Mucosal Addressin Cell Adhesion Molecule 1 Plays an Unexpected Role in the Development of Mouse Guard Hair
2002; Elsevier BV; Volume: 119; Issue: 3 Linguagem: Inglês
10.1046/j.1523-1747.2002.01851.x
ISSN1523-1747
AutoresEri Nishioka, Toshiyuki Tanaka, Hisahiro Yoshida, Kazuyoshi Matsumura, Satomi Nishikawa, Asuka Naito, Jun‐ichiro Inoue, Yoko Funasaka, Masamitsu Ichihashi, Masayuki Miyasaka, Shin‐Ichi Nishikawa,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoThe first wave of coat hair development is initiated around embryonic day 14 in the mouse. Whereas ectodysplasin and ectodermal dysplasia receptor, tumor necrosis factor and tumor necrosis factor receptor family molecules, respectively, were identified to be signals triggering this process, not much was known regarding their downstream molecular targets. In this report, we show that mucosal addressin cell adhesion molecule 1 and intercellular adhesion molecule 1 are induced in the keratinocytes of the hair placode as a direct consequence of ectodermal dysplasia receptor signal, and tumor-necrosis-factor-receptor-associated factor 6 is involved in this mucosal addressin cell adhesion molecule 1 expression. Experiments using an in vitro culture of skin fragments demonstrated that ectodermal-dysplasia-receptor-induced mucosal addressin cell adhesion molecule 1 expression occurs at the initial phase of follicle development before involvement of Sonic hedgehog signal. Follicle development in this culture was also suppressed to some extent, though not completely, by addition of soluble mucosal addressin cell adhesion molecule 1/IgG-Fc chimeric protein, whereas monoclonal antibody that can inhibit mucosal addressin cell adhesion molecule 1 interaction with integrin α4β7 had no effect on this process. These results demonstrated for the first time that the structural proteins, mucosal addressin cell adhesion molecule 1 and intercellular adhesion molecule 1, are induced by ectodermal dysplasia receptor signal and suggested the potential involvement of mucosal addressin cell adhesion molecule 1 in the morphogenesis of follicular keratinocytes. The first wave of coat hair development is initiated around embryonic day 14 in the mouse. Whereas ectodysplasin and ectodermal dysplasia receptor, tumor necrosis factor and tumor necrosis factor receptor family molecules, respectively, were identified to be signals triggering this process, not much was known regarding their downstream molecular targets. In this report, we show that mucosal addressin cell adhesion molecule 1 and intercellular adhesion molecule 1 are induced in the keratinocytes of the hair placode as a direct consequence of ectodermal dysplasia receptor signal, and tumor-necrosis-factor-receptor-associated factor 6 is involved in this mucosal addressin cell adhesion molecule 1 expression. Experiments using an in vitro culture of skin fragments demonstrated that ectodermal-dysplasia-receptor-induced mucosal addressin cell adhesion molecule 1 expression occurs at the initial phase of follicle development before involvement of Sonic hedgehog signal. Follicle development in this culture was also suppressed to some extent, though not completely, by addition of soluble mucosal addressin cell adhesion molecule 1/IgG-Fc chimeric protein, whereas monoclonal antibody that can inhibit mucosal addressin cell adhesion molecule 1 interaction with integrin α4β7 had no effect on this process. These results demonstrated for the first time that the structural proteins, mucosal addressin cell adhesion molecule 1 and intercellular adhesion molecule 1, are induced by ectodermal dysplasia receptor signal and suggested the potential involvement of mucosal addressin cell adhesion molecule 1 in the morphogenesis of follicular keratinocytes. embryonic