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Noggin is a mesenchymally derived stimulator of hair-follicle inductionVladimir A. Botchkarev*††, Natalia V. Botchkareva*, Wera Roth†, Motonobu Nakamura‡§, Ling-Hong Chen*,Wiebke Herzog‡, Gerd Lindner*, Jill A. McMahon¶, Christoph Peters†, Roland Lauster‡, Andrew P. McMahon¶

and Ralf Paus*#***Department of Dermatology, Charité, Humboldt-University Berlin, D-13344 Berlin, Germany

†Department of Internal Medicine, University of Freiburg, D-79106 Freiburg, Germany‡Deutsches Rheumaforschungzentrum, D-10117 Berlin, Germany

§Department of Dermatology, Kyoto University, Kyoto 6068507, Japan¶Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA

#Department of Dermatology, University Hospital Eppendorf, University of Hamburg, D-20246 Hamburg, Germany**e-mail: paus@uke.uni-hamburg.de

††e-mail: vladbotc@bu.edu

The induction of developmental structures derived from the ectoderm, such as the neural tube or tooth, occurs through neutralization of the inhibitory activity of members of the bone-morphogenetic protein (BMP) family by BMP antagonists. Here we show that, during hair-follicle development, the neural inducer and BMP-neutralizing protein Noggin is expressed in the follicular mesenchyme, that noggin-knockout mice show significant retardation of hair-follicle induction, and that Noggin neutralizes the inhibitory action of BMP-4 and stimulates hair-follicle induction in embryonic skin organ culture. As a crucial mesenchymal signal that stimulates hair-follicle induction, Noggin operates through antagonistic interactions with BMP-4, which result in upregulation of the transcription factor Lef-1 and the cell-adhesion molecule NCAM, as well as through BMP4-independent downregulation of the 75 kD neurotrophin receptor in the developing hair follicle.

nductive processes during development and organogenesis aregoverned by intimate interactions between stimulators and inhib-itors, the balance of which determines whether or not induction

occurs. For ectodermal derivatives such as the neural tube, tooth orfeather, potent inhibitors of induction appear to be common andbelong to the bone-morphogenetic protein (BMP) superfamily1–3.The inhibitory activity of BMP-family members is abolished byBMP antagonists, which thus act as stimulators of induction4,5.Noggin, which was first described as a neural inducer1,6, can bindBMP-2 and BMP-4 with high affinity and prevent interaction ofthese proteins with their cell-surface receptors7. Although neural-tube induction clearly occurs even in the absence of Noggin, nog-gin-null mice show substantial abnormalities in neural-tubedevelopment8. Furthermore, neutralization of BMP-4 by ectopi-cally applied Noggin leads to striking changes in tooth phenotypeand to the development of molars instead of incisors9.

Like tooth or feather-bud development, hair-follicle morpho-genesis in embryonic skin is governed by epithelial–mesenchymalinteractions, between hair placode keratinocytes and fibroblasts ofunderlying mesenchymal condensations10–12. Although several mol-ecules, such as β-catenin, Lef-1 and Sonic hedgehog (Shh), areessential for hair-follicle induction and development13–17, the preciserole for BMP-2 and BMP-4 in the hair-follicle inductive signallingcascade is unclear. Mice lacking BMP-2, BMP-4 or BMP receptor-I(BMPR-I) die within ten days of embryonic development becauseof defects in gastrulation and in mesoderm and heart formation18–20,while BMP-4 transgenic mice show a retardation in the develop-ment of whisker hair follicles21.

We proposed that, during hair-follicle morphogenesis, as inneural-tube development1,6,7, Noggin may neutralize the activity ofBMP-2 and BMP-4 expressed by the developing placode andmesenchyme16,22,23, and may thus stimulate hair-follicle induction.To elucidate noggin’s function in hair-follicle induction, we studiedits expression patterns in fetal mouse skin and characteristics ofhair-follicle development in noggin-null mice compared with wild-type controls. We also assessed the effects of administration of Nog-

gin or BMP-4 on hair-follicle development in embryonic skin organculture. Finally, we studied alterations in the expression of differentmorphogens, morphogen receptors, transcription factors andI

Figure 1 Summary of noggin expression during hair-follicle morphogenesis. Those cell populations that express noggin are shown in green. The different stages of hair-follicle development are indicated according ref. 10 with modifications12,50. This summary was derived from analysis of >50 longitudinally sectioned follicles from the back skin of five mice at E18.5. DP, dermal papilla; EP, epidermis; HM, hair matrix; HP, hair placode; HS, hair shaft; IRS and ORS, inner and outer root sheath; MC, mesenchymal condensation; Mel, melanin; PCTS, proximal connective tissue sheath; SG, sebaceous gland.

