Heparan Sulfate Regulates Hair Follicle and Sebaceous Gland Morphogenesis and Homeostasis

Background: Skin appendages play a vital role in keeping the skin healthy.

Results: The loss of heparan sulfate (HS) induces morphogenesis of skin appendages in mature skin.

Conclusion: HS plays an inhibitory role in skin appendage formation in mature skin.

Significance: Manipulation of HS levels in skin could enable the formation of skin appendages after skin injuries and in aged skin.

Abstract

Hair follicle (HF) morphogenesis and cycling are a result of intricate autonomous epithelial-mesenchymal interactions. Once the first HF cycle is complete it repeatedly undergoes cyclic transformations. Heparan sulfate (HS) proteoglycans are found on the cell surface and in the extracellular matrix where they influence a variety of biological processes by interacting with physiologically important proteins, such as growth factors. Inhibition of heparanase (an HS endoglycosidase) in in vitro cultured HFs has been shown to induce a catagen-like process. Therefore, this study aimed to elucidate the precise role of HS in HF morphogenesis and cycling. An inducible tetratransgenic mouse model was generated to excise exostosin glycosyltransferase 1 (Ext1) in keratin 14-positive cells from P21. Interestingly, EXT1^{StEpiΔ/StEpiΔ} mice presented solely anagen HFs. Moreover, waxing the fur to synchronize the HFs revealed accelerated hair regrowth in the EXT1^{StEpiΔ/StEpiΔ} mice and hindered cycling into catagen. The ablation of HS in the interfollicular epidermal cells of mature skin led to the spontaneous formation of new HFs and an increase in Sonic Hedgehog expression resembling wild-type mice at P0, thereby indicating that the HS/Sonic Hedgehog signaling pathway regulates HF formation during embryogenesis and prevents HF formation in mature skin. Finally, the knock-out of HS also led to the morphogenesis and hyperplasia of sebaceous glands and sweat glands in mature mice, leading to exacerbated sebum production and accumulation on the skin surface. Therefore, our findings clearly show that an intricate control of HS levels is required for HF, sebaceous gland, and sweat gland morphogenesis and HF cycling.

Introduction

The hair follicle is a skin appendage that is a composite micro-organ of both epithelial and dermal origin. The follicle is composed of various compartments, namely the outer root sheath (ORS),² inner root sheath (IRS), hair shaft, and extracellular matrix, which are all of epithelial origin, and the dermal papilla and connective tissue sheath, which are both of mesenchymal origin (1). The IRS and ORS are two concentric epithelial cell layers (keratinocytes) that surround the hair follicle. The bulb is located at the proximal end of the hair follicle and contains melanocytes and proximal cells from the ORS. The bulge is a convex extension of the distal ORS and contains the hair follicle stem cells. The dermal papilla is composed of closely packed mesenchymal cells and is engulfed by the bulb during anagen. The connective tissue sheath is composed of fibroblasts, macrophages, and connective tissue tightly attached to the outer side of the hair follicle.

The hair follicle develops as a result of intricate epithelial-mesenchymal interactions between epidermal keratinocytes committed to hair-specific differentiation and a cluster of dermal fibroblasts that form the follicular papilla (2). Hair follicle morphogenesis occurs in three waves, each giving rise to the specific types of fur, and all are completed by postnatal day 20 (3, 4). Thereafter, the budding of new hair follicles ceases, and the existent hair follicles undergo spontaneous hair cycling in a wavelike manner. Hair follicle morphogenesis commences with the appearance of epithelial placodes at embryonic day E14.5 that develop into primary guard hair follicles comprising 1–5% of the adult mouse coat. At embryonic day E16.5, the second wave of placode formation is initiated; these develop into awl and auchene hairs that account for ∼20% of the adult mouse coat. The second wave of placodes develop with an even distribution between the established guard follicles. Finally, at embryonic day E18.5, the third and final wave of placode formation begins; these develop into the zigzag hairs comprising the bulk of the adult coat (3, 4). All mouse hair follicle types have the same basic arrangement.

Once the first hair cycle is completed, hair follicles repeatedly undergo cyclic transformations of rapid growth involving hair shaft production (anagen), apoptosis-driven regression (catagen), and quiescence of mature hair follicles (telogen) (4–6). At the onset of anagen, enhanced proliferation of stem cells (of epithelial origin) located within the bulge region leads to the downward growth of the hair follicles, leading to the formation of the hair shaft, which requires enzymatic degradation of the surrounding extracellular matrix (7). Both hair follicle development and cycling rely on autonomous intricate epithelium-mesenchyme interactions controlled by similar signaling networks involving bone morphogenetic protein (BMP), transforming growth factor (TGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), Sonic Hedgehog (SHH), tumor necrosis factor (TNF), ectodysplasin A (EDA), and Wnt families (8–16). EDA, which is from the TNF superfamily, is involved in hair follicle morphogenesis and hair follicle cycling and has been interposed downstream of inductive Wnt signaling (15). EDA is also required for scale formation in fish and sweat gland development in mammals (15). The growth factors BMP, TGF, EGF, FGF, SHH, TNF, Wnt, and EDA have all been shown previously to be modulated by heparan sulfate (HS) (17–20).

Heparan sulfate proteoglycans (HSPGs) are found on the cell surface and in the extracellular matrix where they influence a variety of biological processes by interacting with physiologically important proteins, such as growth factors, chemokines and cytokines, extracellular matrix proteins, enzymes, and enzyme inhibitors (21). Their activity is due at large to the pattern of sulfated sugar residues along the HS chains covalently bound to the core proteins of the proteoglycans (22–25). The interaction of growth factors with HS protects the growth factors from degradation, creates a storage pool, acts as a co-receptor facilitating the assembly of signaling complexes, regulates growth factor diffusion throughout the tissue, and enables clearance of the growth factors by endocytosis (21, 26). Heparanase is an endoglycosidase that cleaves HS, enabling the release of growth factors bound to HS chains, removal of the physical barrier imposed by HS, and interruption of HS-mediated cell-cell and cell-matrix contacts (27). Therefore, a delicate balance is required between the expression of both HS and heparanase. Previous studies have shown that heparanase expression varies throughout hair follicle cycling with increased expression in anagen hair follicles and decreased expression in catagen follicles (28). Moreover, the overexpression of heparanase accelerates the rate of anagen, and the authors speculate that the release of growth factors upon heparanase cleavage would play a major role (29). Herein we show that it is the absence of HS within the hair follicle that triggers the enhanced rate of anagen and not the release of growth factors upon heparanase digestion. Moreover, we show that HS plays a vital role in hair follicle morphogenesis. Further indications of the important role HS plays in hair follicle homeostasis is that hair abnormalities are solely present in mucopolysaccharidosis types with accumulation of HS, such as MPS I, MPS II, and MPS III; however, no hair abnormalities are found in MPS IV or MPS VI in which HS catabolism is not affected (30).

