articles

Noggin is a mesenchymally derived stimulator of hair-follicle induction

Vladimir 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, Charit, 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 are governed by intimate interactions between stimulators and inhibitors, the balance of which determines whether or not induction occurs. For ectodermal derivatives such as the neural tube, tooth or feather, potent inhibitors of induction appear to be common and belong to the bone-morphogenetic protein (BMP) superfamily13. The inhibitory activity of BMP-family members is abolished by BMP antagonists, which thus act as stimulators of induction4,5. Noggin, which was first described as a neural inducer1,6, can bind BMP-2 and BMP-4 with high affinity and prevent interaction of these proteins with their cell-surface receptors7. Although neuraltube induction clearly occurs even in the absence of Noggin, noggin-null mice show substantial abnormalities in neural-tube development8. Furthermore, neutralization of BMP-4 by ectopically applied Noggin leads to striking changes in tooth phenotype and to the development of molars instead of incisors9. Like tooth or feather-bud development, hair-follicle morphogenesis in embryonic skin is governed by epithelialmesenchymal interactions, between hair placode keratinocytes and fibroblasts of underlying mesenchymal condensations1012. Although several molecules, such as -catenin, Lef-1 and Sonic hedgehog (Shh), are essential for hair-follicle induction and development1317, the precise role for BMP-2 and BMP-4 in the hair-follicle inductive signalling cascade is unclear. Mice lacking BMP-2, BMP-4 or BMP receptor-I (BMPR-I) die within ten days of embryonic development because of defects in gastrulation and in mesoderm and heart formation1820, while BMP-4 transgenic mice show a retardation in the development of whisker hair follicles21. We proposed that, during hair-follicle morphogenesis, as in neural-tube development1,6,7, Noggin may neutralize the activity of BMP-2 and BMP-4 expressed by the developing placode and mesenchyme16,22,23, and may thus stimulate hair-follicle induction. To elucidate noggins function in hair-follicle induction, we studied its expression patterns in fetal mouse skin and characteristics of hair-follicle development in noggin-null mice compared with wildtype controls. We also assessed the effects of administration of Nog-

gin or BMP-4 on hair-follicle development in embryonic skin organ culture. Finally, we studied alterations in the expression of different morphogens, morphogen receptors, transcription factors and
Stage 1 Stage 3

HP MC

EP HP PCTS

EP

PCTS DP

Stage 6
EP

SG

HS IRS ORS Mel HM

PCTS

DP

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.

158

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

articles
adhesion molecules implicated in the control of hair-follicle induction, and of standard markers of keratinocyte proliferation, differentiation and apoptosis under conditions of noggin deletion (in vivo) or administration (in situ). quently, noggin mRNA was seen in the developing dermal papilla and the proximal connective tissue sheath of the hair follicle during developmental stages 46 of pelage hair-follicle morphogenesis (Fig. 2c). In addition, noggin mRNA was prominently expressed in the dermal papilla of embryonic vibrissa hair follicles (Fig. 2d). These expression patterns, strikingly restricted to the developing dermal papilla, a highly specialized fibroblast population that showed hairfollicle-inductive properties in grafting studies24,25, suggested a role for Noggin as a mesenchymally derived modulator of hair-follicle induction and morphogenesis in vivo. Retarded hair-follicle induction and morphogenesis in nogginknockout mice. To determine the functional significance of this prominent and early expression of noggin in a key mesenchymal hairfollicle compartment that controls hair-follicle morphogenesis1012,24, we quantitatively assessed the rate of hair-follicle induction, as well as the progression of the early steps of hair-follicle morphogenesis, in noggin-knockout (noggin/) mice, which were generated as described8. Although we saw no significant differences in hair-follicle development between heterozygous noggin knockout (noggin+/) and

Results
Expression of noggin during hair-follicle development. We studied the expression of noggin in the embryonic back skin of noggin+/ mice at embryonic day (E)17.5E18.5, that is, during the time period in which maximal induction of pelage hair follicles occurs 1012, by analysing -galactosidase activity from a lacZ gene targeted into the noggin locus8 (Figs 1, 2). Prominent expression of noggin messenger RNA was seen in the mesenchymal condensation immediately below the developing hair placode of stage 13 hair follicle (Fig. 2a, b). Subse-

a
EP HP

10 8 6 4 2 0 Wild type (+/+)

b
Hair follicles (%)