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adhesion molecules implicated in the control of hair-follicle induc-tion, and of standard markers of keratinocyte proliferation, differ-entiation and apoptosis under conditions of noggin deletion (invivo) or administration (in situ).

ResultsExpression of noggin during hair-follicle development. We studiedthe expression of noggin in the embryonic back skin of noggin+/– miceat embryonic day (E)17.5–E18.5, that is, during the time period inwhich maximal induction of pelage hair follicles occurs10–12, by ana-lysing β-galactosidase activity from a lacZ gene targeted into the nog-gin locus8 (Figs 1, 2). Prominent expression of noggin messenger RNAwas seen in the mesenchymal condensation immediately below thedeveloping hair placode of stage 1–3 hair follicle (Fig. 2a, b). Subse-

quently, noggin mRNA was seen in the developing dermal papilla andthe proximal connective tissue sheath of the hair follicle during devel-opmental stages 4–6 of pelage hair-follicle morphogenesis (Fig. 2c).In addition, noggin mRNA was prominently expressed in the der-mal papilla of embryonic vibrissa hair follicles (Fig. 2d). Theseexpression patterns, strikingly restricted to the developing dermalpapilla, a highly specialized fibroblast population that showed hair-follicle-inductive properties in grafting studies24,25, suggested a rolefor Noggin as a mesenchymally derived modulator of hair-follicleinduction and morphogenesis in vivo.Retarded hair-follicle induction and morphogenesis in noggin-knockout mice. To determine the functional significance of thisprominent and early expression of noggin in a key mesenchymal hair-follicle compartment that controls hair-follicle morphogenesis10–12,24,we quantitatively assessed the rate of hair-follicle induction, as well asthe progression of the early steps of hair-follicle morphogenesis, innoggin-knockout (noggin–/–) mice, which were generated asdescribed8. Although we saw no significant differences in hair-follicledevelopment between heterozygous noggin knockout (noggin+/–) and

Figure 2 Expression of noggin in the developing hair follicle. Cryosections of heterozygous noggin knockout (noggin+/–) embryos at E18.5 were stained overnight to detect LacZ activity as described41. noggin is expressed in the dermal condensation around stage 1 hair follicles (a, arrows), in the developing dermal papilla of stage 3 hair follicles (b, arrow), in the dermal papilla of stage 6 hair follicles (in the back skin) (c, arrow) and in vibrissa hair follicles (d, arrow). HP, hair placode; HM, hair matrix; MEL, melanin. Scale bars represent 50 µm. Arrowheads in c, d indicate noggin expression in the proximal connective tissue sheath of the hair follicle.

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Figure 3 Retardation of hair-follicle induction and morphogenesis in noggin-knockout mice. Cryosections of noggin-null and wild-type embryos at E17.5 were stained for the detection of endogenous alkaline phosphatase activity as a sensitive marker of the developing dermal papilla51 (a–d), or were double stained for Ki-67/TUNEL as proliferation/apoptosis markers16 (e, f). The number of hair follicles per microscopic field and the percentage of hair follicles at distinct stages of development10,12,50 were evaluated in the back skin. The skin of noggin-null mice shows a significant decrease in the number of hair follicles compared with wild-type skin (a), and a decrease in the percentage of hair follicles in advanced stages of development compared with wild-type mice (b) (values are means±s.e.m.; *P<0.05; **P<0.01). c, d, Representative examples of wild-type and mutant skin at E17.5. Hair follicles at different stages of development are numbered. e, f, Ki-67 (red fluorescence)/TUNEL (green fluorescence) double staining. In noggin-null skin (f), the hair placode (arrows) and epidermis (arrowheads) show TUNEL-positive staining, whereas no TUNEL-positive staining is seen in the hair placode of wild-type skin (e). Cell nuclei are counterstained with Hoechst 33342. DER, dermis; EP, epidermis; HP, hair placode; PCM, panniculus carnosus muscle. Scale bars in c, d represent 100 µm and in e, f represent 25 µm.

