3801DEVELOPMENT AND DISEASE RESEARCH ARTICLE

INTRODUCTIONBMPs were discovered as potent inducers of ectopic bone formationwhen implanted subcutaneously (Urist, 1965). Studies of humandisorders chondrodysplasia (Thomas et al., 1996) and fibrodysplasiaossificans progressiva (Shore et al., 2006) indicate the importanceof BMP signaling in cartilage and muscle, respectively, suggestingthat BMP signaling plays an important role in controllingmineralization in musculoskeletogenesis. Bone is the primarycomponent in skeletogenesis, and osteoblasts are the predominantcell type in bone. However, the mechanism by which BMP signalingin osteoblasts contributes the skeletogenesis has not been fullydescribed.

The majority of bones, including long and ectopic bone, areformed through an endochondral process (Kronenberg, 2003).Condensed mesenchymal cells differentiate into chondrocytes toform a cartilage template that is later replaced by osteoblasts(Mackie et al., 2008; Maes et al., 2007). Mesenchymal cells andchondrocytes respond to BMP signaling to differentiate andmaintain their features in vivo (Bandyopadhyay et al., 2006; Tsuji etal., 2006; Yoon et al., 2005). By contrast, during the alternate processof intramembranous bone formation, mesenchymal cellsdifferentiate directly into osteoblasts without going through acartilaginous phase. In this study, we genetically altered BMP

signaling in osteoblasts using a mouse model. To avoid secondaryeffects from chondrocytes on osteoblasts in the endochondralprocess, we primarily examined intramembranous bone formation(e.g. calvaria) and demonstrated the direct effects that BMPsignaling has on osteoblasts.

BMP receptor type IA (BMPR1A), which is abundantlyexpressed in bone, is activated by major BMP ligands BMP2 andBMP4. Conventional knockout of BMP2, BMP4 and BMPR1A inmice leads to embryonic death before bone development (Mishinaet al., 1995; Winnier et al., 1995; Zhang and Bradley, 1996). Wepreviously disrupted Bmpr1a during adult stages in an osteoblast-specific manner using Og2-Cre mice (Mishina et al., 2004). Thisstudy suggests that the response of osteoblasts to loss of BMPsignaling is age dependent, as bone volume decreased in young micebut increased in old mice. Similarly, the mechanism by which BMPsignaling regulates bone mass is not straightforward, as loss-of-function of BMP2 and gain-of-function of BMP4 both reduce bonemass (Okamoto et al., 2006; Tsuji et al., 2006). Bone mass isdetermined by the balance of bone formation and resorption, andosteoblasts regulate both processes. Thus, we focused on osteoblastsand addressed the complicated effect of BMP signaling on bonemass.

Human genetic studies have shown that loss-of-functionmutations in components of Wnt signaling, such as the Wnt co-receptor low-density lipoprotein receptor-related protein 5 (LRP5),is associated with osteoporosis (Gong et al., 2001; Patel andKarsenty, 2002). Dominant missense LRP5 mutations are associatedwith high bone mass (HBM) diseases (Boyden et al., 2002; Little etal., 2002; Van Wesenbeeck et al., 2003), indicating that canonical/β-catenin Wnt signaling enhances bone mass (Baron et al., 2006; Glassand Karsenty, 2006; Krishnan et al., 2006). In vitro, Wnt signalinginduces BMP expression (Bain et al., 2003; Winkler et al., 2005),whereas BMPs induce Wnt expression (Chen et al., 2007; Rawadi

BMP signaling negatively regulates bone mass throughsclerostin by inhibiting the canonical Wnt pathwayNobuhiro Kamiya1, Ling Ye3, Tatsuya Kobayashi4, Yoshiyuki Mochida5, Mitsuo Yamauchi5,Henry M. Kronenberg4, Jian Q. Feng3 and Yuji Mishina1,2,*

Bone morphogenetic proteins (BMPs) are known to induce ectopic bone. However, it is largely unknown how BMP signaling inosteoblasts directly regulates endogenous bone. This study investigated the mechanism by which BMP signaling through the typeIA receptor (BMPR1A) regulates endogenous bone mass using an inducible Cre-loxP system. When BMPR1A in osteoblasts wasconditionally disrupted during embryonic bone development, bone mass surprisingly was increased with upregulation of canonicalWnt signaling. Although levels of bone formation markers were modestly reduced, levels of resorption markers representingosteoclastogenesis were severely reduced, resulting in a net increase in bone mass. The reduction of osteoclastogenesis wasprimarily caused by Bmpr1a-deficiency in osteoblasts, at least through the RANKL-OPG pathway. Sclerostin (Sost) expression wasdownregulated by about 90% and SOST protein was undetectable in osteoblasts and osteocytes, whereas the Wnt signaling wasupregulated. Treatment of Bmpr1a-deficient calvariae with sclerostin repressed the Wnt signaling and restored normal bonemorphology. By gain of Smad-dependent BMPR1A signaling in mice, Sost expression was upregulated and osteoclastogenesis wasincreased. Finally, the Bmpr1a-deficient bone phenotype was rescued by enhancing BMPR1A signaling, with restoration ofosteoclastogenesis. These findings demonstrate that BMPR1A signaling in osteoblasts restrain endogenous bone mass directly byupregulating osteoclastogenesis through the RANKL-OPG pathway, or indirectly by downregulating canonical Wnt signalingthrough sclerostin, a Wnt inhibitor and a bone mass mediator.

KEY WORDS: BMP receptor IA, Bone mass, Canonical Wnt signaling, Osteoblast, Osteoclastogenesis, Sclerostin, Mouse

Development 135, 3801-3811 (2008) doi:10.1242/dev.025825

1Laboratory of Reproductive and Developmental Toxicology, NIEHS/NIH, ResearchTriangle Park, NC 27709, USA. 2School of Dentistry, University Michigan, Ann Arbor,MI 48109, USA. 3School of Dentistry, University of Missouri-Kansas City, Kansas City,MO 64108, USA. 4Endocrine Unit, Massachusetts General Hospital and HarvardMedical School, Boston, MA 02114, USA. 5Dental Research Center, University ofNorth Carolina, Chapel Hill, NC 27599, USA.

