View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Mucosal Addressin Molecule 1 Plays an Unexpected Role in the Development of Mouse Guard Hair

Eri Nishioka,*²1 Toshiyuki Tanaka,³ Hisahiro Yoshida,* Kazuyoshi Matsumura,* Satomi Nishikawa,*§ Asuka Naito,¶ Jun-ichiro Inoue,** Yoko Funasaka,² Masamitsu Ichihashi,² Masayuki Miyasaka,³ and Shin-Ichi Nishikawa*§ *Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto, Japan; ²Department of Dermatology, Kobe University School of Medicine, Kobe, Japan; ³Laboratory of Molecular and Cellular Recognition, Osaka University Graduate School of Medicine, Suita, Japan; §Center for Developmental Biology, RIKEN, Kobe, Japan; ¶Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan; **Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Japan

The ®rst wave of coat hair development is initiated follicle development before involvement of Sonic around embryonic day 14 in the mouse. Whereas hedgehog signal. Follicle development in this culture ectodysplasin and ectodermal dysplasia receptor, was also suppressed to some extent, though not tumor necrosis factor and tumor necrosis factor completely, by addition of soluble mucosal addressin receptor family molecules, respectively, were identi- 1/IgG-Fc chimeric , ®ed to be signals triggering this process, not much whereas that can inhibit muco- was known regarding their downstream molecular sal addressin cell adhesion molecule 1 interaction targets. In this report, we show that mucosal addres- with integrin a4b7 had no effect on this process. sin cell adhesion molecule 1 and intercellular adhe- These results demonstrated for the ®rst time that the sion molecule 1 are induced in the keratinocytes structural , mucosal addressin cell adhesion of the hair placode as a direct consequence of ecto- molecule 1 and intercellular adhesion molecule 1, are dermal dysplasia receptor signal, and tumor-necro- induced by ectodermal dysplasia receptor signal and sis-factor-receptor-associated factor 6 is involved in suggested the potential involvement of mucosal this mucosal addressin cell adhesion molecule 1 addressin cell adhesion molecule 1 in the morpho- expression. Experiments using an in vitro culture of genesis of follicular keratinocytes. Key words: Edar/ skin fragments demonstrated that ectodermal-dyspla- hypohidrotic ectodermal dysplasia/keratinocyte/tumor necro- sia-receptor-induced mucosal addressin cell adhesion sis factor. J Invest Dermatol 119:632±638, 2002 molecule 1 expression occurs at the initial phase of

t is well established that mammalian hair follicle and avian genetic proteins are involved in the initial process, as positive and feather formation are regulated by a set of signals such as negative regulators, respectively (Song et al, 1996; Jung et al, 1998), bone morphogenetic proteins, Sonic hedgehog (Shh), followed by a subsequent fate speci®cation process involving ®broblast growth factors (FGFs), Wnt, and Notch that Notch/Delta signals (Artavanis-Tsakonas and Simpson, 1991; Iappear successively during organogenesis of hair follicles as Crowe et al, 1998). Likewise, Notch has been implicated in hair well as that of other organs (for reviews see Oro and Scott, 1998; placode development (Powell et al, 1998). Recent studies showed Barsh, 1999). From histologic observations, it was proposed that that conditional deletion of the b-catenin from the epidermis hair follicle formation is initiated when underlying mesenchymal resulted in defects in the formation of the hair placode (Huelsken cells trigger commitment of the overlying epidermis to form the et al, 2001). Moreover, mice lacking the LEF-1 gene displayed placodes (Hardy, 1992; Oro and Scott, 1998). Concerning feather reduction in coat hair number and complete lack of whiskers (van development, it has been shown that FGFs and bone morpho- Genderen et al, 1994). As b-catenin and LEF-1 have been implicated downstream of Wnt signaling, these results indicate a Manuscript received March 20, 2002; accepted for publication May 7, role for Wnt signals in the initial phase of hair follicle development. 2002. Shh, another important signal that is involved repeatedly in many Reprint requests to: Dr. Eri Nishioka, Center for Developmental organogenesis processes (Ekker et al, 1995; Hardy et al, 1995; Biology, RIKEN, 2-2-3 Mnatojima Minamimachi, Chuo-Ku, Kobe, 650- Tanabe et al, 1995), is implicated in the subsequent processes of hair 0047, Japan. Email: [email protected] follicle development after placode formation, particularly in Abbreviations: E, embryonic day; Eda, ectodysplasin; EDA, anhidrotic elongation (St-Jacques et al, 1998; Chiang et al, 1999). Despite ectodermal dysplasia; Edar, ectodermal dysplasia receptor; HED, hypohi- this increasing list of molecules regulating hair follicle development, drotic ectodermal dysplasia; IkB, inhibitor of NF-kB; IKK, IkB kinase; the nature of the initiating signal has remained elusive. MAdCAM, mucosal addressin cell adhesion molecule; NF-kB, nuclear factor kB; TNFR, TNF receptor; TRAF, TNFR-associated factor; Shh, Hypohidrotic (anhidrotic) ectodermal dysplasia [HED (EDA)] Sonic hedgehog; Xedar, X-linked EDA-A2 receptor. stands for a syndrome characterized by hypoplastic development in 1Present address: Center for Developmental Biology, RIKEN, Kobe, ectodermal organs including teeth, hair, and sweat glands and is Japan. composed of X-linked, autosomal recessive and variant forms. After