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wild-type mice at E17.5 (data not shown), homozygous noggin–/–

mice showed a significant decrease (P<0.05) in the number ofinduced hair follicles compared with age-matched wild-type embryos(Fig. 3a). Also, the percentage of hair follicles in advanced stages ofmorphogenesis was significantly lower (P<0.05) in noggin–/– versuswild-type skin (Fig. 3b): at E17.5, about 20% of the hair follicles inwild-type embryos had already reached stage 4 of morphogenesis,whereas stage 1–2 hair follicles predominated (P<0.01) in the noggin-null skin (Fig. 3b–d). Thus, hair-follicle induction and morphogene-sis are both retarded in the absence of the potent BMP-4 antagonistNoggin, even though Noggin is not essential for hair-follicle develop-ment (as shown by the fact that hair-follicle development is not com-pletely absent in noggin–/– mice).

Interestingly, we saw no significant differences in the rate of pro-liferation of hair-placode keratinocytes from noggin–/– and wild-type skin (Fig. 3e, f). However, hair placodes in noggin–/– skin, incomparison with wild-type hair follicles, showed a marked increasein the number of cells that stained for TUNEL, that is, in thenumber of apoptotic cells (Fig. 3e, f). These results indicate that thenormal rate of programmed cell death associated with hair-follicledevelopment26 is enhanced in the absence of Noggin. Noggin operates as a BMP4-neutralizing protein in culture. Totest further whether Noggin stimulates hair-follicle induction, weimplanted Noggin-soaked agarose beads subcutaneously into E13.5embryonic C57Bl/6 murine back-skin biopsies, which we then kept inorgan culture for 48 h (ref. 27). Induction of the first hair follicles inembryonic murine back skin occurs at E14.5 (these hair follicles arethe so-called tylotrich pelage hair follicles)10,28; we therefore used the

back skin of E13.5 embryos, because it shows no induced hair folliclesat the beginning of the experiment. Compared with control skin, Nog-gin-treated biopsies showed a marked increase in the number ofinduced hair follicles (P<0.01) as well as a significant acceleration(P<0.05) in the early steps of hair-follicle development (Fig. 4a, b, d,f). In addition, after Noggin administration, the thickness of the epi-dermis and the proliferation of epidermal cells were markedlyincreased (P<0.05) compared with control skin (Fig. 4c, d–g).

To test whether BMP-4 administration inhibits hair-follicledevelopment, we treated embryonic skin biopsies with beadssoaked in BMP-4. BMP4-treated biopsies showed a lack of hair-fol-licle induction, associated with a dramatic reduction in epidermalthickness and proliferation, compared with controls (P<0.001) orNoggin-treated biopsies (Fig. 4a, c, h, i). Administration of agarosebeads soaked with a mixture of Noggin and BMP-4 significantlyreduced the inhibitory effect of BMP-4 on the epidermal and hair-follicle development induced by Noggin (Fig. 4a, c, j, k). Skin biop-sies treated with a mixture of BMP-4 and Noggin exhibited a fewstage 1 hair follicles (data not shown) and significantly enhancedepidermal thickness and proliferation (P<0.01) compared with thebiopsies treated with BMP-4 alone (Fig. 4c, f–k). These results showthat Noggin may act as a stimulator of hair placode formation, atleast in part by antagonizing the inhibitory effects of BMP-4 onhair-follicle development. noggin deletion affects the expression of Lef-1, NCAM and p75NTR.To explore further the mechanisms by which noggin might act in reg-ulating hair-follicle induction and development, we studied theexpression of several factors implicated in the control of the early

Figure 4 Noggin antagonizes BMP-4 and stimulates hair-follicle induction in embryonic skin organ culture. Skin explants taken from C57Bl/6 mouse embryonic back skin at E13.5 were implanted with beads soaked in Noggin, BMP-4, or a mixture of Noggin and BMP-4 and were incubated for 48 h. The number of induced hair follicles, the percentage of hair follicles at distinct stages of morphogenesis, and epidermal proliferation and thickness were evaluated (values are means±s.e.m.; *P<0.05; **P<0.01; ***P<0.001). a, Number of induced hair follicles in skin explants treated with the indicated proteins. b, Dynamics of hair-follicle development in the

