Establishment of cadherin-based intercellular junctions in the dermal papilla of the developing hair follicle.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 270A:97–102 (2003) Establishment of Cadherin-Based Intercellular Junctions in the Dermal Papilla of the Developing Hair Follicle DAISUKE NANBA, YASUO NAKANISHI, AND YOHKI HIEDA* Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan ABSTRACT During hair follicle development, mesenchymal cells aggregate to form the dermal papilla with hair-inducing activity. However, the cellular mechanisms underlying the aggregative behavior of dermal papilla cells are less known. The present study demonstrates that cadherin-based intercellular junctions interconnect dermal papilla cells in developing hair follicles of mice. It is shown that as mesenchymal cells aggregate to be surrounded by epithelium in developing hair follicles, cadherin-11 comes to exhibit the dotted patterns of distribution. The appearance of the dot-like distribution of the molecule is concomitant with the formation of intercellular junctions in the mesenchymal aggregate, which make a tightly packed population of cells with little extracellular space. At later stages of the development, although extracellular space reappears in the dermal papilla, the cells remain interconnected by well-developed intercellular junctions, where cadherin-11 as well as ␤-catenin is localized. Taking into consideration the normal hair development in cadherin-11 mutant mice, it might be that multiple cadherins are responsible for the establishment of intercellular junctions in the dermal papilla and serve to maintain the aggregative behavior of the cells. Anat Rec Part A 270A:97–102, 2003. © 2003 Wiley-Liss, Inc. Key words: cadherin-11; intercellular junction; cell rearrangement; dermal papilla; hair follicle At the onset of hair follicle development a subpopulation of dermal mesenchymal cells starts to gather just below the epidermal placode. The mesenchymal aggregate is subsequently surrounded by the elongating follicular epithelium to become the dermal papilla with hair-inducing activity (Oliver, 1967, 1970; Ibrahim and Wright, 1977; Jahoda et al., 1984; Horne et al., 1986; Hardy, 1992; Reynolds and Jahoda, 1992; Kamimura et al., 1997). It has been suggested that aggregative behavior is the essential feature of dermal papilla cells associated with the hair-inducing activity. For instance, cultured dermal papilla cells of early passage numbers retain their aggregative and hair-inducing activities, whereas those of later passage numbers lose these activities (Jahoda and Oliver, 1984; Horne et al., 1986; Kishimoto et al., 1999). When dermal papilla cells are cultivated in medium conditioned by keratinocytes, both of the activities can be maintained even after dozens of passages (Inamatsu et al., 1998). © 2003 WILEY-LISS, INC. It was previously reported that deﬁnite junctions with electron-dense materials were present between dermal papilla cells but not between other dermal ﬁbroblasts Grant sponsor: Japan Science Society. Daisuke Nanba’s present address is Department of Medical Biochemistry, School of Medicine, Ehime University, Shitsukawa, Shigenobu-cho, Ehime 791-0025, Japan. *Correspondence to: Yohki Hieda, Ph.D., Department of Biology, Graduate School of Science, Osaka University, 1-16 Machikaneyama, Toyonaka, Osaka 560-0043, Japan. Fax: ⫹81-6-68505817. E-mail: email@example.com Received 21 June 2002; Accepted 21 September 2002 DOI 10.1002/ar.a.10012 98 NANBA ET AL. (Hashimoto, 1970), suggesting that formation of intercellular junctions may contribute to the aggregation of dermal mesenchymal cells, resulting in the dermal papilla formation. A variety of cell adhesion-related molecules have so far been shown to be expressed in mesenchymal aggregates of developing hair follicles, including the neural cell adhesion molecule (NCAM), the ␣1, ␣5, ␣9, and ␤1 subunits of integrins (Kaplan and Holbrook, 1994; Wang et al., 1995; Hardy and Vielkind, 1996; Almond-Roesler et al., 1997; Müller-Röver et al., 1998), and the chondroitin sulfate proteoglycan of versican (Kishimoto et al., 1999; Harris and Jahoda, 2001). No cell adhesion molecule, however, has been associated with the intercellular junctions, leaving the molecular nature of the junctions to be clariﬁed. In addition, detailed ultrastructural information has not been available on how intercellular junctions are established between mesenchymal cells during the dermal papilla formation. Ultrastructurally recognizable intercellular junctions such as adherens junctions and desmosomes in epithelial cells and synapses in neurons are mediated primarily by the cadherin family of cell adhesion molecules, which play crucial roles in tissue morphogenesis and in signaling events (Green and Gaudry, 2000; Takeichi et al., 2000; Yagi and Takeichi, 2000; Nagafuchi, 2001). Although little information is available on the expression proﬁle of cadherins in mesenchymal cells of developing hair