Number of hair follicles per microscope field

70 60 50 40 30 20 10 0 0

** * *
2 4 1 3 Stages of hair-follicle morphogenesis Noggin knockouts (/) Wild type

EP

*
Noggin knockouts (/)

Wild type

Noggin knockouts (/)

c c
2 4 HM MEL 3 2 DER PCM EP 2 EP

d
EP 1 3 2 DER PCM EP
EP EP

e
HP

HM

HP
HP HP

DER

DER
DER DER

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 m. Arrowheads in c, d indicate noggin expression in the proximal connective tissue sheath of the hair follicle.

Figure 3 Retardation of hair-follicle induction and morphogenesis in nogginknockout 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 (ad), 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 meanss.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 m and in e, f represent 25 m.

NATURE CELL BIOLOGY | VOL 1 | JULY 1999 | cellbio.nature.com

1999 Macmillan Magazines Ltd

159

articles
wild-type mice at E17.5 (data not shown), homozygous noggin/ mice showed a significant decrease (P<0.05) in the number of induced hair follicles compared with age-matched wild-type embryos (Fig. 3a). Also, the percentage of hair follicles in advanced stages of morphogenesis was significantly lower (P<0.05) in noggin/ versus wild-type skin (Fig. 3b): at E17.5, about 20% of the hair follicles in wild-type embryos had already reached stage 4 of morphogenesis, whereas stage 12 hair follicles predominated ( P<0.01) in the nogginnull skin (Fig. 3bd). Thus, hair-follicle induction and morphogenesis are both retarded in the absence of the potent BMP-4 antagonist Noggin, even though Noggin is not essential for hair-follicle development (as shown by the fact that hair-follicle development is not completely absent in noggin/ mice). Interestingly, we saw no significant differences in the rate of proliferation of hair-placode keratinocytes from noggin/ and wildtype skin (Fig. 3e, f). However, hair placodes in noggin/ skin, in comparison with wild-type hair follicles, showed a marked increase in the number of cells that stained for TUNEL, that is, in the number of apoptotic cells (Fig. 3e, f). These results indicate that the normal rate of programmed cell death associated with hair-follicle development26 is enhanced in the absence of Noggin. To Noggin operates as a BMP4-neutralizing protein in culture. test further whether Noggin stimulates hair-follicle induction, we implanted Noggin-soaked agarose beads subcutaneously into E13.5 embryonic C57Bl/6 murine back-skin biopsies, which we then kept in organ culture for 48 h (ref. 27). Induction of the first hair follicles in embryonic murine back skin occurs at E14.5 (these hair follicles are the so-called tylotrich pelage hair follicles)10,28; we therefore used the back skin of E13.5 embryos, because it shows no induced hair follicles at the beginning of the experiment. Compared with control skin, Noggin-treated biopsies showed a marked increase in the number of induced 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 epidermis and the proliferation of epidermal cells were markedly increased (P<0.05) compared with control skin (Fig. 4c, dg). To test whether BMP-4 administration inhibits hair-follicle development, we treated embryonic skin biopsies with beads soaked in BMP-4. BMP4-treated biopsies showed a lack of hair-follicle induction, associated with a dramatic reduction in epidermal thickness and proliferation, compared with controls (P<0.001) or Noggin-treated biopsies (Fig. 4a, c, h, i). Administration of agarose beads soaked with a mixture of Noggin and BMP-4 significantly reduced the inhibitory effect of BMP-4 on the epidermal and hairfollicle development induced by Noggin (Fig. 4a, c, j, k). Skin biopsies treated with a mixture of BMP-4 and Noggin exhibited a few stage 1 hair follicles (data not shown) and significantly enhanced epidermal thickness and proliferation (P<0.01) compared with the biopsies treated with BMP-4 alone (Fig. 4c, fk). These results show that Noggin may act as a stimulator of hair placode formation, at least in part by antagonizing the inhibitory effects of BMP-4 on hair-follicle development. noggin deletion affects the expression of Lef-1, NCAM and p75NTR. To explore further the mechanisms by which noggin might act in regulating hair-follicle induction and development, we studied the expression of several factors implicated in the control of the early

a
Number of hair follicles per microscopic field 5 4 3 2 1 0

Number of induced hair follicles

b
Hair follicles (%)

