Role of Sonic hedgehog signaling in epithelial and mesenchymal development of hair follicles in an organ culture of embryonic mouse skin

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1 Develop. Growth Differ. (2003) 45, Role of Sonic hedgehog signaling in epithelial and mesenchymal development of hair follicles in an organ culture of embryonic mouse skin Daisuke Nanba,* Yasuo Nakanishi and Yohki Hieda Department of Biology, Graduate School of Science, Osaka University, 1-16 Machikaneyama-cho, Toyonaka, Osaka , Japan. Studies with gene knockout mice have shown that Sonic hedgehog (Shh) is required for early development of hair follicles, but the role of this gene in the late stages of follicle development is not clear. By using an organ culture system of embryonic mouse skin, the role of Shh signaling in the early and late stages of follicle development was investigated. In the early stage of follicle development, the downward growth of the follicular epithelium was suppressed by cyclopamine, an inhibitor of Shh signaling, and accelerated by recombinant Shh. In addition, cyclopamine impaired dermal papilla formation, accompanied by the rearrangement of papilla cells, but not the elongation of the follicular epithelium at the later stage. These results suggest that Shh signaling is required for the proliferation of epithelial cells in the early development of hair follicles and for the morphogenetic movement of mesenchymal cells at the later stage of follicle development. Key words: dermal papilla, hair follicle, morphogenesis, organ culture, Sonic hedgehog. Introduction Hair follicle morphogenesis depends on sequential and reciprocal interaction between the epithelium and the underlying mesenchyme in embryonic skin. It has been used as an excellent model system for studying epithelial mesenchymal interaction (Hardy 1992; Oro & Scott 1998; Philpott & Paus 1998). The first mesenchymal message instructs the basal layer of the epidermis to form a hair placode. The epidermal message from the developing follicular epithelium then initiates the formation of the mesenchymal dermal papilla. Finally, a second mesenchymal message from the dermal papilla stimulates proliferation and differentiation within the epithelial cells of the developing hair follicles. Many studies have revealed that a number of the signaling molecules and transcription factors that function in signaling pathways of Sonic hedgehog (Shh), bone morphogenetic proteins and Wnts are involved in hair follicle development (Van Genderen et al. 1994; Bitgood & McMahon 1995; *Author to whom all correspondence should be addressed. Present address: Department of Medical Biochemistry, Ehime University School of Medicine, Shitsukawa, Shigenobu-cho, Onsen-gun, Ehime , Japan. nanba@m.ehime-u.ac.jp Received 5 December 2003; revised 21 February 2003; accepted 5 March Zhou et al. 1995; Kratochwil et al. 1996; Gat et al. 1998; St-Jacques et al. 1998; Botchkarev et al. 1999; Chiang et al. 1999; Huelsken et al. 2001). The results of such studies suggest that these intercellular signaling molecules mediate epithelial mesenchymal interaction during hair follicle morphogenesis. Shh is a developmental gene essential for hair follicle morphogenesis. Phenotypes of Shh knockout mice reveal that Shh is required for downward growth of the follicular epithelium and subsequent follicle development, but not for the formation of the initial hair germs comprising epidermal placodes and associated mesenchymal aggregates (St-Jacques et al. 1998; Chiang et al. 1999). Moreover, overexpression of Shh in embryonic skin results in abnormal growth of the follicular epithelium (Oro et al. 1997). Shh is expressed in developing follicular epithelium, and its expression overlaps that of Patched1 (Ptc1) and Gli1, a receptor and a transcription factor, respectively, of hedgehog signaling. Ptc1 and Gli1 are also expressed in the mesenchymal aggregates under the hair placodes and in the subsequent dermal papilla during follicle development (Bitgood & McMahon 1995; Iseki et al. 1996; Oro et al. 1997; Motoyama et al. 1998; St-Jacques et al. 1998; Chiang et al. 1999; Karlsson et al. 1999). These results suggest that the Shh signaling pathway is involved in the development of both epithelium and mesenchyme, and coordinates hair follicle morphogenesis (Callahan & Oro 2001).