follicles, cells of the dermal papilla and dermal sheath have been shown to express mRNA for cadherin-11 (Simonneau et al., 1995). In the present study, we show that intercellular junctions containing cadherin-11 are established in the dermal papilla of the developing hair follicle and that formation of the dermal papilla involves dynamic rearrangements of cells. Cadherin-11 is the only member of the family whose expression has been reported in the dermal papilla. Since cadherin-11 (–/–) mice, however, exhibit normal hair development (Horikawa et al., 1999; Manabe et al., 2000), multiple cadherins are likely involved in the establishment of intercellular junctions during the dermal papilla formation. MATERIALS AND METHODS Mouse Embryos and Organs Embryos were obtained from ddY strain mice (Nihon SLC, Hamamatsu, Japan). The discovery of the vaginal plug was designated as embryonic day 0 (E0). The back skin was dissected from embryos at E15 and E17, and from newborn mice at postnatal day 1 (P1). Antibodies The following primary antibodies were used: mouse monoclonal antibodies speciﬁc for E-cadherin and ␤-catenin (Transduction Laboratories, Lexington, KY), rabbit polyclonal antibody against cadherin-11 (Zymed Laboratories, San Francisco, CA), and rabbit polyclonal antibody to type I collagen (gift from Drs. Yoneda and Kimata, Aichi Medical University, Japan). Secondary antibodies used were rhodamine-conjugated goat anti-mouse immunoglobulin G (IgG) (Chemicon, Temewla, CA), ﬂuorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Cappel, Durham, NC), and 10 nm of colloidal gold-conjugated goat anti-mouse IgG and goat anti-rabbit IgG (British Biocell International, Cardiff, UK). Immunoﬂuorescence Microscopy Freshly prepared skin fragments were embedded in OCT compound and frozen in liquid nitrogen. Tissue sections of 6-m thickness were ﬁxed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C for 10 min or in methanol at –20°C for 10 min. The specimens were treated with 0.5% Triton X-100 in PBS for 10 min and subsequently with 1% bovine serum albumin (BSA). Then they were incubated with primary antibodies for 1 hr, washed in PBS, and incubated with secondary antibodies for 1 hr. After a wash in PBS, the sections were mounted with PBS containing 50% glycerol and 0.1% p-phenylenediamine and examined under an Olympus AX-70 or BX-50 epiﬂuorescence microscope. Electron Microscopy For conventional electron microscopy, freshly prepared skin fragments were ﬁxed in 0.1 M cacodylate buffer (pH 7.3) containing 2.5% glutaraldehyde and 2% paraformaldehyde and were then immersed in 1% osmium tetraoxide in the same buffer. Dehydration in a gradient series of ethanol and propylene oxide was followed by embedding in PolyBed 812 (Polysciences, Warrington, PA). Ultrathin sections were cut on a Reichert ultramicrotome (Leica, Wien, Austria), stained with uranyl acetate for 2 min and with lead citrate for 3 min, and observed with a JEOL 1200EX electron microscope. For immunoelectron microscopy, skin fragments were ﬁxed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) at 4°C overnight. Dehydration in a gradient series of ethanol on ice was followed by embedding in LR white resin (London Resin, Hampshire, UK). Ultrathin sections were cut on an ultramicrotome and mounted on 70-mesh nickel grids supported by Formvar ﬁlms. Incubation in all cases was conducted by ﬂoating grids section side down on drops of reaction or washing media. The sections were treated with 1% BSA in PBS for 30 min. They were then incubated with primary antibodies at 4°C overnight, washed in PBS containing 0.1% Triton X-100, and incubated with 10 nm of colloidal gold-conjugated secondary antibodies for 2 hr. Washes in PBS containing 0.1% Triton X-100 were followed by staining with uranyl acetate for 3 min and with lead citrate for 30 sec. RESULTS We examined the distribution patterns of cadherin-11 and the ultrastructural process of the dermal papilla formation in developing mouse pelage hair follicles at E15, E17, and P1. Immunoﬂuorescence microscopy showed that expression of the cadherin-11 molecule was restricted to the mesenchyme adjacent to epithelium (Fig. 1), being consistent with the expression pattern of the transcripts (Simonneau et al., 1995). Close inspection revealed changes in the distribution pattern of cadherin-11 in mesenchymal cells during formation of the dermal papilla. In the initial mesenchymal aggregate of the E15 developing follicle, cadherin-11 was distributed uniformly along the cell surfaces (Fig. 1A, D). Electron microscopy of the mesenchymal aggregate revealed that junction-like structures had not formed between cells, though the cells had extended numerous protrusions to make contact with their neighbors (Fig. 2A). Collagen ﬁbers were present in the extracellular space, which was conﬁrmed by positive staining for type I collagen in the mesenchymal aggregate, CADHERIN-BASED JUNCTION IN DERMAL PAPILLA 99 Fig. 1. Distribution of cadherin-11 in developing hair