0022-202X/02/$15.00 ´ Copyright # 2002 by The Society for Investigative Dermatology, Inc. 632 VOL. 119, NO. 3 SEPTEMBER 2002 MAdCAM-1 EXPRESSION IN MOUSE HAIR DEVELOPMENT 633 the identi®cation of the gene responsible for X-linked HED (Kere Skin organ culture The organ culture system was adapted from et al, 1996), called EDA, the corresponding mouse gene was cloned Kashiwagi et al (1997). In brief, skin specimens were prepared from the and found to be allelic to the tabby (Ta) locus (Srivastava et al, dorsal coat of E13±13.5 embryos and spread epidermal side up onto a 1997). From the deduced amino acid sequence, the Ta protein and Nuclepore polycarbonate Track Etch Membrane (Nuclepore Europe, Whatman International, U.K.) coated with growth factor reduced EDA product, ectodysplasin (Eda), were identi®ed as tumor Matrigel (Becton Dickinson Biosciences, Bedford, MA). The tissue necrosis factor (TNF) family members (Ezer et al, 1999; Mikkola pieces were incubated at the air±liquid interface, ¯oating on 2 ml of et al, 1999). In complete agreement with this, subsequent cloning of Dulbecco's modi®ed Eagle's medium (Gibco BRL, Grand Island, NY) the gene responsible for downless (dL), which shares a phenotype supplemented with 1% fetal bovine serum, 50 U per ml penicillin, and identical to tabby mice, revealed that the dL gene encodes a novel 50 mg per ml streptomycin, in 35 mm plastic culture dishes with 5% TNF receptor (TNFR) called Edar (for ectodermal dysplasia CO2 at 37°C. EGF, cyclopamine, MAdCAM-1-Ig, and human IgG-Fc receptor) (Headon and Overbeek, 1999). The tabby and downless were added directly to the culture medium. mice (Eda-null and Edar-null mice, respectively) lack primary coat Histology and immunohistochemistry For whole-mount immuno- hair follicles (called guard hair or monotrich) and sweat glands and staining, the embryos and organ culture explants were ®xed in 2% exhibit teeth abnormalities. As the human homolog of dL was paraformaldehyde (pH 7.4) (microwaved for 20 s at 600 W and kept on shown to be mutated in patients with an autosomal recessive form ice for 30 min), and then processed as previously described (Adachi et al, of HED (Monreal et al, 1999), whose phenotype is indistinguish- 1997). In brief, specimens were washed in phosphate-buffered saline (PBS) after ®xation, serially dehydrated with methanol, blocked for able from X-linked HED, this suggested that Eda and Edar might intrinsic peroxidase activities in 0.3% H2O2, and rehydrated. After act as a ligand and a receptor, respectively, in hair development. incubation with PBS-MT (1% skim milk, 0.1% Triton X-100 in PBS) to Eda and Edar were shown to interact in vitro (Tucker et al, 2000; inhibit nonspeci®c binding, specimens were incubated with primary Yan et al, 2000) and subsequent studies investigating the relation- antibodies overnight and washed in PBS-T (0.1% Triton X-100 in PBS). ship between this and other factors strongly suggested Eda/Edar to After incubation with horseradish peroxidase (HRP) conjugated be the most upstream signal in hair follicle development (Huelsken secondary reagents, color reactions were carried out with et al, 2001). diaminobenzidine and nickel chloride. Stained explants and embryos TNF family molecules have been studied most extensively in the were embedded in polyesterwax and sections (7 mm) were counterstained with hematoxylin and eosin as needed. For cryosections used for context of in¯ammation and lympho-hematopoietic organogenesis. immunostaining, embryos ®xed in 2% paraformaldehyde were washed in In these settings, the most common outcome induced by this class PBS, embedded in OCT compound, and then snap frozen in liquid of signals is expression of cell adhesion molecules (CAM). TNF and nitrogen. Cryostat sections (7±10 mm) were washed in PBS and intrinsic have been shown to induce expression of vascular cell peroxidase activity was blocked by treatment in methanol with 0.3% adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 H2O2 on ice for 10 min, followed by PBS washes. After blocking with (ICAM-1), and mucosal addressin cell adhesion molecules PBS-MT, slides were incubated with primary antibodies overnight at (MAdCAM-1) in mesenchymal and endothelial cell populations room temperature and washed in PBS-T. After incubation with HRP- conjugated secondary reagents, color reaction was performed as (Cuff et al, 1998; 1999). These CAMs have been thought to play a mentioned above. For quantifying the lengths and numbers of hair buds, role in maintaining a speci®c set of hematopoietic cells in the those sectioned longitudinally were photographed and their lengths and CAM-positive regions, which might be an important step for numbers were estimated on prints. regulation of cell migration in in¯ammation. Whether CAMs play roles in organogenesis is unclear, however. In this study, we report that two CAMs, MAdCAM-1 and RESULTS ICAM-1, are induced in hair follicle buds at embryonic day (E) MAdCAM-1 is expressed in primary hair buds As Edar is 14±16 and appear to be downstream of Eda/Edar signaling. Using expressed speci®cally in the fraction of epidermal cells that are MAdCAM-1 as an indicator, we succeeded in reproducing this committed to follicular keratinocytes at the beginning of hair Eda/Edar induced process in an organ culture system. Our organ morphogenesis (Headon and Overbeek, 1999), CAMs are expected culture results suggest a role for MAdCAM-1 during hair follicle to be expressed in the same region at the same stage, if they are development. 