control and Noggin-treated biopsies. c, Rate of epidermal proliferation (assessed by Ki-67 staining) and thickness of epidermis in skin explants. d–k, Representative examples, stained by alkaline phosphatase as a sensitive marker of the developing dermal papilla51 (d, f, h, j) or by anti-Ki67 antiserum (e, g, i, k), from the control skin biopsies (d, e) or from biopsies treated with Noggin (f, g), BMP-4 (h, i), or a mixture of Noggin and BMP-4 (j, k). Hair follicles at different stages of development are indicated by numbers (d, f) or arrows (e, g). DER, dermis; EP, epidermis; HP, hair placode. Scale bars in d, f, h, j represent 100 µm and in e, g, i, k represent 50 µm.

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steps of hair-follicle morphogenesis11,12 at E17.5. Although at lowconcentrations noggin is a specific inhibitor of BMP-2 and BMP-4(ref. 7) and regulates bmp-4 transcription29, we found no increase inthe amounts of BMP-2 and BMP-4 mRNAs in the hair placode orunderlying mesenchyme of noggin–/– mice compared with wild-typecontrols (Fig. 5a–d). We also observed no differences in the expres-sion of BMPR-IA (Fig. 5e, f). The expression of shh and β-catenin,key genes that are expressed in the hair placode and which controlhair-follicle morphogenesis15–17, was practically unchanged in nogginmutants compared with wild-type controls, as judged by in situhybridization (Fig. 5g–j). In striking contrast, the expression of thetranscription factor Lef-1, which is critical for hair-follicleinduction13,14,30, was substantially reduced in the hair placodes of nog-gin–/– skin (Fig. 5k, l). Furthermore, we saw no immunoreactivitytowards Lef-1 in the stage 1 hair follicles of noggin–/– mice, whereashair placodes of wild-type mice showed prominent and highly selec-tive nuclear Lef-1 immunoreactivity (Fig. 5m, n).

As neural cell adhesion molecule (NCAM) may be important inthe topobiological control of hair-follicle development28,31,32, we alsocompared immunoreactivity towards NCAM in wild-type and nog-gin–/– skin by immunohistology. NCAM immunoreactivity was sub-stantially decreased in the dermis of noggin mutants compared within wild-type controls (Fig. 5o, p).

The low-affinity neurotrophin receptor p75 (p75NTR; so calledbecause its relative molecular mass is 75,000) is thought to be oneof the first growth-factor receptors that is expressed in the dermalpapilla during hair-follicle morphogenesis33. Hair-follicle morpho-genesis is accelerated in p75NTR-null mice (N.V.B. et al., unpub-lished observations). We observed a marked increase in p75NTR

immunoreactivity in the mesenchyme below the hair placodes in

noggin–/– mice compared with age-matched wild-type controls (Fig.5q, r). Expression of cytokeratin-10 (CK10), a marker of keratinoc-yte differentiation34,35, was reduced in the epidermis of noggin–/– skin(Fig. 5s, t), whereas the expression of cytokeratin-14 (CK14) inbasal epidermal keratinocytes and hair placodes was relativelyunchanged between noggin–/– and wild-type skin (Fig. 5u, v). Effects of Noggin on Lef-1 expression and p75NTR in situ. To testwhether the expression of Lef-1, NCAM and p75NTR, which wasaltered in the developing hair follicle of noggin–/– mice, shows anyabnormalities after exogenous administration of Noggin or BMP-4proteins to normal embryonic mouse skin, we studied skin biopsiescultured in the presence of Noggin or BMP-4 by in situ hybridiza-tion (to assess Lef-1 mRNA expression) and by immunohistology(for expression of Lef-1, NCAM, p75NTR, CK10 or CK14 proteins).Lef-1 mRNA was expressed only weakly in the developing hair pla-code of control biopsies, whereas enhanced lef-1 transcription wasseen in the hair follicles of Noggin-treated biopsies (Fig. 6a, b).Consistent with these in situ hybridization data, immunoreactivitytowards Lef-1 was weak in the control biopsies but prominent in theNoggin-treated hair placodes (Fig. 6d, e). Interestingly, no expres-sion of Lef-1 mRNA and no Lef-1 immunoreactivity was seen in theepidermis of BMP4-treated biopsies (Fig. 6c, f).