*Author for correspondence (e-mail: mishina@umich.edu)

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et al., 2003), suggesting that both BMP and Wnt signaling maysynergistically regulate each other in osteoblast, possibly throughautocrine/paracraine loop. Both BMP and Wnt signaling inducebone mass; however, the mechanism by which BMP and Wntsignaling cooperate to affect bone mass is not well understood,particularly during embryonic development when bone massdramatically increases.

Here, we have employed a tamoxifen-inducible Cre-loxP systemunder the control of a 3.2 kb type I collagen promoter and havedisrupted or upregulated BMP signaling through BMPR1A inosteoblasts during embryonic bone development. We unexpectedlyfound increased bone mass in response to loss of BMPR1A inosteoblasts and a new interaction between BMP and Wnt signalingthrough sclerostin.

MATERIALS AND METHODSMice and tamoxifen administrationMice expressing the tamoxifen (TM)-inducible Cre fusion protein Cre-ERTM

(Danielian et al., 1998; Hayashi and McMahon, 2002) under the control ofa 3.2 kb mouse pro-collagen α1(I) promoter (Col1-CreERTM), which isactive in osteoblasts, odontoblasts and tendon fibroblasts (Rossert et al.,1995), were generated by pronuclear injection and crossed with floxedBmpr1a mice (Mishina et al., 2002). Mice that conditionally express aconstitutively active form of Bmpr1a (caBmpr1a), which has a mutation inthe GS domain of BMPR1A that results in ligand-independent activation ofSmad signaling after Cre recombination, were generated using a transgenicconstruct similar to one previously reported (Fukuda et al., 2006) (see Fig.S4A in the supplementary material). ROSA26 Cre reporter (R26R) (Soriano,1999) and TOPGAL (DasGupta and Fuchs, 1999) mice were obtained fromDr Philippe Soriano and the Jackson Laboratory, respectively. Tamoxifen(TM; Sigma) was dissolved in a small volume of ethanol, diluted with cornoil at a concentration of 10 mg/ml. TM (75 mg/kg) was injectedintraperitoneally daily into pregnant mice (100 to 200 ml/mouse) for at least3 days starting at E13.5.

Histological analysis and skeletal preparationWhole-mount β-gal staining was performed as previously described(Mishina et al., 2004). For histological analysis, fetuses were fixed in 4%paraformaldehyde, embedded in paraffin, and sectioned frontally forcalvariae and sagittally for long bones at 6 μm. Sections were stained withHematoxylin and Eosin or Eosin alone for β-gal stained samples. For vonKossa staining to detect mineral deposition, sections were covered withfiltered 5% silver nitrate (Sigma), exposed to ultraviolet light for 45 minutesand placed in 5% sodium thiosulfate (Sigma) for a few seconds. For BrdU(bromodeoxyuridine) incorporation, 100 μM of BrdU (Roche) was injectedinto pregnant females intraperitoneally 2 hours before collecting calvariae.TRAP (tartrate resistant acid phosphatase) staining was performed using theleukocyte acid phosphatase kit (Sigma). Immunostaining was performedusing primary antibodies against BMPR1A (Orbigen) (Yoon et al., 2005)and phospho-Smad1, -Smad5, -Smad8 (Cell Signaling) and sclerostin(R&D). Alexa Fluor (488, 594, Molecular Probes) and ABC kit (Santa CruzBiotechnology) were used for detection. Frozen frontal sections at 10 μmwere prepared for phospho-Smad1, -Smad5 and -Smad8 antibodies. Forskeletal preparations, mice were dissected and fixed in 100% ethanol, andthen stained with Alcian Blue and Alizarin Red. To count total cell numberin sections, 1 μM of DAPI was treated for 10 minutes.

Quantitative real time RT-PCR (QRT-PCR)RNA was isolated from calvariae using the Micro-FastTrack 2.0 Kit(Invitrogen). cDNA was synthesized using the SuperScript PreamplificationSystem (GIBCO). PCR reactions, data quantification and analysis wereperformed (Applied Biosystems). Values were normalized to Gapdh usingthe 2-ΔΔCt method (Livak and Schmittgen, 2001).

Calvaria and osteoblast cultureFor ex vivo bone culture, newborn calvariae were dissected at the sagittalsuture and cultured in modified BGJ (Invitrogen) supplemented withascorbic acid (Sigma, 50 mg/ml) and 5% fetal bovine serum for the first

24 hours in culture. Hemicalvariae were treated with 4-hydroxyl TM(Sigma, 100 ng/ml) and sclerostin (R&D, 50 ng/ml) in the absence ofserum for 5 days. For in vitro culture, osteoblasts were isolated fromconditional knockout (cKO) and wild-type newborn calvariae, andcultured in the same media with addition of 4-hydroxyl TM (Sigma, 100ng/ml) every other day.

RESULTSTissue specificity and efficiency of Crerecombinase in Col1-CreERTM miceTamoxifen (TM)-inducible Col1-CreERTM mice were mated toROSA26 reporter mice (R26R) to monitor Cre activity assessed bystaining for β-galactosidase (β-gal). When TM was injected atembryonic day (E) 13.5 or earlier (E10.5), β-gal positive cells werefirst visible at E14.5 both in intramembranous and endochondralbones (data not shown). Daily injections for 3 days from E13.5established strong β-gal activity at E18.5 (Fig. 1A). Neither Crenegative littermates nor Cre-positive embryos that did not receiveTM showed β-gal activity (Fig. 1A, data not shown). Cre is activein immature periosteum wrapping growth plates, in osteogeniccenters and in bone collars, but not in chondrocytes (Fig. 1B). By β-gal staining of whole calvariae, the positive areas started at thetemporal border of parietal bones and expanded gradually asintramembranous bone formation progressed from E15.5 to E18.5(Fig. 1C), where Cre was active in osteoblasts by histology. Theseresults demonstrate the specificity and efficiency of Crerecombinase in Col1-CreERTM mice during embryonic bonedevelopment.

Developmental abnormalities in BMPR1A cKObonesCol1-CreERTM mice were bred into mice homozygous for floxedBmpr1a. Bmpr1a cKO fetuses (cKO, Cre+; Bmpr1a fx/fx) andwild-type controls (WT, Cre-; Bmpr1a fx/fx) were collected afterdaily TM injection into pregnant female from E13.5. Grossmorphology of E18.5 cKO was normal (data not shown).Production of BMPR1A was suppressed in osteoblasts andosteocytes in the cKO (Fig. 1D), demonstrating efficient andspecific loss-of-function of BMPR1A in cKO osteoblasts.Phosphorylated Smad 1/5/8 was modestly reduced in cKO (Fig.1E), presumably owing to remaining signals from other type Ireceptors, such as BMPR1B and ACVR1.

