Hair Peg Research Articles

In vitro hair follicle growth model for drug testing

In vitro models of human hair follicle-like tissue could be fundamental tools to better understand hair follicle morphogenesis and hair drug screening. During prenatal development and postnatal cyclic hair regeneration, hair follicle morphogenesis is triggered by reciprocal interactions and the organization of the epithelial and mesenchymal cell populations. Given this mechanism, we developed an approach to induce hair peg-like sprouting in organoid cultures composed of epithelial and mesenchymal cells. Human fetal/adult epithelial and mesenchymal cells were cultured in a medium supplemented with a low concentration of either Matrigel or collagen I. These extracellular matrices significantly enhanced the self-organization capabilities of the epithelial and mesenchymal cells, resulting in spherical aggregation and subsequent hair peg-like sprouting. The length of the hair peg sprouting and associated gene expression significantly increased in the presence of a well-known hair drug, minoxidil. This approach may be beneficial for testing hair growth-promoting drug candidates.

Nestin is a marker of unipotent embryonic and adult progenitors differentiating into an epithelial cell lineage of the hair follicles

Nestin is an intermediate filament protein transiently expressed in neural stem/progenitor cells. We previously demonstrated that outer root sheath (ORS) keratinocytes of adult hair follicles (HFs) in mice descend from nestin-expressing cells, despite being an epithelial cell lineage. This study determined the exact stage when nestin-expressing ORS stem/precursor cells or their descendants appear during HF morphogenesis, and whether they are present in adult HFs. Using Nes-Cre/CAG-CAT-EGFP mice, in which enhanced green fluorescent protein (EGFP) is expressed following Cre-based recombination driven by the nestin promoter, we found that EGFP+ cells appeared in the epithelial layer of embryonic HFs as early as the peg stage. EGFP+ cells in hair pegs were positive for keratin 14 (K14) and K5, but not vimentin, SOX2, SOX10, or S100 alpha 6. Tracing of tamoxifen-induced EGFP+ cells in postnatal Nes-CreERT2/CAG-CAT-EGFP mice revealed labeling of some isthmus HF epithelial cells in the first anagen stage. EGFP+ cells in adult HFs were not immunolabeled for K15, an HF multipotent stem cell marker. However, when hairs were depilated in Nes-CreERT2/CAG-CAT-EGFP mice to induce the anagen stage after tamoxifen injection, the majority of ORS keratinocytes in depilation-induced anagen HFs were labeled for EGFP. Our findings indicate that nestin-expressing unipotent progenitor cells capable of differentiating into ORS keratinocytes are present in HF primordia and adult HFs.

602 Sox13 is a novel marker for hair follicle development and differentiation

The Sry-related high-mobility-group box (Sox) gene encodes a transcription factor family that plays crucial roles in the determination of cell fate and organogenesis. Sox13, a group D member of the Sox family, is expressed in various tissues such as brain, cartilage, hair, kidney, pancreas, liver, kidney and blood vessels. However, knowledge of the biological roles of Sox13 is largely limited. To examine the precise expression domains of Sox13 protein and its role in mouse development, we generated Sox13-LacZ-knock-in mice (Sox13LacZ/+), in which the Sox13 coding sequence was disrupted by an in-frame insertion of the b-galactosidase (LacZ) gene. Using the knock-in mice, we examined spatiotemporal expression patterns of Sox13 in the hair follicle and its role in hair development. X-gal staining in Sox13LacZ/+ embryos revealed that Sox13 is initially expressed in the epithelial portion of the placode at E14.5, and subsequently in the hair germ and the hair peg during hair follicle morphogenesis. Immunohistochemistry with an anti-b-galactosidase antibody and anti-hair keratin antibodies that specifically mark the different layers of the hair follicle revealed that during the postnatal phases of hair development Sox13 was predominantly expressed in the outer root sheath. However, the integumentary structures of Sox13LacZ/LacZ mice were grossly and histologically normal. These results suggest that although Sox13 is dispensable for epidermal and adnexal development, Sox13 is a novel marker for early hair follicle development and differentiation.