2 232 D. Nanba et al. The early arrest of hair follicle development in Shh null mice, however, precludes analysis of the role of the Shh signaling pathway in the later stages of follicle development. Organ cultures of developing tissues are an alternative experimental system: the initial events of hair follicle formation can take place in organ cultures of developing mouse skin (Kashiwagi et al. 1997; Nanba et al. 2000). In the present study, we investigated the role of Shh signaling in developing hair follicles by using an organ culture system of embryonic mouse skin. Here, we report that Shh signaling is required for the proliferation of epithelial cells in the early stage of follicle development and for dermal papilla formation in the later stage. These results suggest that Shh signaling regulates the development of both epithelium and mesenchyme during hair follicle morphogenesis. Materials and Methods Mouse embryos and organs Embryos were obtained from DDY strain mice (Nihon SLC, Hamamatsu, Japan). The time of discovery of the vaginal plug was designated as embryonic day (E)0. The back skin was dissected from embryos at E13 and E15. Chemicals Cyclopamine was a gift from Dr W. Gaffield (Western Regional Research Center, Albany, CA, USA). Tomatidine was purchased from Sigma Chemical (St Louis, MO, USA). The amino-terminal peptide of recombinant mouse Sonic hedgehog (Shh-N) was obtained from R & D Systems (Minneapolis, MN, USA). Organ culture Back skin was taken from E13 and E15 embryos immersed in Hanks balanced salt solution and placed on an ultrathin Millipore filter (JHWP, 0.45 µm pore size) that covered the hole of a plastic plate. In the culture of E15 skin, a pair of latissimus dorsi muscles taken from E15 embryos was placed between the membrane filter and the skin fragments. Medium 199 containing 1% fetal bovine serum and L-ascorbic acid phosphate magnesium salt was applied as a hanging drop to the filter, and the skin explants were cultured in organ tissue culture dishes (Falcon 3037; Becton Dickinson, Franklin Lakes, NJ, USA) at 37 C under an atmosphere of 5% CO 2. The medium was changed every 24 h. The medium was supplemented with 50 µm cyclopamine, tomatidine or 10 µg/ml Shh-N, depending on the experiment. Histochemistry For histological examination, the cultured skin fragments were routinely fixed in Bouin s solution, dehydrated, and embedded in paraffin. Sections were then cut and processed for staining with hematoxylin and eosin. In situ hybridization Mouse Ptc1 cdna obtained by reverse transcription (RT) polymerase chain reaction (PCR) (Podlasek et al. 1999) was cloned into the pcrii vector (Invitrogen, Carlsbad, CA, USA), and antisense digoxigenin (DIG)-labeled riboprobe was synthesized by using a DIG RNA labeling kit according to the manufacturer s instructions (Roche, Mannheim, Germany). The cultured skin fragments were embedded in OCT compound and frozen in liquid nitrogen. Tissue sections of 10 µm thickness were air dried and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 20 min. The sections were incubated in PBS containing diethylpyrocarbonate (DEPC) and equilibrated in 5 standard saline citrate (SSC; 0.75 M NaCl and M Na- Citrate), and were then prehybridized at 58 C for 2 h in the hybridization mix (50% formamide, 5 SSC, 10 mg/ml yeast trna). Probe was added to the hybridization mix, denatured for 5 min at 80 C and cooled on ice. The hybridization reaction was carried out at 58 C for 20 h. After incubation, the sections were washed in 2 SSC at room temperature, in 2 SSC at 65 C, in 0.1 SSC at 65 C, and equilibrated in 0.1 M maleate buffer (ph 7.5). They were then incubated for 2 h at room temperature with alkaline phosphatase-coupled antidigoxigenin antibody (Roche) in maleate buffer containing 0.5% blocking reagent. Excess antibody was removed by several washes in maleate buffer, and the sections were then equilibrated with Buffer 1 (100 mm Tris-HCl, 100 mm NaCl and 50 mm MgCl 2; ph 9.5). Color development was performed at room temperature for 24 h in Buffer 1 containing nitro blue tetrazolium (NBT) and 5-bromo- 4-chloro-3-indolyl phosphate (BCIP; Roche). Staining was stopped by a wash in TE buffer (10 mm Tris and 1 mm ethylenediaminetetraacetic acid (EDTA); ph 8.0), and non-specific staining was removed in 95% ethanol for 1 h. After having been rinsed in water, the sections were mounted in 50% glycerol in PBS.