Mineralization was increased in cKO parietal bones whenassessed by Alizarin Red staining, although skeletal shape in theforelimb and hindlimb were unchanged (Fig. 2A). The ratio ofbone volume to total tissue volume (BV/TV) was 30% higher incKO calvariae and femora at E18.5 when assessed by microcomputed tomography (μCT) (Fig. 2B). Hematoxylin and Eosinstaining of E18.5 calvariae demonstrated that cKO parietal boneswere markedly thicker than wild type (Fig. 2C). The cKO bonyarea positive for Eosin was loose, discontinuous and disorganized,whereas in wild type it was compact and lamellar in structure. VonKossa staining for Ca2+ in mineralized tissue showed increases inmineralized area of cKO calvariae where bone continuity wasdisrupted (Fig. 2D). In cKO femora where bone mass wasincreased (Fig. 2B), woven bone was increased in the primaryspongiosa (Fig. 2E). By BrdU incorporation for detection ofproliferating cells in vivo, positive cells per total cells in bone wereunchanged in cKO calvaria (Fig. 2F). These facts suggest that bonemass of cKO was increased both in calvariae and femora, whichfollow intramembranous and endochondral ossification,respectively.

RESEARCH ARTICLE Development 135 (22)

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Effects of BMPR1A signaling on bone formationand resorption markersBone mass is determined by the balance between bone formationand resorption. We examined changes in bone formation markersusing calvariae and QRT-PCR. Expression of Runx2 and osterix(Sp7), genes that are required for osteoblast differentiation, and bonesialoprotein (Ibsp), which is induced rapidly prior to calcification,were reduced in E16.5 cKO (Fig. 3A). However, expression of thesegenes, as well as of alkaline phosphatase (Akp2; Alpl – MouseGenome Informatics) and osteocalcin (Bglap2) was unchanged atE18.5. In addition, alkaline phosphatase activity was also unchangedat E18.5 (data not shown). These results suggest that bone formationis modestly reduced in E16.5 cKO calvariae, which could impact thephenotype of E18.5.

Among bone resorption markers expressed by osteoclasts,expressions of Mmp9 and cathepsin K (Ctsk) were decreased 25%and 35% in E19.5 cKO calvariae, respectively, and that of tartrateresistant acid phosphatase (Trap) was reduced 30% and 35% atE18.5 and E19.5, respectively (Fig. 3B). Osteoclastogenesis isregulated by RANKL (receptor activator of NFκB ligand), anosteoclast differentiation factor, and OPG (osteoprotegerin), a decoyreceptor for RANKL, both expressed by osteoblasts (Simonet et al.,1997). Rankl expression was reduced 30% at E19.5, and Opg wasincreased 50% and 80% at E18.5 and E19.5, respectively (Fig. 3B),resulting in the reduction of the ratio, Rankl to Opg, by 30% and60% at E18.5 and E19.5, respectively (Fig. 3C). As RANKL

stimulates osteoclastogenesis, whereas OPG inhibits it, thesechanges were consistent with the reduced osteoclastogenesis in cKOcalvariae (Fig. 3B). Consistent with these data, osteoclast activityevaluated by TRAP staining was significantly reduced in cKOcalvariae at E18.5 (Fig. 3D). In addition, expressions of other BMPreceptors type I (Bmpr1b, Acvr1), type II (Bmpr2, Acvr2a, Acvr2b)and their ligands (Bmp2, Bmp4, Bmp6 and Bmp7), were notincreased in cKO calvariae at E18.5 when assessed using QRT-PCR(Fig. 3E), suggesting that a compensation for the loss of BMPR1Aby other BMP receptors or ligands is unlikely. These results suggestthat osteoclastogenesis in cKO calvariae was decreased by loss ofBMPR1A.

Effects of BMPR1A signaling on OPG and RANKL invitroBmpr1a-deficient osteoblasts were reproduced by infectingprimary osteoblasts from Bmpr1a fx/fx calvariae withrecombinant adenovirus expressing Cre protein. Expressions ofBmpr1a and Rankl were reduced 70% and 40%, respectively,whereas Opg increased 2.5-fold in cKO osteoblasts (CRE)compared with control (Mock) (see Fig. S1A in the supplementarymaterial), resulting in reduced ratio of Rankl to Opg by 80% (seeFig. S1A in the supplementary material). These indicate reducedosteoclastogenesis in Bmpr1a-deficient osteoblasts at leastthrough the RANKL-OPG pathway, consistent with in vivo data(Fig. 3).

3803RESEARCH ARTICLESclerostin is a downstream target of BMPR1A

Fig. 1. Tamoxifen (TM)-dependent and osteoblast-specific Cre activity using Cre-ERTM mice. (A) Cre-ERTM transgenic mice mated withROSA26 Cre reporter mice (R26R). TM was injected daily into pregnant females for 3 days from E13.5 to E15.5. Cre-negative and -positivelittermates were stained for β-gal at E18.5. (B) Cre activity in long bones. Forelimbs were stained for β-gal at E18.5. C, chondrocytes; black arrow,periosteum; red arrow, osteogenic center. Scale bars: 50μm. (C) Cre recombination in calvariae at E15.5, E16.5 and E18.5 in whole head (lateraland overhead views) and histology of E18.5 calvariae. Broken line, areas of parietal bones (P). Scale bar: 20μm. (D) BMPR1A as evaluated byimmunohistochemistry using cKO calvariae at E18.5. BMPR1A, green; DAPI (nuclei), blue. Scale bars: 10μm. (E) Immunoblotting for phosphorylatedSmad 1/5/8 using E18.5 calvariae. The membrane was treated with polyclonal rabbit anti-phospho-Smad1/5/8 (1:1000) and monoclonal mouseanti-beta-actin (1:2000), and visualized by ECL.