day ectodysplasin anhidrotic ectodermal dysplasia ectodermal dysplasia receptor hypohidrotic ectodermal dysplasia inhibitor of NF-κB IκB kinase mucosal addressin cell adhesion molecule nuclear factor κB TNF receptor TNFR-associated factor Sonic hedgehog X-linked EDA-A2 receptor It is well established that mammalian hair follicle and avian feather formation are regulated by a set of signals such as bone morphogenetic proteins, Sonic hedgehog (Shh), fibroblast growth factors (FGFs), Wnt, and Notch that appear successively during organogenesis of hair follicles as well as that of other organs (for reviews seeOro and Scott, 1998Oro A.E. Scott M.P. Splitting hairs: dissecting roles of signaling systems in epidermal development.Cell. 1998; 95: 575-578Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar;Barsh, 1999Barsh G. Of ancient tales and hairless tails.Nat Genet. 1999; 22: 315-316Crossref PubMed Scopus (31) Google Scholar). From histologic observations, it was proposed that hair follicle formation is initiated when underlying mesenchymal cells trigger commitment of the overlying epidermis to form the placodes (Hardy, 1992Hardy M.H. The secret life of the hair follicle.Trends Genet. 1992; 8: 55-61Abstract Full Text PDF PubMed Scopus (762) Google ScholarOro and Scott, 1998Oro A.E. Scott M.P. Splitting hairs: dissecting roles of signaling systems in epidermal development.Cell. 1998; 95: 575-578Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Concerning feather development, it has been shown that FGFs and bone morphogenetic proteins are involved in the initial process, as positive and negative regulators, respectively (Song et al., 1996Song H. Wang Y. Goetinck P.F. Fibroblast growth factor 2 can replace ectodermal signaling for feather development.Proc Natl Acad Sci USA. 1996; 93: 10246-10249Crossref PubMed Scopus (73) Google Scholar;Jung et al., 1998Jung H.S. Francis-West P.H. Widelitz R.B. et al.Local inhibitory action of BMPs and their relationships with activators in feather formation: implications for periodic patterning.Dev Biol. 1998; 196: 11-23Crossref PubMed Scopus (285) Google Scholar), followed by a subsequent fate specification process involving Notch/Delta signals (Artavanis-Tsakonas and Simpson, 1991Artavanis-Tsakonas S. Simpson P. Choosing a cell fate: a view from the Notch locus.Trends Genet. 1991; 7: 403-408Abstract Full Text PDF PubMed Scopus (173) Google Scholar;Crowe et al., 1998Crowe R. Henrique D. Ish-Horowicz D. Niswander L. A new role for Notch and Delta in cell fate decisions: patterning the feather array.Development. 1998; 125: 767-775PubMed Google Scholar). Likewise, Notch has been implicated in hair placode development (Powell et al., 1998Powell B.C. Passmore E.A. Nesci A. Dunn S.M. The Notch signalling pathway in hair growth.Mech Dev. 1998; 78: 189-192Crossref PubMed Scopus (82) Google Scholar). Recent studies showed that conditional deletion of the β-catenin gene from the epidermis resulted in defects in the formation of the hair placode (Huelsken et al., 2001Huelsken J. Vogel R. Erdmann B. Cotsarelis G. Birchmeier W. beta-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin.Cell. 2001; 105: 533-545Abstract Full Text Full Text PDF PubMed Scopus (1038) Google Scholar). Moreover, mice lacking the LEF-1 gene displayed reduction in coat hair number and complete lack of whiskers (van Genderen et al., 1994van Genderen C. Okamura R.M. Farinas I. Quo R.G. Parslow T.G. Bruhn L. Grosschedl R. Development of several organs that require inductive epithelial–mesenchymal interactions is impaired in LEF-1-deficient mice.Genes Dev. 1994; 8: 2691-2703Crossref PubMed Scopus (788) Google Scholar). As β-catenin and LEF-1 have been implicated downstream of Wnt signaling, these results indicate a role