Sustained epithelial beta-catenin activity induces precocious hair development but disrupts hair follicle down-growth and hair shaft formation

During embryonic and postnatal development, Wnt/beta-catenin signaling is involved in several stages of hair morphogenesis from placode formation to hair shaft differentiation. Using a transgenic approach, we have investigated further the role of beta-catenin signaling in embryonic hair development. Forced epithelial stabilization of beta-catenin resulted in precocious and excessive induction of hair follicles even in the absence of Eda/Edar signaling, a pathway essential for primary hair placode formation. In addition, the spacing and size of the placodes was randomized. Surprisingly, the down-growth of follicles was suppressed and hair shaft production was severely impaired. Gene and reporter expression analyses revealed elevated mesenchymal Wnt activity, as well as increased BMP signaling, throughout the skin that was accompanied by upregulation of Sostdc1 (Wise, ectodin) expression. Our data suggest that BMPs are downstream of Wnt/beta-catenin and that their interplay may be a critical component in establishing correct patterning of hair follicles through the reaction-diffusion mechanism.     

Characterization of Wnt gene expression in developing and postnatal hair follicles and identification of Wnt5a as a target of Sonic hedgehog in hair follicle morphogenesis

Mutations in WNT effector genes perturb hair follicle morphogenesis, suggesting key roles for WNT proteins in this process. We show that expression of Wnts 10b and 10a is upregulated in placodes at the onset of follicle morphogenesis and in postnatal hair follicles beginning a new cycle of hair growth. The expression of additional Wnt genes is observed in follicles at later stages of differentiation. Among these, we find that Wnt5a is expressed in the developing dermal condensate of wild type but not Sonic hedgehog (Shh)-null embryos, indicating that Wnt5a is a target of SHH in hair follicle morphogenesis. These results identify candidates for several key follicular signals and suggest that WNT and SHH signaling pathways interact to regulate hair follicle morphogenesis.     

Molecular biology of hair morphogenesis: development and cycling

In mammals, hair follicles produce hairs that fulfill a number of functions including thermoregulation, collecting sensory information, protection against environmental trauma, social communication, and mimicry. Hair follicles develop as a result of epithelial-mesenchymal interactions between epidermal keratinocytes committed to hair-specific differentiation and cluster of dermal fibroblasts that form follicular papilla. During postnatal life, hair follicles show patterns of cyclic activity with periods of active growth and hair production (anagen), apoptosis-driven involution (catagen), and relative resting (telogen). During last decade, substantial progress has been achieved in delineating molecular mechanisms that control hair follicle development and cyclic activity. In this review, we summarize the data demonstrating that regulation of hair follicle development in the embryo and control of hair follicle growth during postnatal life are highly conserved and both require involvement of similar molecular mechanisms. Since many of the molecules that control hair follicle development and cycling are also involved in regulating morphogenesis and postnatal biology of other ectodermal derivatives, such as teeth, feathers, and mammary glands, basic principles and molecular mechanisms that govern hair follicle development and growth may also be applicable for other developmental systems.     

Canonical WNT signaling promotes mammary placode development and is essential for initiation of mammary gland morphogenesis

Mammary glands, like other skin appendages such as hair follicles and teeth, develop from the surface epithelium and underlying mesenchyme; however, the molecular controls of embryonic mammary development are largely unknown. We find that activation of the canonical WNT/beta-catenin signaling pathway in the embryonic mouse mammary region coincides with initiation of mammary morphogenesis, and that WNT pathway activity subsequently localizes to mammary placodes and buds. Several Wnt genes are broadly expressed in the surface epithelium at the time of mammary initiation, and expression of additional Wnt and WNT pathway genes localizes to the mammary lines and placodes as they develop. Embryos cultured in medium containing WNT3A or the WNT pathway activator lithium chloride (LiCl) display accelerated formation of expanded placodes, and LiCl induces the formation of ectopic placode-like structures that show elevated expression of the placode marker Wnt10b. Conversely, expression of the secreted WNT inhibitor Dickkopf 1 in transgenic embryo surface epithelium in vivo completely blocks mammary placode formation and prevents localized expression of all mammary placode markers tested. These data indicate that WNT signaling promotes placode development and is required for initiation of mammary gland morphogenesis. WNT signals play similar roles in hair follicle formation and thus may be broadly required for induction of skin appendage morphogenesis.     