Dynamics of hair-follicle development

100 90 80 70 60 50 40 30 20 10 0 Stage
0

Epidermal proliferation/thickness
0 20 Thickness (m) 40 60 80 100

**

Control

*
Stage
1

* *
Stage
2

Noggin BMP-4 BMP-4+ noggin

0 Stage
3

* *** *** * * * *

Stage
4

Stage
5

20 40 60 80 Ki67-positive cells (%)

100

Control

Noggin

BMP-4 Noggin+ BMP-4

Epidermal proliferation

Epidermal thickness

Control

Noggin

Control d 1 Alkaline phosphatase EP DER f 1 3

Noggin EP 2 DER h

BMP-4 j

Noggin+BMP-4 EP DER

EP DER

e HP EP

g HP EP

i
EP

k
EP

Ki67 (red) + Hoechst 33342 (blue)

DER

DER

DER

DER

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 meanss.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 Ki67 staining) and thickness of epidermis in skin explants. dk, 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 m and in e, g, i, k represent 50 m.

160

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

articles
BMP-2 BMP-4 BMPR-IA
EP

Shh

-catenin
EP

Lef-1 mRNA
EP

Lef-1 -IR

a
WT

EP HP

HP

e
HP

EP

HP

HP

HP

EP

m
HP

EP

DER EP

DER DER

DER DER DER HP EP

DER

b
Noggin /

HP

EP

HP

HP

EP

j
HP

EP

l
HP

EP

n
EP HP

DER

DER

DER

DER

DER

DER

DER

NCAM

p75 NTR

CK10
EP

CK14

o
HP

EP

q * * *
DER

s
EP

u
EP HF

WT

DER

*
DER

HF DER

p
Noggin /
DER

EP

r
EP

EP

v
EP

* *
DER DER

DER

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, -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, -catenin mRNA in hair-placode epithelium (arrows). kn, 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 an represent 25 m and in ov represent 100 m.

steps of hair-follicle morphogenesis11,12 at E17.5. Although at low concentrations noggin is a specific inhibitor of BMP-2 and BMP-4 (ref. 7) and regulates bmp-4 transcription29, we found no increase in the amounts of BMP-2 and BMP-4 mRNAs in the hair placode or underlying mesenchyme of noggin/ mice compared with wild-type controls (Fig. 5ad). We also observed no differences in the expression of BMPR-IA (Fig. 5e, f). The expression of shh and -catenin, key genes that are expressed in the hair placode and which control hair-follicle morphogenesis1517, was practically unchanged in noggin mutants compared with wild-type controls, as judged by in situ hybridization (Fig. 5gj). In striking contrast, the expression of the transcription factor Lef-1, which is critical for hair-follicle induction13,14,30, was substantially reduced in the hair placodes of noggin/ skin (Fig. 5k, l). Furthermore, we saw no immunoreactivity towards Lef-1 in the stage 1 hair follicles of noggin/ mice, whereas hair placodes of wild-type mice showed prominent and highly selective nuclear Lef-1 immunoreactivity (Fig. 5m, n). As neural cell adhesion molecule (NCAM) may be important in the topobiological control of hair-follicle development28,31,32, we also compared immunoreactivity towards NCAM in wild-type and noggin/ skin by immunohistology. NCAM immunoreactivity was substantially decreased in the dermis of noggin mutants compared with in wild-type controls (Fig. 5o, p). The low-affinity neurotrophin receptor p75 (p75NTR; so called because its relative molecular mass is 75,000) is thought to be one of the first growth-factor receptors that is expressed in the dermal papilla during hair-follicle morphogenesis33. Hair-follicle morphogenesis is accelerated in p75NTR-null mice (N.V.B. et al., unpublished observations). We observed a marked increase in p75NTR immunoreactivity in the mesenchyme below the hair placodes in
NATURE CELL BIOLOGY | VOL 1 | JULY 1999 | cellbio.nature.com

