Wnt-10b secreted from lymphocytes promotes differentiation of skin epithelial cells

Wnt-10b was originally isolated from lymphoid tissue and is known to be involved in a wide range of biological actions, while recently it was found to be expressed early in the development of hair follicles. However, few studies have been conducted concerning the role of Wnt-10b with the differentiation of skin epithelial cells. To evaluate its role in epithelial differentiation, we purified Wnt-10b from the supernatant of a concanavalin A-stimulated lymphocyte culture using an affinity column and investigated its effects on the differentiation of adult mouse-derived primary skin epithelial cells (MPSEC). MPSEC cultured with Wnt-10b showed morphological changes from cuboidal to spindle-shaped with inhibited proliferation, and also obtained characteristics of the hair shaft and inner root sheath of the hair follicle, represented by red-colored Ayoub Shklar staining, and reactions to AE-13 and AE-15 as seen with immunocytology. Further, RT-PCR analysis demonstrated the expression of mRNA for keratin 1, keratin 2, loricrin, mHa5, and mHb5, in association with a decreased expression of the basal cell marker keratin 5, in Wnt-10b-treated MPSEC. In addition, involvement of the canonical Wnt signal pathway was demonstrated by a TCF reporter (pTOPFLASH) assay. These results suggest that Wnt-10b promotes the differentiation of MPSEC and may play an important role in hair follicle development by promoting differentiation of epithelial cells.     

Wnt-10b, uniquely among Wnts, promotes epithelial differentiation and shaft growth

Although Wnts are expressed in hair follicles throughout life from embryo to adult, and considered to be critical for their development and maturation, their roles remain largely unknown. In the present study, we investigated the effects of Wnts (Wnt-3a, Wnt-5a, Wnt-10b, and Wnt-11) on epithelial cell differentiation using adult mouse-derived primary skin epithelial cell (MPSEC) cultures and hair growth using hair follicle organ cultures. Only Wnt-10b showed evident promotion of epithelial cell differentiation and hair shaft growth, in contrast to Wnt-3a, 5a, and 11. Our results suggest that Wnt-10b is unique and plays an important role in differentiation of epithelial cells in the hair follicle.     

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.     

Wnt-10b promotes differentiation of skin epithelial cells in vitro

To evaluate the role of Wnt-10b in epithelial differentiation, we investigated the effects of Wnt-10b on adult mouse-derived primary skin epithelial cells (MPSEC). Recombinant Wnt-10b protein (rWnt-10b) was prepared using a gene engineering technique and MPSEC were cultured in its presence, which resulted in morphological changes from cuboidal to spindle-shaped and inhibited their proliferation. Further, involvement of the canonical Wnt signal pathway was also observed. MPSEC treated with rWnt-10b showed characteristics of the hair shaft and inner root sheath of the hair follicle, in results of Ayoub Shklar staining and immunocytochemistry. Further, the cells expressed mRNA for differentiated epithelial cells, including keratin 1, keratin 2, loricrin, mHa5, and mHb5, in association with a decreased expression of the basal cell marker keratin 5. These results suggest that Wnt-10b promotes the differentiation of MPSEC.     

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.     

Canonical Wnts,specifically Wnt-10b,show ability to maintain dermal papilla cells

Although Wnts are expressed in hair follicles (HFs) and considered to be crucial for maintaining dermal papilla (DP) cells, the functional differences among them remain largely unknown. In the present study, we investigated the effects of Wnts (Wnt-3a, 5a, 10b, 11) on the proliferation of mouse-derived primary DP cells in vitro as well as their trichogenesis-promoting ability using an in vivo skin reconstitution protocol. Wnt-10b promoted cell proliferation and trichogenesis, while Wnt-3a showed those abilities to a limited extent, and Wnt-5a and 11 had no effects. Furthermore, we investigated the effects of these Wnts on cultured DP cells obtained from versican-GFP transgenic mice and found that Wnt-10b had a potent ability to sustain their GFP-positivity. These results suggest that canonical Wnts, specifically Wnt-10b, play important roles in the maintenance of DP cells and trichogenesis.     

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.     