We found no substantial differences in NCAM immunoreactiv-ity between control and Noggin-treated biopsies. However, NCAMimmunoreactivity was substantially reduced in the biopsies treatedwith BMP-4 (Fig. 6g–i). In contrast to control skin, only weakp75NTR immunoreactivity was seen around hair placodes after treat-ment with Noggin, consistent with the marked upregulation ofp75NTR immunoreactivity in noggin–/– mice (Fig. 5q, r). Interestingly,p75NTR immunoreactivity was absent in the skin mesenchyme after

Figure 5 Expression of transcription factors, adhesion molecules, cytokeratins and morphogens and their receptors in the skin of wild-type and noggin mutant embryos. Skin sections of wild-type (WT) and noggin–/– mice were analysed at E17.5 for expression of BMP-2, BMP-4, β-catenin, Lef-1 and Shh mRNAs by in situ hybridization, or for immunoreactivity towards BMPR-IA, Lef-1, NCAM, p75NTR, CK10 or CK14 by immunohistochemistry. a, b, BMP-2 mRNA in hair placode (arrows). c, d, BMP-4 mRNA expression in the mesenchyme (arrowheads) and in hair-placode epithelium (arrows). e, f, Immunoreactivity towards BMPR-IA in the hair placode (arrows). g, h, Shh mRNA in hair placode (arrows). i, j, β-catenin mRNA in hair-placode epithelium (arrows). k–n, Downregulation of Lef-1 mRNA (k, l) and immunoreactivity towards Lef-1 (Lef-1-IR) (m, n, pink fluorescence) in hair

placodes of noggin-null mice compared with WT mice. In m, n, nuclei are counterstained by Hoechst 33342 (blue fluorescence). o, p, Downregulation of dermal immunoreactivity towards NCAM (arrows) is visible in the skin of noggin–/– mice. q, r, noggin–/– mice show a marked increase, compared with WT mice, in p75NTR protein amounts in the mesenchyme surrounding the hair placode (arrows). Insets show high magnification of the hair follicle in the regions indicated by asterisks. s, t, Compared with WT epidermis, the epidermis of noggin–/– mice shows a decrease in CK10 immunoreactivity (arrowheads). u, v, CK14 immunoreactivity in basal epidermal keratinocytes (arrowheads) and in hair placode (arrow). DER, dermis; EP, epidermis; HP, hair placode. Scale bars in a–n represent 25 µm and in o–v represent 100 µm.

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treatment with BMP-4 (Fig. 6j–l). As epidermal proliferation and thickness were significantly

altered after administration of beads soaked with Noggin and BMP-4 (Fig. 4), we also studied immunoreactivity towards CK10 andCK14, markers of epidermal development, in the correspondingskin biopsies. Interestingly, the thickness of the CK10-immunore-active keratinocyte layer in the epidermis was substantiallyincreased after Noggin treatment compared with control epidermis(Fig. 6m, n). CK10 immunoreactivity was markedly reduced in theepidermis after treatment with BMP-4 (Fig. 6o). In contrast to con-trol skin fragments, CK14 immunoreactivity was also found in the

suprabasal layers of the epidermis in Noggin-treated skin, whereasin BMP4-treated biopsies only weak CK14 immunoreactivity wasseen in basal epidermal keratinocytes (Fig. 6p–r). These resultsindicate that, as well as altering hair-follicle induction and earlymorphogenesis, Noggin and BMP-4 also affect epidermal develop-ment by influencing keratinocyte proliferation and differentiation.

DiscussionHair-follicle morphogenesis occurs as the result of a cascade ofinductive interactions between selected epidermal keratinocytesand underlying dermal fibroblasts. The primary inductive signal isprobably of mesenchymal origin10–12,24. Induction of several ectoder-mal derivatives, including neural tubes, teeth and feathers, occursessentially through neutralization of BMP-family proteins thatinhibit inductive processes2,3,7. We have shown here that the neuralinducer and BMP-neutralizing protein Noggin1 plays an importantpart in hair-follicle induction. Noggin can be considered as one ofthe components of the ‘first dermal message’10 that initiates hair-follicle morphogenesis.

Specifically, Noggin is expressed in the mesenchymal condensa-tion underlying the developing hair placode and in the hair-follicledermal papilla, a structure that has hair-follicle inductiveproperties24,25. Deletion of noggin in mice8 is associated with a signif-icant retardation in hair-follicle induction and in the early steps ofhair-follicle morphogenesis, whereas administration of Noggin-soaked beads stimulates hair-follicle induction in situ.