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. MATERIALS AND METHODS Although we could not detect VCAM-1 expression in embryonic skin at this stage, spots expressing MAdCAM-1 or Mice C57BL/6 mice and tabby mice (B6CBAa-Aw-J/A-Ta/O) were purchased (Nihon SLC, Hamamtsu, Japan, and Jackson Laboratory, Bar ICAM-1 were detected over almost the entire body of the embryo Harbor, ME, respectively); downless mice (dLJ/dLJ) were kindly provided (Fig 1A±C). Among these CAMs, ICAM-1 was expressed in hair by Dr. Paul A. Overbeek (Baylor College of Medicine, Texas). TRAF6- buds, as well as weakly in the surrounding epidermis (Fig 1A). In de®cient (TRAF6±/±) mice were as described previously (Naito et al, contrast, MAdCAM-1 expression was restricted in hair buds at 1999). Matings were conducted overnight, and noon of the day when E14.5 (Fig 1B), which resembles Edar expression at this stage vaginal plugs were observed was designated as E0.5. All animal (Headon and Overbeek, 1999). These patterns suggest that procedures described in this study were performed in accordance with MAdCAM-1 expression correlates more with that of Edar, the guidelines for animal experiments at Kyoto University Graduate whereas ICAM-1 expression might be in¯uenced by other School of Medicine. signals. We thus chose MAdCAM-1 expression as a marker for Materials Antibodies for MAdCAM-1 (MECA-367, MECA-89), correlation with Edar signals. ICAM-1 (3E2), and VCAM-1 (429/MVCAM.A) were purchased (BD To determine the timing of MAdCAM-1 expression, we Pharmingen, San Diego, CA). For immunostaining of MAdCAM-1, examined embryos from E12.5 to E16.5 (Fig 2, and data not MECA-367 was used as a primary antibody. Epidermal growth factor shown). At E12.5, MAdCAM-1 expression was found only in the (EGF) was purchased (Sigma Aldrich, St. Louis, MO) and cyclopamine nasal area where the earliest hair follicles, vibrissa, develop was kindly provided by William Gaf®eld (Department of Agriculture, (Fig 2A). The next MAdCAM-1-positive region observed was Albany, CA). The rat MAdCAM-1/human IgG-Fc chimeric protein eyelids at E13.5 (Fig 2B). At E14.5, MAdCAM-1-positive spots (MAdCAM-1-Ig) was produced and puri®ed as previously described (Iizuka et al, 2000). The organization of rat MAdCAM-1 gene is very appeared over a wide area of the epidermis (Fig 2C). Initially, the similar to that of mouse, with an amino acid identity of 80.5% (Iizuka et MAdCAM-1-positive area appeared homogeneous within hair al, 1998); this MAdCAM-1-Ig can bind to mouse lymphoid cells in vitro, buds, but it condensed rapidly and became concentrated in the binds to integrin a4b7, and lacks L-selectin-reactive carbohydrates central region of the hair follicle (Fig 2D). By E16.5, however, (Iizuka et al, 2000). Human IgG-Fc fragment (ChromPure; Jackson MAdCAM-1 expression could no longer be detected in skin by ImmunoReserach Laboratories, West Grove, PA) was used as a control. whole-mount immunostaining (data not shown). 634 NISHIOKA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 1. Expression of ICAM-1, MAdCAM- 1, and VCAM-1 in mouse embryonic skin. Whole-mount immunostaining of E14.5 C57BL/ 6 embryos with mAbs against ICAM-1 (A), MAdCAM-1 (B), and VCAM-1 (C). The insets are included to show the histology of the ICAM- 1(A) and MAdCAM-1 (B) positive cells. ICAM- 1 is expressed in hair buds, as well as weakly in the surrounding epidermis. MAdCAM-1 expression is restricted to hair buds. VCAM-1 expression is not detected by this method. Arrowheads indicate the expression of each molecule. Scale bar:50mm.

Figure 2. Expression of MAdCAM-1 in C57BL/6 embryonic skin. Whole-mount immunostaining analysis of E12.5 (A), E13.5 (B), E14.5 (C), and E15.5 (D) C57BL6 embryos with mAb against MAdCAM-1. The insets show MAdCAM-1 expression in whiskers (A), eyelids and whiskers (B), and in the spots consistent with hair placodes (C, D). Scale bar: 650 mm(A, B). Scale bar: 1 mm (C, D).

Figure 3. The expression of MAdCAM-1 in embryonic skin is inhibited in tabby, downless, and TRAF6-de®cient (TRAF6±/±) mice. Whole-mount immunostaining analysis of E14.5 tabby (A), E15.5 downless (B), and E14.5 TRAF6±/± (C), and TRAF6+/± (D) with mAb against MAdCAM-1. Whereas MAdCAM-1- positive spots are detected in TRAF6+/± mice, these spots are not detected in tabby, downless, or TRAF6±/± mice. Scale bar: 1 mm.