Inhibition of BMP signaling in P-Cadherin positive hair progenitor cells leads to trichofolliculoma-like hair follicle neoplasias

Background  

Skin stem cells contribute to all three major lineages of epidermal appendages, i.e., the epidermis, the hair follicle, and the sebaceous gland. In hair follicles, highly proliferative committed progenitor cells, called matrix cells, are located at the base of the follicle in the hair bulb. The differentiation of these early progenitor cells leads to specification of a central hair shaft surrounded by an inner root sheath (IRS) and a companion layer. Multiple signaling molecules, including bone morphogenetic proteins (BMPs), have been implicated in this process.     

WNT signaling in the control of hair growth and structure

Characterization of the molecular pathways controlling differentiation and proliferation in mammalian hair follicles is central to our understanding of the regulation of normal hair growth, the basis of hereditary hair loss diseases, and the origin of follicle-based tumors. We demonstrate that the proto-oncogene Wnt3, which encodes a secreted paracrine signaling molecule, is expressed in developing and mature hair follicles and that its overexpression in transgenic mouse skin causes a short-hair phenotype due to altered differentiation of hair shaft precursor cells, and cyclical balding resulting from hair shaft structural defects and associated with an abnormal profile of protein expression in the hair shaft. A putative effector molecule for WNT3 signaling, the cytoplasmic protein Dishevelled 2 (DVL2), is normally present at high levels in a subset of cells in the outer root sheath and in precursor cells of the hair shaft cortex and cuticle which lie immediately adjacent to Wnt3-expressing cells. Overexpression of Dvl2 in the outer root sheath mimics the short-hair phenotype produced by overexpression of Wnt3, supporting the hypothesis that Wnt3 and Dvl2 have the potential to act in the same pathway in the regulation of hair growth. These experiments demonstrate a previously unrecognized role for WNT signaling in the control of hair growth and structure, as well as presenting the first example of a mammalian phenotype resulting from overexpression of a Dvl gene and providing an accessible in vivo system for analysis of mammalian WNT signaling pathways.     

Single-cell sequencing reveals the new existence form of dermal papilla cells in the hair follicle regeneration of cashmere goats

The problem of human hair loss has caused widespread concern, however, such research is difficult because the periodicity is not obvious and the deeper levels knowledge of dermal papilla (DP) stem cells' differentiation are limited. Here, cashmere goats which have obvious periodicity of hair follicles were used, based on unbiased scRNA sequencing, we constructed DP cell lineage differentiation trajectory and revealed the key genes, signals and functions involved in cell fate decisions. And then we revealed the molecular landscape of hair follicle on regeneration. Revealed that DP cells differentiate into four intermediate cell states at different periodicity: Intermediate-cell-10 showed important functions in the growth and maintenance of cashmere; intermediate-cell-1 acting on apoptosis and cashmere shedding; intermediate-cell-0 initiated new follicular cycles, the migration of hair follicles and the occurrence of cashmere; and intermediate-cell-15 are suggested to be DP progenitor cells. In general, we provide new insights for hair regrowth. At the same time, it provides a new research ideas, directions and molecular landscape for the mechanism of dermal papilla cells.     

Transient activation of beta-catenin signalling in adult mouse epidermis is sufficient to induce new hair follicles but continuous activation is required to maintain hair follicle tumours

When beta-catenin signalling is disturbed from mid-gestation onwards lineage commitment is profoundly altered in postnatal mouse epidermis. We have investigated whether adult epidermis has the capacity for beta-catenin-induced lineage conversion without prior embryonic priming. We fused N-terminally truncated, stabilised beta-catenin to the ligand-binding domain of a mutant oestrogen receptor (DeltaNbeta-cateninER). DeltaNbeta-cateninER was expressed in the epidermis of transgenic mice under the control of the keratin 14 promoter and beta-catenin activity was induced in adult epidermis by topical application of 4-hydroxytamoxifen (4OHT). Within 7 days of daily 4OHT treatment resting hair follicles were recruited into the hair growth cycle and epithelial outgrowths formed from existing hair follicles and from interfollicular epidermis. The outgrowths expressed Sonic hedgehog, Patched and markers of hair follicle differentiation, indicative of de novo follicle formation. The interfollicular epidermal differentiation program was largely unaffected but after an initial wave of sebaceous gland duplication sebocyte differentiation was inhibited. A single application of 4OHT was as effective as repeated doses in inducing new follicles and growth of existing follicles. Treatment of epidermis with 4OHT for 21 days resulted in conversion of hair follicles to benign tumours resembling trichofolliculomas. The tumours were dependent on continuous activation of beta-catenin and by 28 days after removal of the drug they had largely regressed. We conclude that interfollicular epidermis and sebaceous glands retain the ability to be reprogrammed in adult life and that continuous beta-catenin signalling is required to maintain hair follicle tumours.     