As Noggin acts as a BMP2/BMP4-neutralizing protein, prevent-ing their interactions with cell-surface receptors7, we propose thatthe stimulation of hair-follicle induction by Noggin is most likelyassociated with its ability to antagonize BMP signalling during hair-follicle development. Specifically, Noggin may prevent interactionsof BMPs produced by the hair mesenchyme (BMP-4) and placode(BMP-2)16,22,23 with BMPR-IA expressed in the hair-follicle epithe-lium (Fig. 5). This idea is supported by our finding that BMP-4 sup-presses hair-follicle induction in embryonic skin organ culture; thissuppression is associated with a complete absence of induced hairfollicles and a dramatic reduction in epidermal thickness and pro-liferation, compared with controls (Fig. 4). Noggin treatment, incontrast, induces an increased number of hair placodes and accel-

Figure 6 Expression of Lef-1, NCAM, p75NTR, CK10 and CK14 in skin explants cultured in the presence of Noggin- and BMP4-soaked beads. Skin biopsies taken from E13.5 embryos were cultured for 48 h with implanted beads soaked in Noggin or BMP-4. Lef-1 mRNA expression was assessed by in situ hybridization, and immunoreactivity (IR) towards Lef-1, NCAM, p75NTR, CK10 and CK14 was studied by immunohistochemistry. a–f, Upregulation of Lef-1 mRNA and protein levels is seen in the Noggin-treated biopsies (b, e, arrows) compared with controls (a, d, arrows) or BMP4-treated skin (c, f, arrow). g–i, Immunoreactivity towards NCAM is reduced in BMP4-treated skin (i) compared with control (g, arrow) or Noggin-treated (h, arrow) skin. j–l, Mesenchymal p75NTR immunoreactivity is reduced in the Noggin-treated hair placodes (k, arrows) and in the BMP4-treated skin (l) compared with controls (j, arrow). m–o, An increase in epidermal thickness and in CK10 immunoreactivity is observed in suprabasal epidermal layers after treatment with Noggin (n, arrowhead), and a dramatic reduction in CK10 immunoreactivity is seen in the epidermis of BMP4-treated biopsies (o, arrowheads), compared with controls (m, arrowhead). p–r, CK14 immunoreactivity appears in suprabasal epidermal layers of Noggin-treated skin (q, arrowheads), and weak CK14 immunoreactivity is seen in the epidermis after BMP-4 treatment (r, arrowheads), compared with control skin (p, arrowheads). DER, dermis; EP, epidermis. Scale bars in a–c, g–r represent 25 µm, and in d–f represent 50 µm.

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Figure 7 Potential molecular targets for Noggin during hair-follicle induction. Mesenchymally derived Noggin, as a component of the ‘first dermal message’10, antagonizes the inhibitory activity of BMP-2 and BMP-4, and, through de-inhibition mechanisms, stimulates Lef-1 and NCAM expression in the developing hair follicle. Independently of BMP-4, Noggin inhibits p75NTR expression in the follicular mesenchyme.

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erates hair-follicle morphogenesis; this phenotype is associated withsignificantly enhanced epidermal thickness and proliferation (Fig.4). The fact that Noggin profoundly antagonizes the inhibitoryeffects of BMP-4 on hair-follicle and epidermal development (Fig.4) further supports our hypothesis that Noggin affects hair-follicleinduction, at least in part, as an antagonist of BMP-4 (Fig. 7).

The retardation of hair-follicle induction and morphogenesis innoggin mutants reported here is associated with an upregulation inthe number of TUNEL-positive cells in the hair placode. This indi-cates that BMP-4 enhances apoptotic cell death in the developinghair-follicle epithelium in the absence of Noggin, a property associ-ated with BMP-4 signalling in other models36,37.

The retardation of hair-follicle induction and early steps of mor-phogenesis in noggin–/– mice is accompanied by substantial down-regulation in the hair placode of Lef-1, a key transcription factorknown to regulate hair-follicle induction and morphogenesis13,14,30.In contrast, increased hair-follicle induction and accelerated hair-follicle development after Noggin administration to embryonicskin organ culture are associated with an upregulation of Lef-1 inthe hair placode. Although we saw no increase in the expression ofBMP-2, BMP-4 and BMPR-IA in the developing hair follicle of nog-gin mutants compared with wild-type controls, our data indicatethat an enhancement of BMP-4 activity in the absence of its antag-onist, Noggin, leads to the downregulation of Lef-1 in the hair pla-code. Presumably, this contributes to the retardation of hair-follicledevelopment (Fig. 7).