3 Role of Shh in hair follicle morphogenesis 233 Antibodies The following primary antibodies were used: mouse monoclonal IgG antibody to E-cadherin (Transduction Laboratories, Lexington, KY, USA); rabbit polyclonal antibody to cadherin-11 (Zymed Laboratories, San Francisco, CA, USA); and rabbit polyclonal antibody to type I collagen (a gift from Dr Yoneda and Dr Kimata, Aichi Medical University, Japan). Secondary antibodies used were rhodamine-conjugated goat antimouse IgG (Chemicon, Temecula, CA, USA) and fluorescein isothiocyanate (FITC)-conjugated goat antirabbit IgG (Cappel, Durham, NC, USA). Immunofluorescence microscopy The cultured skin fragments were embedded in OCT compound and frozen in liquid nitrogen. Tissue sections of 6 µm thickness were air dried and fixed in methanol at 20 C for 10 min, or in 4% paraformaldehyde in PBS at 4 C for 10 min. The sections were first treated with 0.5% Triton X-100 in PBS for 10 min and subsequently with 1% bovine serum albumin. Then they were incubated with primary antibodies for 1 h, washed in PBS, and incubated with secondary antibodies for 1 h. After a wash in PBS, the sections were mounted in PBS containing 50% glycerol and Fig. 1. Effect of cyclopamine on the initial event of hair follicle development in culture. Histological sections show embryonic day (E)13 skin fragments cultured for 1 day (A C), 2 days (D F) and 3 days (G I). (A,D,G) Control treatment; (B,E,H) 50 µm cyclopamine treatment; and (C,F,I) 50 µm tomatidine treatment. A control skin fragment successively showed early morphogenesis of hair follicles. The fragments of control skin and of skin treated with tomatidine formed initial mesenchymal aggregates beneath the hair placodes (arrows) and mesenchymal aggregates under these plugs (arrowheads). Note that the downgrowth of the epithelium and the formation of mesenchymal aggregates was inhibited by cyclopamine (double arrowheads). (J K) Expression of Patched1 (Ptc1) was examined by in situ hybridization to confirm the inhibition of Sonic hedgehog (Shh) signaling by cyclopamine in E13 skin fragments after 2 days in culture. Note that Ptc1 mrna is barely detectable in the hair germs of the cultured skin treated with cyclopamine (K), in contrast to its obvious presence in control skin (J) and in skin treated with tomatidine (I). Dotted lines show the contour of the epidermal tissues. Bar, 25 µm.

4 234 D. Nanba et al. 0.1% p-phenylenediamine and examined under an Olympus BX-50 epifluorescence microscope. Photographs were processed on a computer using Adobe Photoshop software and were printed with Pictrography (Fujifilm, Tokyo, Japan). Results Organ culture studies were carried out to examine the role of Shh signaling in hair follicle morphogenesis. To investigate the role in the early development of hair follicles, we used the back skin of E13 embryos, which have no hair germs. The E13 skin fragments were spread and cultured on a membrane filter. Addition of L-ascorbic acid phosphate magnesium salt to the culture medium remarkably improved the development of the embryonic skin in vitro compared with that achieved by an embryonic mouse skin culture system reported earlier by Kashiwagi et al. (1997; Fig. 1). Hair germs consisting of epidermal placodes and initial mesenchymal aggregates appeared after 1 day in culture (Fig. 1A). The follicular epithelium continuously grew into the dermal mesenchyme and definite mesenchymal aggregates appeared at its base after 2 and 3 days in culture (Fig. 1D,G). For inhibition of the Shh signaling pathway, the plant-derived teratogen cyclopamine, which inhibits the Shh response (Cooper et al. 1998; Incardona et al. 1998), was added to the culture medium. In the presence of 50 µm cyclopamine, although the initial events of follicle development occurred (Fig. 1B), the downward growth of follicular epithelium and the formation of the mesenchymal aggregates were inhibited after 2 and 3 days in culture (Fig. 1E,H). These results were consistent with the phenotypes of the skin in Shhdeficient mice (St-Jacques et al. 1998; Chiang et al. 1999). Tomatidine (50 µm), which is an alkaloid structurally similar to cyclopamine but lacks the teratogenic activity and inhibitory effects on Shh signaling (Cooper et al. 1998; Kim & Melton 1998), did not affect