Journal of Cell Science N322 USA 37232, TN ciiyi eue srfetdi eue oefrainrt n alr odfeetaet auemnrlzn tg.Bp in Bmp2-cKO Bmp2 in stage. reduced mineralizing is mature production a VegfA to marrow. differentiate bone to and failure periosteum and rate the formation in bone angiogenesis reduced controls a indirectly in also reflected osteoblasts as reduced is activity o:10.1242/jcs.118596 doi: 4085–4098 126, Science Cell of Journal 2013 June 10 Accepted oelnsbn omto ovsuaiainand cells vascularization stem to mesenchymal formation bone links bone Bmp2 Article Research h ta. 01 sj ta. 08.Rmvlof Removal 2008). al., et 2006; Tsuji al., 2011; al., et et postnatally, (Bandyopadhyay Shu or development bone embryo family endochondral the BMP in development other whereas cartilage osteoporosis of and and Deletion bone risk 2003). fracture al., members increased et negative with (Styrkarsdottir the plus associated in receptors (Dutko polymorphisms are gremlin Selected and II 2011). noggin Mullins, as type and such are signaling, different pathways BMP of signaling BMP two regulators I BMP type and different 2002). Yoon three involve receptors al., 2009; and ligands et Miyazono, Mullins, BMP for and Zhao physiological and common Watabe 2005; Dutko 2009; al., postnatal al., 2012; et et is al., and Sieber pathway et 2011; developmental signaling (Bonilla-Claudio (BMP) processes for protein morphogenic important bone The Introduction words: Key ( genes marker MSC several of Expression bones. trabecular and 1) periosteum the in bed microvascular of Deletion osteoblasts. cKO new a generated We Summary ` *Deceased 12 11 10 9 8 7 6 5 4 3 2 1 Yang Wuchen sebat n Ssaeavtlln ewe oefrain aclrzto n eecya tmcells. stem mesenchymal and vascularization formation, bone between link vital a are MSCs and osteoblasts rgScott Greg Chen Xiao-Dong abr .Kream E. Barbara uhrfrcrepnec ( correspondence for Author ainlIsiueo niomna elhSine,Rsac ragePr,N 70,USA 27709, NC USA USA Park, 38104, 06030, Triangle TN CN Research Memphis, Farmington, Sciences, Center, Center, Health Science Nashville, Health Environmental Health Center, Connecticut of Tennessee Medical of Institute of University University National USA University Vanderbilt Development, 78229, Medicine, Biology, Skeletal TX of Bone and Antonio, Department for Medicine San Center Regenerative Antonio, Vanderbilt for San and Center at Rehabilitation Center and Science Surgery USA Health 64108, Texas Orthopaedic MO of of City, University Department Kansas Dentistry, City, Comprehensive USA USA USA Kansas of 06030, 10591, 78229, at Department CT NY TX Missouri Farmington, Antonio, Tarrytown, of Center, San Inc., University Antonio, Health Regeneron Biology, San Connecticut Oral at of of Center University Department Science Sciences, Health Craniofacial Texas of of Department University Periodontics, of Department eateto ilgcadMtra cecs colo etsr,Uiest fMcia,AnAbr I419 USA 48109, MI Arbor, Ann Michigan, of USA University 77030, Dentistry, TX of Houston, School Medicine, Sciences, of Material College and Baylor USA Biologic Biophysics, 01701, of and MA Department Physiology Framingham, Molecular Co., of Genzyme Department Science, Renal and Endocrine 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. nvivo in ob sn h .Cla-r rngncmdl oe fBmp2-cKO of Bones model. transgenic 3.6Col1a1-Cre the using ) Bmp7 , Bmp2 nvitro in m2 sebat,Agoeei,Msnhmlse el,VegfA, cells, stem Mesenchymal Angiogenesis, Osteoblasts, Bmp2, 