noggin/ mice compared with age-matched wild-type controls (Fig. 5q, r). Expression of cytokeratin-10 (CK10), a marker of keratinocyte differentiation34,35, was reduced in the epidermis of noggin/ skin (Fig. 5s, t), whereas the expression of cytokeratin-14 (CK14) in basal epidermal keratinocytes and hair placodes was relatively unchanged between noggin/ and wild-type skin (Fig. 5u, v). Effects of Noggin on Lef-1 expression and p75NTR in situ. To test whether the expression of Lef-1, NCAM and p75NTR, which was altered in the developing hair follicle of noggin/ mice, shows any abnormalities after exogenous administration of Noggin or BMP-4 proteins to normal embryonic mouse skin, we studied skin biopsies cultured in the presence of Noggin or BMP-4 by in situ hybridization (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 placode of control biopsies, whereas enhanced lef-1 transcription was seen in the hair follicles of Noggin-treated biopsies (Fig. 6a, b). Consistent with these in situ hybridization data, immunoreactivity towards Lef-1 was weak in the control biopsies but prominent in the Noggin-treated hair placodes (Fig. 6d, e). Interestingly, no expression of Lef-1 mRNA and no Lef-1 immunoreactivity was seen in the epidermis of BMP4-treated biopsies (Fig. 6c, f). We found no substantial differences in NCAM immunoreactivity between control and Noggin-treated biopsies. However, NCAM immunoreactivity was substantially reduced in the biopsies treated with BMP-4 (Fig. 6gi). In contrast to control skin, only weak p75NTR immunoreactivity was seen around hair placodes after treatment with Noggin, consistent with the marked upregulation of p75NTR immunoreactivity in noggin/ mice (Fig. 5q, r). Interestingly, p75NTR immunoreactivity was absent in the skin mesenchyme after
161

1999 Macmillan Magazines Ltd

articles
Control Noggin
EP

BMP-4
EP

Hair placode
BMP-2
EP

a
HP

b
HP

c
Epidermis
HP DER

Lef-1 NCAM Shh NCAM

Lef-1

BMP-2

Lef-1 mRNA
DER

DER

d
HP EP

e
HP

EP HP

f
Dermis
BMP-4 BMP-4

Lef-1-IR
DER DER

g
NCAM
HP

EP

h
HP

EP

i
EP

p75 NTR

Noggin

p75 NTR

Mesenchymal condensation Inhibitory signals

DER

DER

DER

j
HP

EP

k
HP

EP

l
EP

Blockade of BMP-4 inhibitory signals by Noggin (de-inhibition)

Figure 7 Potential molecular targets for Noggin during hair-follicle induction. Mesenchymally derived Noggin, as a component of the first dermal message10, 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.

p75

NTR

DER DER

HP DER

m
CK10

EP

n
EP

o
EP

DER

DER

DER

p
CK14
DER

EP

EP

r
EP

DER

DER

suprabasal layers of the epidermis in Noggin-treated skin, whereas in BMP4-treated biopsies only weak CK14 immunoreactivity was seen in basal epidermal keratinocytes (Fig. 6pr). These results indicate that, as well as altering hair-follicle induction and early morphogenesis, Noggin and BMP-4 also affect epidermal development by influencing keratinocyte proliferation and differentiation.

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. af, 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). gi, Immunoreactivity towards NCAM is reduced in BMP4-treated skin (i) compared with control (g, arrow) or Noggin-treated (h, arrow) skin. jl, 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). mo, 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 BMP4treated biopsies (o, arrowheads), compared with controls (m, arrowhead). pr, 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 ac, gr represent 25 m, and in df represent 50 m.