Reconstruction of proliferative activity of hair follicle cells based on the geometric parameters of the hair shaft

Proliferative activity and differentiation rate of hair follicle cells determine the shape of the hair and parameters of hair growth. The hair growth rate and changes of geometric parameters of the hair shaft during its elongation (in time) are analyzed to reconstruct the proliferation dynamics of follicle matrix cells. The reconstruction demonstrated that the number of cells of hair follicle matrix increases in a typical manner, including the phase of exponential growth and the stationary phase.     

BMP signaling in the control of skin development and hair follicle growth

Bone morphogenetic proteins (BMPs), their antagonists, and BMP receptors are involved in controlling a large number of biological functions including cell proliferation, differentiation, cell fate decision, and apoptosis in many different types of cells and tissues during embryonic development and postnatal life. BMPs exert their biological effects via using BMP-Smad and BMP-MAPK intracellular pathways. The magnitude and specificity of BMP signaling are regulated by a large number of modulators operating on several levels (extracellular, cytoplasmic, nuclear). In developing and postnatal skin, BMPs, their receptors, and BMP antagonists show stringent spatio-temporal expressions patterns to achieve proper regulation of cell proliferation and differentiation in the epidermis and in the hair follicle. Genetic studies assert an essential role for BMP signaling in the control of cell differentiation and apoptosis in developing epidermis, as well as in the regulation of key steps of hair follicle development (initiation, cell fate decision, cell lineage differentiation). In postnatal hair follicles, BMP signaling plays an important role in controlling the initiation of the growth phase and is also involved in the regulation of apoptosis-driven hair follicle involution. However, additional efforts are required to fully understand the mechanisms and targets involved in the realization of BMP effects on distinct cell population in the skin and hair follicle. Progress in this area of research will hopefully lead to the development of new therapeutic approaches for using BMPs and BMP antagonists in the treatment of skin and hair growth disorders.     

Multi-potentiality of a new immortalized epithelial stem cell line derived from human hair follicles

We previously demonstrated that keratin 15 expressing cells present in the bulge region of hair follicles exhibit properties of adult stem cells. We have now established and characterized an immortalized adult epithelial stem cell line derived from cells isolated from the human hair follicle bulge region. Telogen hair follicles from human skin were microdissected to obtain an enriched population of keratin 15 positive skin stem cells. By expressing human papillomavirus 16 E6/E7 genes in these stem cells, we have been able to culture the cells for >30 passages and maintain a stable phenotype after 12 mo of continuous passage. The cell line was compared to primary stem cells for expression of stem cell specific proteins, for in vitro stem cell properties, and for their capacity to differentiate into different cell lineages. This new cell line, named Tel-E6E7 showed similar expression patterns to normal skin stem cells and maintained in vitro properties of stem cells. The cells can differentiate into epidermal, sebaceous gland, and hair follicle lineages. Intact beta-catenin dependent signaling, which is known to control in vivo hair differentiation in rodents, is maintained in this cell line. The Tel-E6E7 cell line may provide the basis for valid, reproducible in vitro models for studies on stem cell lineage determination and differentiation.     

beta-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin

beta-Catenin is an essential molecule in Wnt/wingless signaling, which controls decisive steps in embryogenesis. To study the role of beta-catenin in skin development, we introduced a conditional mutation of the gene in the epidermis and hair follicles using Cre/loxP technology. When beta-catenin is mutated during embryogenesis, formation of placodes that generate hair follicles is blocked. We show that beta-catenin is required genetically downstream of tabby/downless and upstream of bmp and shh in placode formation. If beta-catenin is deleted after hair follicles have formed, hair is completely lost after the first hair cycle. Further analysis demonstrates that beta-catenin is essential for fate decisions of skin stem cells: in the absence of beta-catenin, stem cells fail to differentiate into follicular keratinocytes, but instead adopt an epidermal fate.