In mouse coat hair development, it has been known that the MAdCAM-1 is induced speci®cally in Eda/Edar-dependent primary hair follicle (guard hair) develops at around E14 and the follicles, but not in follicles controlled by other signals. secondary hair follicle (nonguard hair, such as awl, auchen, and TRAF is a molecule that is involved in signal transmission of a zigzag) develops at E17±19. So, to see whether or not MAdCAM-1 number of TNF family molecules (for reviews see Inoue et al, 2000; is expressed in secondary hair follicles, and to con®rm the Bradley and Pober, 2001). TRAF6±/± mice share some character- disappearance of MAdCAM-1 expression from developing follicles, istics with downless and tabby mice, including a bald patch behind which is observed in the results of whole-mount immunostaining, the ear and a kinky tail, and they show defects in guard hair, sweat the immunostaining of cryostat sections was performed. Outside of glands, and teeth (Naito et al, unpublished data). This suggests vibrissa, MAdCAM-1 was detected in cryostat sections of hair strongly that TRAF6 is involved in transmitting the Eda/Edar placodes in E14.5±15.5 embryos, and also in newly formed hair signal. To test this possibility, we investigated the MAdCAM-1 placodes at E16.5, but not in hair placodes at E17.5±18.5 nor in expression in TRAF6±/± mice. As shown in Fig 3, MAdCAM-1 elongating hair follicles at E16.5±18.5 (data not shown). expression could not be detected in embryonic skin of TRAF6±/± mice (Fig 3C), whereas TRAF6+/± mice had levels of MAdCAM- MAdCAM-1 expression is regulated by Eda/Edar signaling 1 expression comparable with C57BL/6 (Figs 1B,3D). and requires TNFR-associated factor 6 (TRAF6) In order to investigate whether or not MAdCAM-1 is induced directly by Expression of MAdCAM-1 is reproduced in vitro and Edar, MAdCAM-1 expression in the embryonic keratinocytes of unaffected by Shh inhibitor To further dissect the process of downless (Edar-null) and tabby (Eda-null) mice was investigated. As Eda/Edar-induced hair follicle development, we adopted the skin we expected, no MAdCAM-1 expression was detected in organ culture system developed by Kashiwagi et al (1997) to the embryonic skin of these mice at any stage including E14.5 investigate whether MAdCAM-1 expression at the initial phase of or E15.5 (Fig 3A, B), and Eda/Edar-independent hair follicles follicular development is reproduced in vitro. As it has been shown develop normally in these mice. These results demonstrate that that EGF inhibits the induction phase of follicular development VOL. 119, NO. 3 SEPTEMBER 2002 MAdCAM-1 EXPRESSION IN MOUSE HAIR DEVELOPMENT 635

Figure 4. The expression of MAdCAM-1 in embryonic skin is reproduced in skin organ culture. The back skin of E13±13.5 embryos, in which MAdCAM-1 was not yet expressed, was harvested and cultured ¯oating on Dulbecco's modi®ed Eagle's medium/1% fetal bovine serum (A, and see Materials and Methods). Dotted lines denote the areas from which skin was excised. These skin specimens were cultured for 24 h (B, control), with EGF (100 ng per ml, C), or with cyclopamine (2 mM, D), and then subjected to whole-mount immunostaining with mAb against MAdCAM-1. EGF inhibits the MAdCAM-1 expression, whereas cyclopamine does not affect this process. Scale bar: 1 mm.

Figure 5. Histologic analysis of cultured embryonic skin. Skin specimens taken from E13±13.5 embryos were cultured for 72 h without added factors (A, control) or with EGF (100 ng per ml, B), a mixture of MECA-89 and MECA-367 (50 mg per ml, respectively, C), cyclopamine (2 mM, D), control IgG-Fc (50 mg per ml, E), or MAdCAM-1-Ig (50 mg per ml, F), and then subjected to histologic analysis. EGF completely inhibits the hair follicle induction. Cyclopamine-treated skin shows only the hair placode-like structure. MAdCAM-1-Ig-treated skin shows the hair placode-like structure. Polyesterwax-embedded specimens were sectioned at 7 mm and stained with hematoxylin and eosin. Scale bar: 100 mm.

(Iizuka et al, 1998) and that cyclopamine, an inhibitor of Shh signal carbohydrates that are recognized by mAb MECA-79 when (Incardona et al, 1998), can inhibit the late phase of follicular MAdCAM-1 is expressed in high endothelial venule cells development (Jordan and Jackson, 2000), we used these two (Hemmerich et al, 1994; Streeter et al, 1988b). We found by inhibitors for determining the chronologic relationship of Eda/ immunohistochemistry that developing hair follicles are reactive Edar-induced signals during the process of hair follicle development with mAbs MECA-367 and MECA-89 but not MECA-79 (data in vitro. Skin fragments of E13±13.5 embryos were dissected and not shown), indicating that epidermal MAdCAM-1 contains the cultured (Fig 4A). After 24 h cultivation, the fragments were ®xed two N-terminal Ig domains and lacks L-selectin-binding and stained by monoclonal antibody to MAdCAM-1. Expression carbohydrates. of MAdCAM-1 in the hair placodes was observed in vitro as occurs Given that MAdCAM-1 is induced in the earliest hair placode, in the embryo (Fig 4B). In vitro induction of MAdCAM-1 in the the next important question is whether or not MAdCAM-1 plays hair placode was not inhibited by cyclopamine, which inhibits the any roles in hair follicle development. To address this issue, we next Shh-dependent late phase of follicle development (Fig 4D). investigated the effects of neutralizing anti-MAdCAM-1 mAb and Addition of EGF in the culture, however, completely inhibited MAdCAM-1-Ig on hair follicle development in vitro in comparison this MAdCAM-1 expression in skin (Fig 4C). Thus, Eda/Edar- with those of EGF and