follicles. Cadherin-11 expression is restricted to mesenchyme at all the stages examined. At E15, cadherin-11 shows relatively uniform distribution along cell surfaces in the mesenchymal aggregate (A, arrow; D, for closer inspection). The dotted distribution of cadherin-11 is noted in the dermal papilla at E17 (B, arrow; E, for closer inspection) and becomes more evident at P1 (C, arrow; F, for closer inspection). Mesenchymal cells surrounding the follicular epithelium at E17 and P1 are also stained in a dot-like pattern (arrowheads). No signal was detected when the primary antibodies were omitted (data not shown). Arrows indicate the mesenchymal aggregate and dermal papilla. Scale bars ⫽ 25 m (A–C); 5 m (D–F). though the staining intensity was weaker than that in other dermal mesenchymes (Fig. 3A). In the developing hair follicles at E17, cadherin-11 tended to exhibit the dot-like patterns of distribution in the presumptive dermal papilla, which had just become surrounded by the epithelium, and in the dermal sheath (Fig. 1B, E). Noticeably, concomitant with the appearance of the dot-like distribution of the molecule, small but ultrastructurally recognizable intercellular junctions were found between the cells (Fig. 2B). The dermal papilla cells were tightly packed or stuck along their entire surfaces, with little extracellular space. Staining for type I collagen was hardly detected (Fig. 3B). The dot-like distribution of cadherin-11 became more evident in the dermal papilla of P1 hair follicles (Fig. 1C, F). Ultrastructurally, although the extracellular space had widened again, dermal papilla cells kept their adherence to each other at the tips of their extensions, where deﬁnite intercellular junctions with electron-dense materials had become established (Fig. 2C). The extracellular space of the dermal papilla was abundant in amorphous materials rather than in collagen ﬁbrils. Only limited staining was observed for type I collagen (Fig. 3C). Immunoelectron microscopy revealed cadherin-11 as well as ␤-catenin to be indeed localized at the intercellular junctions in the dermal papilla (Fig. 4). The present study aimed to clarify the morphological and molecular bases underlying the aggregation of mesenchymal cells during dermal papilla formation. It was reported earlier that junction-like structures were present between dermal papilla cells but not between other dermal ﬁbroblasts (Hashimoto, 1970) and that cadherin-11 mRNA was expressed in the dermal papilla (Simonneau et al., 1995). We showed here that cadherin-11 expressed in mesenchymal aggregates became distributed in dot-like patterns during dermal papilla formation, which was coincident with the appearance of junctional structures between the cells. As revealed by immunoelectron microscopy, cadherin-11 as well as ␤-catenin was localized at the intercellular junctions established in the dermal papilla. These ﬁndings demonstrate that dermal papilla cells in developing hair follicles become interconnected by cadherin-based intercellular junctions. Although mesenchymal cells of the initial aggregate, before it was surrounded by epithelium, made contact with their neighbors by extending protrusions, junctionlike structures were not detected at cell contact sites, and cadherin-11 was distributed uniformly along cell surfaces. Ultrastructurally recognizable intercellular junctions ﬁrst appeared at the subsequent stage when the dermal papilla DISCUSSION 100 NANBA ET AL. Fig. 2. Electron micrographs of aggregating mesenchymal cells in the developing hair follicles. A: Cells of the mesenchymal aggregate at E15 have extended numerous protrusions to make contact with their neighbors; however, almost no junction-like structure has formed between cells. In the extracellular space, collagen ﬁbrils are present (arrowheads). B: The dermal papilla at E17 makes a tightly packed population of cells. Note that junctionlike structures have formed between the cells (arrows). C: The extracellular space has been restored in the dermal papilla at P1, where amorphous materials rather than collagen ﬁbrils are abundant. Note, however, that the cells remain connected by the developed intercellular junctions (arrows). Insets, higher magniﬁcation of boxed regions. Scale bars ⫽ 1 m; 200 nm in insets. made a tightly packed population of cells with little extracellular space. Together with the concomitant redistribution of cadherin-11 to dot-like patterns at cell boundaries, cadherin-mediated adhesion appears to become activated or promoted in the cells. It is not clear at present whether the formation of cadherin-based intercellular junctions is responsible for the “compaction” of the cells. Alternatively, it would also be possible that degradation of the extracellular matrix, as revealed by electron microscopy and immunostaining for interstitial collagens, results in close apposition of cell surfaces, allowing the cells to develop the intercellular junctions. The compaction of dermal papilla cells was