View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Modulation of BMP Signaling by Noggin is Required for Induction of the Secondary (Nontylotrich) Hair Follicles Vladimir A. Botchkarev, Natalia V. Botchkareva, Andrei A. Sharov, Keiko Funa,* Otmar Huber,² and Barbara A. Gilchrest Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts, U.S.A.; *Department of Anatomy and Cell Biology, University of Gothenburg, Gothenburg, Sweden; ²Department of Clinical Chemistry and Pathobiochemistry, University Hospital Benjamin Franklin, Free University, Berlin, Germany Increasing evidence suggests that morphogenesis of arrest of primary hair follicle development prior to the distinct developmental structures derived from hair shaft formation. The loss of noggin activity was the same organ-committed epithelium is controlled associated with failure to express genes that specify by differential mechanisms. As was recently shown hair follicle cell fates in the epidermis (Lef-1, b-cate- in mice with mutations in the downless (dL) gene, nin, Shh) and dermal papilla (p75 kDa neurotrophin induction of primary or tylotrich hair follicles is receptor, alkaline phosphatase). This suggests that strikingly dependent of signaling through the Tnf regulation of BMP2/4 signaling by noggin is essential receptor homologue, Edar. Here, we show that dor- for the induction of secondary hair follicles, as well sal skin of murine embryos with constitutive deletion as for advanced stages of development in primary of the BMP2/4 antagonist noggin, after transplanta- hair follicles. Key words: appendage/bone morphogenetic tion into SCID mice, is characterized by the lack of proteins/development/hair follicle/Lef-1/morphogenesis/ induction of secondary hair follicles, and by the Shh/skin/Wnt. J Invest Dermatol 118:3±10, 2002 he hair follicle (HF) represents a hair shaft-producing Several indications suggest that induction of primary and mini-organ, which development is governed by the secondary HF may require different signaling pathways. As inductive interactions between epidermal keratino- shown in mice with spontaneous mutation in the downless (dL) cytes committed to HF-speci®c differentiation and a gene (which are characterized by lack of the primary HF and Tpopulation of mesenchymal cells selected to form the apparently normal development of the secondary HF), induction of connective tissue compartment of developing HF ± the dermal primary HF is strikingly dependent of signaling through the Tnf papilla (Hardy, 1992; Philpott and Paus, 1998; Fuchs et al, 2001). receptor homologue, Edar (Headon and Overbeek, 1999). These interactions led to the construction of hair bulb, in which The precise molecular mechanisms initiating the inductive keratinocyte proliferate and differentiate into six distinct cell process of the secondary HF, however, remain to be elucidated. populations, forming the medulla, cortex, and cuticle of the hair The sites of induction of the secondary HF in embryonic epidermis shaft, as well as the cuticle, Huxley, and Henle layers of the inner show expression of Lef-1 transcription factor, b-catenin, and root sheath. Inner root sheath separates the hair shaft from the outer transforming growth factor-b receptor type II, implicating import- root sheath, which forms the external concentric layer of epithelial ance of the Wnt and transforming growth factor-b signaling cells in the HF (Sengel, 1976). pathways for these processes (Zhou et al, 1995; Paus et al, 1997; In mice, fur consists of a number of different types of HF Botchkarev et al, 1999a; DasGupta and Fuchs, 1999; Paus et al, characterized by differences in their morphology and time-course 1999). Indeed, Lef-1 and transforming growth factor-b2 knockout of induction during embryogenesis (Lyon et al, 1996; Vielkind and mice are characterized by the signi®cantly reduced number of HF Hardy, 1996). Primary or tylotrich (guard) HF, which make up and retardation of HF development (van Genderen et al, 1994; approximately 5±10% of all HF in mouse dorsal skin and are Foitzik et al, 1999). Instead, Lef-1 overexpression leads to the characterized by a large hair bulb, long straight hair, and two ectopic HF formation in the oral epithelium, and overexpression of sebaceous glands, begin to be induced at embryonic day 14.5 b-catenin, the upstream effector of Lef-1 in the Wnt signaling (E14.5) (Vielkind and Hardy, 1996; Philpott and Paus, 1998; Paus pathway, induces HF neogenesis in postnatal skin (Zhou et al, 1995; and Cotsarelis, 1999). Induction of the secondary or nontylotrich Gat et al, 1998). (awl and zigzag) HF producing thinner and shorter hair shafts and Recent data have proven previous observations suggesting a having one sebaceous gland, occurs from E16.5 to postnatal day 0.5 fundamental role for Wnt/b-catenin/Lef-1 signaling pathway in (P0.5). hair development (reviewed in Fuchs et al, 2001; Cotsarelis and Millar, 2001). Wnt 10a and Wnt 10b