ILK modulates epithelial polarity and matrix formation in hair follicles

Hair follicle morphogenesis requires coordination of multiple signals and communication between its epithelial and mesenchymal constituents. Cell adhesion protein platforms, which include integrins and integrin-linked kinase (ILK), are critical for hair follicle formation. However, their precise contribution to this process is poorly understood. We show that in the absence of ILK, the hair follicle matrix lineage fails to develop, likely due to abnormalities in development of apical–basal cell polarity, as well as in laminin-511 and basement membrane assembly at the tip of the hair bud. These defects also result in impaired specification of hair matrix and absence of precortex and inner sheath root cell lineages. The molecular pathways affected in ILK-deficient follicles are similar to those in the absence of epidermal integrin β1 and include Wnt, but not sonic hedgehog, signaling. ILK-deficient hair buds also show abnormalities in the dermal papilla. Addition of exogenous laminin-511 restores morphological and molecular markers associated with hair matrix formation, indicating that ILK regulates hair bud cell polarity and functions upstream from laminin-511 assembly to regulate the developmental progression of hair follicles beyond the germ stage.     

Interactions between Shh, Sostdc1 and Wnt signaling and a new feedback loop for spatial patterning of the teeth

Each vertebrate species displays specific tooth patterns in each quadrant of the jaw: the mouse has one incisor and three molars, which develop at precise locations and at different times. The reason why multiple teeth form in the jaw of vertebrates and the way in which they develop separately from each other have been extensively studied, but the genetic mechanism governing the spatial patterning of teeth still remains to be elucidated. Sonic hedgehog (Shh) is one of the key signaling molecules involved in the spatial patterning of teeth and other ectodermal organs such as hair, vibrissae and feathers. Sostdc1, a secreted inhibitor of the Wnt and Bmp pathways, also regulates the spatial patterning of teeth and hair. Here, by utilizing maternal transfer of 5E1 (an anti-Shh antibody) to mouse embryos through the placenta, we show that Sostdc1 is downstream of Shh signaling and suggest a Wnt-Shh-Sostdc1 negative feedback loop as a pivotal mechanism controlling the spatial patterning of teeth. Furthermore, we propose a new reaction-diffusion model in which Wnt, Shh and Sostdc1 act as the activator, mediator and inhibitor, respectively, and confirm that such interactions can generate the tooth pattern of a wild-type mouse and can explain the various tooth patterns produced experimentally.     

Polyclonal origin and hair induction ability of dermal papillae in neonatal and adult mouse back skin

Hair follicle development and growth are regulated by Wnt signalling and depend on interactions between epidermal cells and a population of fibroblasts at the base of the follicle, known as the dermal papilla (DP). DP cells have a distinct gene expression signature from non-DP dermal fibroblasts. However, their origins are largely unknown. By generating chimeric mice and performing skin reconstitution assays we show that, irrespective of whether DP form during development, are induced by epidermal Wnt activation in adult skin or assemble from disaggregated cells, they are polyclonal in origin. While fibroblast proliferation is necessary for hair follicle formation in skin reconstitution assays, mitotically inhibited cells readily contribute to DP. Although new hair follicles do not usually develop in adult skin, adult dermal fibroblasts are competent to contribute to DP during hair follicle neogenesis, irrespective of whether they originate from skin in the resting or growth phase of the hair cycle or skin with β-catenin-induced ectopic follicles. We propose that during skin reconstitution fibroblasts may be induced to become DP cells by interactions with hair follicle epidermal cells, rather than being derived from a distinct subpopulation of cells.     

Smad7-induced beta-catenin degradation alters epidermal appendage development

To assess whether Smad signaling affects skin development, we generated transgenic mice in which a Smad antagonist, Smad7, was induced in keratinocytes, including epidermal stem cells. Smad7 transgene induction perturbed hair follicle morphogenesis and differentiation, but accelerated sebaceous gland morphogenesis. Further analysis revealed that independent of its role in anti-Smad signaling, Smad7 bound beta-catenin and induced beta-catenin degradation by recruiting an E3 ligase, Smurf2, to the Smad7/beta-catenin complex. Consequently, Wnt/beta-catenin signaling was suppressed in Smad7 transgenic hair follicles. Coexpression of the Smurf2 and Smad7 transgenes exacerbated Smad7-induced abnormalities in hair follicles and sebaceous glands. Conversely, when endogenous Smad7 was knocked down, keratinocytes exhibited increased beta-catenin protein and enhanced Wnt signaling. Our data reveal a mechanism for Smad7 in antagonizing Wnt/beta-catenin signaling, thereby shifting the skin differentiation program from forming hair follicles to sebaceous glands.     

Wls Is Expressed in the Epidermis and Regulates Embryonic Hair Follicle Induction in Mice

Wnt proteins are secreted molecules that play multiple roles during hair follicle development and postnatal hair cycling. Wntless (Wls) is a cargo protein required for the secretion of various Wnt ligands. However, its role during hair follicle development and hair cycling remains unclear. Here, we examined the expression of Wls during hair follicle induction and postnatal hair cycling. We also conditionally deleted Wls with K14-cre to investigate its role in hair follicle induction. K14-cre;Wls^c/c mice exhibited abnormal hair follicle development, which is possibly caused by impaired canonical Wnt signaling. Meanwhile, Wnt5a is also expressed in embryonic epidermis, but Wnt5a null mice showed no significant defect in embryonic hair follicle morphogenesis. Therefore, Wls may regulate hair follicle induction by mediating the Wnt/β-catenin pathway.     