hair follicle development (Fig. 1C,F,I). To confirm the inhibition of the Shh signaling pathway in cultured skin by cyclopamine, we examined the expression of Ptc1 by in situ hybridization. Ptc1 is one of target genes of hedgehog signaling and Shh induces the transcription of Ptc1 in receiving cells (Callahan & Oro 2001). Therefore, expression of Ptc1 gives an indication of which cells are directly responding to hedgehog ligands. It has been reported that expression of Ptc1 mrna is much reduced in hair germs of Shh null mice (St-Jacques et al. 1998; Chiang et al. 1999). In the E13 skin fragments after 2 days of cultivation, expression of Ptc1 mrna was observed in the epithelial and mesenchymal components of the hair plugs (Fig. 1J). Cyclopamine, however, almost completely inhibited Ptc1 mrna expression (Fig. 1K). Ptc1 mrna was detected in the presence of tomatidine (Fig. 1L). These results reveal that cyclopamine inhibits Shh signaling pathway in organ cultures of embryonic mouse skin. Aberrant activation of Shh signaling caused abnormal growth of the epithelium in developing hair folli- Fig. 2. Aberrant activation of Sonic hedgehog (Shh) signaling in embryonic day (E)13 skin fragments after 3 days in culture by the amino-terminal peptide of recombinant mouse Shh (Shh-N). Histological sections show E13 skin fragments cultured for 3 days in control medium (A), medium containing 10 µg/ml Shh-N (B) and medium containing a mixture of 10 µg/ml Shh-N and 50 µm cyclopamine (C). A control skin fragment showed development of hair plugs with mesenchymal aggregates (A; arrow). Note the marked proliferation of epithelial cells and absence of mesenchymal aggregates in the cultured skin treated with Shh-N (B; arrowhead). Cyclopamine offset the effect of Shh-N on epithelial cell proliferation, but the absence of the mesenchymal aggregates was not reversed (C; arrowhead). Bar, 25 µm.

5 Role of Shh in hair follicle morphogenesis cles when E13 skin fragments were cultured in medium containing Shh-N. After 3 days of cultivation, hair follicle development proceeded normally in the untreated (control) cultured skin (Fig. 2A). In contrast, skin fragments incubated with 10 µg/ml Shh-N revealed a marked proliferation of the follicular epithelium and failure of formation of mesenchymal aggregates (Fig. 2B). Cyclopamine inhibited hyperplasia of follicular epithelium in the cultured skin treated with Shh-N (Fig. 2C). Shh signaling was also found to be required for hair follicle morphogenesis at the stage of dermal papilla 235 formation when we examined the role of Shh signaling in the stages after initial follicle development. We cultured the back skin of E15 embryos with hair plugs consisting of an epithelial bud and the underlying mesenchymal aggregate. When the E15 skin fragments were cultured on a membrane filter, downward growth of the hair plugs in the skin was inhibited, or both epithelial and mesenchymal cells of the distal region of the elongating follicles died (data not shown). The skin fragments tightly adhered and spread out on the membrane filter during culture, which caused a loss of space for the developing Fig. 3. Effect of cyclopamine on dermal papilla formation in culture. Histological sections show embryonic day (E)15 skin fragments cultured for 3 days in control medium (A), medium containing 50 µm cyclopamine (B) and medium supplemented with 50 µm tomatidine (C). The dermal papilla (arrows) is evident in the skin fragments of control skin and skin treated with tomatidine. Note that the mesenchymal aggregate (arrowhead) is not surrounded by follicular epithelium in the presence of cyclopamine. Bar, 25 µm. Fig. 4. Immunofluorescence microscopy showing expression of cadherin-11 in embryonic day (E)15 skin fragments after 3 days in culture. The sections were double-stained for cadherin-11 (green) and E-cadherin (red). Staining for E-cadherin shows the epithelium of developing hair follicles. Cadherin-11 was detected in the dermal papilla (arrow) in vitro (A). In the presence of cyclopamine, cells expressing cadherin-11 were detected under the follicular epithelium, but the number of intensely stained dot-like structures was reduced (B; arrowhead). Tomatidine did not affect the expression pattern of cadherin-11 (C; arrow). Bar, 25 µm.