9 aa Ray Manas , notolsso eisemadtrabecular and periosteum of osteoblasts in and 1,2 sciia o hnrct ucinand function chondrocyte for critical is F sasaddlto of deletion and assays CFU aogGuo Dayong , 5 Bmp4 hre Skinner Charles , Bmp2 [email protected] 7 ai .Rowe W. David , Bmp2 ugssta hypa io oe in roles minor play they that suggests odtoa-ncotall ihu e asteta eoe the removes that cassette neo a without allele conditional-knockout 9 notolssas ed odfciemsnhmlse el MC) hc orltswt h reduced the with correlates which (MSCs), cells stem mesenchymal defective to leads also osteoblasts in .Liu S. , ) 3,4 10 ai .Harris A. Marie , ae .Martin F. James , 5 efyS Nyman S. Jeffry , 7 v Kalajzic Ivo , m2i vitro in Bmp2 Bmp2 BMP2 nmouse in 1 gene 7 ogCui Yong , a David Val , 11 6 in ae .Edwards R. James , uiMishina Yuji , a -SMA 09.Rcnl,Luadclege Lue l,2012) of al., progression et the in (Liu required colleagues al., is (Osterix et precursors Zhang and VegfA osteoblast 2012; al., that Liu et Maes (Deckers demonstrated 2011; Recently, Runx2/Hif1a al., et and 2009). Kwon 2002; Osterix osteoblasts al., associated in through et signaling and BMP VegfA bone 2010a). new bone al., increases of increased et formation (Maes is treatment, vessels to blood leads bone overexpression marrow VegfA glucocorticoid in bone 2010). in al., brittleness et al., or Weinstein and et 2010; (Weinstein, Schipani reduced aging 2012; is Schipani, al., angiogenesis et and During Maes (Araldi 2008; healing 2009). al., fracture et Jacobsen to 2010; and formation bone and risk 2006). al., fracture et increased adult (Tsuji an but heal show formation, to that failure bone bones small endochondral have during minor mice a Bmp2 shows embryos for d.p.c. role 10 in cells precursor mesenchymal aclrzto n nignssaelne ocriaeand cartilage to linked are angiogenesis and Vascularization 1 + 8 a ob eiaGluhak-Heinrich Jelica , arlL Quarles L. Darryl , SA cells -SMA+ Ssspotorcnlsos rtclrlsof roles Critical conclusions. our support MSCs ieaetinr ihicesdbitees Osteoblast brittleness. increased with thinner, are mice 12 n tpe .Harris E. Stephen and 6 rgr .Mundy R. Gregory , Bmp2 8 + eer Villareal Demetri , a omtr osteoblasts. mature to ) SA D4 n Angiopoietin- and CD146 -SMA, eefo sebat (Bmp2- osteoblasts from gene 1 ujeWu Junjie , 1, 6, ` ,Ae Lichtler Alex *, 5 , 1 , Bmp2 4085 in ob 7 , Journal of Cell Science mroi hntps sw aeson(ig ta. 2008). al., et (Singh in shown role have a would we play as which can phenotypes, Bmp2 phenotypes, Maternal embryonic phenotype. bone bone stage the to minimize placenta embryo the crosses potential development. Bmp2 rescue maternal proper that is for possibility Another tissues the Col1a1-expressing that embryonic suggests this sebat nBmp2-cKO in osteoblasts i.SE(otmrgt n i.S bto aes] ihtime, With length panels)]. (bottom in material S2 Fig. [supplementary decrease and Hets) right) 5% (bottom or a S1E (WT Fig. with controls with vertebrae compared unfused smaller and Ssta ifrnit nootolss(rei ta. 2012). al., et (Grcevic osteoblasts into Osterix differentiate that MSCs ae) lzrnRdadAca lesann fnewborns Bmp2-cKO of the staining in Blue normal was Alcian skeleton and the Red showed Alizarin panel). ise htepestp olgn(1–1)a eadothers embryonic and vertebrae we in Rosa26– as the with active (E15–E19) collagen observed of is 1 have type 3.6Col1a1-Cre size (supplementary express The that controls overall tissues S2). WT the Fig. or in material heterozygotes decrease with slight compared a with rnin tt,mv nobn arwi soito with SMA association in 2012). 