Discussion
Hair-follicle morphogenesis occurs as the result of a cascade of inductive interactions between selected epidermal keratinocytes and underlying dermal fibroblasts. The primary inductive signal is probably of mesenchymal origin1012,24. Induction of several ectodermal derivatives, including neural tubes, teeth and feathers, occurs essentially through neutralization of BMP-family proteins that inhibit inductive processes2,3,7. We have shown here that the neural inducer and BMP-neutralizing protein Noggin1 plays an important part in hair-follicle induction. Noggin can be considered as one of the components of the first dermal message10 that initiates hairfollicle morphogenesis. Specifically, Noggin is expressed in the mesenchymal condensation underlying the developing hair placode and in the hair-follicle dermal papilla, a structure that has hair-follicle inductive properties24,25. Deletion of noggin in mice8 is associated with a significant retardation in hair-follicle induction and in the early steps of hair-follicle morphogenesis, whereas administration of Nogginsoaked beads stimulates hair-follicle induction in situ. As Noggin acts as a BMP2/BMP4-neutralizing protein, preventing their interactions with cell-surface receptors7, we propose that the stimulation of hair-follicle induction by Noggin is most likely associated with its ability to antagonize BMP signalling during hairfollicle development. Specifically, Noggin may prevent interactions of BMPs produced by the hair mesenchyme (BMP-4) and placode (BMP-2)16,22,23 with BMPR-IA expressed in the hair-follicle epithelium (Fig. 5). This idea is supported by our finding that BMP-4 suppresses hair-follicle induction in embryonic skin organ culture; this suppression is associated with a complete absence of induced hair follicles and a dramatic reduction in epidermal thickness and proliferation, compared with controls (Fig. 4). Noggin treatment, in contrast, induces an increased number of hair placodes and accel-

treatment with BMP-4 (Fig. 6jl). As epidermal proliferation and thickness were significantly altered after administration of beads soaked with Noggin and BMP4 (Fig. 4), we also studied immunoreactivity towards CK10 and CK14, markers of epidermal development, in the corresponding skin biopsies. Interestingly, the thickness of the CK10-immunoreactive keratinocyte layer in the epidermis was substantially increased after Noggin treatment compared with control epidermis (Fig. 6m, n). CK10 immunoreactivity was markedly reduced in the epidermis after treatment with BMP-4 (Fig. 6o). In contrast to control skin fragments, CK14 immunoreactivity was also found in the
162

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

articles
erates hair-follicle morphogenesis; this phenotype is associated with significantly enhanced epidermal thickness and proliferation (Fig. 4). The fact that Noggin profoundly antagonizes the inhibitory effects of BMP-4 on hair-follicle and epidermal development (Fig. 4) further supports our hypothesis that Noggin affects hair-follicle induction, at least in part, as an antagonist of BMP-4 (Fig. 7). The retardation of hair-follicle induction and morphogenesis in noggin mutants reported here is associated with an upregulation in the number of TUNEL-positive cells in the hair placode. This indicates that BMP-4 enhances apoptotic cell death in the developing hair-follicle epithelium in the absence of Noggin, a property associated with BMP-4 signalling in other models36,37. The retardation of hair-follicle induction and early steps of morphogenesis in noggin/ mice is accompanied by substantial downregulation in the hair placode of Lef-1, a key transcription factor known to regulate hair-follicle induction and morphogenesis13,14,30. In contrast, increased hair-follicle induction and accelerated hairfollicle development after Noggin administration to embryonic skin organ culture are associated with an upregulation of Lef-1 in the hair placode. Although we saw no increase in the expression of BMP-2, BMP-4 and BMPR-IA in the developing hair follicle of noggin mutants compared with wild-type controls, our data indicate that an enhancement of BMP-4 activity in the absence of its antagonist, Noggin, leads to the downregulation of Lef-1 in the hair placode. Presumably, this contributes to the retardation of hair-follicle development (Fig. 7). It has been reported that, during tooth development, ectopically expressed BMP-2 and BMP-4 stimulate lef-1 expression in the mesenchyme30,38. However, it is not known whether BMPs alter lef-1 expression in the mandibular epithelium; such an alteration is functionally important for tooth development30. Perhaps the effects of BMP-4 on lef-1 expression during hair-follicle and tooth development depend on the developmental origin of the respective BMP-4 targets (epithelial hair placode versus mandibular mesenchyme). The expression of the upstream effector of lef-1 signalling, -catenin, which is essential for hair-follicle induction and development15,39, is not altered in noggin mutants. The expression of Shh, which is important in the morphogenesis of the epithelial placode into mature hair follicle16,17, is also unaffected in noggin mutants. In contrast to these two markers, the adhesion molecule NCAM, which is expressed in hair placode and mesenchyme during hair-follicle development28,31,32, is important for the formation of the dermal condensation during feather-bud morphogenesis40, and is substantially reduced in the dermis of noggin/ mice. As BMP-4 downregulates dermal NCAM immunoreactivity in skin organ culture, we propose that the decline in NCAM immunoreactivity in noggin mutants is BMP4-dependent rather than associated with direct effects of Noggin on NCAM expression. Indeed, no differences in NCAM immunoreactivity are seen between Noggintreated and control skin biopsies. We have also shown that noggin mutants exhibit a striking upregulation of p75NTR in the mesenchyme surrounding hair placodes33. Mesenchymal p75NTR expression indeed has an important inhibitory role in hair-follicle development, as p75NTR-null mice show a significant 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 hair placode in noggin mutants, together with the downregulation of p75NTR expression by Noggin treatment, indicates that Noggin may be important in the control of p75NTR expression in vivo. As BMP4treated skin biopsies do not show any increase in mesenchymal p75NTR 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-follicle mesenchyme acts as an important modulator of epithelialmesenchymal interactions during hair-follicle development. Noggin stimulates hair-follicle induction predominantly through antagonistic
NATURE CELL BIOLOGY | VOL 1 | JULY 1999 | cellbio.nature.com