followed by restoration of extracellular space in the dermal papilla, where amorphous materials rather than collagen ﬁbrils were abundant. Cells of the dermal papilla remained connected, however, by well-developed intercellular junctions containing cadherin-11 and ␤-catenin, suggesting that establishment of cadherin-based intercellular junctions serves to keep the cells from separating from one another. It has been well documented that the aggregative behavior of dermal papilla cells is closely coupled to their hair-inducing activity (Jahoda and Oliver, 1984; Horne et al., 1986; Inamatsu et al., 1998; Kishimoto et al., 1999). The aggregative and hair-inducing activities of cultured dermal papilla cells can be maintained by conditioned medium from keratinocyte cultures and by Wnt ligands (Inamatsu et al., 1998; Kishimoto et al., 1999, 2000) and that the Wnt signaling components such as frizzled and ␤-catenin are expressed in dermal papilla cells (Kishimoto et al., 2000). In addition, Wnt signaling was shown to regulate cadherin-mediated cell adhesion (Bradley et al., CADHERIN-BASED JUNCTION IN DERMAL PAPILLA Fig. 3. Distribution of type I collagen in developing hair follicles. Tissue sections were double-stained for type I collagen (green) and E-cadherin (red). The mesenchymal aggregate at E15 is positive for type I collagen (A, arrow). However, note that the staining intensity in the Fig. 4. Localization of cadherin-11 and ␤-catenin at intercellular junctions in the dermal papilla. Immunoelectron microscopy was performed by using 10 nm of colloidal gold-conjugated secondary antibodies to visualize the localization of cadherin-11 (A) and ␤-catenin (B) in the developing hair follicle at P1. Speciﬁc labeling is observed for both molecules at intercellular junctions in the dermal papilla. Scale bar ⫽ 100 nm. 1993; Hinck et al., 1994). Taken these and our present ﬁndings together, it might be suggested that regulation of cadherin-mediated adhesion of dermal papilla cells by Wnt signaling is involved in the acquisition of their hairinductive activity. Cadherin-11 is the only member of the family whose expression has been reported in the dermal papilla (Simonneau et al., 1995) and confers adhesive activity on cells (Okazaki et al., 1994; Kimura et al., 1995; Shibata et al., 1996; Horikawa et al., 1999; Pishvaian et al., 1999; Shimoyama et al., 2000). Mice deﬁcient in cadherin-11 were recently shown, however, to exhibit normal hair development, surviving to the adult stage (Horikawa et al., 1999; Manabe et al., 2000). This ﬁnding may suggest that multiple cadherins are expressed in the dermal papilla to be involved in hair follicle development. Although cadherin-11 was reported to cooperate with N-cadherin in somitogenesis (Horikawa et al., 1999) and to exhibit not only homophilic binding but also heterophilic binding with cadherin-8 (Shimoyama et al., 2000), neither N-cadherin nor cadherin-8 is detectable in the dermal papilla (Nanba 101 aggregate is weaker than that in other dermal mesenchymes. At E17 (B) and P1 (C), the collagen staining is hardly detectable or very limited in the dermal papilla (arrows). Scale bar ⫽ 10 m. and Hieda, unpublished data). Identiﬁcation of the repertoire of cadherins expressed in the dermal papilla should provide a clue to elucidating the mechanism by which the aggregative and hair-inducing activities are coupled. The establishment of cadherin-based intercellular junctions in dermal papilla cells with aggregative behavior suggests that their intercellular adhesivity is indeed increased. On the other hand, as we previously showed, epithelial cells of the hair matrix-containing region, which overlie to surround the dermal papilla, lose intercellular junctions and show the decreased expression of desmosomal molecules, including the cadherin members desmogleins and desmocollins (Nanba et al., 2000). In contrast to dermal papilla cells, these epithelial cells of the developing hair follicle likely have decreased intercellular adhesivity. As demonstrated in our recent study, the epithelial adhesion system is also downregulated in the mammary bud, where the mammary epithelial cells have very decreased intercellular adhesivity in comparison with the overlying epidermal cells, which have a well-developed adhesion system (Nanba et al., 2001). These observations together support our proposal that the morphology of the distal portion of the hair follicle, where the epithelium surrounds the dermal papilla, results from engulfment of dermal papilla cells with increased intercellular adhesivity by the overlying epithelial cells with decreased adhesivity (Nanba et al., 2000). ACKNOWLEDGMENTS This work was supported in part by a Sasakawa Scientiﬁc Research Grant from the Japan Science Society to D.N. LITERATURE CITED Almond-Roesler B, Schön M, Schön MP, Blume-Peytavi U, Sommer C, Löster K, Orfanos CE. 1997. Cultured dermal papilla cells of the rat vibrissa follicle. 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