are expressed in placodes at the onset of HF morphogenesis, and Wnt 5a is expressed in the Manuscript received July 16, 2001; revised September 19, 2001; developing dermal papilla (Reddy et al, 2001). Conditional accepted for publication October 10, 2001. disruption of b-catenin in epidermis leads to the failure in Reprint requests to: Dr. V. A. Botchkarev, Department of Dermatology, Boston University School of Medicine, 609 Albany Street, Boston, MA induction of both primary and secondary HF (Huelsken et al, 02118. Email: [email protected] 2001). Furthermore, overexpression of dominant-negative Lef-1 0022-202X/01/$15.00 ´ Copyright # 2002 by The Society for Investigative Dermatology, Inc. 3 4 BOTCHKAREV ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Figure 1. Alterations of the HF induction and development in the noggin null embryos and in the noggin null skin transplanted on to SCID mice. Dorsal skin of homozygous (±/±) noggin knockout embryos (n = 8) and corresponding wild-type embryos (n = 10) was studied at E16, E18.5, and 3 wk after transplantation on to 7±8 wk old SCID female mice (n = 8). Skin sections were processed for histoenzymatic detection of alkaline phosphatase to visualize HF dermal papilla. (A)Number of induced HF per mm of epidermal length in the wild-type and noggin null embryos and skin grafts (Student's t test, **p < 0.01, ***p < 0.001). (B, C) E16: wild-type (B)and noggin null skin (C)show presence of single primary HF (arrows). (D, E) E18.5: wild-type skin (D)shows numerous HF, whereas only single HF are seen in the noggin null skin (E, arrow). (F±H) Three weeks after transplantation: wild-type skin grafts show hair emerging through the epidermis (F, animal on left, arrow) and fully developed HF (G, arrows), whereas the noggin null skin grafts show no hairs (F, animal on the right, arrow) and arrest of development in single induced HF at stage 4 is seen (H, arrow). (G, H) HF of the albino host can be distinguished by their lack of black melanin granules and are indicated by arrowheads.(B±E, H) The stage of HF development for every HF is indicated by the Arabic numerals. Scale bars: 100 mm. driven by K14 promoter leads to the suppression of hair During induction of the secondary HF, BMP2 and BMP differentiation and gives rise to sebocyte differentiation (Merrill et receptor IA (BMPR-IA) are found in the hair placode, i.e., in the al, 2001). sites of Lef-1/b-catenin expression, whereas BMP4 and BMP Several indications suggest that during development, Wnt antagonist noggin are expressed in cells of the mesenchymal signaling could be modulated by members of the bone morpho- condensation (Bitgood and McMahon, 1995; St-Jacques et al, genetic protein (BMP) superfamily (Chuong et al, 1996; 1998; Botchkarev et al, 1999a). In a previous study, we showed that Kratochwill et al, 1996; Dassule and McMahon, 1998; Noramly in the E17.5 noggin knockout embryos, the number of induced HF and Morgan, 1998; Baker et al, 1999; Noramly et al, 1999; Miyazaki is signi®cantly decreased and HF development is retarded, com- et al, 2000). In turn, BMP2 and BMP4 represent important pared with wild-type embryos (Botchkarev et al, 1999a). This was downstream components of b-catenin during hair placode forma- accompanied by downregulation of Lef-1 in the hair placodes tion (Huelsken et al, 2001). BMP ful®ll multiple functions during (Botchkarev et al, 1999a) suggesting that neutralization of BMP2/ development (Hogan, 1996, 1999). In particular, BMP2 and BMP4 BMP4 by its antagonist noggin stimulates HF induction, at least in inhibit the induction of ectodermal derivatives, such as neural tube, part, via activation of Wnt pathway; however, as at E17.5 skin tooth, feather, and HF (Lamb et al, 1993; Neubuser et al, 1997; already contains both primary and secondary HF (Vielkind and Jung et al, 1998; Botchkarev et al, 1999a), and BMP4 transgenic Hardy, 1996; Philpott and Paus, 1998), in a previous study we were mice show a retardation of HF development (Blessing et al, 1993). unable to identify precisely a role for BMP2/BMP4 and noggin in VOL. 118, NO. 1 JANUARY 2002 NOGGIN STIMULATES THE SECONDARY HAIR FOLLICLE INDUCTION 5 the control of induction of the secondary HF (Botchkarev et al, histochemical detection of endogenous alkaline phosphatase activity was 1999a). used, as this highlights the dermal papilla as a useful morphologic marker In this study, we explore the roles for BMP2, BMP4, and their for staging HF development and cycling (Handjiski et al, 1994; Paus et al, antagonist noggin in the control of induction of the secondary 1999). All sections were analyzed at 100±2003 magni®cation, mean and SEM were calculated from pooled data. Differences were judged as (nontylotrich) HF. Speci®cally, we compare the rate of HF signi®cant if the p < 0.05, as determined by the independent Student's t induction between wild-type and noggin null embryos of different test for unpaired samples.