interactions with BMP-4, preventing its interactions with BMPR1A expressed in the hair placode; this leads to the upregulation of lef-1 and NCAM in the developing hair follicle. In addition, Noggin downregulates p75NTR in the hair-follicle mesenchyme, most likely through BMP4-independent pathways (Fig. 7). Neutralization of the inhibitory activity of BMP-family members by BMP antagonists may therefore represent an important mechanism in the inductive signalling cascade induced by the first dermal message10 during hair-follicle morphogenesis.

Methods
Animal 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 (TusseTek), 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 g ml1 L-glutamine and an antibioticantimycotic 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 described 1. Recombinant human BMP4 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 SDSPAGE. Briefly, 200 l Affigel blue beads (Bio-Rad, 100 m in diameter) were soaked with 200 l mouse normal serum (control), 200 l 10 g ml1 Noggin, 10 g ml1 BMP-4, or a mixture of 10 g ml1 Noggin and 10 g ml1 BMP-4 as described3,9. Per experimental group, 46 randomized skin explants, derived from the back skin of 23 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 airliquid 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 described 41, and -galactosidase staining of embryonic limbs of noggin+/ and wildtype 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 -catenin mRNAs16,46 was done as described27. Immunohistochemical detection of BMPR-IA, Lef-1, NCAM, p75NTR, CK10 and CK14 was performed as described31,4749 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 5060 longitudinal hair-follicle sections in 5060 microscopic fields derived from three noggin mutant animals, or from 46 skin biopsies of every experimental group, were analysed and compared to those of 150200 hair follicles from three age-matched wild-type mice or to 5060 hair follicles from skin biopsies of the control group. The thickness of interfollicular epidermis was assessed in cryostat sections of 46 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 100200 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 Students t-test for unpaired samples.
RECEIVED 16 MARCH 1999; REVISED 28 APRIL 1999; ACCEPTED 26 MAY 1999; PUBLISHED 11 JUNE 1999.

1. Lamb, T. M. et al. Neural induction by the secreted polypeptide noggin. Science 262, 713718 (1993). 2. Neubser, A., Peters, H., Balling, R. & Martin, G. R. Antagonistic interactions between FGF and BMP signaling pathways: a mechanism for positioning the sites of tooth formation. Cell 90, 247255 (1997). 3. Jung, H. S. et al. Local inhibitory action of BMPs and their relationships with activators in feather formation: implications for periodic patterning. Dev. Biol. 196, 1123 (1998). 4. Tanabe, Y. & Jessel, T. M. Diversity and pattern in the developing spinal cord. Science 274, 11151123 (1996). 5. Harland, R. & Gerhart, J. Formation and function of Spemann's organizer. Annu. Rev. Cell Dev. Biol. 13, 611667 (1997). 6. Harland, R. M. Neural induction in Xenopus. Curr. Opin. Genet. Dev. 4, 543549 (1994). 7. Zimmerman, L. B., De Jesus Escobar, J. M. & Harland, R. M. The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4. Cell 86, 599606 (1996). 8. McMahon, J. A. et al. Noggin-mediated antagonism of BMP signaling is required for growth and