cyclopamine. After 72 h cultivation of induced events occur at the early phase of hair follicle development. embryonic skin fragments, involuting hair follicle buds were seen in the control specimens (Fig 5A), which are similar to those at MAdCAM-1-Ig chimeric protein decreased the number E16±16.5 in vivo except for the thickening epidermis (Iizuka et al, and length of hair follicles in embryonic skin 1998). As reported, EGF almost completely inhibited hair follicle cultures MAdCAM-1 is a multidomain cell adhesion molecule, development (Fig 5B), and cyclopamine inhibited this process at comprising two N-terminal Ig domains and the membrane later stages after formation of hair follicle buds (Fig 5D). mAb proximal mucin domain. The N-terminal Ig domains 1 and 2, MECA-367, which inhibits MAdCAM-1 binding to integrin which are recognized by mAbs MECA-367 and MECA-89, a4b7, did not signi®cantly affect the hair follicle development respectively (Berlin et al, 1993; Streeter et al, 1988a), are both process in vitro (Fig 5C) even in the presence of another anti- required for ef®cient binding of MAdCAM-1 to its lymphocyte MAdCAM-1 mAb MECA-89. In accordance with this observa- counter-receptor, integrin a4b7 (Briskin et al, 1996). The mucin tion, no integrin a4b7-expressing cells were detected in the domain of MAdCAM-1 can be modi®ed by L-selectin-binding epidermis and dermis by immunohistochemistry (data not shown). 636 NISHIOKA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

the basic machinery available in keratinocytes for TNF family signal transduction is similar to that of ®broblasts and endothelial cells. Two classes of molecules, TRAF family and nuclear factor kB (NF-kB)/rel family molecules, have been implicated downstream to TNFR signaling. With regard to keratinocytes and Edar, recent studies demonstrated that inhibitors of NF-kB(IkB) and IkB kinase g (IKK-g) (NEMO) are involved in the activation of NF-kB downstream of Edar signaling (Zonana et al, 2000; Dof®nger et al, 2001; Schmidt-Ullrich et al, 2001). It has been indicated that TRAF family molecules are required for transmitting TNFR signals to the IKK/NF-kB pathway. To date, TRAF2, TRAF5, and TRAF6 have been shown to be able to activate the NF-kB signal (for review see Inoue et al, 2000). Using MAdCAM-1 expression as an indicator of Edar stimulation, we identi®ed TRAF6 as a potential downstream molecule transmitting Edar signals to the IKK/NF-kB pathway. In fact, TRAF6±/± mice share hair defects identical to the downless (Edar-null) mice (Naito et al, unpublished data). TRAF6 and IKK/NF-kB have already been implicated in CD40, osteoprotegerin ligand, and IL-1 signaling of hematopoietic cells (Lomaga et al, 1999; Naito et al, 1999). As MAdCAM-1 and ICAM-1 have been known to be activated by Figure 6. MAdCAM-1-Ig decreases the number and length of NF-kB, our study suggests that Eda signal may be transmitted hair follicles in skin organ cultures in a dose-dependent manner. sequentially from Edar, TRAF6, NEMO, IkB, and NF-kBto Skin specimens taken from E13±13.5 embryos were cultured for 72 h activate transcription of MAdCAM-1. This notion is inconsistent without factors (control) or with a mixture of MECA-89 and MECA- with recent studies, however, showing that Edar does not bind to 367 (50 mg per ml, respectively), MAdCAM-1-Ig, and control IgG-Fc, any known TRAF family molecules including TRAF6, though its and then subjected to histologic analysis. (A) The length from the stimulation can trigger NF-kB activation (Yan et al, 2000; Kumar et basement membrane of epidermis to the tip of hair follicle buds was al, 2001). measured. n = 50. (B) The number of hair follicle buds per 1 mm2. n = 25. Tissue sections were randomly selected from three independent The two longest forms of Eda, EDA-A1 and EDA-A2, differing experiments and subjected to quantitative analysis. For quantitating the by only two amino acids in the extracellular domain, activate NF- number per square millimeter, the number of hair follicle buds was kB-mediated transcription by binding to distinct receptors, Edar counted on prints and squared to score. The results portrayed represent and X-linked EDA-A2 receptor (Xedar), respectively (Yan et al, the mean of the length and the number of hair follicles, 2000). Xedar was shown to be able to bind to TRAF1, TRAF3, respectively, 6 SD. *Signi®cantly different from control, by Student's t and TRAF6, and implicated in hair follicle development, though test, p < 0.001. **MAdCAM-1-Ig, mg per ml. ®nal proof of its role requires gene knockout studies. If this is the case, Xedar should function at the same time as Edar does, as MAdCAM-1 expression that appears to be dependent on TRAF6 is expressed then. It has been shown, however, that expression of If the extracellular portion of MAdCAM-1 were functionally Xedar starts at E16±17, distinctly later than the expression of involved in primary hair follicle development, the soluble form of MAdCAM-1 in the hair placodes (Yan et al, 2000). In addition to MAdCAM-1 may interfere with this pathway. To test this these results, recent transgenic studies have revealed that EDA-A1, possibility, MAdCAM-1-Ig was directly added to the skin organ which can bind to Edar but not to Xedar, was suf®cient to rescue culture. Interestingly, exogenously added MAdCAM-1-Ig attenu- the hair phenotype in tabby mice (Srivastava et al, 2001). Thus, it is ated hair follicle development in the skin organ culture system unlikely that Xedar is involved in TRAF6-dependent MAdCAM- (Fig 5F). Human IgG-Fc was used as a control to this chimeric 1 expression at E14. Recently, TROY, another TNFR family