for Wnt signals in the initial phase of hair follicle development. Shh, another important signal that is involved repeatedly in many organogenesis processes (Ekker et al., 1995Ekker S.C. Ungar A.R. Greenstein P. von Kessler D.P. Porter J.A. Moon R.T. Beachy P.A. Patterning activities of vertebrate hedgehog proteins in the developing eye and brain.Curr Biol. 1995; 5: 944-955Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar;Hardy et al., 1995Hardy A. Richardson M.K. Francis-West P.H. Rodriguez C. Izpisua-Belmonte J.C. Duprez D. Wolpert L. Gene expression, polarising activity and skeletal patterning in reaggregated hind limb mesenchyme.Development. 1995; 121: 4329-4337PubMed Google Scholar;Tanabe et al., 1995Tanabe Y. Roelink H. Jessell T.M. Induction of motor neurons by Sonic hedgehog is independent of floor plate differentiation.Curr Biol. 1995; 5: 651-658Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), is implicated in the subsequent processes of hair follicle development after placode formation, particularly in elongation (St-Jacques et al., 1998St-Jacques B. Dassule H.R. Karavanova I. et al.Sonic hedgehog signaling is essential for hair development.Curr Biol. 1998; 8: 1058-1068Abstract Full Text Full Text PDF PubMed Google Scholar;Chiang et al., 1999Chiang C. Swan R.Z. Grachtchouk M. et al.Essential role for Sonic hedgehog during hair follicle morphogenesis.Dev Biol. 1999; 205: 1-9Crossref PubMed Scopus (403) Google Scholar). Despite this increasing list of molecules regulating hair follicle development, the nature of the initiating signal has remained elusive. Hypohidrotic (anhidrotic) ectodermal dysplasia [HED (EDA)] stands for a syndrome characterized by hypoplastic development in ectodermal organs including teeth, hair, and sweat glands and is composed of X-linked, autosomal recessive and variant forms. After the identification of the gene responsible for X-linked HED (Kere et al., 1996Kere J. Srivastava A.K. Montonen O. et al.X-linked anhidrotic (hypohidrotic) ectodermal dysplasia is caused by mutation in a novel transmembrane protein.Nat Genet. 1996; 13: 409-416Crossref PubMed Scopus (562) Google Scholar), called EDA, the corresponding mouse gene was cloned and found to be allelic to the tabby (Ta) locus (Srivastava et al., 1997Srivastava A.K. Pispa J. Hartung A.J. et al.The Tabby phenotype is caused by mutation in a mouse homologue of the EDA gene that reveals novel mouse and human exons and encodes a protein (ectodysplasin-A) with collagenous domains.Proc Natl Acad Sci USA. 1997; 94: 13069-13074Crossref PubMed Scopus (243) Google Scholar). From the deduced amino acid sequence, the Ta protein and EDA product, ectodysplasin (Eda), were identified as tumor necrosis factor (TNF) family members (Ezer et al., 1999Ezer S. Bayes M. Elomaa O. Schlessinger D. Kere J. Ectodysplasin is a collagenous trimeric type II membrane protein with a tumor necrosis factor-like domain and co-localizes with cytoskeletal structures at lateral and apical surfaces of cells.Hum Mol Genet. 1999; 8: 2079-2086Crossref PubMed Scopus (110) Google Scholar;Mikkola et al., 1999Mikkola M.L. Pispa J. Pekkanen M. Paulin L. Nieminen P. Kere J. Thesleff I. Ectodysplasin, a protein required for epithelial morphogenesis, is a novel TNF homologue and promotes cell-matrix adhesion.Mech Dev. 1999; 88: 133-146Crossref PubMed Scopus (104) Google Scholar). In complete agreement with this, subsequent cloning of the gene responsible for downless (dL), which shares a phenotype identical to tabby mice, revealed that the dL gene encodes a novel TNF receptor (TNFR) called Edar (for ectodermal dysplasia receptor) (Headon and Overbeek, 1999Headon D.J. Overbeek P.A. Involvement of a novel Tnf receptor homologue in hair follicle induction.Nat Genet. 