1999 Macmillan Magazines Ltd

163

articles
patterning of the neural tube and somite. Genes Dev. 12, 14381452 (1998). 9. Tucker, A. S., Matthews, K. L. & Sharpe, P. T. Transformation of tooth type induced by inhibition of BMP signaling. Science 282, 11361138 (1998). 10. Hardy, M. H. The secret life of the hair follicle. Trends Genet. 8, 5561 (1992). 11. Oro, A. E. & Scott, M. P. Splitting hairs: dissecting roles of signaling systems in epidermal development. Cell 96, 575578 (1998). 12. Philpott, M. P. & Paus, R. in Molecular Basis of Epithelial Appendage Morphogenesis (ed. Chuong, C. M.) 75103 (Landes Bioscience, Austin, 1998). 13. van Genderen, C. et al. Development of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient mice. Genes Dev. 15, 26912703 (1994). 14. Zhou, P., Byrne, C., Jacobs, J. & Fuchs, E. Lymphoid enhancer factor 1 directs hair follicle patterning and epithelial cell fate. Genes Dev. 9, 700713 (1995). 15. Gat, U., DasGupta, R., Degenstein, L. & Fuchs, E. De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin. Cell 95, 605614 (1998). 16. St-Jacques, B. et al. Sonic hedgehog signalling is essential for hair development. Curr. Biol 24, 1058 1068 (1998). 17. Chiang, C. et al. Essential role for Sonic hedgehog during hair follicle morphogenesis. Dev. Biol. 205, 19 (1999). 18. Winnier, G., Blessing, M., Labosky, P. A. & Hogan, B. L. Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev. 9, 21052116 (1995). 19. Mishina, Y., Suzuki, A., Ueno, N. & Behringer, R. R. Bmpr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis. Genes Dev. 9, 3027 3037 (1995). 20. Hogan, B. L. Bone morphogenetic proteins in development. Curr. Opin. Genet. Dev. 6, 432438 (1996). 21. Blessing, M., Nanney, L. B., King, L. E., Jones, C. M. & Hogan, B. L. Transgenic mice as a model to study the role of TGF-beta-related molecules in hair follicles. Genes Dev. 7, 204215 (1993). 22. Lyons, K. M., Pelton, R. W. & Hogan, B. L. Organogenesis and pattern formation in the mouse: RNA distribution patterns suggest a role for bone morphogenetic protein-2A (BMP-2A). Development 109, 833844 (1990). 23. Bitgood, M. J. & McMahon, A. P. Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Dev. Biol. 172, 126138 (1995). 24. Jahoda, C. A., Horne, K. A. & Oliver, R. F. Induction of hair growth by implantation of cultured dermal papilla cells. Nature 311, 560562 (1984). 25. Jahoda, A. B. & Reynolds, A. J. Dermal-epidermal interactions adult follicle-derived cell populations and hair growth. Dermatol. Clin. 14, 573583 (1996). 26. Polakowska, R. R., Piacentini, M., Bartlett, R., Goldsmith, L. A. & Haake, A. R. Apoptosis in human skin development: morphogenesis, periderm, and stem cells. Dev. Dyn. 199, 176188 (1994). 27. Botchkarev, V. A. et al. A new role for neurotrophins: involvement of brain-derived neurotrophic factor and neurotrophin-4 in hair cycle control. FASEB J. 13, 395410 (1999). 28. Hardy, M. H. & Vielkind, U. Changing patterns of cell adhesion molecules during mouse pelage hair follicle development. 1. Follicle morphogenesis in wild-type mice. Acta Anat. 157, 169182 (1996). 29. Kim, J. et al. Transcriptional regulation of BMP-4 in the Xenopus embryo: analysis of genomic BMP4 and its promoter. Biochem. Biophys. Res. Commun. 250, 516530 (1998). 30. Kratochwill, K., Dull, M., Farinas, I., Galceran, J. & Grosscheldl, R. Lef1 expression is activated by BMP-4 and regulates inductive tissue interactions in tooth and hair development. Genes Dev. 10, 13821394 (1996). 31. Mller-Rver, S., Peters, E. M. J., Botchkarev, V. A., Panteleyev, A. A. & Paus, R. Distinct patterns of N-CAM expression are associated with defined stages of murine hair follicle morphogenesis and regression. J. Histochem. Cytochem. 