protein and it did not affect the hair follicle development (Fig 5E). molecule that is closely related to Edar, was cloned (Kojima et al, As summarized in Fig 6, MAdCAM-1-Ig decreased the density 2000). As TROY is expressed in the hair placode of E14 embryos of follicles per unit space, and reduced the average length of follicles (data not shown), TROY might be the TRAF6 activator during in the culture in a dose-dependent manner. These results early follicle development. Alternatively, Edar is directly involved collectively suggest potential involvement of MAdCAM-1 in hair in TRAF6 activation, but probably through collaboration with an follicle development by mechanisms distinct from what has been adaptor molecule. TRAF6 activation by IL-1 or nerve growth shown already in hematopoietic±endothelial cell interaction. factor has been known to require adaptor proteins (Inoue et al, 2000; Bradley and Pober, 2001). Indeed, the gene responsible for the crinkled mutant that shows the same phenotype as tabby and DISCUSSION downless mice was shown to encode an adaptor protein with a death domain (Headon et al, 2001; Yan et al, 2002). This adaptor, The initial aim of this study was to investigate downstream factors Edaradd (for Edar-associated death domain) (Headon et al, 2001), in Edar signaling during hair follicle development. Edar is a TNF was shown to interact with the death domain of Edar, and with family molecule that is expressed in epidermal keratinocytes, and TRAF1, TRAF2, and TRAF3. It has not been shown whether provides a unique opportunity for investigating the outcome of this Edaradd can interact with TRAF6. class of signal in the context of keratinocytes. Histologically, placode formation consists of epidermal cell shape In in¯ammation, CAMs and various chemokines are represen- change from ¯at cuboidal to spheroidal and formation of multi- tative of molecules induced by TNFR signals (Paul and Seder, layered aggregates of epidermal cells. Thus, the Edar signal should 1994; Sacca et al, 1997). Indeed, we could show that both ICAM-1 induce a set of molecules involved in this morphogenesis. In and MAdCAM-1 but not VCAM-1 are induced in keratinocytes addition to keratin, keratinocytes have the potential to produce only when Edar signaling is active. As the spatiotemporal cytokines and chemokines (Barker et al, 1991), and previous studies expression pattern of these CAMs, especially that of MAdCAM- demonstrated that NF-kB activation in adult keratinocytes resulted 1, was almost identical to that of Edar during the primary hair in expression of these molecules (Goebeler et al, 1995). More developmental period, it is likely that expression of these CAMs is interestingly, Benoliel et al (1997) reported that migration of the direct outcome of stimulation by Edar. This result indicates that keratinocytes is induced by NF-kB activation. Given that VOL. 119, NO. 3 SEPTEMBER 2002 MAdCAM-1 EXPRESSION IN MOUSE HAIR DEVELOPMENT 637 activation of NF-kB is an outcome of Edar signaling, some of these REFERENCES events may be induced by Edar activation. In this sense, Adachi S, Yoshida H, Kataoka H, Nishikawa S: Three distinctive steps in Peyer's MAdCAM-1 and ICAM-1 would be the ®rst structural proteins patch formation of murine embryo. Int Immunol 9:507±514, 1997 to be linked to Edar and TRAF6 signaling in the context of hair Artavanis-Tsakonas S, Simpson P: Choosing a cell fate: a view from the Notch locus. follicle formation. To understand the molecular basis underlying Trends Genet 7:403±408, 1991 Barker JN, Mitra RS, Grif®ths CE, Dixit VM, Nickoloff BJ: Keratinocytes as this morphogenic activity, it is important to know the molecules initiators of in¯ammation. Lancet 337:211±214, 1991 induced by Edar signal. In this respect, our observation that surface Barsh G: Of ancient tales and hairless tails. Nat Genet 22:315±316, 1999 molecules such as MAdCAM-1 and ICAM-1 are induced specif- Benoliel AM, Kahn-Peries B, Imbert J, Verrando P: Insulin stimulates haptotactic ically in keratinocytes that are activated by Edar indicates the migration of human epidermal keratinocytes through activation of NF-kappa B transcription factor. J Cell Sci 110:2089±2097, 1997 potential to use those surface markers to sort keratinocytes in the Berlin C, Berg EL, Briskin MJ, et al: Alpha 4 beta 7 integrin mediates lymphocyte placode. binding to the mucosal vascular addressin MAdCAM-1. Cell 74:185, 1993 A question remaining is whether or not the CAMs induced by Bradley JR, Pober JS: Tumor necrosis factor receptor-associated factors (TRAFs). Oncogene 20:6482±6491, 2001 Edar play a role in the induction of hair placodes. It has been Briskin MJ, Rott L, Butcher EC: Structural requirements for mucosal vascular reported that, in normal postnatal mouse skin, ICAM-1 is expressed addressin binding to its lymphocyte receptor alpha 4 beta 7. Common themes regularly in endothelial cells and transiently in some selected among integrin±Ig family interactions. J Immunol 156:719±726, 1996 epidermal and follicular keratinocytes (Muller-Rover et al, 2000). Chiang C, Swan RZ, Grachtchouk M, et al: Essential role for Sonic hedgehog during hair follicle morphogenesis. Dev Biol 205:1±9, 1999 In catagen of the hair cycle, over 80% of keratinocytes constituting Crowe R, Henrique D, Ish-Horowicz D, Niswander L: A new role for Notch and outer root sheath are ICAM-1-positive and ICAM-1-de®cient Delta in cell fate decisions: patterning the feather array. Development mice showed signi®cant catagen acceleration compared to wild- 125:767±775, 1998 type control (Muller-Rover et al, 2000). This suggested that Cuff CA, Schwartz J, Bergman