To study the role of HS in hair follicle homeostasis and cycling, a transgenic mouse model was generated to knock out exostosin glycosyltransferase 1 (Ext1) using a tissue-specific inducible system. Ext1 was knocked out of cells of epithelial origin using a keratin 14 (K14)-rtTA driver system after the completion of the first hair cycle, generating EXT1^{StEpiΔ/StEpiΔ} mice (excision of Ext1 in stratified epithelium). Therefore, with the Ext1 knock-out system, HS was specifically ablated from the ORS, IRS, and hair shaft of these mice but remained in the hair follicle components of dermal origin. Interestingly, the ablation of HS in the hair follicle presents a more severe phenotype when compared with the heparanase overexpression mouse model by accelerating anagen, enhancing active hair growth, and enabling faster hair regrowth upon waxing, thereby suggesting that HS plays an important barrier function during rapid hair follicle growth and hair shaft production. The absence of HS also impeded the formation of catagen follicles in EXT1^{StEpiΔ/StEpiΔ} mice, resulting in sequestration of all hair follicles in anagen upon cycling. Moreover, EXT1^{StEpiΔ/StEpiΔ} mature mice induced at all time points analyzed (P20, P23, P25, and P35) presented spontaneous hair follicle budding upon induction, revealing the important role HS plays in both hair follicle morphogenesis and homeostasis. Both manipulations of the HF cycling and the ability of inducing morphogenesis present great pharmaceutical potential.

EXPERIMENTAL PROCEDURES

Mouse Strains and Genotyping

Transgenic mouse lines K14-rtTA (stock number 008099) (31), tetO-cre (TC) (stock number 006224) (32), and RosamTmG/mTmG (stock number 007576) (33) were purchased from The Jackson Laboratory (Bar Harbor, ME). The floxed mice utilized were Ext1^flox/flox (34). Compound transgenic mice were generated by mating. All the mice were bred at the Animal Facility of the University of Cincinnati Medical Center. Experimental procedures for handling the mice were approved by the Institutional Animal Care and Use Committee, University of Cincinnati/College of Medicine. The identification of each transgene allele was performed by PCR genotyping with tail DNA.

Induction by Administration of Doxycycline Chow

Administration of doxycycline chow was utilized to induce K14-driven persistent and irreversible excision of Ext1 in tetratransgenic mice (K14-rtTA/TC/RosaLSL/Ext1). Transgenic mice at P20 or older were fed with doxycycline chow (1 g of doxycycline/kg of chow; Custom Animal Diets LLC, Bangor, PA) ad libitum. Control animals were either double transgenic or triple transgenic heterozygous littermates.

Skin Collection

Skin samples were obtained from EXT1^{StEpiΔ/StEpiΔ} mice and control littermates. Rectangular pieces of skin from the left and right sides of both the dorsal and abdominal regions were collected parallel to the vertebral line (∼3.5 × 1 cm, length × width) and processed for further paraffin or cryoembedding or protein extraction.

Agarose Gel Electrophoresis

The skin samples were minced in acetone and centrifuged. The precipitate was dried and subjected to proteolysis with subtilisin (Sigma-Aldrich), and protein products were removed by trichloroacetic (TCA) acid (Sigma-Aldrich) precipitation. The GAGs were then precipitated overnight with methanol followed by centrifugation. The skin levels of GAG were first analyzed by 0.6% agarose gel electrophoresis in 0.05 m propanediamine acetate buffer, pH 9 as described previously (35). Following electrophoresis, the gels were submerged in 0.2% Cetavlon (cetyltrimethylammonium bromide, Sigma-Aldrich) for 1 h at room temperature; dried; stained with 0.1% toluidine blue prepared in a solution of 1% acetic acid, 50% ethanol, and 49% water; and destained with the same solution without toluidine blue.

Dimethylmethylene Blue Assay

To quantify HS levels, chondroitin sulfate and dermatan sulfate were removed from total skin GAGs by Chondroitinase ABC (Sigma) digestion followed by centrifugation using Microcon® centrifugal filters (10,000; Millipore). The HS content was then measured using dimethylmethylene blue reagent. The HS content was calculated using a standard curve prepared with porcine mucosa HS (Neoparin, Alameda, CA).

Histochemistry

Tissues were fixed for 12 h in 4% buffered paraformaldehyde, washed five times with PBS, sequentially dehydrated, immersed in paraffin overnight, and subsequently mounted. The blocks were sectioned at 7 μm, and sections were collected on poly-l-lysine-treated slides. Upon use, tissue sections were deparaffinized, rehydrated in PBS, and stained with hematoxylin and eosin. Images were captured using a Zeiss Observer Z1 inverted microscope or Zeiss LSM-710 confocal microscope, and images were analyzed using LSM Image Browser 3.2 software (Zeiss, Germany).

Immunohistochemistry

Tissues were fixed for 12 h in 4% buffered paraformaldehyde, treated for 15 min in 0.1% sodium borohydride, and embedded in Tissue-Tek® embedding medium for cryosectioning. 10-μm sections were cut using a cryostat (Cryostar NX70, Thermo Scientific) and collected on Fisherbrand® Superfrost®Plus Gold microscope slides (Thermo Scientific). Upon use, sections were incubated for 30 min at 37 °C, and excess tissue embedding medium was removed with PBS. Nonspecific protein binding sites were blocked with 5% fetal bovine serum (FBS). Sections were then incubated with primary antibody anti-K14 (Covance, PRB-155P), anti-keratin 15 (K15) (Thermo Scientific, MS-1068), anti-syndecan 1 (Abcam, ab34164), anti-syndecan 2 (Abcam, ab79978), anti-syndecan 3 (Abcam, ab63932), anti-syndecan 4 (Abcam, ab24511), anti-EXT1 (HPA044394, Atlas), anti-EXT1 (TA323730, Origene), anti-HS (clone 10E4, US Biological) anti-Wnt1 (Abcam, ab15251), anti-Wnt2 (Abcam, ab27794), anti-BMP4 (Millipore, MAB1049), anti-EDA, anti-SHH (Sigma, AV44235), or anti-β-catenin (Cell Signaling Technology, 9582). Sections were washed and incubated with appropriate secondary antibodies produced in donkey labeled with Alexa Fluor® 647 (Invitrogen) for 1 h at 18 °C. Subsequently, sections were washed, and nuclei were stained with DAPI. Sections were mounted in Fluoromount-G® (Electron Microscopy Sciences). Images were captured using a Zeiss Observer Z1 inverted microscope or Zeiss LSM-710 confocal microscope, and images were analyzed using LSM Image Browser 3.2 software (Zeiss).

Oil Red O Staining

For the detection of neutral lipids, cryosections were washed with PBS and then stained with 0.5% oil red O (Sigma) for 15 min, rinsed with PBS, and counterstained with hematoxylin. Sections were mounted in FluoromountG. Images were captured using a Nikon Eclipse E800 microscope coupled with Axiocam ICc5 and processed using Axiovision 4.8 (Zeiss).

Induction of Hair Cycle

The left dorsal fur of mice was removed using unscented commercial hair-removing wax strips, leading to the synchronized development of anagen hair follicles. Skin tissue samples were collected 5, 7, and 25 days after hair removal. Five mice were used per group for each time point. The samples were fixed in 4% paraformaldehyde and processed for histology and immunohistochemistry.

Iodine Sweat Test

Iodine/alcohol (1 g of iodine/100 ml of ethanol) was smeared on the plantar surface of the rear paw with the use of a paint brush and air-dried. Thereafter, a starch oil suspension (50 g of starch/100 ml of castor oil) was smeared over the iodine solution, and black dots formed, revealing the opening of sweat glands. Images were captured 5 s after the starch oil suspension was applied to the foot pad.