46, 14011409 (1998). 32. Mller-Rver, S. & Paus, R. in Molecular Biology of Epithelial Appendage Morphogenesis (ed. Chuong, C. M.) 283314 (Landes Bioscience, Austin, 1998). 33. Holbrook, K. A. & Minami, S. I. Hair follicle embryogenesis in the human. Characterization of events in vivo and in vitro. Ann. NY Acad. Sci. 642, 167196 (1991). 34. Byrne, C., Tainsky, M. & Fuchs, E. Programming gene expression in developing epidermis. Development 120, 23692383 (1994). 35. Eckert, R. L., Crish, J. F. & Robinson, N. A. The epidermal keratinocyte as a model for the study of gene regulation and cell differentiation. Physiol. Rev. 77, 397424 (1997). 36. Schmidt, C., Christ, B., Patel, K. & Brand-Saberi, B. Experimental induction of BMP-4 expression leads to apoptosis in the paraxial and lateral plate mesoderm. Dev. Biol. 202, 253263 (1998). 37. Buckland, R. A., Collinson, J. M., Graham, E., Davidson, D. R. & Hill, R. E. Antagonistic effects of FGF4 on BMP induction of apoptosis and chondrogenesis in the chick limb bud. Mech. Dev. 71, 143 150 (1998). 38. Dassule, H. & McMahon, A. P. Analysis of epithelial-mesenchymal interactions in the initial morphogenesis of the mammalian tooth. Dev. Biol. 202, 215227 (1998). 39. Chan, E. F., Gat, U., McNiff, J. M., & Fuchs, E. A common human skin tumour is caused by activating mutations in -catenin. Nature Genet. 21, 410413 (1999). 40. Jiang, T. X. & Chuong, C.-M. Mechanism of skin morphogenesis. I. Analyses with antibodies to adhesion molecules tenascin, N-CAM, and integrin. Dev. Biol. 150, 8298 (1992). 41. Brunet, L. J., McMahon, J. A., McMahon, A. P. & Harland, R. M. Noggin, cartilage morphogenesis, and joint formation in the mammalian skeleton. Science 280, 14551457 (1998). 42. Paus, R., Hofmann, U., Eichmller, S. & Czarnetzki, B. M. Distribution and changing density of gamma-delta T cells in murine skin during the induced hair cycle. Br. J. Dermatol. 130, 281289 (1994). 43. Cerletti, N. et al. Process for the production of biologically active protein (TGF-). European Patent Application No. 0433 225 A1; filed June 19, 1991. 44. Li, L., Paus, R., Slominski, A. & Hoffman, R. Skin histoculture assay for studying the hair cycle. In Vitro Cell. Dev. Biol. 28, 695698 (1992). 45. Botchkarev, V. A. et al. A new role for neurotrophin-3: involvement in the regulation of hair follicle regression (catagen). Am. J. Pathol. 153, 705719 (1998). 46. Ohsugi, M. et al. Expression and cell membrane localization of catenins during mouse preimplantation development. Dev. Dyn. 206, 391402 (1996). 47. Huber, O. et al. Nuclear localization of beta-catenin by interaction with transcription factor LEF-1. Mech. Dev. 59, 310 (1996). 48. Yamada, N. et al. Bone morphogenetic protein type IB receptor is progressively expressed in malignant glioma tumours. Br. J. Cancer 73, 624629 (1996). 49. Lindner, G. et al. Analysis of apoptosis during hair follicle regression (catagen). Am. J. Pathol. 151, 16011617 (1997). 50. Paus, R., Foitzik, K., Welker, P., Bulfone-Paus, S. & Eichmller, S. Transforming growth factor-beta receptor type I and type II expression during murine hair follicle development and cycling. J. Invest. Dermatol. 109, 518526 (1997). 51. Handjiski, B., Eichmller, S., Hofmann, U., Czarnetzki, B. M. & Paus, R. Alkaline phosphatase activity and localisation during the murine hair cycle. Br. J. Dermatol. 131, 303310 (1994). 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

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

Copyright of Nature Cell Biology is the property of Nature Publishing Group and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.