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Monocytic cells, precursors of osteoclasts, were isolated from adultcKO and wild-type spleens, and were induced by RANKL and M-CSF. There was no difference both in osteoclast number and Bmpr1aexpression levels between wild-type and cKO cells (see Fig. S1B,Cin the supplementary material), suggesting that cKO osteoclasts areintact and able to fully accomplish osteoclastogenesis by respondingto these cytokines. Next, a mixture of osteoblasts and osteoclasts wasisolated from adult cKO and wild-type bone marrow cellsindependently, and induced by 1α, 25-dihydroxyvitamin D3. The cKOmixture was defective in osteoclast number and Bmpr1a expressionlevels (see Fig. S1B,C in the supplementary material). There was noCre activity in osteoclasts in bone marrow (data not shown). Theseresults suggest that osteoclasts in cKO bone marrow are intact butcannot accomplish osteoclastogenesis, partly because of a defect inRANKL-OPG signaling in Bmpr1a-deficient osteoblasts. Takentogether, these data suggest that the reduced osteoclastogenesis incKO calvaria (Fig. 3) is primarily caused by defects in osteoblasts.

Upregulation of canonical Wnt signaling in cKObonesBoth BMP and Wnt signaling (Baron et al., 2006; Glass andKarsenty, 2006; Hartmann, 2006; Krishnan et al., 2006) regulatebone mass. However, there is no described interaction between the

two in either mouse models or human mutation. We firstinvestigated canonical Wnt signaling in cKO bones using TOPGALWnt reporter mice. The Wnt signaling assessed by β-gal stainingwas increased in cKO calvariae at E17.5 (Fig. 4A). Relative numberof β-gal-positive cells was 8.5 times higher in cKO calvariaecompared with wild type, which was significant (Fig. 4C). Althoughit is known that endogenous levels of canonical Wnt signalingassessed using TOPGAL mice are low in osteoblasts duringembryonic bone development (Hens et al., 2005), the intensity of β-gal staining was dramatically increased in cKO long bonescompared with wild type (Fig. 4D,E). These suggest that canonicalWnt signaling in cKO mice was stimulated during bothendochondral and intramembranous ossification. Thus, Bmpr1adeficiency in osteoblasts upregulates Wnt signaling at least throughthe canonical pathway.

Downregulation of sclerostin in cKO bonesAlternation of Wnt-related genes were further examined.Sclerostin (Sost) expression was consistently reduced by 95% incKO calvariae from E16.5 to E18.5 when assessed with QRT-PCR(Fig. 5A) and was the most severely downregulated at E18.5 onmicroarray data (–5.68-fold, P=3.27E-24) (see Fig. S2 in thesupplementary material). Expression levels of Wnt target genes

RESEARCH ARTICLE Development 135 (22)

Fig. 2. Increased bone mass in Bmpr1a cKO mice. (A) Skeletal preparation of the skull and long bones at E18.5 using Alizarin Red/Alcian Bluestaining. Broken line, areas of parietal bones (P). (B) Bone volume over total tissue volume (BV/TV) obtained fromμCT analysis on parietal bones ofthe skull (left) and femur (right) at E18.5. Values are expressed as mean±s.d. (wild type, n=5; cKO, n=4, Student’s t-test; *P<0.01). (C) Hematoxylinand Eosin staining of E18.5 calvariae. Boxed areas in frontal sections were magnified. Scale bars: 25μm. (D) Von Kossa staining for Ca2+ usingE18.5 calvariae. Scale bars: 25μm. (E) Hematoxylin and Eosin staining of E18.5 humerus. Boxed areas in sagittal sections are magnified. Scale bars:100μm. (F) BrdU incorporation using E18.5 calvariae. BrdU-positive cells (arrows) per total cells in bone were unchanged. Scale bars: 25μm.

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Axin2 and Ctgf were significantly increased in cKO calvariae atE18.5 when assessed with QRT-PCR (Fig. 5B), but those of Wntligands (Wnt3a, Wnt5a, Wnt7a, Wnt7b and Wnt9a), other Wntinhibitors [dickkopf 1 (Dkk1), Dkk2 and secreted frizzled-relatedproteins] and co-receptor Lrp5 were unchanged both whenassessed with microarray and QRT-PCR at E18.5 (Fig. 5A, datanot shown). Immunohistochemistry using E17.5 cKO calvariaeconfirmed that Bmpr1a deficiency in osteoblasts and osteocytescorrelated with increased levels of β-gal staining and reducedproduction of sclerostin at cellular levels (Fig. 5C,D). Thereduction of sclerostin was reproduced in vitro using primaryosteoblasts from newborn cKO mice (Fig. 5E). Similarly, Sostexpression was 90% reduced by loss of BMPR1A when assessedusing adenoviral Cre infection in vitro (see Fig. S1A in thesupplementary material). These results suggest that sclerostin is adownstream effector of BMPR1A on Wnt signaling, and thatBmpr1a deficiency in osteoblasts increased canonical Wntsignaling at least by the suppression of sclerostin.

Wnt inhibitor sclerostin regulates bone massThe effect of sclerostin on Wnt signaling and bone mass was furtherexamined using Bmpr1a-deficient calvariae treated with 4-hydroxyl TM for 5 days ex vivo. Cre-dependent DNArecombination and upregulated Wnt signaling were confirmed byβ-gal staining using R26R and cKO:TOPGAL newborn calvariae,

respectively (Fig. 6A, parts a,b). Expression levels of Bmpr1a andSost were significantly reduced by 80% and by 90%, respectively,but Dkk1, Dkk2 and Lrp5 were unchanged (Fig. 6A, parts c,d).After treatment with 4-hydroxyl TM for 5 days, expression levelsof bone resorption markers Mmp9, Ctsk and Trap were significantlyreduced when assessed by QRT-PCR (Fig. 6A, part d), consistentwith in vivo results (Fig. 4A, Fig. 5A). Next, when sclerostin wasadded in the culture, both canonical Wnt signaling and bonethickness were reduced in cKO calvariae (cKO+Scl, Fig. 6B), andthe ratio of β-gal positive cells was significantly reduced (cKO,65.3%; cKO+Scl, 17.2%). The expression levels of osteoclastmarkers Rankl and Opg, and the ratio of Rankl to Opg werepartially restored in the cKO compared with wild type (Fig. 6C).Moreover, TRAP staining pattern in the cKO resembled that in thewild type in response to sclerostin treatment (Fig. 6D, compare withFig. 3D). These results suggest that exogenous sclerostin inBmpr1a-deficienct calvariae partially restore morphologicalchanges, Wnt signaling and osteoclastogenesis. In addition, Noggintreatment ex vivo reduced Sost expression by 85% and upregulatedthe Wnt signaling (see Fig. S3A,B in the supplementary material).Noggin antagonizes both BMP2 and BMP4, which are potentligands of BMPR1A. Thus, we conclude that loss of BMP signalingreduces Sost expression, and sclerostin inhibits canonical Wntsignaling as a bone mass regulator during embryonic bonedevelopment.