6 236 D. Nanba et al. follicles. To overcome this problem, we laid a pair of latissimus dorsi muscles from E15 embryos on a membrane filter and the E15 skin fragments were spread out and cultured on it. This method avoided the adhering and spreading of the skin fragments on the membrane filter during culture, and enabled the downward growth of the follicular epithelium and subsequent dermal papilla formation (Fig. 3). After 3 days in culture, the follicular epithelium elongated and showed a horseshoe-like shape at its base, and the dermal papilla was surrounded by epithelial tissue (Fig. 3A). In contrast, dermal papilla formation in culture was inhibited by treatment with 50 µm cyclopamine. Although in the presence of cyclopamine elongation of the follicular epithelium and maintenance of the mesenchymal aggregates was observed, the aggregates were not surrounded by follicular epithelium at its base, and the epithelium appeared to have a rod-like shape (Fig. 3B). E15 skin fragments cultured with 50 µm tomatidine showed normal hair follicle development in culture (Fig. 3C). We next examined the effect of aberrant activation of Shh signaling on E15 skin cultures by using Shh-N, but found that no marked effects on hair follicle morphogenesis or skin development (data not shown). Our previous study indicated that dermal papilla formation from the mesenchymal aggregate was accompanied by the establishment of intercellular junctions containing cadherin-11 and the loss of extracellular matrix, including collagen I (Nanba et al. 2003). Therefore, expression of cadherin-11 and collagen I was examined by immunofluorescence microscopy. Cadherin-11 was localized to the dermal papilla (the mesenchymal aggregate) at the base of the follicular epithelium in the cultured skin treated with normal (Fig. 4A), cyclopamine-containing (Fig. 4B) or tomatidine-containing (Fig. 4C) medium, but intense dot-like signals of cadherin-11 between the cells, which were an indication of intercellular junctions containing cadherin-11 (Nanba et al. 2003), were reduced in number in the mesenchymal aggregates of the cultured skin treated with cyclopamine (Fig. 4B). Collagen I was barely able to be detected in the region of the dermal papilla of elongating follicles in the E15 skin fragments after 3 days of cultivation with normal (Fig. 5A) or tomatidinecontaining medium (Fig. 5C). In the presence of cyclopamine, however, collagen I was detected in the mesenchymal aggregate at the base of rod-shaped follicular epithelium (Fig. 5B). Discussion Shh null mice revealed that hair follicle morphogenesis was blocked at the hair placode stage (St-Jacques et al. 1998; Chiang et al. 1999). Overexpression of Shh in the skin resulted in abnormal growth of the developing follicles (Oro et al. 1997). The organ culture study presented here revealed that Shh signaling regulated initial epidermal downward growth during hair follicle development, but not elongation of the follicular epithelium. In addition, our study showed that Shh signaling was involved in dermal papilla formation. These results indicate that the Shh signaling pathway stimulates the initial downward growth of follicular epithelium and morphogenetic movements of mesenchymal cells. The early epithelial tissues in developing hair follicles have a histology very similar to that of basal cell Fig. 5. Immunofluorescence microscopy showing expression of collagen I in embryonic day (E)15 skin fragments after 3 days in culture. The sections were double-stained for collagen I (green) and E-cadherin (red). Staining for E-cadherin shows the epithelium of developing hair follicles. Staining for collagen I is barely detectable in the dermal papilla (arrows) of the elongating follicles of the control skin (A) and in skin treated with tomatidine (C). In the presence of cyclopamine, however, collagen I is seen at the base of elongating follicles where the mesenchymal aggregate is observed (B; arrowhead). Bar, 25 µm.

