecia around the age of 3-4 weeks Very long hairs. No defects in hair follicle morphology or hair structure Hairless Angora Nude Balding Lethal spotting General alopecia, except for whiskers Focal alopecia due to separation of inner and outer root sheath in anagen follicles White spots in pelage hair. Affects pigmentation Point mutation in endothelin-3 [100] [62] [102] [103] [104] [105] Review R135 Induction Stage 0 Organogenesis Stage 1 (placode) Stage 2 (germ) Cytodifferentiation Stage 3–5 (peg) Stage 6–8 (bulbous peg) See Table 2 for molecular controls of ORS, IRS and hair shaft formation, polarity, shaping, innervation etc. See Table 2 for molecular controls of ORS, IRS and hair shaft formation, polarity, shaping, innervation etc. Epidermis Dermis Interacting gradients of activators and inhibitors creating an inductive field in the epidermis (pre-germ). Specialised dermal fibroblasts gather underneath pre-germ. Visible hair germ (placode). Promotion of placode growth: Wnt/ β-catenin. EdaA1/EdaR/NF-κB. Noggin/Lef-1. CTGF? Ectodin? P-cadherin. Proliferation of epidermal hair germ cells: Shh/Smo/Gli2, Wnt (10b,10a)/Lef-1, FGFs/FGF2R-IIIb. TGFβ2? Follistatin? Inhibition of placode fate in surrounding cells and placode growth: Dkks (Dkk1, 2, and 4). BMPs (2, 4 and 7) Formation of dermal papilla: Shh/Smo/Gli2, PDGF-A. Current Biology Figure 2. Embryonic pelage hair follicle development in mice. Murine hair follicle development can be divided into three phases: Induction, organogenesis and cytodifferentiation. The morphological characteristics of the most important stages (0–8) of prenatal hair follicle development and their molecular controls are depicted. Primary guard hair development starts as early as stage E14. Secondary hair follicle development comes in two major waves: At E16.5 for the intermediate awl and auchene hairs, and around P0 for the downy zigzag hairs. Induction (left column): At stage 0, prior to visible hair follicle placode formation, interaction between the epidermis and the underlying dermis, which is the likely source of the unknown first inductive signal, involves local hair follicle activators (such as Wnt/b-catenin signaling) overriding hair follicle inhibitors, thereby creating an inductive field. In subsequent stages, the hair placode becomes visible to the eye and downward growth is initiated. At these stages the dermal condensate and future dermal papilla form. Within the developing placode, BMP signaling has to be down-regulated by specific BMP antagonists in order to allow for placode growth. Organogenesis (middle column) and Cytodifferentiation (right column): In the following stages 3–8, the orientation of the follicle is defined (peg stage), and the different hair lineages develop (bulbous peg). Colors of cells do not correspond to particular signals. ORS: outer root sheath; IRS: inner root sheath. induction [21,22]. Conversely, forced expression of a stabilized form of b-catenin causes strongly enhanced placode formation, because epidermal keratinocytes globally adopt a hair follicle fate [23,24]. While forced epidermal expression of secreted Dkk1 does not rule out that the Wnt signal may originate in the dermis, the absence of hair follicle placode formation in mice with epithelial lack, or forced expression of an activated b-catenin strongly suggests that Wnt signalling in the surface ectoderm is essential for hair follicle fate [21,24]. Once hair follicle fate has been adopted by regionally defined groups of specialized keratinocytes, placode formation and growth are initiated. Mouse pelage hair follicle development takes place in two waves, starting with the primary (guard) hairs at E14.5, followed by secondary intermediate (awl, auchene) and downy (zigzag) hairs between E17 and P0. Importantly, placode formation signals vary significantly between the different hair types (Figure 2) [7]. Growth of primary guard hair follicles requires Eda-A1, its receptor Edar and their downstream effector, the transcription factor NF-kB [7]. Stimulation of the Eda-A1/Edar/NF-kB pathway upregulates Shh and cyclin D1 expression leading to placode growth [7,25]. In contrast, secondary awl hair follicle development is independent of Eda-A1/EdaR/NF-kB signalling, but requires the BMP antagonist Noggin and the transcription factor Lef-1 [26–28]. Inductive signals for zigzag hair placodes remain unknown, but mice with suppressed NF-kB activity fail to develop zigzag placodes [29]. Epithelial placode cells signal to the underlying mesenchyme to form the dermal condensate which will subsequently give rise to the dermal