3805RESEARCH ARTICLESclerostin is a downstream target of BMPR1A

Fig. 3. Alteration of bone formation and resorption in Bmpr1a cKO mice. (A) QRT-PCR for bone formation markers (Runx2, Sp7, Ibsp,Akp2 and Bglap2 using E16.5 and E18.5 calvariae). Values are expressed relative to wild type at E16.5. (B) QRT-PCR for bone resorptionmarkers expressed by osteoclasts (Mmp9, Ctsk and Trap), and Rankl and Opg expressed by osteoblasts using E18.5 and E19.5 calvariae. Valuesare expressed relative to wild type at E18.5. (C) Relative ratio of Rankl to Opg expression levels calculated from Fig. 3B. Values are expressedrelative to wild type at E18.5. (D) Evaluation of osteoclast activity by TRAP staining using E18.5 calvariae. The positive cells localized randomlyin cKO calvariae compared with wild type (left two panels, red arrows). The percent of TRAP-positive cells per total cells in bone area detectedby DAPI was significantly reduced in cKO calvariae (wild type, 13.9%; cKO, 6.4%, right panel). Scale bars: 50 μm. (E) QRT-PCR for BMP type Ireceptors (Bmpr1b, Acvr1), type II receptors (Bmpr2, Acvr2a and Acvr2b) and potential ligands for these receptors (Bmp2, Bmp4, Bmp6 andBmp7) using E18.5 calvariae. Values in A-E represent mean±s.d. from a minimum of three independent experiments using wild-type and cKObones. Student’s t-test; *P<0.05.

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Smad-dependent BMPR1A signaling upregulatessclerostin expression in vivoTo reveal the effects of Smad-dependent BMPR1A signaling onsclerostin expression and bone morphology in vivo, we generatedinducible transgenic mice expressing caBmpr1a (constitutivelyactivated Bmpr1a) in osteoblasts (see Fig. S4A in the supplementarymaterial). After daily TM injection from E13.5 to E17.5, grossmorphology of caBmpr1a fetuses was relatively normal at E18.5(data not shown). Histology showed moderately reduced thicknessin caBmpr1a calvariae (Cre+, caBmpr1a+) at E18.5, where levels ofphosphorylated Smads were enhanced compared with controls(Cre-, caBmpr1a+) (Fig. 7A). Sost expression when assessed byQRT-PCR increased approximately sevenfold in the transgeniccalvariae at E18.5, indicating that Smad signaling positively controlsSost expression. Expressions of bone resorption markers were allincreased over fourfold, and Rankl levels increased four times whileOpg levels decreased 40% (Fig. 7B), resulting in a 6.5-fold increase

in the ratio of Rankl to Opg (Fig. 7C). Expression of bone formationmarkers was also increased in the caBmpr1a (see Fig. S4B in thesupplementary material). These changes observed in the caBmpr1awere the opposite of those seen in Bmpr1a cKO calvariae (Fig. 3).We next generated rescue mice expressing caBmpr1a on a Bmpr1acKO background (Cre+, caBmpr1a+, Bmpr1afx/fx) and comparedthem with littermate Bmpr1a cKO mice (cKO: Cre+, caBmpr1a-,Bmpr1afx/fx). In E18.5 calvariae from rescued mice, expression ofSost, Rankl and osteoclast markers increased over four times (Fig.7D), and the ratio of Rankl to Opg increased about 2.5-fold (Fig. 7E)when compared with the cKO. There was also a reduction inmorphological changes observed in littermate Bmpr1a cKO mice(Fig. 7F). These results strongly suggest that Sost expression isregulated by BMPR1A signaling at least through the Smad pathway.

DISCUSSIONThis study showed that loss of BMPR1A increased bone mass andleads to two possibilities: (1) that BMPR1A signaling directlyregulates osteoclastogenesis through RANKL-OPG pathway; or (2)indirectly regulates them via secondary mediator sclerostin as a Wntinhibitor (Fig. 8). Both indicate that BMP signaling in osteoblastsrestrains bone mass.

BMP signaling, bone formation and bone massBy using a 3.2 kb mouse pro-collagen α1(I) promoter (Rossert et al.,1995), we demonstrate that reduction in BMP signaling throughBMPR1A in osteoblasts increased bone mass during embryonicstages. This is consistent with our previous report that showedincreased bone volume (BV/TV) at 10 months by loss of BMPsignaling in osteoblasts, using Og2-Cre mice in which osteoblastsinitiate Cre expression postnatally. However, it is not consistent with3-month-old mice, which showed decreased bone mass (Mishina etal., 2004). The bone phenotype observed in Bmpr1a-deficient micewith Og2-Cre was milder than that with Col1-CreERTM. Thesediscrepancies may be due to differences in the timing ofrecombination between promoters. As characteristics of osteoblastschange as they mature (Aubin, 1998), the effects of disruptingBMPR1A signaling may be influenced by the stages of osteoblastmaturation. In Col1-CreERTM mice, Cre activity was detected inimmature periosteum that wraps growth plates, in osteogenic centersand in bone collars (Fig. 1B), indicating that Cre activation occursjust after commitment of mesenchymal cells towards osteoblasticcells. Therefore, Col1-CreERTM mice can induce recombinationearlier in osteoblastogenesis, including in preosteoblasts, comparedwith Og2-Cre mice, which could explain why the Bmpr1a-deficientmice using Og2-Cre mice did not change expression levels of earlyosteogenic markers Runx2 and Bsp, and showed mild bonephenotype (Mishina et al., 2004).