1999; 22: 370-374Crossref PubMed Scopus (291) Google Scholar). The tabby and downless mice (Eda-null and Edar-null mice, respectively) lack primary coat hair follicles (called guard hair or monotrich) and sweat glands and exhibit teeth abnormalities. As the human homolog of dL was shown to be mutated in patients with an autosomal recessive form of HED (Monreal et al., 1999Monreal A.W. Ferguson B.M. Headon D.J. Street S.L. Overbeek P.A. Zonana J. Mutations in the human homologue of mouse dl cause autosomal recessive and dominant hypohidrotic ectodermal dysplasia.Nat Genet. 1999; 22: 366-369Crossref PubMed Scopus (315) Google Scholar), whose phenotype is indistinguishable from X-linked HED, this suggested that Eda and Edar might act as a ligand and a receptor, respectively, in hair development. Eda and Edar were shown to interact in vitro (Tucker et al., 2000Tucker A.S. Headon D.J. Schneider P. Ferguson B.M. Overbeek P. Tschopp J. Sharpe P.T. Edar/Eda interactions regulate enamel knot formation in tooth morphogenesis.Development. 2000; 127: 4691-4700PubMed Google Scholar;Yan et al., 2000Yan M. Wang L.C. Hymowitz S.G. et al.Two-amino acid molecular switch in an epithelial morphogen that regulates binding to two distinct receptors.Science. 2000; 290: 523-527Crossref PubMed Scopus (229) Google Scholar) and subsequent studies investigating the relationship between this and other factors strongly suggested Eda/Edar to be the most upstream signal in hair follicle development (Huelsken et al., 2001Huelsken J. Vogel R. Erdmann B. Cotsarelis G. Birchmeier W. beta-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin.Cell. 2001; 105: 533-545Abstract Full Text Full Text PDF PubMed Scopus (1038) Google Scholar). TNF family molecules have been studied most extensively in the context of inflammation and lympho-hematopoietic organogenesis. In these settings, the most common outcome induced by this class of signals is expression of cell adhesion molecules (CAM). TNF and lymphotoxin have been shown to induce expression of vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and mucosal addressin cell adhesion molecules (MAdCAM-1) in mesenchymal and endothelial cell populations (Cuff et al., 1998Cuff C.A. Schwartz J. Bergman C.M. Russell K.S. Bender J.R. Ruddle N.H. Lymphotoxin alpha3 induces chemokines and adhesion molecules: insight into the role of LT alpha in inflammation and lymphoid organ development.J Immunol. 1998; 161: 6853-6860PubMed Google Scholar;Cuff et al., 1999Cuff C.A. Sacca R. Ruddle N.H. Differential induction of adhesion molecule and chemokine expression by LTalpha3 and LTalphabeta in inflammation elucidates potential mechanisms of mesenteric and peripheral lymph node development.J Immunol. 1999; 162: 5965-5972PubMed Google Scholar). These CAMs have been thought to play a role in maintaining a specific set of hematopoietic cells in the CAM-positive regions, which might be an important step for regulation of cell migration in inflammation. Whether CAMs play roles in organogenesis is unclear, however. In this study, we report that two CAMs, MAdCAM-1 and ICAM-1, are induced in hair follicle buds at embryonic day (E) 14-16 and appear to be downstream of Eda/Edar signaling. Using MAdCAM-1 as an indicator, we succeeded in reproducing this Eda/Edar induced process in an organ culture system. Our organ culture results suggest a role for MAdCAM-1 during hair follicle development. C57BL/6 mice and tabby mice (B6CBAa-Aw-J/A-Ta/O) were purchased (Nihon SLC, Hamamtsu, Japan, and Jackson Laboratory, Bar Harbor, ME, respectively); downless mice (dLJ/dLJ) were kindly provided by Dr. Paul A. Overbeek (Baylor College of Medicine, Texas). TRAF6-deficient (TRAF6–/–) mice were as described previously (Naito et al., 1999Naito A. Azuma S. Tanaka S. et al.Severe osteopetrosis, defective interleukin-1 signalling and lymph node organogenesis in TRAF6-deficient mice.Genes Cells. 