papilla. Sonic hedgehog (Shh) is a pivotal growth signal for dermal papilla maturation and growth (Figure 2) [30,31]. The dermal condensate itself sends out specific growth signals to the epidermis allowing placode growth into the underlying mesenchyme, and further reciprocal epithelial–mesenchymal signalling will eventually lead to maturation and formation of the different hair follicle lineages (Figure 2 and Table 2). Not every epidermal keratinocyte will become a follicular keratinocyte, which results in hairless spaces between the regular array of hair follicles. This implies that there must be a mechanism which promotes or blocks hair follicle fate. Local gradients of hair follicle activators and inhibitors are believed to account for this mechanism. Eventually hair follicle activating signals will create a morphogenetic field by locally overriding hair follicle inhibitors [7,32–34]. While Wnt, Eda-A1 and Noggin are bona fide hair placode-inducing signals, the hair follicle inhibiting signals and their mechanisms of action remain to be clarified. Results from studies with embryonic mouse skin explants show that BMP is able to inhibit placode growth [28,35]. This is further confirmed by the hair follicle type-specific expression of BMP antagonists within hair follicle placodes: In guard hairs, Eda-A1/Edar/NF-kB up-regulates connective tissue growth factor (CTGF) expression [25,35], while secondary hair Current Biology Vol 19 No 3 R136 Table 2. Summary of the best defined molecular regulators of hair follicle morphogenesis. Hair Follicle Morphogenesis Step Gene Product Outer root sheath Sox9 Shh/Smo Inner root sheath GATA3 Cutl1 (CDP) BMPs/BMPR1a Shh/Smo Hair shaft Notch1/Jagged/ Delta/RBP-Jk for IRS fate maintenance b-catenin, Wnt’s and Lef-1 VDR (Vitamin D receptor) (only in postnatal hair cycle)* BMPs/BMPR1a Shh/Smo msx1 and 2 FoxN1/Nude HoxC13 Notch1/Jagged/Delta/RBP-Jk (only in postnatal hair cycle)* Polarity, shaping Shh (asymmetric, polarized and bending expression pattern in hair matrix) Igfbp5 Eda A1 Krox-20 FoxE1 Runx3 Sox18 Innervation Pigmentation Bulge region/ hair stem cell maintenance b-catenin NCAM FoxN1/Nude SCF/c-kit Notch BMPs/BMPR1a Lhx2 Sox9 NFATc1 p63 Tcf3 (w/o Wnt activity!) Rac1 integrins a3b1 and a6b4 References [82] [106], and references therein [107] [108] [36–38] [106], and references therein Reviewed in [52] [23,24] [109] [36–38] [106], and references therein [110] [102] Reviewed in [111] Reviewed in [52] [112] [113] Reviewed in [7] [114] [115] [116] [117] [24] [118] [92] [90] Reviewed in [119] Reviewed in [8,9] Reviewed in [8] [79,82] [78] Reviewed in [9] [60] Reviewed in [80] Reviewed in [9] Transcription factors in Italics. placode development depends on Noggin [27]. However, mice deficient in epidermal BMP activity have relatively normal placode numbers, suggesting that inhibition of epidermal BMP activity alone is insufficient to stimulate hair follicle placode formation, and that BMP inhibition may be most important within the developing placode in order to allow for growth [36–38]. The regular pattern of hair follicle placodes may reflect the Turing model of reaction–diffusion systems [32–34], as previously described for feather development [39]. Both, Wnt and Dkks are secreted into the extracellular space and can therefore participate in such a reaction–diffusion system (Figure 2). Given the key role of Wnt in hair follicle fate induction [21], its activity within and around the developing hair placode must be tightly controlled. Limiting Wnt activity may also be important to avoid skin tumour formation, which occurs in mice with overactive epidermal Wnt/b-catenin signalling [40]. Due to its smaller molecular size, the Wnt inhibitor Dkk4 could easily diffuse into placode surroundings, thereby inhibiting Wnt signalling in the interfollicular epidermis proximal to the developing hair follicle placode, while the larger Wnt molecules would remain within the placode [34]. Thus, moderate overexpression of the hair follicle activator Wnt would increase hair follicle density, while moderate overexpression of hair follicle inhibitor Dkk4 would decrease hair follicle density [21,23,24,34]. Hair Follicle Cycling The hair follicle undergoes regular cycles of involution and regeneration throughout life. Once a first ‘test’ hair shaft has been generated during continued postnatal hair follicle morphogenesis, in mice, hair follicle cycling is initiated about 17 days post partum. At this time, hair follicles undergo rapid organ involution (catagen; Figure 3). This first catagen lasts two to three days, and is