2010b; marrow al., al., et et (Maes bone Park development bone into during structures move vascular state, transient Dvsua aso h aclrbdi ohteprotu and periosteum the performed both in (5) bed marrow; created vascular bone the (4) and of biology; maps genes vascular vascular and key 3D performed osteoblast the (3) in involved quality; of proteins bone immunocytochemistry evaluate and tested to hybridization mechanically bones long (2) the histomorphometry; including marrow, Results osteoblasts; of into differentiate performed to (6) capacity and their and number MSC tgso segnss agn rmarqieetfrterminal for Bmp2-cKO requirement MSCs. a new of formation from and vascularization ranging driving to differentiation osteogenesis, of stages the we observations, above that vessel the bone From blood hypothesize site. new new distraction by the rapid followed at formation, first, formation osteogenesis MSC is candidate distraction There associated 2012). and during al., activated et (Matsubara is that angiogenesis aeilFg 1,tppnl.Hmrslnt hwda5% Bmp2- and a control showed between change (supplementary length no months Humerus with cKO Bmp2-cKO weeks panel). 6 2 the at top to decrease of S1D, assayed Fig. weight as in material reduced little body greatly changed also The mouse were (Yang 1B). levels bones (Fig. protein trabecular osteoblasts Bmp2 and 2012). periosteum 1 al., in days, et 5 months that at 4 osteoblasts show and the in S1C month 85–90% Fig. by reduced material was expression supplementary and 1A Fig. after phenotype Skeletal 4086 Cia ta. 08 rei ta. 02 acet ta. 2007). bone al., et in Sacchetti 2012; structures al., et microvascular Grcevic a 2008; with al., et associated (Crisan are and CD146 2010; express MSCs al., and et 2008) (Bianco Kronenberg, osteoblasts and Schipani of progenitors are marrow, bone -SMA w ek fe it,Bmp2-cKO birth, after weeks Two m2epeso satvtdi Ss(mohmsl cells muscle (smooth MSCs in activated is expression Bmp2 nteesuisw:()caatrzdtebn hntp na in phenotype bone the characterized (1) we: studies these In eecya tmcls(Ss rmbt h eisemand periosteum the both from (MSCs) cells stem Mesenchymal Bmp2 ob + nnwidcdbodvsesdrn oefrainand formation bone during vessels blood induced new on ) + t24mnh splmnaymtra i.SD bottom S1D, Fig. material (supplementary months 2–4 at + nerce MSCs. enriched in el sltdb ieg rcn tde r ls of class a are studies tracing lineage by isolated cells ora fCl cec 2 (18) 126 Science Cell of Journal rotolss hc ersn non-proliferating a represent which preosteoblasts, ob oe nbt h eisemadi h bone the in and periosteum the both in model nvitro in Bmp2 Bmp2 etn fBp ucinb deletion by function Bmp2 of testing Bmp2 eehsmlil oe tdifferent at roles multiple has gene ob loxP nvitro in mice eei iial eurdin required minimally is gene –Stop– eealto rmearly from ablation gene ob et o elatnm of autonomy cell for tests iehdtinrbones thinner had mice loxP-–lacZ sas and assays, ob animals, a -SMA. nsitu in Bmp2 a ob - eisemo eusas hwda4%dces n2-month- in decrease 40% trabeculae a the Bmp2-cKO in showed marrow BFR old also (BFR). femurs bone rate to formation of leading bone the bottom), periosteum in 1D, decrease both (Fig. overall periosteum 40% the in a in deposition) and 20% top) mineral 1D, by new (Fig. of reduced rate (MAR, were rate apposition mineral h oeotoei-iepeoye ihtinrbnsi the in bones thinner with phenotype, Bmp2-cKO