Sox13 is a novel early marker for hair follicle development

Sox13, a group D member of the Sry-related high-mobility group box (Sox) transcription factor family, is expressed in various tissues including the hair follicle. However, its spatiotemporal expression patterns in the hair follicle and its role in hair development remain to be elucidated. To address these questions, we generated Sox13-LacZ-knock-in mice (Sox13LacZ/+), in which the LacZ reporter gene was inserted in-frame into exon 2, which contains the translation initiation codon. X-gal staining in Sox13LacZ/+ embryos revealed that Sox13 is initially expressed in the epithelial portion of the placode, and subsequently in the hair germ and the hair peg during early hair follicle development. In postnatal catagen and anagen, Sox13 was detected in the epithelial sheath, whereas in telogen, Sox13 was localized in the bulge region, where hair follicle stem cells reside. Immunohistochemistry with an anti-β-galactosidase antibody and anti-hair keratin antibodies that specifically mark the different layers of the hair follicle revealed that Sox13 was predominantly expressed in the outer root sheath in anagen. However, the integumentary structures of Sox13LacZ/LacZ mice were grossly and histologically indistinguishable from those of wild type mice. These results suggest that although Sox13 is dispensable for epidermal and adnexal development, Sox13 is a useful marker for early hair follicle development.

Early stages of hair follicle development: a step by step microarray identity.

Hair follicle morphogenesis requires an epithelial-mesenchymal cross-talk during development, from hair placode to hair peg, and finally hair follicle formation. During this step, factors known as activators and inhibitors allow the patterning distribution of hair follicle within the skin. Our goal was to investigate the modulation of expression of various factors already known to be part of the hair placode formation, and to identify novel factors involved during the initiation of this process. In mice, primary hair follicles arise in utero from E12.5 mouse embryos. Back skin RNA were extracted from E12.5 to E14.5 embryos to perform microarray analysis (Affymetrix). We identified four new Wnt related genes which could be involved in hair follicle initiation because of their maximum expression at E12.5, namely two activators: Wnt-2 and Zic-1 and two inhibitors: Dkk-2 and Dact-1. Real-time quantitative polymerase chain reactions confirmed their expression. Our data provide a more precise view of transcript expressions involved during induction of HF morphogenesis, particularly the hair primordium formation.

Rat hair follicle-constituting cells labeled by a newly-developed somatic stem cell-recognizing antibody: a possible marker of hair follicle development.

A3 was generated as an antibody recognizing somatic stem cells in rat tissues. We investigated the distribution of A3-positive cells in developing rat hair follicles by immunolabeling. A3-positive cells began to be seen in the hair germ and peg in fetuses and neonates; the positive cells were epithelial cells above basal cells. Furthermore, A3-positive cells were seen in the outer root sheath adjacent to the bulge in mature hair follicles. Double immunofluorescence revealed that these A3-positive epithelial cells reacted to E-cadherin (for all epithelial elements) but not to CK15 (for basal cells/epithelial stem cells) or to nestin (for stem cells), indicating that A3-positive epithelial cells are suprabasal cells in the developing epidermic hair follicle. Additionally, spindle-shaped mesenchymal cells surrounding the hair peg and mature hair follicle reacted to A3; in double immunofluorescence, the A3-positive cells were located outside collagen type IV-positive glassy membrane, and reacted to vimentin (for mesenchmal cells), Thy-1 (for immature mesenchymal cells), CD34 (for stem cells) and nestin, but not to α-smooth muscle actin (for myofibroblasts); the positive cells were regarded as immature mesenchymal cells with stem cell nature in the connective tissue sheath of developing hair follicles. A3-positive epithelial and mesenchymal cells did not show proliferating activity. Collectively, it is considered that A3-positive cells seen in developing rat hair follicles may be quiescent post-progenitor cells with the potential to differentiate into either highly-differentiated epithelial or mesenchymal cells. A3 would become a useful antibody to know the kinetics of rat hair follicle-constituting cells.

The function of TRPS1 in the development and differentiation of bone, kidney, and hair follicles.

TRPS1 is a gene involved in Tricho-rhino-phalangeal syndrome (TRPS), an autosomal dominant skeletal disorder. TRPS1 encodes a GATA-type transcription factor that has nine zinc-finger motifs. A variety of mutations in TRPS1 including deletions and insertions, have been found in patients with TRPS type I and III. The functions of each domain of TRPS1 have been clarified from study of these mutations. Further studies on the localization and the function of TRPS1 have been performed using TRPS1Δgt and Trps1-deficient mice, which allow examination of the development and differentiation of all tissues with Trps1 expression. These studies suggest that TRPS1 exhibits a variety of functions in cartilage, kidneys, and hair follicles. In the growth plate cartilage, TRPS1 regulates the differentiation, proliferation, and apoptosis of chondrocytes through interaction of several signaling molecules. In addition, TRPS1 has a function downstream of BMP7, which regulates the mesenchymal-epithelial transition when nephrons are formed in renal development. Furthermore, TRPS1 suppresses the epithelial-mesenchymal transition and renal fibrosis induced by unilateral ureteral obstruction by decreasing Arkadia expression. Finally, TRPS1 is expressed in the dermal papillae and the mesenchymal cells surrounding the hair pegs, and the loss of TRPS1 largely influences the development of hair follicles. The molecular mechanisms of the function of TRPS1 in cartilage, kidneys, and hair follicles are discussed in this review.