CM, Russell KS, Bender JR, Ruddle NH: Lymphotoxin alpha3 induces chemokines and adhesion molecules: insight into ICAM-1 might be implicated in catagen control. On the other the role of LT alpha in in¯ammation and lymphoid organ development. hand, during the wound healing process, ICAM-1 expression is J Immunol 161:6853±6860, 1998 induced in keratinocytes located at the edges of the wound and Cuff CA, Sacca R, Ruddle NH: Differential induction of adhesion molecule and chemokine expression by LTalpha3 and LTalphabeta in in¯ammation ICAM-1-de®cient mice show delays due to impairment of elucidates potential mechanisms of mesenteric and peripheral lymph node keratinocyte migration (Nagaoka et al, 2000). Whereas the authors development. J Immunol 162:5965±5972, 1999 of this paper ascribed the delay of wound healing in ICAM-1- Dof®nger R, Smahi A, Bessia C, et al: X-linked anhidrotic ectodermal dysplasia with de®cient mice to the functional defect in endothelial cells, it is also immunode®ciency is caused by impaired NF-kappaB signaling. Nat Genet 27:277±285, 2001 possible that ICAM-1 in keratinocytes contributes directly to Ekker SC, Ungar AR, Greenstein P, von Kessler DP, Porter JA, Moon RT, Beachy keratinocyte migration. PA: Patterning activities of vertebrate hedgehog proteins in the developing eye ICAM-1-de®cient and MAdCAM-1-de®cient mice, however, and brain. Curr Biol 5:944±955, 1995 do not show remarkable abnormalities in hair follicle development Ezer 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 (Sligh et al, 1993; Muller, personal communication). Moreover, and co-localizes with cytoskeletal structures at lateral and apical surfaces of cells. mice harboring K14-ICAM-1 and K14-MAdCAM-1 transgenes, Hum Mol Genet 8:2079±2086, 1999 which express high levels of ICAM-1 and MAdCAM-1, respect- van Genderen C, Okamura RM, Farinas I, Quo RG, Parslow TG, Bruhn L, Grosschedl R: Development of several organs that require inductive epithelial± ively, in keratinocytes, display no phenotype in hair follicle mesenchymal interactions is impaired in LEF-1-de®cient mice. Dev development (Williams and Kupper, 1994; Nishioka, unpublished 8:2691±2703, 1994 data). Thus, ICAM-1 and MAdCAM-1 may not be requisite Goebeler M, Roth J, Brocker EB, Sorg C, Schulze-Osthoff K: Activation of nuclear molecules for the induction phase of hair placodes, but, as factor-kappa B and gene expression in human endothelial cells by the common haptens nickel and cobalt. J Immunol 155:2459±2467, 1995 mentioned above, might play important roles in the ability of Hardy A, Richardson MK, Francis-West PH, Rodriguez C, Izpisua-Belmonte JC, keratinocytes to compose speci®c structures. Duprez D, Wolpert L: Gene expression, polarising activity and skeletal Notwithstanding these negative results in genetically de®cient patterning in reaggregated hind limb mesenchyme. Development 121:4329± mice, the MAdCAM-1 role in hair placode development is 4337, 1995 Hardy MH: The secret life of the hair follicle. Trends Genet 8:55±61, 1992 detectable in the in vitro culture system. As expected, mAbs that can Headon DJ, Overbeek PA: Involvement of a novel Tnf receptor homologue in hair block MAdCAM-1 interaction to its hematopoietic cell-speci®c follicle induction. Nat Genet 22:370±374, 1999 ligands showed no effect. MAdCAM-1-Ig decreased the length and Headon DJ, Emmal SA, Ferguson BM, et al: Gene defect in ectodermal dysplasia the number of hair follicles in vitro, however. As this inhibitory implicates a death domain adapter in development. Nature 414:913±916, 2001 Hemmerich S, Butcher EC, Rosen SD: Sulfation-dependent recognition of high effect appeared to be speci®c to MAdCAM-1-Ig, we believe that in endothelial venules (HEV)-ligands by L-selectin and MECA 79, and adhesion- a setting such as skin organ culture the molecular redundancy may blocking monoclonal antibody. J Exp Med 180:2219±2226, 1994 be reduced to such a level that it allows us to discern the function of Huelsken J, Vogel R, Erdmann B, Cotsarelis G, Birchmeier W: beta-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin. MAdCAM-1. If this is the case, these data indicate the presence of Cell 105:533±545, 2001 an as yet unidenti®ed ligand for MAdCAM-1 in keratinocytes. Iizuka T, Koike R, Miyasaka N, Miyasaka M, Watanabe T: Cloning and In conclusion, this work identi®ed, for the ®rst time, characterization of the rat MAdCAM-1 cDNA and gene. Biochim Biophys MAdCAM-1 and ICAM-1 as molecules that are induced as a Acta 1395:266±270, 1998 Iizuka T, Tanaka T, Suematsu M, et al: Stage-speci®c expression of mucosal addressin direct outcome of Edar signaling in keratinocytes and de®nes the cell adhesion molecule-1 during embryogenesis in rats. J Immunol 164:2463± molecules that link Edar and expression of MAdCAM-1. Whereas 2471, 2000 the role of those adhesion molecules in hair follicle development Incardona JP, Gaf®eld W, Kapur RP, Roelink H: The teratogenic Veratrum alkaloid remains to be investigated, these ®ndings provide clues for hair cyclopamine inhibits sonic hedgehog signal transduction. Development 125:3553±3562, 1998 placode puri®cation, as well as a valuable insight into the molecular Inoue J, Ishida T, Tsukamoto N, Kobayashi N, Naito A, Azuma S, Yamamoto T: mechanisms regulating the morphogenic activity of follicular Tumor necrosis factor receptor-associated factor (TRAF) family: adapter keratinocytes. proteins that mediate cytokine signaling. Exp Cell Res 254:14±24, 2000 Jordan SA, Jackson IJ: MGF (KIT ligand) is a chemokinetic factor for melanoblast migration into hair follicles. Dev Biol 225:424±436, 2000 Jung HS, Francis-West PH, Widelitz RB, et