Protein Extraction from Skin

Skin samples were homogenized in modified radioimmune precipitation assay lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mm EDTA) containing 1 mm phenylmethylsulfonyl fluoride and 1× protease inhibitor mixture (Sigma, P8340). The samples were homogenized on ice and then centrifuged at 13,000 × g for 10 min at 4 °C to obtain a soluble fraction. Protein content was assayed using the Bradford method (36). Western blot analysis was performed as described previously (37, 38). Briefly, protein (25 μg) was separated on a gradient SDS-polyacrylamide gels (4–20%) by SDS-PAGE and transferred by electrical current to polyvinylidene difluoride (PVDF) membranes. The membranes were developed with anti-syndecan 1, anti-syndecan 3, anti-syndecan 4, anti-SHH, and anti-β-catenin followed by secondary antibodies coupled with Alexa Fluor 555. For syndecan, Western blotting analysis samples were previously treated with heparinase II (Hepase II) (IBEX) and heparinase III (Hepase III) (IBEX) for 24 h at 37 °C. A loading control was performed with goat anti-β-actin followed by anti-goat secondary antibody coupled with Alexa Fluor 488.

Statistical Analysis

All values are presented as mean ± S.D. The difference between two groups was compared by unpaired Mann-Whitney test. p < 0.05 was considered to be statistically significant. Statistical analysis was performed with the GraphPad Prism version 5 software package (GraphPad Software, San Diego, CA).

RESULTS

Ext1 Excision of the Stratified Epithelium and Hair Follicle

Tetratransgenic mice (K14-rtTA/tetO-cre-EXT1^flox-mTmG) were generated to specifically excise Ext1 upon doxycycline induction in keratin 14-expressing cells; hence, HS was not expressed in the stratified epithelium (StEpi), generating EXT1^{StEpiΔ/StEpiΔ} mice. Prior to doxycycline induction, the K14-rtTA/tetO-cre-EXT1^flox-mTmG mice exhibited no macroscopic or microscopic abnormalities of the skin. EXT1^{StEpiΔ/StEpiΔ} mice at P60 began to lose weight, and by P120, the mice showed severe signs of dehydration and had to be culled.

K14 Expression in the Hair Follicle

K14 staining was performed to determine the population of hair follicle cells that express K14 (Fig. 1). Anti-K14 staining of telogen hair follicles revealed that cells in the ORS, IRS, IRS cone, hair bulb, and sebaceous gland all express K14 (Fig. 1). A similar K14 expression pattern was observed in developing hair follicles and anagen hair follicles in P0 and P20 mice. The hair follicles from the skin of EXT1^{StEpiΔ/StEpiΔ} mice at P55 also showed K14 staining in the ORS, IRS, IRS cone, hair bulb, and sebaceous gland (Fig. 1). Therefore, all of the hair follicle components of epithelial origin express K14, and thus, in our mouse model, these cells would lack Ext1 and consequently be HS-deficient. EXT1 staining was also performed on the skin of EXT1^{StEpiΔ/StEpiΔ} and littermate control mice at P55, revealing a loss in EXT1 expression in the hair follicles of EXT1^{StEpiΔ/StEpiΔ} mice (Fig. 1D).

FIGURE 1.

A, K14 staining (red) can be observed in the bulb, ORS, and IRS of hair follicles and in the sebaceous gland from littermate control mice at P0 (panel a), P20 (panel b), and P55 (panel c) and EXT1^{StEpiΔ/StEpiΔ} mice at P55 (panels d and e). Nuclei were stained with DAPI (blue). Images were captured using a Zeiss LSM-710 confocal microscope. B, the dermatan sulfate level in the skin of EXT1^{StEpiΔ/StEpiΔ} and littermate control mice was determined by agarose gel electrophoresis of skin GAG extracts. C, the HS level in the skin of EXT1^{StEpiΔ/StEpiΔ} and littermate control mice was determined by dimethylmethylene blue assay after Chondroitinase ABC treatment of skin GAG extracts. D, panels a–c, loss of EXT1 staining can be observed in the hair follicle of EXT1^{StEpiΔ/StEpiΔ} mice (b and c) compared with littermate control (a). Scale bars, 20 μm. Error bars represent S.D. CS, chondroitin sulfate; DS, dermatan sulfate.

To further evaluate which components of the hair follicle of EXT1^{StEpiΔ/StEpiΔ} mice would lack HS, the mice were bred with the reporter mTmG gene, which expresses strong red fluorescence in all tissues; however, in cells expressing Cre recombinase, the mT cassette is deleted, enabling the expression of the downstream cassette, membrane-targeted enhanced GFP (mG). Therefore, in all cells that present K14-driven Cre recombinase activity, both the floxed Ext1 gene and mT cassette will be deleted, and cells will consequently express membrane-bound GFP. The reporter mG expression driven by K14 expression presented an expression pattern similar to that of anti-K14 staining wherein the IRS, IRS cone, hair bulb, and sebaceous gland all presented strong GFP expression (Fig. 2), further reinforcing that the EXT1^{StEpiΔ/StEpiΔ} mouse model lacks HS expression in the hair follicle in all cells of epithelial origin. Therefore, based on reporter expression, the hair follicles of EXT1^{StEpiΔ/StEpiΔ} mice lack HS in the IRS, IRS cone, and hair bulb but retain normal HS expression in the hair follicle components of mesenchymal origin (Fig. 2). HS staining was further performed using anti-HS (clone 10E4) confirming the knock-out of HS in the hair follicle components of epithelial origin (Fig. 2). Moreover, GAGs were extracted from the skin of EXT1^{StEpiΔ/StEpiΔ} and littermate control mice and subjected to Chondroitinase ABC treatment followed by dimethylmethylene blue assay, which further confirmed the decrease in skin HS (Fig. 1C). Moreover, the extracted GAGs were also subjected to agarose gel electrophoresis, which revealed no changes in the skin levels of dermatan sulfate between EXT1^{StEpiΔ/StEpiΔ} and littermate control mice (Fig. 1B).

FIGURE 2.

HS staining (white) was performed with anti-HS (clone 10E4) on skin cryosections from littermate control mice at P0 (a, b, and c), P20 (d, e, and f), and P55 (g, h, and i) and EXT1^{StEpiΔ/StEpiΔ} mice at P55 (j, k, and l). Mice were bred with the reporter RosamTmG gene; thereby K14-positive cells that expressed Cre recombinase upon induction express mG representing EXT1-deficient cells in EXT1^{StEpiΔ/StEpiΔ} mice. EXT1^{StEpiΔ/StEpiΔ} mice present HS staining solely in the mT-positive cells, and therefore, Cre recombinase successfully excised Ext1 from the cells of epithelial origin. The interfollicular epithelium (Epi) is oriented upward in the images. a, d, g, and j, merged mT, mGFP, HS, and DAPI images. b, e, h, and k, HS and DAPI. c, f, i, and l, HS and mT. Nuclei were stained with DAPI (blue). Scale bars, 40 μm.