P75 Neurotrophin receptor differentiates between morphoeic basal cell carcinoma and desmoplastic trichoepithelioma: insights into the histogenesis of adnexal tumours based on embryology and hair follicle biology

Tumour development is frequently described in the basic pathology literature as a recapitulation of embryogenesis. However, a link between the embryology of the skin and the histogenesis of adnexal tumours has been largely overlooked. The low-affinity p75 neurotrophin receptor (p75NTR) has a profound role in hair follicle biology. We therefore speculated that it is involved in the histogenesis of follicular adnexal tumours. One of the most challenging diagnoses in dermatopathology is differentiating morphoeic basal cell carcinoma from desmoplastic trichoepithelioma. To describe the expression pattern of p75NTR during cutaneous embryogenesis, in the adult hair follicle and in morphoeic basal cell carcinoma and desmoplastic trichoepithelioma. Evaluation of the staining pattern for p75NTR was performed using standard immunohistochemical techniques. For comparison, we examined staining for cytokeratin 20 which highlights Merkel cells. All 17 desmoplastic trichoepitheliomas were immunoreactive with > 80% of the cells stained, whereas 12 of the 14 (86%) morphoeic basal cell carcinomas were p75NTR negative. In the two positive cases of morphoeic basal cell carcinoma < 30% of cells were labelled. In the late bulbous hair peg stage and in the postnatal anagen hair follicle p75NTR highlights the outer root sheath. Our results support the classification of desmoplastic trichoepithelioma as a follicular hamartoma mimicking the outer root sheath. In contrast, the lack of p75NTR expression in morphoeic basal cell carcinoma favours a concept of this tumour as a more primitive follicular lesion with the characteristics of a carcinoma and not a hamartoma. We suggest including p75NTR as a tool in the differential diagnosis between morphoeic basal cell carcinoma and desmoplastic trichoepithelioma.

Basal cell carcinoma and pilomatrixoma mirror human follicular embryogenesis as reflected by their differential expression patterns of SOX9 and β-catenin

The current classification schemes of adnexal tumours are predominantly based on morphological and immunophenotypical similarities to adult skin structures, whereas a link between the embryology of the skin and the histogenesis of adnexal tumours has been largely neglected. To describe the expression patterns of two proteins with proven relevance for hair follicle homeostasis (SOX9 and β-catenin) during human cutaneous embryogenesis and to compare the findings with their expression in basal cell carcinoma (BCC) and pilomatrixoma. Immunohistochemical evaluation with monoclonal antibodies against SOX9 and β-catenin was carried out in embryonic and adult human scalp skin, and BCC and pilomatrixoma samples. We found that the expression patterns of SOX9 and β-catenin during human hair follicle embryogenesis mirror the patterns in BCC and pilomatrixoma in spatial distribution within the various follicular subcompartments. Beginning with the hair peg stage, nucleocytoplasmic immunoreactivity of β-catenin is exclusively confined to the emerging matrix (comparable to pilomatrixoma), whereas SOX9 is restricted to the primordial outer root sheath (comparable to BCC). An appropriate immunophenotyping validated within the conceptual framework of cutaneous developmental biology allows a logical classification of adnexal neoplasms. Expanding this approach further has the potential to revise the current classification schemes so that not only BCC and pilomatrixoma but all adnexal tumours can be categorized logically.