1999; 4: 353-362Crossref PubMed Scopus (516) Google Scholar). Matings were conducted overnight, and noon of the day when vaginal plugs were observed was designated as E0.5. All animal procedures described in this study were performed in accordance with the guidelines for animal experiments at Kyoto University Graduate School of Medicine. Antibodies for MAdCAM-1 (MECA-367, MECA-89), ICAM-1 (3E2), and VCAM-1 (429/MVCAM.A) were purchased (BD Pharmingen, San Diego, CA). For immunostaining of MAdCAM-1, MECA-367 was used as a primary antibody. Epidermal growth factor (EGF) was purchased (Sigma Aldrich, St. Louis, MO) and cyclopamine was kindly provided by William Gaffield (Department of Agriculture, Albany, CA). The rat MAdCAM-1/human IgG-Fc chimeric protein (MAdCAM-1-Ig) was produced and purified as previously described (Iizuka et al., 2000Iizuka T. Tanaka T. Suematsu M. et al.Stage-specific expression of mucosal addressin cell adhesion molecule-1 during embryogenesis in rats.J Immunol. 2000; 164: 2463-2471Crossref PubMed Scopus (45) Google Scholar). The organization of rat MAdCAM-1 gene is very similar to that of mouse, with an amino acid identity of 80.5% (Iizuka et al., 1998Iizuka T. Koike R. Miyasaka N. Miyasaka M. Watanabe T. Cloning and characterization of the rat MAdCAM-1 cDNA and gene.Biochim Biophys Acta. 1998; 1395: 266-270Crossref PubMed Scopus (10) Google Scholar); this MAdCAM-1-Ig can bind to mouse lymphoid cells in vitro, binds to integrin α4β7, and lacks L-selectin-reactive carbohydrates (Iizuka et al., 2000Iizuka T. Tanaka T. Suematsu M. et al.Stage-specific expression of mucosal addressin cell adhesion molecule-1 during embryogenesis in rats.J Immunol. 2000; 164: 2463-2471Crossref PubMed Scopus (45) Google Scholar). Human IgG-Fc fragment (ChromPure; Jackson ImmunoReserach Laboratories, West Grove, PA) was used as a control. The organ culture system was adapted fromKashiwagi et al., 1997Kashiwagi M. Kuroki T. Huh N. Specific inhibition of hair follicle formation by epidermal growth factor in an organ culture of developing mouse skin.Dev Biol. 1997; 189: 22-32Crossref PubMed Scopus (39) Google Scholar. In brief, skin specimens were prepared from the dorsal coat of E13-13.5 embryos and spread epidermal side up onto a Nuclepore polycarbonate Track Etch Membrane (Nuclepore Europe, Whatman International, U.K.) coated with growth factor reduced Matrigel (Becton Dickinson Biosciences, Bedford, MA). The tissue pieces were incubated at the air–liquid interface, floating on 2 ml of Dulbecco's modified Eagle's medium (Gibco BRL, Grand Island, NY) supplemented with 1% fetal bovine serum, 50 U per ml penicillin, and 50 μg per ml streptomycin, in 35 mm plastic culture dishes with 5% CO2 at 37°C. EGF, cyclopamine, MAdCAM-1-Ig, and human IgG-Fc were added directly to the culture medium. For whole-mount immuno staining, the embryos and organ culture explants were fixed in 2% paraformaldehyde (pH 7.4) (microwaved for 20 s at 600 W and kept on ice for 30 min), and then processed as previously described (Adachi et al., 1997Adachi S. Yoshida H. Kataoka H. Nishikawa S. Three distinctive steps in Peyer's patch formation of murine embryo.Int Immunol. 