Role of HS in Hair Follicle Morphogenesis and Cycling

Histology of the EXT1^{StEpiΔ/StEpiΔ} mice induced from P20 to P55 revealed a 4-fold increase in the number of hair follicles compared with littermate controls (Fig. 3, a, b, c, and e). Thus, hair follicle morphogenesis occurs in EXT1^{StEpiΔ/StEpiΔ} mice upon induction at P20. The drastic increase in overall hair follicle number in animals induced at all time frames led to the irregular spacing of hair follicles with some fused hair follicles that merged at the outer epithelial layers; however, each individual hair follicle possessed its own hair bulb, dermal papilla, hair shaft, and sebaceous gland. EXT1^{StEpiΔ/StEpiΔ} mice beyond P55 presented such a dense distribution of hair follicles that three or four were fused together in some locations. Moreover, in vivo confocal microscopy of the abdomen of the mice revealed an overall 4-fold increase in the quantity of fur in the EXT1^{StEpiΔ/StEpiΔ} mice when compared with the littermate control (Fig. 3, f and g). These data indicate that the absence of HS in the epithelium leads to the budding of hair follicles in the mature skin of mice.

FIGURE 3.

Skin sections from littermate control (a and c) and EXT1^{StEpiΔ/StEpiΔ} mice (b and e) at P55 were double stained with hematoxylin and eosin and visualized under a light microscope. Telogen hair follicles can be observed in the skin sections from littermate control mice, and anagen hair follicles can be observed in the skin sections from EXT1^{StEpiΔ/StEpiΔ} mice. EXT1^{StEpiΔ/StEpiΔ} mice at P55 present excessive accumulation of sebum on the skin and fur (d) due to an increase in the number and size of sebaceous glands. f and g, in vivo confocal microscopy of the mouse abdomen was performed to assess the fur density of littermate control (f) and EXT1^{StEpiΔ/StEpiΔ} (g) mice at P55. An overall increase in hair density number was observed in EXT1^{StEpiΔ/StEpiΔ} mice compared with littermate controls.

Interestingly, the EXT1^{StEpiΔ/StEpiΔ} mice at all time frames solely presented anagen hair follicles; however, the littermate control mice presented telogen hair follicles at the same time frame (Fig. 3). Previous studies have shown that the first mouse fur cycle starts around P28 and is concluded by approximately P49, and the second fur cycle commences around P84 (1). Therefore, mice analyzed at P55 should present mainly telogen hair follicles as observed in the littermate controls. This suggests that the lack of HS in hair follicles impedes cycling into the catagen phase and consequently the telogen phase in EXT1^{StEpiΔ/StEpiΔ} mice.

Syndecan and Perlecan Expression during Hair Follicle Morphogenesis and Cycling

Previous studies have shown that hair follicles express syndecan 1. To elucidate the expression profile of syndecan 1 in hair follicles, anti-syndecan 1 staining was performed on the skin of control mice at P0 and P20. During hair follicle morphogenesis (P0), high levels of syndecan 1 expression were observed in the stroma proximal to the epithelium; hence, syndecan 1 could play a role in stroma-epithelium cross-talk during hair follicle formation (Fig. 4A, panels a and b). Moreover, hair follicles at P0 and P20 also presented syndecan 1 expression in the IRS and ORS (Fig. 4A, panels a–d). By P20, there was a drastic decrease in stromal syndecan 1 expression, which was present solely adjacent to the epithelium and in the ORS (Fig. 4A, panels c and d). In the mature hair follicles of the littermate control at P55, syndecan 1 expression was restricted to the ORS and stroma adjacent to the epithelium (Fig. 4A, panels e and f). Interestingly, EXT1^{StEpiΔ/StEpiΔ} mice induced from P20 to P55 presented strong stromal syndecan 1 expression similar to that observed in the P0 mice (Fig. 4A, panels g–j). Moreover, there was a 6-fold increase in syndecan 1 expression in the ORS of EXT1^{StEpiΔ/StEpiΔ} mice compared with that of control mice at P0, P20, and P55 (quantification of immunofluorescence staining performed using Zen, Zeiss). An increase of 2-fold in syndecan 1 expression was further confirmed by Western blotting analysis (Fig. 4, B and C).

FIGURE 4.

A, syndecan 1 staining (white) was performed in skin cryosections from littermate control mice at P0 (panels a and b), P20 (panels c and d), and P55 (panels e and f) and EXT1^{StEpiΔ/StEpiΔ} mice at P55 (panels g–j). The interfollicular epithelium (Epi) is oriented upward in the images. Control mice at P0 and P20 present syndecan 1 expression in the stroma adjacent to the epithelium, ORS, IRS, and bulb; however, mature skin with telogen hair follicles presents syndecan 1 distributed throughout the interfollicular epithelium and ORS. EXT1^{StEpiΔ/StEpiΔ} mice present syndecan 1 distribution similar to that of P0 and P20 control mice; however, a 20-fold increase is observed in the ORS. Panels a, c, e, g, and i, merged mT (red), mGFP (green), syndecan 1, and DAPI images. Panels b, d, f, h, and j, syndecan 1 and DAPI. Nuclei were stained with DAPI (blue). Scale bars, 40 μm. B, protein was extracted from the skin of EXT1^f/f and EXT1^{StEpiΔ/StEpiΔ} mice, submitted to separation by SDS-PAGE, and transferred by current to a PVDF membrane. The membrane was developed with anti-syndecan 1 followed by anti-rabbit secondary antibody conjugated with Alexa Fluor 555. C, the syndecan 1-positive band from the Western blot was quantified, and overall content was divided by that quantified for β-actin, which was developed using anti-β-actin followed by anti-goat secondary antibody conjugated with Alexa Fluor 488. Results are presented as a graph. Error bars represent S.D. RelUF, relative units of fluorescence.

The expression of syndecans 2, 3, and 4 was also investigated in the hair follicles of EXT1^{StEpiΔ/StEpiΔ} mice. The lack of HS in the EXT1^{StEpiΔ/StEpiΔ} mice led to an induction/increase in the expression of syndecan 3 in the ORS and sebaceous glands of EXT1^{StEpiΔ/StEpiΔ} mice (Fig. 5A, panels g–j). Western blotting analysis confirmed a 2-fold increase of syndecan 3 expression in EXT1^{StEpiΔ/StEpiΔ} mice when compared with littermate controls (Fig. 5, B and C). Syndecan 4 expression was not present in control mice at P0; however, weak staining was present in the ORS and IRS at P20 and P55. EXT1^{StEpiΔ/StEpiΔ} mice also presented weak syndecan 4 expression in the ORS and IRS at P55 (Fig. 6). No significant syndecan 2 expression was observed in any of the samples analyzed (results not shown).

FIGURE 5.