followed by the first phase of relative quiescence (telogen; Figure 3). In mice, hair follicle morphogenesis proceeds throughout early postnatal life, which is why it is routinely misinterpreted as ‘first anagen’. Instead, the first real growth phase of the hair follicle cycle (anagen; Figure 3) does not occur until 4 weeks after birth [41]. In anagen, the hair matrix keratinocytes, which represent transient amplifying cells derived from epithelial hair follicle stem cells in the bulge, proliferate intensively and then differentiate into distinct epithelial hair lineages (Table 2) [2,8,13,41]. During catagen, the lower two-thirds of the hair follicle rapidly regress, mainly by apoptosis of matrix, inner root sheath and outer root sheath keratinocytes, while bulge hair follicle stem cells escape apoptosis. Eventually, the lower hair follicle becomes reduced to an epithelial strand, bringing the dermal papilla into close proximity of the bulge (Figure 3) [2,41]. In mice, the old hair shaft (club hair) normally remains in its canal even when the new hair emerges through the same orifice, and the telogen club hair can rest in its socket during several cycles, thereby contributing to the density of the coat. This leads to bulging of the outer root sheath around the club (Figure 3). While old hair shafts can be shed passively by mechanical forces, shedding may mainly be an active process, termed exogen, the molecular controls of which remain to be clarified [2]. Upon catagen completion, hair follicles enter a phase of relative quiescence (telogen), lasting several days in the first telogen and usually more than 3 weeks in the second telogen. With each additional cycle telogen duration expands, and hair follicle cycling slows down considerably in aging animals. Moreover, while the initial waves of pelage hair follicle cycling in mice are highly synchronized, patches of coordinated hair follicle cycling will eventually form distinct hair cycle domains, which become more scattered as the mouse ages [42]. In contrast, in humans, synchronized fetal hair follicle cycling becomes asynchronous soon after birth, when each hair follicle starts to follow its own mosaic cycling pattern [3,11]. The molecular mechanisms that drive hair follicle cycling remain obscure, although multiple molecular regulators have been identified through mouse mutants with defects in hair follicle cycling, and by characterizing gene expression profiles of distinct murine hair cycle stages [2,11,43]. Mouse mutants have demonstrated that Wnt/b-catenin, BMP antagonists (e.g. Noggin) and Shh act as anagen-inducing signals, while FGF5 is a key inducer of catagen [2,3,11,44]. FGF5-deficient mice have a prolonged anagen phase resulting in an angora hair phenotype [44]. In addition to FGF5, TGF-b1, Review R137 Permanent Start of hair follicle cycling Stage 8 Stage 1 DC SG Stage 5 Stage 3 SC Placode Cycling Epidermis Catagen APM HS Bulge IRS cone MC DP Regressing epithelial column ge n Hair follicle morphogenesis Ca ta Anagen VI Anagen SG Telogen Teloge n APM Bulge Bulge Club hair DP Anagen IV IRS Anagen II HS ORS Bulge Bulb New hair germ DP New hair DP Current Biology Figure 3. Key stages of the hair cycle. The hair cycle is divided into three phases: Anagen (growth phase), catagen (regression phase) and telogen (resting phase). Postnatal hair morphogenesis leads to elongation of the follicle and production of the hair fiber, which emerges from the skin. Once the hair follicle has matured, it enters the regression phase, during which the lower, cycling portion of the hair follicle is degraded. This process brings the dermal papilla into close proximity of the bulge, where the hair stem cells (HSCs) reside. The molecular interaction between the HSCs and the dermal papilla is essential to form a new hair follicle. The proximity between bulge and dermal papilla is maintained throughout telogen, the resting phase. Only when a critical concentration of hair growth activating signals is reached, anagen phase is entered and a new hair is regrown. The first postnatal hair cycle is initiated and passed by all hair follicles at the same time point, while subsequent cycles are no longer synchronized. Stages 1–8 of embryonic hair development are depicted (upper left), demonstrating the continuous transition between hair follicle development and the first postnatal hair cycle. APM: arrector pili muscle; DC, dermal condensate (green); DP: dermal papilla (green); HS: hair shaft (brown); IRS: inner root sheath (blue); MC: melanocytes; ORS: outer root sheath; SC: sebocytes (yellow); SG: sebaceous