Perturbed desmosomal cadherin expression in grainy head-like 1-null mice

In Drosophila, the grainy head (grh) gene plays a range of key developmental roles through the regulation of members of the cadherin gene family. We now report that mice lacking the grh homologue grainy head-like 1 (Grhl1) exhibit hair and skin phenotypes consistent with a reduction in expression of the genes encoding the desmosomal cadherin, desmoglein 1 (Dsg1). Grhl1-null mice show an initial delay in coat growth, and older mice exhibit hair loss as a result of poor anchoring of the hair shaft in the follicle. The mice also develop palmoplantar keratoderma, analogous to humans with DSG1 mutations. Sequence analysis, DNA binding, and chromatin immunoprecipitation experiments demonstrate that the human and mouse Dsg1 promoters are direct targets of GRHL1. Ultrastructural analysis reveals reduced numbers of abnormal desmosomes in the interfollicular epidermis. These findings establish GRHL1 as an important regulator of the Dsg1 genes in the context of hair anchorage and epidermal differentiation, and suggest that cadherin family genes are key targets of the grainy head-like genes across 700 million years of evolution.

The chromatin remodeler Mi-2β is required for establishment of the basal epidermis and normal differentiation of its progeny

Using conditional gene targeting in mice, we show that the chromatin remodeler Mi-2beta is crucial for different aspects of skin development. Early (E10.5) depletion of Mi-2beta in the developing ventral epidermis results in the delayed reduction of its suprabasal layers in late embryogenesis and to the ultimate depletion of its basal layer. Later (E13.5) loss of Mi-2beta in the dorsal epidermis does not interfere with suprabasal layer differentiation or maintenance of the basal layer, but induction of hair follicles is blocked. After initiation of the follicle, some subsequent morphogenesis of the hair peg may proceed in the absence of Mi-2beta, but production of the progenitors that give rise to the inner layers of the hair follicle and hair shaft is impaired. These results suggest that the extended self-renewal capacity of epidermal precursors arises early during embryogenesis by a process that is critically dependent on Mi-2beta. Once this process is complete, Mi-2beta is apparently dispensable for the maintenance of established repopulating epidermal stem cells and for the differentiation of their progeny into interfollicular epidermis for the remainder of gestation. Mi-2beta is however essential for the reprogramming of basal cells to the follicular and, subsequently, hair matrix fates.

Prenatal exclusion of harlequin ichthyosis; potential pitfalls in the timing of the fetal skin biopsy

Harlequin ichthyosis (HI) is a severe and usually fatal congenital skin disorder with autosomal recessive inheritance. Several cases of HI prenatal diagnosis have been performed using fetal skin biopsy, mainly at around 23 weeks estimated gestational age (EGA), and reported in the literature. However, prenatal testing must be done earlier than 21 weeks EGA in several countries including Japan where the present HI families live, because termination is legally allowed only until 22 weeks EGA. We report the successful prenatal exclusion of HI in two fetuses from two independent families and discuss the technical difficulties and potential pitfalls in the prenatal exclusion of HI at early gestation stages. Fetal skin biopsy specimens and amniotic fluid samples at 19 and 20 weeks EGA from two fetuses at risk of HI were examined by light and electron microscopy. For the prenatal diagnosis in case 1, the fetal skin biopsy samples were obtained at 20 weeks EGA and showed normal keratinization in the hair canals; no abnormalities were observed in the keratinized cells. In case 2, the interfollicular epidermis and the hair follicles in the samples obtained at 19 weeks EGA had not differentiated enough to show proper keratinization. However, lamellar granules were normally formed in the inner root sheath cells of the late bulbous hair pegs. From these ultrastructural findings, the case 1 fetus was diagnosed as unaffected with HI, and the case 2 fetus was diagnosed as unlikely to be affected. Subsequently, both were born as healthy, unaffected babies. The timing of biopsies at 19 weeks EGA is not ideal for fetal skin biopsy because the samples are not always sufficiently differentiated for the prenatal diagnosis of HI. However, morphological observations of lamellar granules gives us important additional information useful for HI prenatal diagnosis.

Apoptosis coordinates with proliferation and differentiation during human hair follicle morphogenesis