al: Local inhibitory action of BMPs and We thank Emi K. Nishimura and Mariko Moriyama for technical assistance; their relationships with activators in feather formation: implications for periodic William Gaf®eld and Paul A. Overbeek for providing reagent, and mice, patterning. Dev Biol 196:11±23, 1998 respectively; and Ruth Yu for critical reading of the manuscript. This work was Kashiwagi M, Kuroki T, Huh N: Speci®c inhibition of hair follicle formation by epidermal growth factor in an organ culture of developing mouse skin. Dev Biol supported by grants from the Ministry of Education, Culture, Sports, Science, and 189:22±32, 1997 Technology of Japan (12219209), and a grant from the Center of Developmental Kere J, Srivastava AK, Montonen O, et al: X-linked anhidrotic (hypohidrotic) Biology, RIKEN, Japan. ectodermal dysplasia is caused by mutation in a novel transmembrane protein. Nat Genet 13:409±416, 1996 638 NISHIOKA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Kojima T, Morikawa Y, Copeland NG, Gilbert DJ, Jenkins NA, Senba E, Kitamura Song H, Wang Y, Goetinck PF: Fibroblast growth factor 2 can replace ectodermal T: TROY, a newly identi®ed member of the tumor necrosis factor receptor signaling for feather development. Proc Natl Acad Sci USA 93:10246±10249, superfamily, exhibits a homology with Edar and is expressed in embryonic skin 1996 and hair follicles. J Biol Chem 275:20742±20747, 2000 Srivastava AK, Pispa J, Hartung AJ, et al: The Tabby phenotype is caused by mutation Kumar A, Eby MT, Sinha S, Jasmin A, Chaudhary PM: The ectodermal dysplasia in a mouse homologue of the EDA gene that reveals novel mouse and human receptor activates the nuclear factor-kappaB, JNK, and cell death pathways and exons and encodes a protein (ectodysplasin-A) with collagenous domains. Proc binds to ectodysplasin A. J Biol Chem 276:2668±2677, 2001 Natl Acad Sci USA 94:13069±13074, 1997 Lomaga MA, Yeh WC, Sarosi I, et al: TRAF6 de®ciency results in osteopetrosis and Srivastava AK, Durmowicz MC, Hartung AJ, et al: Ectodysplasin-A1 is suf®cient to defective interleukin-1, CD40, and LPS signaling. Genes Dev 13:1015±1024, rescue both hair growth and sweat glands in Tabby mice. Hum Mol Genet 1999 10:2973±2981, 2001 Mikkola ML, Pispa J, Pekkanen M, Paulin L, Nieminen P, Kere J, Thesleff I: St-Jacques B, Dassule HR, Karavanova I, et al: Sonic hedgehog signaling is essential Ectodysplasin, a protein required for epithelial morphogenesis, is a novel TNF for hair development. Curr Biol 8:1058±1068, 1998 homologue and promotes cell±matrix adhesion. Mech Dev 88:133±146, 1999 Streeter PR, Berg EL, Rouse BT, Bargatze RF, Butcher EC: A tissue-speci®c Monreal AW, Ferguson BM, Headon DJ, Street SL, Overbeek PA, Zonana J: endothelial cell molecule involved in lymphocyte homing. Nature 331:41±46, Mutations in the human homologue of mouse dl cause autosomal recessive and 1998a dominant hypohidrotic ectodermal dysplasia. Nat Genet 22:366±369, 1999 Streeter PR, Rouse BT, Butcher EC: Immunohistologic and functional Muller-Rover S, Bulfone-Paus S, Handjiski B, et al: Intercellular adhesion molecule- characterization of a vascular addressin involved in lymphocyte homing into 1 and hair follicle regression. J Histochem Cytochem 48:557±568, 2000 peripheral lymph nodes. J Cell Biol 107:1853±1862, 1998b Nagaoka T, Kaburagi Y, Hamaguchi Y, et al: Delayed wound healing in the absence Tanabe Y, Roelink H, Jessell TM: Induction of motor neurons by Sonic hedgehog is of intercellular adhesion molecule-1 or L-selectin expression. Am J Pathol 157:237±247, 2000 independent of ¯oor plate differentiation. Curr Biol 5:651±658, 1995 Naito A, Azuma S, Tanaka S, et al: Severe osteopetrosis, defective interleukin-1 Tucker AS, Headon DJ, Schneider P, Ferguson BM, Overbeek P, Tschopp J, Sharpe signalling and lymph node organogenesis in TRAF6-de®cient mice. Genes PT: Edar/Eda interactions regulate enamel knot formation in tooth Cells 4:353±362, 1999 morphogenesis. Development 127:4691±4700, 2000 Oro AE, Scott MP: Splitting hairs: dissecting roles of signaling systems in epidermal Williams IR, Kupper TS: Epidermal expression of intercellular adhesion molecule 1 development. Cell 95:575±578, 1998 is not a primary inducer of cutaneous in¯ammation in transgenic mice. Proc Paul WE, Seder RA: Lymphocyte responses and cytokines. Cell 76:241±251, 1994 Natl Acad Sci USA 91:9710±9714, 1994 Powell BC, Passmore EA, Nesci A, Dunn SM: The Notch signalling pathway in hair Yan M, Wang LC, Hymowitz SG, et al: Two-amino acid molecular switch in an growth. Mech Dev 78:189±192, 1998 epithelial morphogen that regulates binding to two distinct receptors. Science Sacca R, Cuff CA, Ruddle NH: Mediators of in¯ammation. Curr Opin Immunol 290:523±527, 2000 9:851±857, 1997 Yan M, Zhang Z, Brady JR, Schilbach S, Fairbrother WJ, Dixit VM: Identi®cation Schmidt-Ullrich R, Aebischer T, Hulsken J, Birchmeier W, Klemm U, Scheidereit of a novel death domain-containing adaptor molecule for ectodysplasin-A C: Requirement of NF-kappaB/Rel for the development of hair follicles and receptor that is mutated in crinkled mice. Curr Biol 12:409±413, 2002 other epidermal appendices. Development 128:3843±3853, 2001 Zonana J, Elder ME, Schneider LC, et al: A novel X-linked disorder of immune Sligh JE Jr, Ballantyne CM, Rich SS, Hawkins HK, Smith CW, Bradeley A, Beaudet de®ciency and hypohidrotic ectodermal dysplasia is allelic to incontinentia AL: In¯ammatory and immune responses are impaired in mice de®cient in pigmenti and due to mutations in IKK-gamma (NEMO). Am J Hum Genet intercellular adhesion molecule 1. Proc Natl Acad Sci USA 90:8529±8533, 1993 67:1555±1562, 2000