gland. interleukin-1b, the neurotrophins NT-3, NT-4 and BDNF, BMP2/4 and TNF-a have been described to induce catagen [2,11]. Conversely, insulin-like growth factor-1 (IGF-1), hepatic growth factor (HGF), and vascular endothelial growth factor (VEGF) are thought to be important for anagen maintenance [11,45]. Furthermore, the molecular crosstalk between downstream effectors of TNF-a signalling and keratin17 (K17) may be in part responsible for controlling catagen entry by regulating the rate of apoptosis [45]. Additional molecules implicated in controlling anagen– catagen transformation include the vitamin D receptor (VDR), the transcriptional repressor Hairless and the retinoic acid receptor [11,46–49]. Interestingly, Hairless interacts with VDR to regulate transcription [50], and in mice lacking these regulators, hair follicles disintegrate into epithelial sacs and dermal cysts upon catagen entry [46–49]. The concomitant detachment of the dermal papilla from the hair follicle epithelium disrupts the essential interaction between hair follicle stem cells and the inductive hair follicle mesenchyme [46–49]. Although traditionally described as the resting phase of the hair follicle cycle, telogen coincides with major changes in gene activity [43]. Indeed, some regulatory proteins, like the estrogen receptor, are markedly up-regulated in telogen [51]. Therefore, telogen is not at all quiescent, and probably represents a key stage in hair cycle control. This is supported Current Biology Vol 19 No 3 R138 Stem cell Activation stab. β-catenin, Wnts? ↑c-myc Maintenance LHX2, Sox9, low c-myc, integrins α3β1 and α6β4. Quiescence BMPs, TCF3 and other Wnt inhibitors, NFATc1 ↑ BMP Transient amplifying cell 2–5 divisions. ↓ integrins and hemidesmosomes Hair follicle Differentiation Wnt/β-cat, TGFβ, Notch, BMPs, Shh, etc. ↑ BMP Figure 4. Schematic presentation of the temporal interplay of the molecular players in hair stem cell activation. Upper panel: List of molecules required for stem cell maintenance/quiescence, activation and differentiation. Lower panel: Comparison between the relative amounts of BMP (blue) and Wnt/b-catenin (orange) activity needed for quiescence, activation and differentiation of hair follicle stem cells. For more details see main text. and Wnt activation together with stabilization of b-catenin (Figure 4) [8– 10,40,57,58]. In addition, both pathways may temporally regulate the activities of each other: During quies↓ BMP Wnt/β-cat W nttWnt/β-cat / β -catt ↓ BMP B MP cence, BMP may indirectly prevent – + b-catenin stabilization [59]. However, Quiescence Differentiation HF Activation at anagen onset, increasing Wnt sigCurrent Biology nalling may induce b-catenin stabilization and the local expression of BMP by the recent discovery that telogen can be divided into inhibitors in the dermal papilla [8,10]. Nevertheless, it a phase that is refractory to hair follicle growth stimuli and currently remains unexplained how Wnt signalling is that is characterized by upregulation and activation of switched on. During early anagen development, the bulge progressively BMP2/4, and a competent phase in which bulge stem cells become highly sensitive to anagen-inducing factors [42]. In moves away from the dermal papilla (Figure 3), and epithelial the competent phase, BMP signalling is turned off while hair follicle stem cells re-adopt quiescence. However, the Wnt/b-catenin signalling is turned on to reach its optimal stem cell progeny in the hair matrix (transient amplifying activity in early anagen. Interestingly, there are cyclic changes cells) maintains active Wnt signalling and concomitant stabiin BMP2 and BMP4 expression in extrafollicular skin, namely lization of b-catenin throughout anagen. More distally in the in subcutaneous adipocytes [42]. This extrafollicular system precortical hair matrix, these cells stop proliferating and must interact with the autonomous, intrafollicular ‘hair cycle initiate terminal differentiation into the different lineages of hair follicle epithelium (Figure 1D and Table 2). By now, clock’ to regulate individual hair follicle cycles [11]. In telogen, the dermal papilla comes to rest immediately many molecular regulators of hair lineages have been below the bulge, which allows direct interactions between discovered, such as GATA3, Cutl1 and BMPs for inner root bulge stem cells and the dermal papilla. The dermal papilla sheath formation, Sox9 and SHH for outer root sheath formais essential for stem cell activation and initiation of a new tion, and Wnt/b-catenin, VDR, Notch, BMPs and Foxn1 for hair cycle. Once a