osteopenic-like bone the RAwsfrtntda al s5dy,adtedfeec in difference the and days, 5 as Bmp2-cKO and early control as between expression noted first was mRNA h Bmp2-cKO the xrsini eue y8%i euso -a-l Bmp2- 5-day-old of femurs in 80% by cKO reduced is expression respectively. right), S3A, Fig. material supplementary erae 5 nBmp2-cKO in 35% decreased il ogns nBmp2-cKO in toughness yield 0 eraei oevlm oprdwt otos but controls, with compared in shown). not decrease volume (data 20% thickness bone cortical a 30– in similar as a showed was decrease well humerus (BV/TV) and as 40% femur volume tibia, region, The total thickness. trabecular trabecular to the volume in bone found in reduction 40% t2 ek n 41%dces ntbaadfmro Bmp2- of femur and tibia in decrease region) aBMD 14–16% L4–L6 cKO a in in and decrease weeks decrease (20% 25 spine progressive at the in a especially age, demonstrated with age of months nraei o-ieqaiyo rbcleo h Bmp2-cKO the of trabeculae of quality rod-like in increase ieaiigsrae(MS/BS surface mineralizing in analysis. similar X-ray on were by focused phenotypes males We and and 1C). females (Fig. evaluation, age statistical of for year males 1 to up radiography digital uodlplrzdotolssi Bmp2-cKO in mature osteoblasts fewer and polarized osteoblasts immature cuboidal and flat many differentiation were osteoblast There in failure a analysis demonstrates expression gene and Histomorphometric ( fracture to work in in decrease decrease or 50% 40% stress–strain and a resulting stiffness demonstrated and we femurs curves, of force–displacement bending three-point By Bmp2- that indicate cKO properties material and Mechanical e ru)sosvrereaesalrb 5 nteBmp2- the in 35% by smaller are vertebrae cKO shows group) per .CB nteBmp2-cKO and OC.S/BS the in decrease weeks 10–15% in 2 a noted after we N.OC/BS (N.OC/BS) weeks, surface 2 At bone age. number of of multinucleated the unit or per (TRAP+ (OC.S/BS) osteoclasts surfaces of osteoclasts bone covering the stain) red in cells, no and observed change left We bottom respectively. significant 3A, per S3A), (Fig. osteoblasts Fig. material N.OB/BS the mature supplementary and of covering OB.S/BS number osteoblasts surface, decreased an mature bone a is of and there region surface weeks, the bone 3A 8 in (Fig. By months decrease S3A). 6 80% Fig. to material days 5 supplementary from and assayed as littermates, control h pn BDwsnoted. was aBMD spine the Fg C otmrgt,dmntaigta h oe r much are bones the that brittle. demonstrating more right), bottom 2C, (Fig. sei,Clae ye11(o11 n Osteocalcin and (Col1a1) 1a1 Type Collagen Osterix, paetbn iea est aM)fo ek o8 to weeks 2 from (aBMD) density mineral bone Apparent saaye ydnmcbn omto ae both rate, formation bone dynamic by analyzed As m Taayi fvrereadln oe t4mnh ( months 4 at bones long and vertebrae of analysis CT ob ob ob ob iha nue oslrgo Fg A.W bevdan observed We 2A). (Fig. region dorsal unfused an with Fg B.A n ots iia 0 eraein decrease 20% similar a months, 8 and 6 At 2B). (Fig. Fg B.Dcesdepeso of expression Decreased 3B). (Fig. oe r oebrittle more are bones ob eaeporsieyavne,a sae by assayed as advanced, progressively became ob ob oe.Oealtuheso h oe a also was bones the of toughness Overall bones. oprdwt otos(aantshown). not (data controls with compared ob 5 ob ob oe Fg C o ih) Post- right). top 2C, (Fig. bones ciesraeae)adthe and area) surface active oe a erae y75% by decreased was bones Fg A otmrgtand right bottom 3A, (Fig. ob Col1a1 