Apoptosis sculptures the most complicated skin appendage, feathers, out of epidermal layers by playing a variety of roles (1). Human hair follicle formation is different from feathers in growth direction and pattern formation of proliferative zone. To delineate the apoptotic events together with proliferation and differentiation in developing human hair follicle and emphasis on the mechanism and biological meaning of epidermal hair canal. We used TUNEL to examine apoptosis and Ki-67, involucrin, filaggrin immuno-localization to examine proliferation and differentiation. In hair germs, apoptosis was diffuse in periderm, basal keratinocytes, and mesenchymal cells with high Ki-67 expression, but spared follicular germinative cells with low Ki-67 and high bcl-2 expression. In hair pegs, apoptosis was active in high Ki-67 expression area, like outer root sheath, hair follicle sheath, but spared dermal papilla with low Ki-67 and high bcl-2 expression. In bulbous pegs, apoptosis appeared in companion layer, precortical area, inner root sheath and outer root sheath, but spared bulge area with high bcl-2 expression. Apoptosis resulted in epidermal and subepidermal hair canal formation. Filaggrin and involucrin were expressed in the lining cells of hair canal. CD1a+ cells were densely distributed alone the hair canal before its opening. During human hair follicle morphogenesis, apoptosis coordinates with proliferation to shape the growth zone, creates space to free the hair shaft from follicular wall, and directs a driving force on hair shaft extension. Apoptosis accompanies the terminal differentiation of epidermal hair canal. The bottom becomes interfollicular epidermis after roof shedding and hair exposure. Langerhans cells also populate in the hair canal before its opening. The biological meaning of epidermal hair canal is supposed to prepare the barrier when hair perturbing the intact of epidermis.

Epithelium-Mesenchyme Interactions Control the Activity of Peroxisome Proliferator-Activated Receptor β/δ during Hair Follicle Development

Hair follicle morphogenesis depends on a delicate balance between cell proliferation and apoptosis, which involves epithelium-mesenchyme interactions. We show that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) and Akt1 are highly expressed in follicular keratinocytes throughout hair follicle development. Interestingly, PPARbeta/delta- and Akt1-deficient mice exhibit similar retardation of postnatal hair follicle morphogenesis, particularly at the hair peg stage, revealing a new important function for both factors in the growth of early hair follicles. We demonstrate that a time-regulated activation of the PPARbeta/delta protein in follicular keratinocytes involves the up-regulation of the cyclooxygenase 2 enzyme by a mesenchymal paracrine factor, the hepatocyte growth factor. Subsequent PPARbeta/delta-mediated temporal activation of the antiapoptotic Akt1 pathway in vivo protects keratinocytes from hair pegs against apoptosis, which is required for normal hair follicle development. Together, these results demonstrate that epithelium-mesenchyme interactions in the skin regulate the activity of PPARbeta/delta during hair follicle development via the control of ligand production and provide important new insights into the molecular biology of hair growth.

Gap junction development in the human fetal hair follicle and bulge region.

Gap junctions, composed of connexin (Cx) subunits, are channels that allow intercellular communication between adjacent cells and are thought to play a key role in the regulation of cell proliferation and differentiation. The Cx expression pattern and formation of gap junctions in human fetal hair follicles has yet to be clarified, including the prominent follicular bulge region that is believed to be a site rich in stem cells. To study the expression of two major Cxs, Cx26 and Cx43, in developing hair follicles in skin samples from a series of human fetuses of estimated gestational age (EGA) 88-163 days, and to determine quantitatively the presence of gap junctions. We used immunofluorescence labelling to investigate the sequential expression pattern of Cx26 and Cx43 in developing human hair follicles. Gap junction formation was observed by electron microscopy and the numbers of gap junctions were analysed quantitatively. Results Both Cx26 and Cx43 expression were observed at 88 days' EGA in the inner part of the hair peg. At 135 days' EGA, Cx26 was expressed in the outer root sheath (ORS) and the inner root sheath (IRS), while Cx43 was expressed chiefly in the IRS, hair matrix and sebaceous glands. At 163 days' EGA, Cx26 expression was most intense in the outermost layer of the ORS, in contrast to Cx43 expression which was in the inner part of the ORS. In the bulge region, only Cx43 was expressed in a subset of cells in the bulge. Ultrastructurally, gap junctions were observed at 102 days' EGA in the hair peg, and the number of gap junctions increased as the hair follicle matured. Gap junctions were also observed between the bulge cells in considerable numbers. The changing expression patterns of Cx26 and Cx43 and the increasing gap junction numbers suggest a close association of Cx expression and gap junction formation with hair follicle morphogenesis. In addition, the present ultrastructural observations demonstrate that considerable numbers of the bulge cells, a putative site rich in hair follicle stem cells, form gap junctions during human hair follicle development.

Transdifferentiation of corneal epithelium: evidence for a linkage between the segregation of epidermal stem cells and the induction of hair follicles during embryogenesis.