It has been reported that, during tooth development, ectopicallyexpressed BMP-2 and BMP-4 stimulate lef-1 expression in themesenchyme30,38. However, it is not known whether BMPs alter lef-1expression in the mandibular epithelium; such an alteration is func-tionally important for tooth development30. Perhaps the effects ofBMP-4 on lef-1 expression during hair-follicle and tooth develop-ment depend on the developmental origin of the respective BMP-4targets (epithelial hair placode versus mandibular mesenchyme).

The expression of the upstream effector of lef-1 signalling, β-cat-enin, which is essential for hair-follicle induction anddevelopment15,39, is not altered in noggin mutants. The expression ofShh, which is important in the morphogenesis of the epithelial pla-code into mature hair follicle16,17, is also unaffected in nogginmutants. In contrast to these two markers, the adhesion moleculeNCAM, which is expressed in hair placode and mesenchyme duringhair-follicle development28,31,32, is important for the formation of thedermal condensation during feather-bud morphogenesis40, and issubstantially reduced in the dermis of noggin–/– mice. As BMP-4downregulates dermal NCAM immunoreactivity in skin organ cul-ture, we propose that the decline in NCAM immunoreactivity innoggin mutants is BMP4-dependent rather than associated withdirect effects of Noggin on NCAM expression. Indeed, no differ-ences in NCAM immunoreactivity are seen between Noggin-treated and control skin biopsies.

We have also shown that noggin mutants exhibit a striking upreg-ulation of p75NTR in the mesenchyme surrounding hair placodes33.Mesenchymal p75NTR expression indeed has an important inhibitoryrole in hair-follicle development, as p75NTR-null mice show a signifi-cant acceleration of hair-follicle morphogenesis (N.V.B. et al.,unpublished observations). Thus, the marked increase in p75NTR

immunoreactivity in the mesenchyme directly adjacent to the hairplacode in noggin mutants, together with the downregulation ofp75NTR expression by Noggin treatment, indicates that Noggin may beimportant in the control of p75NTR expression in vivo. As BMP4-treated skin biopsies do not show any increase in mesenchymalp75NTR expression, we propose that the effects of Noggin on p75NTR

expression are largely BMP4 independent.Although hair-follicle induction clearly occurs in the absence of

Noggin, our data indicate that Noggin produced by the hair-folliclemesenchyme acts as an important modulator of epithelial–mesen-chymal interactions during hair-follicle development. Noggin stim-ulates hair-follicle induction predominantly through antagonistic

interactions with BMP-4, preventing its interactions with BMPR-1A expressed in the hair placode; this leads to the upregulation oflef-1 and NCAM in the developing hair follicle. In addition, Noggindownregulates p75NTR in the hair-follicle mesenchyme, most likelythrough BMP4-independent pathways (Fig. 7). Neutralization ofthe inhibitory activity of BMP-family members by BMP antagonistsmay therefore represent an important mechanism in the inductivesignalling cascade induced by the ‘first dermal message’10 duringhair-follicle morphogenesis. h

MethodsAnimal models and tissue collection.noggin-knockout mice were generated as described8,41. For the analysis of hair-follicle morphogenesis,

embryonic skin of noggin–/– (n=3), noggin+/– (n=5) and wild-type (n=3) mice was collected at E17.5. For

the analysis of noggin expression, skin of noggin+/– embryos was studied at E18.5. For organ culture

experiments, C57Bl/6 mouse embryos were used at E13.5. In all experiments, embryos or biopsy samples

of the back skin were flash-frozen in liquid nitrogen and were embedded in OCT compound (Tusse-

Tek), using a special technique for obtaining longitudinal cryosections through the hair follicle from one

defined site42.