rgesdwt age with progressed ob W oprdwith compared f Fg C in 2C) (Fig. ) and Osterix ob n 5 .A 5 Journal of Cell Science itesann fpopoSa158wsosre nteBmp2- the in signal. observed cKO phospho-Smad1/5/8 was phospho-Smad1/5/8 pink) of to staining (purple Little nuclear robust a had otmpanels). bottom Bmp2-cKO the in localization nuclear mice or control cytoplasmic of little osteoblasts in and (purple) staining nuclear anti-phospho-Smad1/5/ an antibody with 8 Fig. noted material we (supplementary mice, tibia 6-week-old in In weeks S4). 2 at and bone alveolar aclrascae el rudsalbodvses( the vessels of blood 20% small Also, around panels). cells associated top vascular S5, Fig. material (supplementary Bmp2-cKO from bones in Smad5 (p-Smad1/5/8) Smad1, Smad8 in phosphorylated or decrease for 70% positive osteoblasts a of was number There S3B). Fig. material (supplementary ob lo esl splmnaymtra i.S,mdl and middle S5, Fig. material (supplementary vessels blood ob t5dy in days 5 at , 30 m m), ob efso ( perfusion oe fteBmp2-cKO the of Bones the in VegfA Bmp2-cKO decreased and vascularization Decreased hr r eue eeso D1i lo esl nteBmp2- the in vessels blood in CD31 of cKO 4A). levels (Fig. reduced immunohistochemistry IV are Col There with assayed as vessels einada7%dces nbodvse oueprtrabecular Bmp2-cKO per volume the bone vessel diaphysis blood in the in volume in decrease 75% vessels We a blood 2009). and in al., region decrease et 50% (David Methods a and observed Materials the in described eisemo ogbns eue h BaSO the used and We diaphysis bones. long metaphysis, of the periosteum in structure vascular the of mapping ob Bmp2 splmnaymtra i.S) epromdvascular performed We S6). Fig. material (supplementary n 5 ob notolssadlnst Ss4087 MSCs to links and osteoblasts in ,cnrladBmp2-cKO and control 3, mice ob ob r esrdadso eue blood reduced show and red less are Fg B.I h eisem we periosteum, the In 4B). (Fig. cKO Bmp2- and control of litters Two independent (bottom). periosteal and (top) trabecular for images magnification and Bmp2-cKO control 4-month-old from in vertebrae periosteum and region in trabecular BFR (D) shown. a X-ray with representative evaluated, were ages different as 20 bars: means are are Results rates shown. BFR and MS/BS relative ffmr t5dy n ot.OB, month. 1 and days 5 at femurs of region. trabecular and periosteum in deletion Osteoblast-selective 1. Fig. t4mnh n ero oto and control of year 1 Bmp2-cKO and months spine 4 lower at and arms of Radiograph (C) section. per with images sections multiple 2–3 age with of evaluated month were 1 at litters two independent from Femurs surface. on bone osteoblasts OB, expression. protein Bmp2 of Immunocytochemistry (B) evaluated. were femurs from section 2–3 per and photographs sections 2–3 age, at each litters independent Two osteoblast. 40 m ob (D). m nml eeeautd n the and evaluated, were animals (A) m ob AB;1 m(C); mm 10 (A,B); m ob ob ots and months) 7 , nsitu In nml.High- animals. of litters 200 Over mice. 4 efso ehdas method perfusion yrdzto (blue) hybridization 6 Bmp2 ..Scale s.d. m T3D CT Journal of Cell Science 0 eraei lo eslvlm obn ufc nteBmp2- the in surface bone cKO to a volume volume, vessel total blood to in volume decrease vessel 60% blood in decrease 75% a observed 4088 ufc Fg C.Bodvsesgetrta 20 than greater Bmp2-cKO the vessels in severely Blood affected 4C). (Fig. surface ob n 5 eraei lo eslnme e bone per number vessel blood in decrease 75% a and ora fCl cec 2 (18) 126 