Corneal epithelium transdifferentiation into a hair-bearing epidermis provides a particularly useful system for studying the possibility that transient amplifying (TA) cells are able to activate different genetic programs in response to a change in their fibroblast environment, as well as to follow the different steps of rebuilding an epidermis from induced stem cells. Corneal stem and TA cells are found in different locations - stem cells at the periphery, in the limbus, and TA cells more central. Moreover, the TA cells already express the differentiating corneal-type keratin pair K3/K12, whereas the limbal keratinocytes express the basal keratin pair K5/K14. In contrast, suprabasal epidermal keratinocytes express keratin pair K1-2/K10, and basal keratinocytes the keratin pair K5/K14. The results of tissue recombination experiments show that adult central corneal cells are able to respond to specific information originating from embryonic dermis. First, the cells located at the base of the corneal epithelium show a decrease in expression of K12 keratin, followed by an increase in K5 expression; they then proliferate and form hair follicles. The first K10 expressing cells appear at the junction of the new hair follicles and the covering corneal epithelium. Their expansion finally gives rise to epidermal strata, which displace the corneal suprabasal keratinocytes. Corneal TA cells can thus be reprogrammed to form epidermal cells, first by reverting to a basal epithelial-type, then to hair pegs and probably concomitantly to hair stem cells. This confirms the role of the hair as the main reservoir of epidermal stem cells and raises the question of the nature of the dermal messages which are both involved in hair induction and stem cell specification.

Expression of Cyclooxygenase Isozymes During Morphogenesis and Cycling of Pelage Hair Follicles in Mouse Skin: Precocious Onset of the First Catagen Phase and Alopecia upon Cyclooxygenase-2 Overexpression

Cyclooxygenase (COX)-1 and -2 catalyze the key reaction in prostaglandin biosynthesis. Whereas COX-1 is found in most tissues, COX-2, with a few exceptions, is not expressed in normal tissues but becomes transiently induced in the course of inflammatory reactions. In many neoplastic epithelia, COX-2 is constitutively overexpressed. Here we show that COX isozymes are spatiotemporally expressed during morphogenesis of dorsal skin epithelium of NMRI mice. COX-1 and COX-2 mRNA and protein were detected in embryonic and postnatal epidermal tissue by RT-PCR, northern blot, and immunoblot analysis indicating that both isoforms may contribute to prostaglandin production. Being barely detectable in interfollicular epidermis and resting hair follicles of adult mice, COX-2 protein appeared in embryonic skin first in epidermal precursor cells and later on in the basal cells and the peridermal layer of the stratified epidermis. In the course of pelage hair follicle morphogenesis, COX-2 remained expressed in the basal interfollicular compartment and, in addition, became apparent in elongated hair germs and hair pegs and later on in the outer root sheath cells of the distal and proximal hair follicles as well as in basal sebaceous gland cells. During the subsequent synchronous phases of hair cycling, COX-2 expression declined in catagen, was barely detectable in telogen, and was reinduced in the basal outer root sheath and basal sebaceous gland cells of anagen hair follicles. COX-1 immunosignals were detected predominantly in the interfollicular spinous and granular layers of the developing, neonatal, and adult epidermis but not in follicular epithelial cells of developing or cycling hair follicles. Dendritic cells in the interfollicular epidermis and distal hair follicles were also COX-1-positive. Transgenic overexpression of COX-2 under the control of a keratin 5 promoter in basal cells of the interfollicular and follicular epidermis induced a precocious entry into the first catagen stage of postnatal hair follicle cycling and a subsequent disturbance of hair follicle phasing. Furthermore, transgenic mice developed an alopecia. Inhibition of transgenic COX-2 activity by feeding the specific COX-2 inhibitor valdecoxib suppressed the development of alopecia, indicating that COX-2-mediated prostaglandin synthesis is involved in hair follicle biology.

Formation of cornified cell envelope in human hair follicle development.