critical concentration of stem cell activa- the expression of selected hair shaft keratins and/or hair tors has been achieved, anagen is initiated [11]. Despite the shaft development (Table 2). In contrast, TCF3 operates as many morphological and molecular parallels between the a general inhibitor of all epithelial lineages [60]. Many important questions in hair cycle research remain interactions of the bulge region with the dermal papilla and the hair follicle placode with the dermal condensate during unanswered. For instance, it remains unknown how only hair follicle development, there are also marked molecular a few selected stem cells are activated during each cycle differences (Figure 3, Table 2). For example, some members and how the other stem cells are kept in a quiescent state. of the TGF-b and nerve growth factor families have opposing And how exactly is the responsiveness of stem cells to Wnt functions during hair follicle development and cycling: While regulated? The most important challenge, however, is to both promote development, they stimulate regression (cata- dissect the molecular nature of the autonomous intrafollicugen) in mature follicles [11]. Other signals, like VDR, hairless lar oscillator system that regulates the cyclic transformation and Notch, are dispensable for hair follicle development, of each hair follicle [11]. While many genes controlling epithelial hair follicle stem cell activity and hair follicle cycling have but essential for anagen induction (Table 2) [48,52]. The molecular details of stem cell activation remain been identified, the intrafollicular clock seems to be unknown. However, a critical threshold concentration of controlled by a more elementary molecular machinery, which one or several activator molecules must be reached to may even incorporate differential ‘clock gene’ activities. This trigger anagen onset, while inhibitors have to be switched hair cycle clock must not be confused with signals that arise off. Some molecular players that control the balance from extrafollicular skin to alter the cycling of large groups of between stem cell quiescence/maintenance and activation hair follicles in order to induce wave and domain pattern have already been identified by gene expression profiling formation [42]. Mouse genetics and further hair cycle gene of the hair cycle and the hair follicle compartment in mice expression profiling appear most promising approaches and humans [15,43,53–56]. While stem cell quiescence towards elucidating the enigmatic ‘‘hair cycle clock’’ appears to be maintained by activation of BMP signalling [11,43]. A more detailed analysis of mice with greatly accelin combination with Wnt inhibition by TCF3 and DKKs, erated hair follicle cycling, such as fuzzy mice, which carry stem cell activation is thought to require BMP inhibition a mutation in sgk3, may be a good start [61,62]. Review R139 Hair Follicle Stem Cells As a self-regenerating system, the hair follicle and its connective tissue sheath are not only a rich source of epithelial, but also of melanocyte, mesenchymal and neural stem cells [9,14,63–65]. Making use of their slow cycling, labelretaining experiments have located adult epithelial hair follicle stem cells in the hair follicle bulge region of mice and humans [14,63]. This led to the ‘bulge activation hypothesis’: At the onset of anagen, a subset of hair follicle stem cells is activated and starts to divide. Their daughter cells migrate to the base of the follicle where they start to proliferate and become transiently activated matrix cells. In vivo labelling and transplantation studies confirmed the ability of bulge stem cells to give rise to all cell lineages of the mature hair follicle [66,67]. In accordance with this hypothesis, major inflammatory damage of the bulge can result in permanent, cicatricial alopecia [68,69]. Unless the skin is injured, bulge stem cells exclusively contribute to hair follicle maintenance and regeneration [70–73], although they are also capable of generating sebaceous glands and the interfollicular epidermis [55,56]. Conversely, at least in mice, epithelial cells may assume a hair follicle stem cell phenotype under certain wounding conditions, which may lead to hair follicle neogenesis in adult skin [4]. Apart from their ability to generate hair follicles, sebaceous glands and interfollicular epidermis, epithelial hair follicle stem cells also hold a ‘dark side’: Their long life span exposes them to multiple genetic mutations, and their quiescent character may facilitate retention of carcinogens, thus rendering them more