Expression levels of bone formation markers (Runx2, Sp7, Ibsp,Akp2 and Bglap2) were increased more than twofold in caBmpr1amice (see Fig. S4B in the supplementary material), indicating thatBMP signaling through Smad1/5/8 induces osteoblastogenesis, as iswell known in vitro (Chen et al., 2004). Some of these markers(Runx2, Sp7 and Ibsp) were reduced in cKO bones as expected (Fig.3A), consistent with the modest decrease in Smad phosphorylationlevels (Fig. 1E) and our previous report that loss of BMP signalingdecreases bone formation rate over bone surface (BFR/BS) duringadulthood (Mishina et al., 2004). It is also suggested that normalbones respond to endogenous BMPs by phosphorylating Smads ata very low level, which is difficult to detect by immunostaining (Fig.7A). It is possible that proliferation of osteoblasts is increased incKO bones, which could influence increased bone mass; however,

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Fig. 4. Upregulation of canonical Wnt signaling in Bmpr1a cKOmice. (A) Canonical Wnt signaling assessed by TOPGAL mice usingE17.5 calvariae (upper panel, lateral view; lower panel, top view). Twoindependent parietal bones from two littermates in each group afterdissection are shown in the right-hand panel. Red arrow indicatesenhanced Wnt signaling. Broken line, areas of parietal bones (P).(B) Histological analysis assessed by TOPGAL mice using E17.5 calvariae.Scale bars: 50μm. (C) Relative cell number of DAPI and β-gal positivecells. Cells were counted in 50 fields of E17.5 calvariae from cKO (n=4)and wild type (n=4). Total cell number was obtained by counting DAPI-positive nuclei in bone. Cell number of wild type is set as 1.0. Therewere 2.3 times more total cells in cKO calvariae in a given section thanin wild type owing to the thicker tissues, but the number of β-gal-positive cells was 19.7 times greater, resulting in an 8.5-fold increase inthe proportion of β-gal positive cells in cKO mice. (D) Canonical Wntsignaling assessed by TOPGAL mice using humerus, ulna and radius atE17.5. Red arrow indicates enhanced Wnt signaling. (E) Histologicalanalysis assessed by TOPGAL mice using radius at E17.5. Asterisks,growth plates. Scale bars: 100μm.

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this is less likely because the number of BrdU-positive cells per totalcells in bone was unchanged (Fig. 2E). Histomorphometric analysisof bone is an established method to assess bone formation but istechnically unfeasible in fetuses. Thus, we appliedhistomorphometry to adult cKO mice, where we confirmedosteoblasts failed to support osteoclastogenesis (see Fig. S1B in thesupplementary material). Bone volume (BV/TV) was significantlyincreased, but formation rate (BFR/BS) and osteoclast number perbone area (N.Oc/T.Ar) were significantly decreased and osteoblastsurface per bone surface (Ob.S/BS) were unchanged in the adultcKO (Kamiya et al., 2008). Similar to embryonic cKO data (Fig. 3),expression levels of osteoclast markers and the ratio of Rankl to Opgwere significantly reduced when assessed by QRT-PCR in the adultcKO (Kamiya et al., 2008). These results suggest that osteoblastnumber is unchanged and bone formation is modestly reduced,while resorption markedly decreases in cKO bones, resulting in a netincrease in bone mass. In addition, BMP signaling can control boneformation and resorption by inducing both osteoblastogenesis andosteoclastogenesis in vivo.

RANKL-OPG pathway involved in BMP signalingThis study found that osteoblasts respond to BMP signaling tosupport differentiation of osteoclasts at least through the RANKL-OPG pathway. The link between BMP signaling and the RANKL-OPG pathway has been reported in vitro, showing that BMP2enhances osteoclastogenesis by upregulating Rankl (Itoh et al.,2001; Usui et al., 2008). However, the link is not detected in thosemice overexpressing Noggin or Bmp4 in osteoblasts using a 2.3 kbtype I collagen promoter (Okamoto et al., 2006) or by knocking outBMPR1A using an osteocalcin promoter (Og2-Cre) (Mishina et al.,2004), whereas osteoclastogenesis is upregulated by BMP signaling.As the 2.3 kb type I collagen and osteocalcin promoter are firstactivated in mature osteoblasts (Ducy and Karsenty, 1995; Kalajzicet al., 2002), immature osteoblasts, where the 3.2 kb type I collagen

promoter is active, may be crucial for regulating osteoclastogenesisthrough the RANKL-OPG pathway. Furthermore, it is possible thatBMP signaling directly controls osteoclastogenesis through theRANKL-OPG signaling pathway probably by downregulating Opgexpression in addition to upregulating Rankl, because BMP responseelements were identified in the Opg promoter by EvolutionaryConserved Regions (ECR) browser search (data not shown). Furtheranalysis is necessary to clarify the direct regulation of RANKL-OPGpathway by BMP signaling.

It is also possible that secondary mediators such as Wnts regulateexpression of RANKL and OPG. Two recent studies have suggestedthat Wnt signaling in osteoblasts through the canonical β-cateninpathway not only boosts bone formation by fostering osteoblastactivity but can also inhibit bone resorption by affecting osteoclasts(Goldring and Goldring, 2007). One study provided evidence thatthe Wnt pathway positively regulates osteoblast expression ofosteoprotegerin (OPG) by overexpressing stabilized β-catenin inosteoblasts in mice, resulting in decreased osteoclast differentiationand increased bone volume (Glass et al., 2005). Another studyshowed that osteoblasts lacking the β-catenin gene exhibitedimpaired maturation and mineralization with elevated expression ofRankl and diminished Opg, suggesting that the Wnt pathway cansuppress osteoclast-mediated bone resorption (Holmen et al., 2005).In our study, expression levels of Opg and Rankl as well asosteoclast markers (Mmp9, Ctsk and Trap) were partially restoredby exogenous sclerostin ex vivo with a concomitant reduction inWnt signaling (Fig. 6). Similarly, Wnt inhibitors Dkk1 and Dkk2 caninduce osteoclastogenesis by changing the RANKL-OPG pathwayin vitro (Fujita and Janz, 2007). These facts suggest that Wntinhibitors in the canonical/β-catenin pathway enhanceosteoclastogenesis. In addition, Wnt/β-catenin-responsive LEF1-binding sites were identified both in the Opg promoter and the Ranklpromoter by an ECR browser search (data not shown). These factsstrongly suggest that the changes in Bmpr1a-deficient bones are due