Cornified cell envelope (CCE) formation is an important step in the final stage of keratinization, in which CCE precursor proteins including involucrin and loricrin are cross-linked by keratinocyte transglutaminases (TGases) to the inner surface of the plasma membrane of cornified cells, while the outer surface is coated with material derived from secreted lamellar granules. Skin samples from human fetuses of a series of estimated gestational age (EGA) (49-163 days) were studied for the prescence of precursor proteins. Methods TGase activity was studied by in situ TGase activity assay, and ultrastructural features of CCE formation were observed at each stage of hair follicle development. We used immunofluorescent labelling to investigate the time and site of expression of CCE precursor proteins involucrin and loricrin, TGases 1, 2 and 3, and a 25-kDa lamellar granule-associated protein (LGP) in developing human hair follicles. In the hair germ (65-84 days EGA) (corresponding to the stages 1-2 of murine hair follicle morphogenesis), only TGase 2 was observed in the entire hair germ, where in situ TGase activity was weakly positive, although thickening of cell membrane was not seen ultrastructurally. In the hair peg (85-104 days EGA) (corresponding to the stage 3 of murine hair follicle morphogenesis), loricrin and TGase 2 were seen in cells of the upper part of the hair peg while TGase 1, 3 and LGP were observed in the inner cells of the hair peg. In situ TGase activity was weakly positive in the upper part and inner cells of the hair peg. In the bulbous hair peg (105-135 days EGA) (corresponding to the stages 4-6 of murine hair follicle morphogenesis) and differentiated lanugo hair follicle (> 135 days EGA) (corresponding to the stages 7-8 of murine hair follicle morphogenesis), immunoreactivities of involucrin, loricrin, TGase 1, 2, 3, in situ TGase activity and LGP were detected in the inner root sheath cells, hair canals and inner cells of the outer root sheath in the region of the isthmus. Ultrastructurally, thickening of cell membrane was already seen in the inner root sheath cells of the bulbous hair peg and electron-dense, thick CCE was observed in the hair cuticle and hair canal of differentiated lanugo hair follicle. These data indicate that, in terms of CCE formation, certain portions of the developing human hair follicle have already been determined in differentiation of the hair canal and cuticle at the hair peg stage.

Inhibition of Hair Follicle Growth by a Laminin-1 G-Domain Peptide, RKRLQVQLSIRT, in an Organ Culture of Isolated Vibrissa Rudiment

We established a serum-free organ culture system of isolated single vibrissa rudiments taken from embryonic day 13 mice. This system allowed us to test more than 30 laminin-derived cell adhesive peptides to determine their roles on the growth and differentiation of vibrissa hair follicles. We found that the RKRLQVQLSIRT sequence (designated AG-73), which mapped to the LG-4 module of the laminin-alpha1 chain carboxyl-terminal G domain, perturbed the growth of hair follicles in vitro. AG-73 is one of the cell-binding peptides identified from more than 600 systematically synthesized 12 amino acid peptides covering the whole amino acid sequence of the laminin-alpha1, -beta1, and -gamma1 chains, by cell adhesion assay. Other cell-adhesive laminin peptides and a control scrambled peptide, LQQRRSVLRTKI, however, failed to show any significant effects on the growth of hair follicles. The AG-73 peptide binds to syndecan-1, a transmembrane heparan-sulfate proteoglycan. Syndecan-1 was expressed in both the mesenchymal condensation and the epithelial hair peg of developing vibrissa, suggesting that AG-73 binding to the cell surface syndecan-1 perturbed the epithelial-mesenchymal interactions of developing vibrissa. The formation of hair bulbs was aberrant in the explants treated with AG-73. In addition, impaired basement membrane formation, an abnormal cytoplasmic bleb formation, and an unusual basal formation of actin bundles were noted in the AG-73-treated-hair matrix epithelium, indicating that AG-73 binding perturbs various steps of epithelial morphogenesis, including the basement membrane remodeling. We also found a region-specific loss of the laminin-alpha1 chain in the basement membrane at the distal region of the invading hair follicle epithelium, indicating that laminins play a part in hair morphogenesis.

Neuron-specific PGP9.5 expression in rat hair follicle development and cycle

Protein gene product PGP9.5 is a neuron-specific ubiquitin C-terminal hydrolase. We found that it also has immunoreactivity in the hair follicle of the Wistar rat dorsal skin and its expression patterns change with the development and cycle. During the morphogenesis, the PGP9.5 was expressed in the hair germ and hair peg elongated from the epidermis, and became restricted in the outer root sheath as the development progressed. In catagen, however, the PGP9.5 was detected in the tailing epithelial strand of the regressing proximal follicle epithelium, and in the keratinocytes directly contacted with the club hair, but rarely in the outer root sheath. With the beginning of the anagen of the second hair follicle, the PGP9.5 was again expressed in the second hair germ, and in the keratinocytes surrounding the remaining club hair and of distal follicle of the first hair. These findings showed that PGP9.5 is not specific to the neuron but is also involved in the hair follicle, and should provide new insight into the development and regression of the hair follicle.