7 Role of Shh in hair follicle morphogenesis 237 carcinomas, which are the most frequent cancer of the skin and have a low metastatic potential but invade deeply into the surrounding tissues. It has been demonstrated that inappropriate activation of the Shh signaling pathway leads to basal cell carcinomas (Hahn et al. 1996; Johnson et al. 1996; Unden et al. 1996; Dahmane et al. 1997; Fan et al. 1997; Oro et al. 1997; Xie et al. 1998). Furthermore, Shh opposes p21 Cip1 -induced cell cycle arrest that occurs before keratinocyte terminal differentiation, and causes continued proliferation of growth-arrested suprabasal cells in the epidermis (Fan & Khavari 1999). These findings suggest that activation of the Shh signaling pathway leads to epidermal growth into the underlying dermal tissues by continued proliferation of the suprabasal cells detached from the basal layer of the epidermis. An earlier study of ours showed that desmosomal and hemidesmosomal adhesion systems were downregulated in the cells of hair placodes and hair plugs (Nanba et al. 2000). Other studies have shown the same phenomenon to occur in basal cell carcinomas (Zelickson 1962; McNutt 1976; Kumakiri & Hashimoto 1978; Luzi et al. 1987; Savoia et al. 1993; Fairly et al. 1995; Kore-eda et al. 1998). We proposed earlier that the downward growth of the epidermal tissues into the dermal tissues is driven by segregation or exclusion of the detaching and proliferating cell population, with weaker cell adhesiveness from the neighboring, more tightly adhering epidermal cells (Nanba et al. 2000, 2001). Downregulation of desmosomes and hemidesmosomes was observed in the hair placode treated with cyclopamine (Nanba, Nakanishi and Hieda, unpubl. data, 2000), which suggests that Shh signaling is involved in the proliferation of epithelial cells but not in the downregulation of desmosomal and hemidesmosomal adhesion systems in the early development of hair follicles. The elongation of hair plugs in spite of the presence of cyclopamine, as shown in this study, implies that the proliferation of follicular epithelial cells during the follicle elongation stage is not stimulated by the Shh signaling pathway. This idea is supported by the data that show that activation of Shh signaling did not affect hair follicle morphogenesis in E15 skin cultured with Shh-N. At the late stages of follicle development, Shh and Ptc1 were expressed in the hair matrix region of elongating follicular epithelium (Karlsson et al. 1999), and ectopic expression of Shh by adenovirus in the adult skin resulted in the promotion of hair growth (Sato et al. 1999). Therefore, the data suggest that Shh signaling is not involved in epithelial morphogenesis of hair follicles at late stages, but stimulates proliferation of hair matrix cells and hair growth in mature hair follicles. Expression patterns of Shh signaling components suggest that the mesenchymal cells of developing hair follicles respond to Shh secreted by the follicular epithelium (Bitgood & McMahon 1995; Iseki et al. 1996; Oro et al. 1997; Motoyama et al. 1998; St- Jacques et al. 1998; Chiang et al. 1999; Karlsson et al. 1999). The present study revealed that Shh signaling was also required for the mesenchymal development of hair follicles. Initial aggregation of mesenchymal cells was seen beneath the placodes in the cultured skin treated with cyclopamine as well as beneath the placodes in Shh-deficient skin (St- Jacques et al. 1998; Chiang et al. 1999). However, a definite structure of the mesenchymal aggregate under the hair plug was not observed in the presence of cyclopamine or Shh-N. At the stage of dermal papilla formation, cyclopamine inhibited follicular epithelium surrounding the mesenchymal aggregates. Our previous study demonstrated that the mesenchymal aggregate makes a tightly packed population of cells with little extracellular space during dermal papilla formation (Nanba et al. 2003). Immunostaining for cadherin-11 and collagen I revealed that the compaction of mesenchymal aggregates was inhibited by cyclopamine. Migration and adhesion of mesenchymal cells and matrix remodeling are involved in the formation of the mesenchymal aggregate and the dermal papilla (Nanba et al. 2003). 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