3807RESEARCH ARTICLESclerostin is a downstream target of BMPR1A

Fig. 5. Inhibition of canonical Wntsignaling by sclerostin in Bmpr1a cKOmice. (A) QRT-PCR analysis for Sost, Dkk1,Dkk2 and Lrp5 using calvariae at E16.5,E17.5 and E18.5. Values are expressedrelative to wild type at E16.5. Student’s t-test; *P<0.05. (B) QRT-PCR analysis forWnt target gene Axin2 and Ctgf usingE18.5 calvariae. Expression levels of Axin2and Ctgf were significantly increased incKO calvariae. Student’s t-test; *P<0.05.(C) Immunohistochemical staining ofsclerostin (brown) counterstained withHematoxylin (blue) using E17.5 calvariae.Broken line, osteoblasts. Scale bars:50μm, left panel; 10μm, right panel.(D) Detection of BMPR1A (green),canonical Wnt signaling (blue) andsclerostin (red) using E17.5 calvariae.Canonical Wnt signaling was assessed byβ-gal staining using TOPGAL mice. Nucleiwere stained with DAPI (blue). Brokenline, osteoblasts. Scale bars: 20μm.(E) Immunohistochemical staining forsclerostin (red) in primary osteoblasts fromBmpr1a cKO and wild-type control. Nucleiwere stained with DAPI (blue). Scale bars:20μm. D

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to decreased bone resorption and osteoclastogenesis through theRANKL-OPG pathway, and that sclerostin is presumably involvedin the pathway as a Wnt signaling inhibitor (Fig. 8).

Sclerostin, a downstream target of BMPR1ASclerostin, which is encoded by Sost, has homology to the DANfamily of BMP antagonists, which can control both BMP and Wntsignaling in Xenopus (Bell et al., 2003; Itasaki et al., 2003; Piccoloet al., 1999). However, although sclerostin directly binds BMPs(Kusu et al., 2003; Winkler et al., 2003), its antagonistic effects onBMP activity in mammals are still controversial. Resent in vitrostudies showed that sclerostin is not a BMP antagonist (vanBezooijen et al., 2004), as it binds only weakly to BMPs and doesnot inhibit direct BMP-induced responses (ten Dijke et al., 2008).Together with the evidence from human mutations in SOST or its co-receptor LRP5, in vitro studies revealed that sclerostin is a Wntinhibitor that binds LRP5 (Li et al., 2005; Semenov et al., 2005; vanBezooijen et al., 2007). Loss-of-function and hypomorphicmutations in SOST cause sclerosteosis (Balemans et al., 2001;Brunkow et al., 2001) and Van Buchem disease (Balemans et al.,2002; Staehling-Hampton et al., 2002), respectively, and bone massis increased in these disorders, similar to Sost KO mouse (Li et al.,

2008). In human and mouse, these Sost mutants share the high bonemass (HBM) phenotype with humans who have gain-of-functionmutations in LRP5 (Boyden et al., 2002; Little et al., 2002; VanWesenbeeck et al., 2003) and by overexpression of Lrp5 in mice(Babij et al., 2003). By contrast, loss-of-function of LRP5 leads toosteoporosis pseudoglioma syndrome in humans with low bonemass (Gong et al., 2001; Patel and Karsenty, 2002), which is similarto the bone phenotype of mice that overexpress Sost (Winkler et al.,2003). Consistent with these facts, Bmpr1a cKO mice show a HBMphenotype with lack of sclerostin expression. In addition, sclerostindiffers from the BMP antagonist Noggin with respect to effects onWnt signaling, as sclerostin inhibits Wnt (Fig. 6) whereas Nogginupregulates it (see Fig. S3A in the supplementary material). Thus,our study strongly suggests that sclerostin physiologically acts as aninhibitor of canonical Wnt signaling in vivo during embryonic bonedevelopment, and loss of sclerostin largely directs the Bmpr1a cKObone phenotype.

The phenotype of Sost KO mice (Li et al., 2008) is different fromthat observed in Bmpr1a cKO with respect to the morphology of thebone, as Bmpr1a cKO mice showed disorganized bone structure(Fig. 2), which is not described in Sost KO mice (Li et al., 2008) orSOST human disorders. This fact implies that BMPR1A is not only

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Fig. 6. Suppressed expression of sclerostin by loss of BMP signaling ex vivo. (A) Newborn mouse calvariae were cultured for 5 days treatedwith 4OH-TM (100 ng/ml). (a) Confirmation of Cre activity in the calvariae from CreER:R26R mice assessed by β-gal staining. (b) Upregulation ofcanonical Wnt signaling in the calvariae from CreER:Bmpr1afx/fx:TOPGAL mice assessed by β-gal staining. Broken lines, areas of parietal bones (P).(c) Expression of Wnt inhibitors Sost, Dkk1, Dkk2 and Lrp5 assessed by QRT-PCR. (d) Expressions of bone resorption markers Mmp9, Ctsk, TRAP andBmpr1a. Values are expressed relative to those of wild type (Cre-, TM-). (B) Sclerostin treatment of wild-type (WT+Scl) and cKO (cKO+Scl) calvariaeex vivo. The ratio of β-gal to DAPI-positive cells was evaluated from 50 fields in frontal sections (n=3, in each condition). Broken line, parietal bones.Scale bars: 50μm. (C) Expression of bone resorption markers Rankl and Opg, and relative ratio of Rankl to Opg using wild-type and cKO calvariae insclerostin treated versus untreated groups. Values are expressed relative to those of wild type without sclerostin treatment. (D) TRAP staining of cKOcalvariae treated with sclerostin and non-treated ex vivo. Values in A-C represent mean±s.d. from three independent experiments. Student’s t-test;*P<0.05.

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an upstream effector of sclerostin but also has other roles beyondregulating Sost in the osteoblasts. In the Sost KO there is no increasein osteoclast number or erosion surface, which is consistent withmice overexpressing LRP5 with HBM phenotype (Babij et al.,2003), suggesting that bone formation and resorption are uncoupledin these mutants. However, as we discussed earlier, loss of BMPsignaling reduced both bone formation and resorption together. Inaddition, the diameter of collagen fibrils in Bmpr1a cKO bones were

heterogeneous, aggregations of collagen fibers were disorganizedand MMP expression levels were reduced (data not shown),suggesting that BMP signaling additionally regulates proper bonestructure and turnover. In support of our speculation, reduction inosteoclastogenesis was not fully restored by sclerostin treatment exvivo (Fig. 6C). These facts suggest that BMP signaling in osteoblastsdirectly regulates bone resorption independently of Wnt signalingthrough sclerostin, which in turn maintains proper bone structureand bone mass.