1997; 9: 507-514Crossref PubMed Scopus (166) Google Scholar). In brief, specimens were washed in phosphate-buffered saline (PBS) after fixation, serially dehydrated with methanol, blocked for intrinsic peroxidase activities in 0.3% H2O2, and rehydrated. After incubation with PBS-MT (1% skim milk, 0.1% Triton X-100 in PBS) to inhibit nonspecific binding, specimens were incubated with primary antibodies overnight and washed in PBS-T (0.1% Triton X-100 in PBS). After incubation with horseradish peroxidase (HRP) conjugated secondary reagents, color reactions were carried out with diaminobenzidine and nickel chloride. Stained explants and embryos were embedded in polyesterwax and sections (7 μm) were counterstained with hematoxylin and eosin as needed. For cryosections used for immunostaining, embryos fixed in 2% paraformaldehyde were washed in PBS, embedded in OCT compound, and then snap frozen in liquid nitrogen. Cryostat sections (7–10 μm) were washed in PBS and intrinsic peroxidase activity was blocked by treatment in methanol with 0.3% H2O2 on ice for 10 min, followed by PBS washes. After blocking with PBS-MT, slides were incubated with primary antibodies overnight at room temperature and washed in PBS-T. After incubation with HRP-conjugated secondary reagents, color reaction was performed as mentioned above. For quantifying the lengths and numbers of hair buds, those sectioned longitudinally were photographed and their lengths and numbers were estimated on prints. As Edar is expressed specifically in the fraction of epidermal cells that are committed to follicular keratinocytes at the beginning of hair morphogenesis (Headon and Overbeek, 1999Headon D.J. Overbeek P.A. Involvement of a novel Tnf receptor homologue in hair follicle induction.Nat Genet. 1999; 22: 370-374Crossref PubMed Scopus (291) Google Scholar), CAMs are expected to be expressed in the same region at the same stage, if they are induced by this signal. To test this possibility, we carried out whole-mount immunostaining of E14.5 embryos with monoclonal antibodies (mAbs) to VCAM-1, ICAM-1, or MAdCAM-1. Although we could not detect VCAM-1 expression in embryonic skin at this stage, spots expressing MAdCAM-1 or ICAM-1 were detected over almost the entire body of the embryo (Figure 1a–c). Among these CAMs, ICAM-1 was expressed in hair buds, as well as weakly in the surrounding epidermis (Figure 1a). In contrast, MAdCAM-1 expression was restricted in hair buds at E14.5 (Figure 1b), which resembles Edar expression at this stage (Headon and Overbeek, 1999Headon D.J. Overbeek P.A. Involvement of a novel Tnf receptor homologue in hair follicle induction.Nat Genet. 1999; 22: 370-374Crossref PubMed Scopus (291) Google Scholar). These patterns suggest that MAdCAM-1 expression correlates more with that of Edar, whereas ICAM-1 expression might be influenced by other signals. We thus chose MAdCAM-1 expression as a marker for correlation with Edar signals. To determine the timing of MAdCAM-1 expression, we examined embryos from E12.5 to E16.5 (Figure 2 and data not shown). At E12.5, MAdCAM-1 expression was found only in the nasal area where the earliest hair follicles, vibrissa, develop (Figure 2a). The next MAdCAM-1-positive region observed was eyelids at E13.5 (Figure 2b). At E14.5, MAdCAM-1-positive spots appeared over a wide area of the epidermis (Figure 2c). Initially, the MAdCAM-1-positive area appeared homogeneous within hair buds, but it condensed rapidly and became concentrated in the central region of the hair follicle (Figure 2d). By E16.5, however, MAdCAM-1 expression could no longer be detected in skin by whole-mount immunostaining (data not shown). In mouse coat hair development, it has been known that the primary hair follicle (guard hair) develops at around E14 and the secondary hair follicle (nonguard hair, such as awl, auchen, and zigzag) develops at E17-19. So, to see whether or not MAdCAM-1 is expressed in secondary hair follicles, and to confirm the disappearance of MAdCAM-1 expression from developing follicles, which is observed in the results of whole-mount immunostaining, the immunostaining of cryostat sections was performed. Outside of vibrissa, MAdCAM-1 was detected in cryostat sections of hair placodes in