A, syndecan 3 staining (white) was performed in skin cryosections from littermate control mice at P0 (panels a and b), P20 (panels c and d), and P55 (panels e and f) and EXT1^{StEpiΔ/StEpiΔ} mice at P55 (panels g–j). The interfollicular epithelium (Epi) is oriented upward in the images. Syndecan 3 expression is observed primarily in the sebaceous glands, and EXT1^{StEpiΔ/StEpiΔ} mice present an increase in expression. Panels a, c, e, g, and i, merged mT (red), mGFP (green), syndecan 3, and DAPI (blue) images. Panels b, d, f, h, and j, syndecan 3 and DAPI. Scale bars, 40 μm. B, protein was extracted from the skin of EXT1^f/f and EXT1^{StEpiΔ/StEpiΔ} mice, submitted to separation by SDS-PAGE, and transferred by current to a PVDF membrane. The membrane was developed with anti-syndecan 3 followed by anti-rabbit secondary antibody conjugated with Alexa Fluor 555. C, the syndecan 3-positive band from the Western blot was quantified, and overall content was divided by that quantified for β-actin, which was developed using anti-β-actin followed by anti-goat secondary antibody conjugated with Alexa Fluor 488. Results are presented as a graph. Error bars represent S.D. RelUF, relative units of fluorescence.

FIGURE 6.

A, syndecan 4 staining (white) was performed in skin cryosections from littermate control mice at P0 (panels a and b), P20 (panels c and d), and P55 (panels e and f) and EXT1^{StEpiΔ/StEpiΔ} mice at P55 (panels g–h). The interfollicular epithelium (Epi) is oriented upward in the images. Panels a, c, e, and g, merged mT (red), mGFP (green), syndecan 4, and DAPI (blue) images. Panels b, d, f, and h, syndecan 4 and DAPI (blue). Scale bars, 40 μm. B, protein was extracted from the skin of EXT1^f/f and EXT1^{StEpiΔ/StEpiΔ} mice, submitted to separation by SDS-PAGE, and transferred by current to a PVDF membrane. The membrane was developed with anti-syndecan 4 followed by anti-rabbit secondary antibody conjugated with Alexa Fluor 555.

Strong perlecan staining was present in the stroma of control mice at P0; by P20, the expression decreased and was solely present in the stroma cells adjacent to the hair follicles; and by P55, there was an overall decrease in perlecan staining. EXT1^{StEpiΔ/StEpiΔ} mice presented strong perlecan staining similar to that observed in littermate control mice at P0 (Figure 7). The perlecan staining colocalized with the mT-expressing cells surrounding the hair follicles.

FIGURE 7.

Perlecan staining (white) was performed in skin cryosections from littermate control mice at P0 (a and b), P20 (c and d), and P55 (e and f) and EXT1^{StEpiΔ/StEpiΔ} mice at P55 (g and h). The interfollicular epithelium (Epi) is oriented upward in the images. Perlecan expression is observed primarily in connective tissue sheath, sebaceous glands, and stroma of both EXT1^f/f mice at P0 and EXT1^{StEpiΔ/StEpiΔ} mice at P55. a, c, e, and g, merged mT (red), mGFP (green), perlecan, and DAPI (blue) images. b, d, f, and h, perlecan and DAPI (blue). Nuclei were stained with DAPI (blue). Scale bars, 40 μm.

Hair Follicle Differentiation

K15 is at large considered to be a marker for hair follicle cells (39). To further investigate the role of HS in hair follicle differentiation, and consequently cycling, anti-K15 staining was performed. Hair follicles at P0 presented faint K15 staining along the hair follicle shaft; however, by P20, the hair follicles displayed dense K15 staining in the ORS, IRS, and sebaceous gland (Fig. 8, a–d). The telogen hair follicles in the littermate control mice also presented intense K15 staining in the ORS, IRS, bulge, and sebaceous gland (Fig. 8, e and f). In the EXT1^{StEpiΔ/StEpiΔ} mice induced from P20 to P55, there was an overall loss of K15 expression in the hair follicles with subtle staining solely in the ORS and some newly developing sebaceous glands (Fig. 8, g–i). Therefore, the lack of HS in the EXT1^{StEpiΔ/StEpiΔ} mice leads to altered differentiation of the hair follicle cells. This supports the notion that the lack of HS leads to altered differentiation of anagen hair follicles that could hinder the cycling into catagen.

FIGURE 8.

K15 staining (white) was performed in skin cryosections from EXT1^f/f mice at P0 (a and b), P20 (c and d), and P55 (e and f) and EXT1^{StEpiΔ/StEpiΔ} mice at P55 (g–i). The interfollicular epithelium (Epi) is oriented upward in the images. Control mice at P0 present dispersed K15 expression in the epithelium; however, P0 and P55 control mice present K15 expression in the dermal epithelium, ORS, IRS, and bulb. EXT1^{StEpiΔ/StEpiΔ} mice present a loss of K15 expression in the ORS, IRS, and bulb but retain K15 expression in the dermal epithelium and sebaceous glands and in the ORS solely in some areas. a, c, e, g, and i, merged mT (red), mGFP (green), K15, and DAPI images. b, d, f, and h, K15 and DAPI. Nuclei were stained with DAPI (blue). Scale bars, 40 μm.

Wnt1, EDA, and SHH Signaling during Hair Follicle Morphogenesis and Cycling

In an attempt to elucidate the role of HS in hair follicle morphogenesis and cycling, immunostaining was performed for Wnt1, Wnt2, BMP4, β-catenin, EDA, and SHH. No changes in Wnt1, Wnt2, BMP4, and EDA expression and localization were detected between the EXT1^{StEpiΔ/StEpiΔ} mice and littermate controls (results not shown). EDA expression was detected in the ORS, and Wnt1 expression was detected in the IRS and bulb of the hair follicles in adult EXT1^{StEpiΔ/StEpiΔ} mice (Fig. 9). Wnt1 expression was localized to the bulb, in the matrix, in the IRS cone in the bulb region, and along the proximal IRS of the anagen hair follicles in the EXT1^{StEpiΔ/StEpiΔ} mice (Fig. 9). Wnt2 staining was very weak and localized primarily to the ORS of the EXT1^{StEpiΔ/StEpiΔ} mice anagen hair follicles (results not shown). EDA expression was predominantly located along the proximal IRS of the EXT1^{StEpiΔ/StEpiΔ} mice anagen hair follicles.

FIGURE 9.

EDA (a, b, and c) and Wnt 1 (d, e, and f) staining (white) was performed in skin cryosections from EXT1^{StEpiΔ/StEpiΔ} mice at P55. EXT1^{StEpiΔ/StEpiΔ} mice present EDA staining in the ORS and Wnt1 staining in the proximal IRS and bulb. a, merged mT (red), mGFP (green), EDA, and DAPI images. b and e, merged mT, mGFP, and DAPI images. c, EDA and DAPI images. d, merged mT (red), mGFP (green), Wnt1, and DAPI images. f, Wnt1 and DAPI images. Nuclei were stained with DAPI (blue).

A drastic increase in SHH expression was observed in the EXT1^{StEpiΔ/StEpiΔ} mice (Fig. 10). Littermate control mice at P0 presented SHH expression in the interfollicular epidermal cells, whereas SHH was no longer detected in the interfollicular epithelium of mice at P20 or P55, corroborating previous findings that SHH plays a role in hair follicle formation (Fig. 10A, panels a–f). In contrast, there was a 20-fold increase in SHH expression in the interfollicular epidermal cells of EXT1^{StEpiΔ/StEpiΔ} mice when compared with littermate control mice at P0 (determined by quantification of immunofluorescence) (Fig. 10A, panels a, b, and g–j). Moreover, the dermal stroma adjacent to the epithelium and the sebaceous glands also presented strong SHH staining in EXT1^{StEpiΔ/StEpiΔ} mice (Fig. 10A, panels g–j). Therefore the loss of HS leads to an increase in SHH expression (Fig. 10). These results were further confirmed through Western blotting analysis, which revealed an ∼7-fold increase in SHH expression in the EXT1^{StEpiΔ/StEpiΔ} mice when compared with littermate control mice at P55.