susceptible to tumour development, such as basal cell carcinoma. [14]. Keratin 15 (K15) and CD34 are typical markers for rodent hair follicle epithelial stem cells [14,74,75], and K15 has been used to isolate putative murine bulge stem cells and to establish their gene expression profile [54–56]. When compared with epidermal basal layer stem cells, genes typically upregulated in the bulge include stem cell markers such as c-kit ligand, ephrin tyrosine kinase receptors and CD34, but also many known bulge markers like the homeobox genes and transcription factors Barx2, Sox9, Lhx2, and the transcription factor TCF3 [54–56]. As expected, down-regulated genes include proliferation-associated proteins such as Ki67and Cdc25C. Genes involved in Wnt pathway activation are largely repressed in murine bulge stem cells, while the expression of Wnt inhibitors such as Srfp1, Dab2, and TCF3 is increased as compared to non-bulge keratinocytes [54–56,60]. This is in line with the concept that Wnt signalling induces epithelial hair follicle stem cells to differentiate and to adopt a hair fate [22,58,60]. Hair follicle stem cell quiescence requires BMP signalling [8,10]. In mice lacking BMPR1a, hair follicle stem cells are continuously activated and start proliferating aberrantly, resulting in the eventual loss of slow cycling cells in these mice [36–38,76,77]. As expected, these mice reveal increased and aberrant levels of Lef-1 and stabilized b-catenin in the stem cell niche, suggesting that BMP signalling may be important for decelerating the cell cycle and maintaining the hair follicle stem cell population by preventing activation of the Wnt pathway (Figure 4). In addition to BMPs, stem cell quiescence appears to be regulated by the Wnt inhibitor TCF3 and the calcium-dependent transcription factor NFATc1 [60,76,78]. Stem cell maintenance is ensured by slow cycling, and controlled by low levels of c-myc and by the expression of the transcription factors LHX2 and Sox9 (Figure 4) [79–82]. Conversely, activation of stem cells during the telogen–anagen transition requires BMP inhibition by BMP antagonists, Wnts and stabilized b-catenin, as well as increased levels of c-myc, and Runx1 (Figure 4) [8,10,83]. Distinct subpopulations of epithelial hair follicle stem cells and their progeny are already present at the earliest stages of hair follicle development prior to birth [79]. Sox9-positive stem cells appear to be required for the postnatal formation of a normal matrix, outer root sheath, bulge and sebaceous gland [79,82]. By contrast, Lhx2-positive stem cells are found in the early placode and germ, but disappear later in hair follicle development. These cells may represent transient amplifying cells that participate in prenatal hair follicle initiation and development [79]. In human hair follicles, characteristic hair stem cell markers remain to be identified [64]. However, up-regulated KRT15, KRT19 and CD200 expression, combined with the absence of CD34, nestin, and connexin 43 expression may indicate the presence of human bulge epithelial stem cells [84]. Finally, transcriptional profiling studies of human bulge cells have revealed similarities and important disparities between mouse and human bulge cells [53]: Similar to mouse hair stem cells, inhibitors of the Wnt signalling pathway and CD200 are transcriptionally up-regulated, while the mouse bulge stem cell marker CD34 is not expressed [14,64,84]. Hair Pigmentation Hair shaft pigmentation serves multiple purposes, which vary between species: skin protection against UV irradiation, thermoregulation, camouflage and sexual signals. Furthermore, the hair pigment melanin, which is produced in hair follicle melanocytes, is a potent free-radical scavenger. Melanin production inside the metabolically highly active anagen hair bulb may, therefore, help to buffer cell stress induced by reactive oxygen species. In addition, the transfer of melanin from melanocytes to keratinocytes may promote differentiation of the melanin-receiving keratinocytes situated in the precortical hair matrix [85]. In contrast to the continuous melanogenesis observed in epidermal melanocytes, follicular melanogenesis is a strictly anagen-coupled, cyclic phenomenon. It is shut off very early during the anagen–catagen transition, beginning with the down-regulation of key enzymes of melanogenesis, followed by hair follicle melanocyte apoptosis [85]. Hair follicle melanocytes and their precursors reside in the hair matrix and along the outer root sheath of anagen hair follicles. However, production of hair pigment (black eumelanin