Sost expression was dramatically downregulated by removal ofBmpr1a in vivo, whereas it was upregulated by enhancing Smad-dependent BMPR1A signaling using caBmpr1a. It is reported thatboth BMP2 and BMP4, potent ligands of BMPR1A, induce Sostexpression in mouse and human osteoblasts (Ohyama et al., 2004;Sutherland et al., 2004). Although sclerostin is produced primarilyby osteocytes in adult bones (Poole et al., 2005; van Bezooijen et al.,2004), it is also detected in osteoblasts before birth (Fig. 5),suggesting the positive regulatory role of BMPR1A signaling in Sostexpression both in osteoblasts and osteocytes during embryonicbone development. Interestingly, the promoter region of the Sostgene contains putative BMP response elements that were detectedby an ECR browser search (data not shown), suggesting possibleregulation of Sost by BMP signaling at the transcriptional level.

Interaction of BMP and Wnt signaling in boneAccumulating evidence suggests that both BMP and Wnt signalingmay regulate each other in a context and age-dependent manner(Barrow et al., 2003; Guo et al., 2004; He et al., 2004; Huelsken etal., 2001). Only a few cascades such as Pten/Akt (Zhang et al., 2006)and Smad1/Dvl1 (Liu et al., 2006) are reported in intracellularcrosstalk between the BMP and Wnt pathways. However, as in bone,the interaction between BMP and Wnt signaling is not welldescribed generally in vivo, partly because alteration of BMP

3809RESEARCH ARTICLESclerostin is a downstream target of BMPR1A

Fig. 7. Enhanced BMPR1Asignaling upregulates bothsclerostin and osteoclastogenesis.(A) Constitutively active Bmpr1a(caBmpr1a) mouse fetuses at E18.5induced daily by TM injection fromE13.5. Hematoxylin and Eosinstaining showed moderately reducedthickness in the caBmpr1a calvariae(Cre+, caBmpr1a+) where levels ofphosphorylated Smad1/5/8 (brown)were enhanced compared withlittermate controls (Cre-,caBmpr1a+). Scale bars: 50 μm.(B) Expressions of Sost and the boneresorption markers Rankl and Opgassessed by QRT-PCR using caBmpr1aand control calvariae at E18.5. Valuesare expressed relative to control.(C) Relative ratio of Rankl to Opgcalculated based on the expressionlevels in Fig. 7B. (D) Expressions ofSost, bone resorption markers, Rankland Opg by QRT-PCR using rescued(black bar: Cre+, caBmpr1a+, Bmpr1afx/fx) and littermate cKO (gray bar:Cre+, caBmpr1a-, Bmpr1a fx/fx) calvariae at E18.5. Values of rescued mice are expressed relative to littermate cKO mice. (E) Relative ratio ofRankl to Opg calculated based on expression levels in Fig. 7D. (F) Hematoxylin and Eosin staining of rescued and cKO calvariae at E18.5. Scalebars: 100 μm. Values in B-E are mean±s.d. from three independent experiments. Student’s t-test; *P<0.05.

Fig. 8. A model of the relationship between BMPR1A andcanonical Wnt signaling in mouse bone. BMPR1A signalingupregulates sclerostin expression, leading to an inhibition of canonicalWnt signaling and a decrease in bone mass by upregulatingosteoclastogenesis through the RANKL-OPG pathway. Sclerostin, theSOST gene product, acts as a downstream effector of BMPR1Asignaling, an inhibitor of canonical Wnt signaling and a bone mass-determining factor. Broken line indicates another possibility: that BMPsignaling directly upregulates osteoclastogenesis through the RANKL-OPG pathway. D

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signaling has not been studied in Wnt signaling mutants and viceversa. Some in vitro studies have demonstrated that both BMP andWnt pathways synergistically regulate each other possibly throughautocrine/paracraine loop, as BMPs induce Wnts in C2C12 cells andprimary osteoblasts (Chen et al., 2007; Rawadi et al., 2003), and Wntsignaling, on the contrary, enhances BMPs expression inC3H10T1/2 cells (Bain et al., 2003; Winkler et al., 2005). Thesestudies indicate that BMP signaling may upregulate Wnt signaling.

By contrast, our in vivo study suggests that BMP signalingdownregulates Wnt signaling, as loss of BMPR1A signalingupregulates Wnt signaling by inhibiting Sost expression. Thesediscrepancies are partly due to the differences of cell type examined,as both osteoblasts and osteoclasts always affect each other ascoupling factors in vivo, and coupling effects on the two signalingpathways are difficult to address using osteoblasts alone in vitro.Furthermore, it is also difficult for in vitro studies to address age-dependent changes in both signaling pathways and their interactions.Similar to our results, other studies have shown antagonisticinteraction of BMP and Wnt signaling in lung (Dean et al., 2005),intestine (He et al., 2004), hair (Zhang et al., 2006) and joints (Guoet al., 2004). Moreover, Noggin treatment ex vivo upregulatedcanonical Wnt signaling and downregulated Sost expression (seeFig. S3A,B in the supplementary material), presumably byantagonizing BMP2 and BMP4, ligands for BMPR1A. Thus, ourstudy strongly suggests that BMPR1A signaling in osteoblastsregulates negatively canonical Wnt signaling through downstreameffector sclerostin during embryonic bone development.

In conclusion, we demonstrate a new interaction between theBMP and Wnt signaling pathways in osteoblasts through sclerostin,a Wnt inhibitor and a bone mass regulator. BMP signaling viaBMPR1A directs osteoblasts to reduce bone mass in part byupregulating sclerostin expression and supportingosteoclastogenesis through the RANKL-OPG pathway.

We gratefully thank Tomokazu Fukuda and Greg Scott for generation ofcaBmpr1a mouse line and Donald Lucas for critical reading of this manuscript.This work was supported by NIH grants P01 DK56246 (H.K.), R01 AR051587(J.F.), R21 AR052824 (M.Y.), ES071003-11 and a conditional gift from RIKENBrain Science Institute (Y.M.) and Lilly Fellowship Foundation (N.K.).

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/135/22/3801/DC1

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3811RESEARCH ARTICLESclerostin is a downstream target of BMPR1A

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