Dermal β-catenin activity in response to epidermal Wnt ligands is required for fibroblast proliferation and hair follicle initiation

Dermal fibroblasts are required for structural integrity of the skin and for hair follicle development. Uniform Wnt signaling activity is present in dermal fibroblast precursors preceding hair follicle initiation, but the functional requirement of dermal Wnt signaling at early stages of skin differentiation and patterning remains largely uncharacterized. We show in mice that epidermal Wnt ligands are required for uniform dermal Wnt signaling/β-catenin activity and regulate fibroblast cell proliferation and initiation of hair follicle placodes. In the absence of dermal Wnt signaling/β-catenin activity, patterned upregulation of epidermal β-catenin activity and Edar expression are absent. Conversely, forced activation of β-catenin signaling leads to the formation of thickened dermis, enlarged epidermal placodes and dermal condensates that result in prematurely differentiated enlarged hair follicles. These data reveal functional roles for dermal Wnt signaling/β-catenin in fibroblast proliferation and in the epidermal hair follicle initiation program.     

Essential roles of BMPR-IA signaling in differentiation and growth of hair follicles and in skin tumorigenesis

Hair differentiation and growth are controlled by complex reciprocal signaling between epithelial and mesenchymal cells. To better understand the requirement and molecular mechanism of BMP signaling in hair follicle development, we performed genetic analyses of bone morphogenetic protein receptor 1A (BMPR-IA) function during hair follicle development by using a conditional knockout approach. The conditional mutation of Bmpr1a in ventral limb ectoderm and its derivatives (epidermis and hair follicles) resulted in a lack of hair outgrowth from the affected skin regions. Mutant hair follicles exhibited abnormal morphology and lacked hair formation and pigment deposition during anagen. The timing of the hair cycle and the proliferation of hair matrix cells were also affected in the mutant follicles. We demonstrate that signaling via epithelial BMPR-IA is required for differentiation of both hair shaft and inner root sheath from hair matrix precursor cells in anagen hair follicles but is dispensable for embryonic hair follicle induction. Surprisingly, aberrant de novo hair follicle morphogenesis together with hair matrix cell hyperplasia was observed in the absence of BMPR-IA signaling within the affected skin of adult mutants. They developed hair follicle tumors from 3 months of age, indicating that inactivation of epidermal BMPR-IA signaling can lead to hair tumor formation. Taken together, our data provide genetic evidence that BMPR-IA signaling plays critical and multiple roles in controlling cell fate decisions or maintenance, proliferation, and differentiation during hair morphogenesis and growth, and implicate Bmpr1a as a tumor suppressor in skin tumorigenesis.     

Coordinated activation of Wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration

Melanocyte stem cells (McSCs) intimately interact with epithelial stem cells (EpSCs) in the hair follicle bulge and secondary hair germ (sHG). Together, they undergo activation and differentiation to regenerate pigmented hair. However, the mechanisms behind this coordinated stem cell behavior have not been elucidated. Here, we identified Wnt signaling as a key pathway that couples the behavior of the two stem cells. EpSCs and McSCs coordinately activate Wnt signaling at the onset of hair follicle regeneration within the sHG. Using genetic mouse models that specifically target either EpSCs or McSCs, we show that Wnt activation in McSCs drives their differentiation into pigment-producing melanocytes, while EpSC Wnt signaling not only dictates hair follicle formation but also regulates McSC proliferation during hair regeneration. Our data define a role for Wnt signaling in the regulation of McSCs and also illustrate a mechanism for regeneration of complex organs through collaboration between heterotypic stem cell populations.     

Notch1 is essential for postnatal hair follicle development and homeostasis

Notch genes encode evolutionarily conserved large, single transmembrane receptors, which regulate many cell fate decisions and differentiation processes during fetal and postnatal life. Multiple Notch receptors and ligands are expressed in both developing and adult epidermis and hair follicles. Proliferation and differentiation of these two ectodermal-derived structures have been proposed to be controlled in part by the Notch pathway. Whether Notch signaling is involved in postnatal hair homeostasis is currently unknown. Here, we investigate and compare the role of the Notch1 receptor during embryonic hair follicle development and postnatal hair homeostasis using Cre-loxP based tissue specific and inducible loss-of-function approaches. During embryonic development, tissue-specific ablation of Notch1 does not perturb formation and patterning of hair follicle placodes. However, Notch1 deficient hair follicles invaginate prematurely into the dermis. Embryonic as well as postnatal inactivation of Notch1 shortly after birth or in adult mice results in almost complete hair loss followed by cyst formation. The first hair cycle of Notch1 deficient mice is characterized by shortened anagen and a premature entry into catagen. These data show that Notch1 is essential for late stages of hair follicle development during embryogenesis as well as for post-natal hair follicle development and hair homeostasis.