FIGURE 10.

A, SHH staining (white) was performed in skin cryosections from littermate control mice at P0 (panels a and b), P20 (panels c and d), and P55 (panels e and f) and EXT1^{StEpiΔ/StEpiΔ} mice at P55 (panels g–j). Panels i and j are enlarged images of the boxed area in panel g. The interfollicular epithelium (Epi) is oriented upward in the images. Control mice at P0 present SHH expression in the dermal epithelium that is absent in the P20 and P55 control mice. EXT1^{StEpiΔ/StEpiΔ} mice present strong SHH expression in the dermal epithelium, stroma adjacent to the epithelium, ORS, IRS, and sebaceous glands. Panels a, c, e, g, and i, merged mT, mGFP, SHH, and DAPI images. Panels b, d, f, h, and j, SHH and DAPI. Panels i and j, enlarged images from the boxed area in panel g. Nuclei were stained with DAPI (blue). Scale bars, 40 μm. B, protein was extracted from the skin of EXT1^f/f and EXT1^{StEpiΔ/StEpiΔ} mice, submitted to separation by SDS-PAGE, and transferred by current to a PVDF membrane. The membrane was developed with anti-SHH followed by anti-rabbit secondary antibody conjugated with Alexa Fluor 555. C, the SHH positive band from the Western blot was quantified, and overall content was divided by that quantified for β-actin, which was developed using anti-β-actin followed by anti-goat secondary antibody conjugated with Alexa Fluor 488. Results are presented as a graph. Error bars represent S.D. RelUF, relative units of fluorescence.

The expression and localization of β-catenin was also investigated in the EXT1^{StEpiΔ/StEpiΔ} mice (Fig. 11). Control mice at P0 presented strong β-catenin staining in the IRS of the hair follicles and in the interfollicular epidermal cells. The expression of β-catenin decreases once hair follicle morphogenesis ceases, and the hair follicles of control mice at P20 and P55 presented low levels of β-catenin staining. Conversely, EXT1^{StEpiΔ/StEpiΔ} mice presented strong β-catenin in the dermal epithelium, stroma, bulb, dermal papilla, and ORS at P55. The increase in β-catenin expression in EXT1^{StEpiΔ/StEpiΔ} mice was further confirmed by Western blotting, which revealed a ∼4.5-fold increase.

FIGURE 11.

A, β-catenin staining (white) was performed in skin cryosections from littermate control mice at P0 (panels a and b), P20 (panels c and d), and P55 (panels e and f) and EXT1^{StEpiΔ/StEpiΔ} mice at P55 (panels g and h). The interfollicular epithelium (Epi) is oriented upward in the images. Control mice at P0 present strong β-catenin expression in the interfollicular epithelium, IRS, and bulb that is absent in the P20 and P55 control mice. EXT1^{StEpiΔ/StEpiΔ} mice present strong β-catenin expression in the dermal epithelium, stroma adjacent to the epithelium, ORS, IRS, and sebaceous glands. Panels a, c, e, and g, merged mT (red), mGFP (green), β-catenin, and DAPI images. Panels b, d, f, and h, β-catenin and DAPI. Nuclei were stained with DAPI (blue). Scale bars, 40 μm. B, protein was extracted from the skin of EXT1^f/f and EXT1^{StEpiΔ/StEpiΔ} mice, submitted to separation by SDS-PAGE, and transferred by current to a PVDF membrane. The membrane was developed with anti-β-catenin followed by anti-rabbit secondary antibody conjugated with Alexa Fluor 555. C, the β-catenin-positive band from the Western blot was quantified, and overall content was divided by that quantified for β-actin, which was developed using anti-β-actin followed by anti-goat secondary antibody conjugated with Alexa Fluor 488. Results are presented as a graph. Error bars represent S.D. RelUF, relative units of fluorescence.

Role of HS in Sebaceous Gland Morphogenesis

EXT1^{StEpiΔ/StEpiΔ} mice induced from P20 to P55 presented a 4-fold increase in sebaceous gland number compared with littermate controls, which is in accordance with the increase in hair follicle number. Moreover, EXT1^{StEpiΔ/StEpiΔ} mice induced at all time frames presented sebaceous gland hyperplasia. Interestingly, EXT1^{StEpiΔ/StEpiΔ} mice induced from P20 presented excessive sebum production to the extent that by P55 macroscopically the fur looked wet (Fig. 3d). Oil red O staining of the skin further revealed the hyperplastic sebaceous glands with altered morphology presenting irregular shapes and thickening of the sebaceous gland canal (Fig. 12A).

FIGURE 12.

A, oil red O staining of EXT1^f/f mice at P0 and P55 and EXT1^{StEpiΔ/StEpiΔ} mice at P55. Sections were counterstained with hematoxylin. EXT1^{StEpiΔ/StEpiΔ} mice present an increase in oil red O staining, revealing hyperplastic sebaceous glands with an irregular shape. B, iodine/starch test reveals sweat glands of littermate control mice at P55 and EXT1^{StEpiΔ/StEpiΔ} mice at P35, P55, and P120. EXT1^{StEpiΔ/StEpiΔ} mice present an increase in sweat gland duct openings. C, sections of the hind paws from EXT1^f/f and EXT1^{StEpiΔ/StEpiΔ} mice at P55 were double stained with hematoxylin and eosin and visualized under a light microscope. A 2.5-fold increase in sweat gland (asterisks) number was observed in EXT1^{StEpiΔ/StEpiΔ} mice compared with littermate controls. Scale bars, 100 μm.

Role of HS in Sweat Gland Morphogenesis

Mice present sweat glands solely on the plantar surface of their paws; therefore, to determine whether HS plays a role in sweat gland morphogenesis, the iodine/starch test was performed on the foot pads of the hind paws. There was an overall increase in the number of sweat glands in EXT1^{StEpiΔ/StEpiΔ} mice when compared with littermate control mice (Fig. 12B). Moreover, there was a gradual increase in overall sweat gland number over time in the EXT1^{StEpiΔ/StEpiΔ} mice (Fig. 12B). These results were further confirmed by histochemistry of the hind paws that revealed a 2.5-fold increase in sweat gland number in EXT1^{StEpiΔ/StEpiΔ} mice compared with littermate control mice at P55 (Fig. 12C).