E14.5–15.5 embryos, and also in newly formed hair placodes at E16.5, but not in hair placodes at E17.5–18.5 nor in elongating hair follicles at E16.5–18.5 (data not shown). In order to investigate whether or not MAdCAM-1 is induced directly by Edar, MAdCAM-1 expression in the embryonic keratinocytes of downless (Edar-null) and tabby (Eda-null) mice was investigated. As we expected, no MAdCAM-1 expression was detected in the embryonic skin of these mice at any stage including E14.5 or E15.5 (Figure 3a, b), and Eda/Edar-independent hair follicles develop normally in these mice. These results demonstrate that MAdCAM-1 is induced specifically in Eda/Edar-dependent follicles, but not in follicles controlled by other signals. TRAF is a molecule that is involved in signal transmission of a number of TNF family molecules (for reviews seeInoue et al., 2000Inoue J. Ishida T. Tsukamoto N. Kobayashi N. Naito A. Azuma S. Yamamoto T. Tumor necrosis factor receptor-associated factor (TRAF) family: adapter proteins that mediate cytokine signaling.Exp Cell Res. 2000; 254: 14-24Crossref PubMed Scopus (343) Google Scholar;Bradley and Pober, 2001Bradley J.R. Pober J.S. Tumor necrosis factor receptor-associated factors (TRAFs).Oncogene. 2001; 20: 6482-6491Crossref PubMed Scopus (490) Google Scholar). TRAF6–/– mice share some characteristics with downless and tabby mice, including a bald patch behind the ear and a kinky tail, and they show defects in guard hair, sweat glands, and teeth (Naito et al, unpublished data). This suggests strongly that TRAF6 is involved in transmitting the Eda/Edar signal. To test this possibility, we investigated the MAdCAM-1 expression in TRAF6–/– mice. As shown in Figure 3, MAdCAM-1 expression could not be detected in embryonic skin of TRAF6–/– mice (Figure 3c), whereas TRAF6+/– mice had levels of MAdCAM-1 expression comparable with C57BL/6 (Figure 1b,Figure 3d). To further dissect the process of Eda/Edar-induced hair follicle development, we adopted the skin organ culture system developed byKashiwagi et al., 1997Kashiwagi M. Kuroki T. Huh N. Specific inhibition of hair follicle formation by epidermal growth factor in an organ culture of developing mouse skin.Dev Biol. 1997; 189: 22-32Crossref PubMed Scopus (39) Google Scholar to investigate whether MAdCAM-1 expression at the initial phase of follicular development is reproduced in vitro. As it has been shown that EGF inhibits the induction phase of follicular development (Iizuka et al., 1998Iizuka T. Koike R. Miyasaka N. Miyasaka M. Watanabe T. Cloning and characterization of the rat MAdCAM-1 cDNA and gene.Biochim Biophys Acta. 1998; 1395: 266-270Crossref PubMed Scopus (10) Google Scholar) and that cyclopamine, an inhibitor of Shh signal (Incardona et al., 1998Incardona J.P. Gaffield W. Kapur R.P. Roelink H. The teratogenic Veratrum alkaloid cyclopamine inhibits sonic hedgehog signal transduction.Development. 1998; 125: 3553-3562Crossref PubMed Google Scholar), can inhibit the late phase of follicular development (Jordan and Jackson, 2000Jordan S.A. Jackson I.J. MGF (KIT ligand) is a chemokinetic factor for melanoblast migration into hair follicles.Dev Biol. 2000; 225: 424-436Crossref PubMed Scopus (87) Google Scholar), we used these two inhibitors for determining the chronologic relationship of Eda/Edar-induced signals during the process of hair follicle development in vitro. Skin fragments of E13-13.5 embryos were dissected and cultured (Figure 4a). After 24 h cultivation, the fragments were fixed and stained by monoclonal antibody to MAdCAM-1. Expression of MAdCAM-1 in the hair placodes was observed in vitro as occurs in the embryo (Figure 4b). In vitro induction of MAdCAM-1 in the hair placode was not inhibited by cyclopamine, which inhibits the Shh-dependent late