and/or the reddish pheomelanin) only occurs in the specialized hair follicle pigmentary unit, located above and around the dermal papilla during anagen III–VI. Melanin synthesis is confined to lysosome-related organelles, melanosomes, under the control of tyrosinase. In the precortical matrix, these melanosomes are actively exported and transferred to the recipient hair shaft keratinocytes, resulting in the formation of a pigmented hair shaft [85]. In mice, neural-crest derived melanoblasts populate the hair follicle epithelium in defined developmental waves, a process regulated by c-KIT and its ligand, stem cell factor (SCF) [86]. The melanocytes and melanoblasts that come to reside in the outer root sheath normally do not in engage in pigment production, yet can replace lost epidermal melanocytes. The hair follicle also contains melanocyte stem cells, which are located in the bulge and in the secondary hair Current Biology Vol 19 No 3 R140 germ region (i.e. below the telogen club hair), where they serve as a cell pool for the cyclic regeneration of the hair follicle pigmentary unit [85]. Melanocyte stem cells are needed for replacing the vast majority of differentiated, melanotic hair follicle melanocytes that undergo apoptosis during catagen [85,87]. Preservation of the hair follicle melanocyte stem cell pool and regulation of the sensitive balance between maintenance and differentiation appears to depend on the transcription factors Pax3 and Mitf [88,89]. The correct localization and activity of melanocytes within different skin compartments is directed by paracrine signals. In murine pelage hair follicles, these include SCF and hepatocyte growth factor [90,91]. Recently, expression of the forkhead family transcription factors, Foxn1, and activation of its downstream target FGF2 in pigment-recipient keratinocytes were identified as the major regulators in determining the patterning of hair pigmentation [92]. Thus, hair pigmentation patterning appears to depend on the melanin-receiving hair follicle keratinocytes, which specifically attract hair follicle melanocytes into their close proximity by FGF2 via Foxn1 [92]. Graying of hair (canities) is the direct result of a marked loss in active melanocytes in the hair follicle pigmentary unit, and usually begins between the mid-30s and the mid-40s. The chief cause of graying has been attributed to a depletion of the melanocyte stem cell reservoir. However, damage to the hair follicle pigmentary unit by reactive oxygen species probably also plays an important role in graying, independent of, and in addition to its deleterious effects on hair follicle melanocyte stem cells [85,93]. It is not clear yet, however, why hair follicle melanocytes and their progenitors appear to age faster than their epidermal counterparts. regenerative medicine [6,64,95]. Beyond the topics covered in this review, emerging areas in hair research — for example, understanding the immune privilege of the hair follicle bulb and bulge and the complex (neuro-)endocrine activities of the hair follicle — may revolutionize our understanding of hair follicle biology in the near future [69,96,97]. Acknowledgments Writing of this review was made possible in part by grants from the Deutsche Forschungsgemeinschaft (DGF) to M.R.S. (GRK1029), R.S.U. (SCHM 855/3-1) and R.P. (Pa 345/12-1). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Conclusions and Perspectives Proper functioning of the hair follicle comprises perfectly coordinated principles of developmental, stem cell, and tumour biology, tissue regeneration, and the control of cell growth, migration, death, and differentiation. As such, the hair follicle is an accessible and clinically relevant model. From the biological point of view, further detailed studies are needed to unravel the signalling cascades and crosstalk that lead to induction of hair follicle development and subsequent morphogenesis. It will also be interesting to further dissect the molecular differences between fetal hair follicle induction and development and the hair follicle cycle. From the clinical perspective, the key challenge will be to translate the insights from hair follicle biology into treatments for disorders of the human hair follicle, such as androgenetic alopecia, telogen effluvium, alopecia areata, hirsutism, or graying. This may be achieved by specifically targeting some of the key molecular controls in hair follicle cycling and melanogenesis, perhaps even to the point of de novo induction of hair follicles in adult human skin [5,6]. 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