Skin organ culture.Dorsal skin of E13.5 embryos was dissected in DMEM medium (Gibco) containing 10% fetal calf serum,

50 µg ml–1 L-glutamine and an antibiotic–antimycotic mixture (Sigma), and beads soaked with Noggin,

BMP-4 or a mixture of Noggin and BMP-4 were implanted subcutaneously. Noggin protein was isolated

from the supernatants of Noggin-producing CHO B3.A4 cells as described1. Recombinant human BMP-

4 was expressed in Escherichia coli with a carboxy-terminal histidine tag using a modified pQE vector

(Qiagen) and was purified under denaturing conditions on Ni-NTA agarose (Qiagen). BMP-4 was then

dimerized as described43, and protein concentration was determined by SDS–PAGE. Briefly, 200 µl Affi-

gel blue beads (Bio-Rad, 100 µm in diameter) were soaked with 200 µl mouse normal serum (control),

200 µl 10 µg ml–1 Noggin, 10 µg ml–1 BMP-4, or a mixture of 10 µg ml–1 Noggin and 10 µg ml–1 BMP-4 as

described3,9. Per experimental group, 4–6 randomized skin explants, derived from the back skin of 2–3

different embryos, were placed dermis down on gelatin sponges (Gelfoam, Upjohn) in 35-mm Petri

dishes and were cultured in the same medium at the air–liquid interface at 37 °C in 100% humidity and

5% CO2 atmosphere for 48 h as described44,45. At the end of the incubation, all skin fragments were washed

repeatedly in PBS buffer at 4 °C, flash-frozen in liquid nitrogen and embedded as described above.

In situ hybridization and immunohistochemistry.To study noggin expression in skin, we stained cryosections of noggin+/– embryos at E18.5 overnight to

detect LacZ activity as described41, and β-galactosidase staining of embryonic limbs of noggin+/– and wild-

type mice were used as positive and negative controls, respectively8. In situ hybridization using

digoxygenin-labelled riboprobes for BMP-2, BMP-4, Lef-1, Shh and β-catenin mRNAs16,46 was done as

described27. Immunohistochemical detection of BMPR-IA, Lef-1, NCAM, p75NTR, CK10 and CK14 was

performed as described31,47–49 using corresponding antisera. Double immunovisualization of Ki67- and

TUNEL-stained cells was done as described16. In the selected immunofluorescence procedures, nuclei

were counterstained by Hoechst 33342 (ref. 45).

Quantitative histomorphometry.The number of hair follicles per length unit of epidermis was calculated in skin cryostat sections of noggin

mutants (n=3) at E17.5, and was compared with that of age-matched wild-type controls (n=3). To be

sure that each assessed microscopic field contained new hair follicles, we analysed only every tenth

section from each animal. The percentage of hair follicles in different stages of morphogenesis was

assessed and defined on the basis of accepted morphological criteria10,12,50. To identify the defined

substages of hair-follicle morphogenesis as precisely as possible, we used histochemical detection of

endogenous alkaline phosphatase activity51, as this highlights the developing dermal papilla as a useful

morphological marker for staging hair-follicle development12,50. At least 50–60 longitudinal hair-follicle

sections in 50–60 microscopic fields derived from three noggin mutant animals, or from 4–6 skin biopsies

of every experimental group, were analysed and compared to those of 150–200 hair follicles from three

age-matched wild-type mice or to 50–60 hair follicles from skin biopsies of the control group. The

thickness of interfollicular epidermis was assessed in cryostat sections of 4–6 skin biopsies from every

experimental group and in control biopsies, and at least 100 measurements for every group were taken

using digital image analysis (ISIS Metasystems). All sections were analysed at ×100–200 magnification;

means and s.e.m.s were calculated from pooled data. Differences were judged significant if P<0.05, as

determined by the independent Student’s t-test for unpaired samples.

RECEIVED 16 MARCH 1999; REVISED 28 APRIL 1999; ACCEPTED 26 MAY 1999; PUBLISHED 11 JUNE 1999.

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ACKNOWLEDGEMENTS

We thank R. Pliet for technical assistance; R. Harland for providing the Noggin-producing cell line; A.

Vortkamp and E. Chelkovnikova for the purification of Noggin protein; and O. Huber and K. Funa for

supplying plasmids and antisera. This study was supported by a grant from the Deutsche

Forschungsgemeinschaft to R.P. (Pa 345/8-2). Work in A.P.M.’s laboratory is supported by grants from

the NIH.

Correspondence and requests for materials should be addressed to R.P. or V.A.B.

164 NATURE CELL BIOLOGY | VOL 1 | JULY 1999 | cellbio.nature.com© 1999 Macmillan Magazines Ltd