phase of follicle development (Figure 4d). Addition of EGF in the culture, however, completely inhibited this MAdCAM-1 expression in skin (Figure 4c). Thus, Eda/Edar-induced events occur at the early phase of hair follicle development. MAdCAM-1 is a multidomain cell adhesion molecule, comprising two N-terminal Ig domains and the membrane proximal mucin domain. The N-terminal Ig domains 1 and 2, which are recognized by mAbs MECA-367 and MECA-89, respectively (Berlin et al., 1993Berlin C. Berg E.L. Briskin M.J. et al.Alpha 4 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1.Cell. 1993; 74: 185Abstract Full Text PDF PubMed Scopus (1226) Google Scholar;Streeter et al, 1988a), are both required for efficient binding of MAdCAM-1 to its lymphocyte counter-receptor, integrin α4β7 (Briskin et al., 1996Briskin M.J. Rott L. Butcher E.C. Structural requirements for mucosal vascular addressin binding to its lymphocyte receptor alpha 4 beta 7. Common themes among integrin–Ig family interactions.J Immunol. 1996; 156: 719-726PubMed Google Scholar). The mucin domain of MAdCAM-1 can be modified by L-selectin-binding carbohydrates that are recognized by mAb MECA-79 when MAdCAM-1 is expressed in high endothelial venule cells (Hemmerich et al., 1994Hemmerich S. Butcher E.C. Rosen S.D. Sulfation-dependent recognition of high endothelial venules (HEV)-ligands by L-selectin and MECA 79, and adhesion-blocking monoclonal antibody.J Exp Med. 1994; 180: 2219-2226Crossref PubMed Scopus (245) Google Scholar;Streeter et al, 1988b). We found by immunohistochemistry that developing hair follicles are reactive with mAbs MECA-367 and MECA-89 but not MECA-79 (data not shown), indicating that epidermal MAdCAM-1 contains the two N-terminal Ig domains and lacks L-selectin-binding carbohydrates. Given that MAdCAM-1 is induced in the earliest hair placode, the next important question is whether or not MAdCAM-1 plays any roles in hair follicle development. To address this issue, we next investigated the effects of neutralizing anti-MAdCAM-1 mAb and MAdCAM-1-Ig on hair follicle development in vitro in comparison with those of EGF and cyclopamine. After 72 h cultivation of embryonic skin fragments, involuting hair follicle buds were seen in the control specimens (Figure 5a), which are similar to those at E16-16.5 in vivo except for the thickening epidermis (Iizuka et al., 1998Iizuka T. Koike R. Miyasaka N. Miyasaka M. Watanabe T. Cloning and characterization of the rat MAdCAM-1 cDNA and gene.Biochim Biophys Acta. 1998; 1395: 266-270Crossref PubMed Scopus (10) Google Scholar). As reported, EGF almost completely inhibited hair follicle development (Figure 5b), and cyclopamine inhibited this process at later stages after formation of hair follicle buds (Figure 5d). mAb MECA-367, which inhibits MAdCAM-1 binding to integrin α4β7, did not significantly affect the hair follicle development process in vitro (Figure 5c) even in the presence of another anti-MAdCAM-1 mAb MECA-89. In accordance with this observation, no integrin α4β7-expressing cells were detected in the epidermis and dermis by immunohistochemistry (data not shown). If the extracellular portion of MAdCAM-1 were functionally involved in primary hair follicle development, the soluble form of MAdCAM-1 may interfere with this pathway. To test this possibility, MAdCAM-1-Ig was directly added to the skin organ culture. Interestingly, exogenously added MAdCAM-1-Ig attenuated hair follicle development in the skin organ culture system (Figure 5f). Human IgG-Fc was used as a control to this chimeric protein and it did not affect the hair follicle development (Figure 5e). As summarized in Figure 6, MAdCAM-1-Ig decreased the density of follicles per unit space, and
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