Induction of Hair Cycle

To determine whether anagen hair follicles present in the EXT1^{StEpiΔ/StEpiΔ} mice are able to cycle into the telogen phase, fur was removed by waxing to synchronize hair follicles and induce entry of the hair follicles into the anagen phase in mutant and littermate mice. Thereafter, the animals were left for 25 days to complete a full hair cycle and enter the telogen phase. Interestingly, EXT1^{StEpiΔ/StEpiΔ} mice at all time points analyzed presented black skin upon waxing, which represents the anagen phase, whereas EXT1^f/f waxed at P35 and P55 presented pink skin, which is representative of the telogen phase, and at P40 presented gray skin, which is representative of the catagen phase (Fig. 13), thereby indicating that all EXT1^{StEpiΔ/StEpiΔ} mice were arrested in the anagen phase. Within 5 days of waxing, EXT1^{StEpiΔ/StEpiΔ} mice waxed at all time points presented fully grown fur in the waxed region, thereby no longer presenting a bald patch, whereas littermate control mice only presented signs of fur growing back 15 days after waxing (Fig. 14). The skin of the waxed animals was harvested 25 days after waxing upon completion of a full hair cycle. The waxed skin of littermate control mice presented primarily telogen hair follicles; however, EXT1^{StEpiΔ/StEpiΔ} mice presented solely anagen hair follicles, further demonstrating that the absence of HS in hair follicle epithelial cells impedes the progression of hair follicles into the catagen and, consequently, telogen phases (Fig. 14). Moreover, a gradual increase in syndecan 1 expression was detected upon waxing, further demonstrating that syndecan 1 plays an important role in hair follicle cycling (Fig. 14C). To evaluate whether the ablation of HS in stratified epithelial cells accelerates fur regrowth during the anagen phase, animals were induced at P23 or P30 and waxed either 7 days or immediately after induction, respectively, and the skin was analyzed 5 days after waxing. Interestingly, when the mice were induced 7 days before waxing, the fur of P30 EXT1^{StEpiΔ/StEpiΔ} mice grew back within 5 days (results not shown), and histology revealed a high density of anagen hair follicles throughout the waxed region, whereas control mice presented no visual signs of fur regrowth, and histology revealed regressing hair follicles as well as hair follicles in the initial anagen phase (results not shown). When the mice were waxed immediately after induction, the fur of the EXT1^{StEpiΔ/StEpiΔ} mice grew back within 12 days, whereas control mice presented no visual signs of fur regrowth until 15 days after waxing (results not shown).

FIGURE 13.

EXT1^f/f and EXT1^{StEpiΔ/StEpiΔ} mice were waxed at P55, exposing the skin and revealing that EXT1^f/f mice present pink skin and EXT1^{StEpiΔ/StEpiΔ} present black/gray skin.

FIGURE 14.

A, accelerated fur regrowth in EXT1^{StEpiΔ/StEpiΔ} mice. EXT1^{StEpiΔ/StEpiΔ} and littermate control mice were induced at P23, waxed at P30, and imaged at P35. EXT1^{StEpiΔ/StEpiΔ} mice present fur regrowth 5 days after waxing; however, littermate control mice present no signs of hair regrowth at the same time frame. B, EXT1^{StEpiΔ/StEpiΔ} mice present hair follicles sequestered in anagen. EXT1^{StEpiΔ/StEpiΔ} and littermate control mice were waxed to synchronize hair follicles and left to complete a full hair cycle. EXT1^{StEpiΔ/StEpiΔ} mice present no catagen or telogen hair follicles, whereas littermate control mice present primarily telogen hair follicles. Tissue sections were double stained with hematoxylin and eosin and visualized under a Nikon light microscope. C, protein was extracted from naïve skin and skin 25 days after waxing from EXT1^f/f and EXT1^{StEpiΔ/StEpiΔ} mice, submitted to separation by SDS-PAGE, and transferred by current to a PVDF membrane. The membrane was developed with anti-syndecan 1 followed by anti-rabbit secondary antibody conjugated with Alexa Fluor 555. D, the syndecan 1-positive band from the Western blot was quantified, and overall content was divided by that quantified for β-actin, which was developed using anti-β-actin followed by anti-goat secondary antibody conjugated with Alexa Fluor 488. Results are presented as a graph. Error bars represent S.D. RelUF, relative units of fluorescence.

To evaluate the long term effects of excessive hair follicle formation and hair follicles being sequestered in the anagen phase, animals were induced at P20 and waxed at P81. Indeed, fur had regrown on the EXT1^{StEpiΔ/StEpiΔ} mice 5 days after waxing; however, the fur grew back in patches, and several areas remained furless. 25 days after waxing (P105), the EXT1^{StEpiΔ/StEpiΔ} mice still presented irregular fur and bald patches in the waxed region. Animals at P120 presented general hair loss and areas of melanin incontinence within the balding regions (Fig. 15). EXT1^{StEpiΔ/StEpiΔ} mice beyond P55 presented a dense distribution of hair follicles throughout the skin with significantly enlarged sebaceous glands, which seemed to constrict the hair shaft and could possibly have led to the hair loss in mice beyond P55 (Fig. 15). However, the lack of hair cycling due to hair follicles being sequestered in the anagen phase could lead to hair loss from an exhausted anagen hair follicle, and an overall saturated number of hair follicles could impede the development of new hair follicles.

FIGURE 15.

EXT1^{StEpiΔ/StEpiΔ} mice present hair loss beyond P55 (a and b), melanin incontinence beyond P120 (c), and fused hair follicles and sebaceous glands (d and e). e represents the boxed area in d at higher magnification. Tissue sections were double stained with hematoxylin and eosin and visualized under a Nikon light microscope.

DISCUSSION

Epithelial-mesenchymal interactions trigger the development of hair follicles during embryogenesis. The extracellular matrix composition and the coordination between cells and the extracellular matrix play a major role in epithelial-mesenchymal interactions (40). HS, a major component of the extracellular matrix (ECM), is present on the cell surface covalently bound to syndecan 1, 2, 3, or 4 or in the ECM, for example, bound to perlecan. HS present on the cell surface plays a fundamental role in cell-cell and cell-matrix interactions (21). HS has been shown to modulate various cytokines involved in cell differentiation and/or proliferation, such as FGFs, VEGF, SHH, BMP, and Wnts (41, 42). During hair follicle morphogenesis, there is the formation of an epithelial placode, after which initially fibroblasts from the underlying mesenchyme differentiate into rudimentary dermal papillae (43). For this process, intricate cross-talk between the epithelial cells and underlying mesenchyme must take place. The precise events that regulate hair follicle morphogenesis remain elusive; however, EDA/EDA receptor (member of the FGF family), SHH, Wnt1, Wnt2, and β-catenin have been shown to play a role in promoting placode formation, whereas BMP2 and BMP4 have been shown to play an inhibiting role in placode formation (44–48). Interestingly, the ablation of HS in the interfollicular epidermal cells of mice at P20, P23, P25, and P35 led to the formation of new hair follicles. EDA plays an important role in the early stages of hair follicle morphogenesis during initial mesenchymal-ectodermal interactions. After induction, the EXT1^{StEpiΔ/StEpiΔ} mice presented persistent formation of new hair follicles, leading to an excessive distribution of hair follicles throughout the skin. The developing hair follicles in the EXT1^{StEpiΔ/StEpiΔ} mice presented EDA expression at P55, confirming the formation of hair follicles in mature skin with epithelial cells lacking HS. Moreover, the population of stem cells that maintain the epidermis are also K14-positive and therefore in our mouse model also undergo the ablation of the Ext1 gene and therefore lack HS (49, 50). Previous studies have shown that during homeostasis the epidermal stem cells simply maintain tissue integrity; however, upon wounding or specific genetic modifications, the stem cells can give rise to any differentiated epidermal lineage (49, 50). Therefore, our studies demonstrate that upon HS ablation epidermal stem cells differentiate into hair follicle, sebaceous gland cells, and sweat glands; thereby HS is an important regulator of the epidermal stem cells, and HSPGs are promising stem cell markers for the epidermis and epidermal appendages.

Previous studies have shown that hair follicles express high levels of syndecan 1 (51, 52). We hereby show that during hair follicle morphogenesis high levels of syndecan 1 expression were observed in the stroma proximal to the epithelium, and hence, syndecan 1 could play a role in stroma-epithelium cross-talk during hair follicle formation. Syndecan 1 is the major syndecan expressed by epithelial cells, and it plays a vital role during wound healing; however, no hair phenotype has been described in syndecan 1-overexpressing or knock-out mice to date (38). Syndecan 1, syndecan 3, and syndecan 4 knock-out studies have revealed that these mice present subtle phenotypes when compared with knock-out mice lacking enzymes involved in HS biosynthesis that could be attributed to compensatory mechanisms between syndecans (53). Accordingly, we hereby show that the ablation of HS in solely the interfollicular epidermis led to a phenotype not present in the syndecan knock-out mice. In contrast, the perlecan knock-out mice (Hspg2^−/−) present a severe phenotype, and the mice die from embryonic day 10.5 to just after birth. It remains to be elucidated whether knocking out perlecan using an inducible system would reproduce the phenotype observed in the EXT1^{StEpiΔ/StEpiΔ} mice.

HS chains can bind a plethora of growth factors, acting as a reservoir of soluble factors in the ECM. To investigate which signaling pathways were affected by the absence of HS, we analyzed the expression profile of cytokines that govern hair follicle morphogenesis. HS has been shown previously to bind and modulate the activity of Wnt and BMP family cytokines; however, no alteration in their expression profile was observed. SHH is a morphogen that mediates many developmental processes, including hair follicle morphogenesis and cycling, and requires HS for normal distribution and signaling activity (54–56). During embryogenesis, SHH is expressed at the distal tip of the developing follicle by the proliferating cells. Interestingly, EXT1^{StEpiΔ/StEpiΔ} mice presented strong SHH staining in the interfollicular epidermal cells at P55 that in control mice was solely present during hair follicle morphogenesis. Thus, SHH expressed by the interfollicular epidermal cells may play an intricate role in the epithelial-mesenchymal cross-talk regulating the formation of hair follicles, which is mediated through HSPGs. Thereby, we can hypothesize that the HS/SHH balance in the interfollicular epidermal cells of mature skin hinders the formation of new hair follicles, and solely the ablation of HS from these cells in the adult mice triggered formation of new hair follicles. Thus, pharmaceutically targeting HS levels in skin epithelial cells could enable engineering of de novo skin appendages in mature skin after skin burns and scarring.

Hair follicles maintain hair health by undergoing repeated cycles of growth (anagen), regression (catagen), and finally quiescence of mature hair follicles (telogen). During anagen, the matrix cells at the base of hair follicles derived from stem cells in the bulge region undergo rapid proliferation. Thereafter, the downward growth of anagen hair follicles requires enzymatic degradation of the surrounding ECM. Therefore, previous studies with heparanase (HS endoglycosidase) overexpression in mice revealed an increased hair growth rate during the anagen phase, suggesting that heparanase plays an important role in degrading the ECM during this downward growth (27, 28). Interestingly, EXT1^{StEpiΔ/StEpiΔ} mice showed a drastic increase in hair growth rate; however, these mice have intact HS in the ECM surrounding the hair follicle. However, the bulge region and the stem cells in the bulge region, which undergo rapid proliferation and are responsible for the downward growth, lack HS, which could potentially eliminate a physical barrier imposed by HS within the hair follicle, thereby facilitating the migration of stem cells undergoing rapid proliferation from the bulge region to the base of the hair follicle.

The absence of HS in hair follicles also led to altered differentiation of the cells during anagen, which could hinder regression into catagen. SHH and β-catenin have been shown to also play an important role in regulating hair follicle cycling (10). The absence of HS in the hair follicles led to an increase in SHH and β-catenin expression, which could directly affect the differentiation of these cells, hindering them in the anagen phase by affecting the signaling pathways of SHH and β-catenin, thus altering the signaling cues for catagen.

Heparanase has been detected previously in the ORS of murine hair follicles. Heparanase overexpression has been reported to improve mouse hair regrowth (28). Heparanase has been shown to be distributed in hair follicles, primarily located in the IRS of human hair follicles strictly in the anagen phase (28). Moreover, the inhibition of heparanase in in vitro cultured hair follicles induces a catagen-like process (28). Our findings are in accordance with the absence of HS impeding progression into catagen. Moreover, Malgouries et al. (29) showed previously that there is an increase in heparanase expression in human anagen hair follicles; however, at the onset of catagen, hair follicle heparanase expression ceases, further supporting our findings. Therefore, our results clearly reveal the importance of fluctuations in the HS levels of hair follicle epithelial cells, which dictate hair follicle cycling and thus play a vital role in hair follicle homeostasis.

The sebaceous gland plays a vital role in hair and skin homeostasis by producing sebum, which is deposited on the hair within the follicle and is brought to the surface through the hair shaft. Sebum protects the hair and skin by maintaining lubrication and preventing dryness, infections, and irritation. Lipid components of sebum have also been speculated to play a role in waterproofing the skin and hair. The effects of hair follicle cycling on sebaceous gland homeostasis and whether the sebaceous gland itself plays a role during hair follicle cycling remain elusive. EXT1^{StEpiΔ/StEpiΔ} mice presented a drastic increase in the overall number of hair follicles, which consequently led to an increase in the number of sebaceous glands. The ablation of HS in sebaceous glands led to hyperplasia, and the glands presented an overall altered morphology (enlarged with an irregular shape). From P55, the animals presented a significant increase in sebum secretion and excessive sebum accumulation on the skin and fur. As the animals aged, the size of sebaceous glands increased to the extent that they constrained the hair shaft, possibly blocking the surfacing of hair from the hair follicles, and this could have led to the hair loss observed beyond P55.

Taken together, our findings clearly show that an intricate control of HS levels is required for hair follicle and sebaceous gland cycling. A gradual decrease in HS is required for anagen, and thereafter an increase in HS expression is required for catagen. Moreover, HS plays a vital role in the early stages of hair follicle formation during mesenchymal-ectodermal interactions. Our findings are summarized in Fig. 16. Studies on hair follicle morphogenesis may be transposed to other appendages, such as tooth formation and mammary gland formation, which require similar mesenchymal-epithelial interactions (57). The fundamental signaling cues for hair morphogenesis are evolutionarily conserved across species and similar for other types of skin appendages, such as feathers and scales (58).

FIGURE 16.

Schematic of the hair follicle morphogenesis and cycling in EXT1^{StEpiΔ/StEpiΔ} mice. Top left panel, events that take place during placode formation during normal hair follicle morphogenesis. Bottom left panel, signaling pathways and protein expression in telogen hair follicles at P55 in littermate control mice. Right panel, signaling pathways and protein expression present in hair follicles of EXT1^{StEpiΔ/StEpiΔ} mice at P55 revealing similarities with early morphogenesis. Syn, syndecan.