Science Cell of Journal ob oee,i h metaphysis, the in However, . m eenot were m nteesalbn-soitdmcoesl nteBmp2-cKO the in microvessels small-bone-associated these in esta 15 than less Fg D ih ie.A3 eosrto fterdcdsmall reduced the of demonstration 3D A side). right 4D, (Fig. ihtemn ml ailre n iuodlrgosi close in 50 than regions (less sinusoidal bone and the capillaries with small association many the with eut r means are Results eue nBmp2-cKO in reduced n ogbnsadmcaia etn ffmr rmcontrol from femurs of testing Bmp2-cKO mechanical and and bones long and rcueaddaaial eue otyedtuhes Analysis on toughness. done post-yield was reduced dramatically and fracture nml ftetogntpsa ahae C he-on edn to bending Three-point on (C) Bmp2-cKO done age. indicates each was fracture at Analysis genotypes months. two the 8 of to animals weeks 2 from assayed eue vrl oevlm ottlvlm.Aayi a done was Analysis volume. total to with volume and bone size reduced overall arrow), reduced (yellow process ventral open have vertebrae 2. Fig. n m 5 ndaee) eosre akd9%decrease 90% marked a observed we diameter), in m m nml fcnrlo Bmp2-cKO or control of animals 5 Taayi fvrere M nlsso vertebrae of analysis BMD vertebrae, of analysis CT n 5 –1mc fec eoye otso age. of months 4 genotype, each of mice 6–11 ob 6 animals. ..Saebr mm. 1 bar: Scale s.d. ob nbt h etba n h ogbns as bones, long the and vertebrae the both in (A) ob iehv eue tfns,wr to work stiffness, reduced have mice m Tsosta Bmp2-cKO that shows CT m wyfo oeand bone from away m ob eoye.()BDis BMD (B) genotypes. n 5 ob 3–6 ob Journal of Cell Science rbclrrgo.Cnoa nlsso h aclrwall vascular the of on number analysis reduced a Confocal demonstrated region. trabecular eeosre nteprotu f5dyodad1-month-old and 5-day-old of periosteum Bmp2-cKO the cells CD146 in of observed numbers Reduced were top). 6B, (Fig. cells endothelial SMA h rbclrrgo eerdcdby reduced were region trabecular the h Bmp2-cKO the a Bmp2-cKO MSCs the for in genes marker of Expression Bmp2-cKO the in vessels blood fteprotu Fg A.C16o CMwsrdcdby reduced was MCAM or CD146 6A). , (Fig. periosteum the of Ag1 eeswr eue y6%i h oemro of marrow bone the 1 in Angiopoietin 63% bottom). by 6B, reduced Bmp2-cKO (Fig. tibia were vessel levels blood (Ang-1) of length unit esl ihtetaeua oei hw nsupplementary in shown is Bmp2-cKO bone 1. trabecular Movie material the with vessels SApoen(muoeciiy a eue y6–0 in 60–70% by reduced was (immunoreactivity) protein -SMA 0 nteBmp2-cKO the in 60% Using + el e nto arwvlm a bevdi the in observed was volume marrow of unit per cells a SACer eotrmc,a7%rdcinin reduction 70% a mice, reporter -SMA-Cherry ob ob ieb 08%(i.5) ef rti eesin levels protein VegfA 5A). (Fig. 60–80% by mice oprdwt otos(aantshown). not (data controls with compared ob ob ii rbclrrgo n ntecmillayer cambial the in and region trabecular tibia ob oprdwt otos ssoni i.6C. Fig. in shown as controls, with compared VegfA mice ob RAepeso a erae in decreased was expression mRNA aclrascae el ett the to next cells vascular-associated ob n ls soito fblood of association close and a , -SMA 0 Fg 5B). (Fig. 70% nvivo in + Cer el per cells –Cherry r reduced are a - cultures 07 Fg D ih) 0 euto ntenme fCFU-F of al., number the et the in (Chen Bmp2-cKO using reduction the Methods 70% in clones A measured and right). 6D, Materials as (Fig. from the 2007) animals, in cultures described wild-type with methods compared or (CFU-OB) heterozygous matrix mineralized sn xrclua arx MC ananterse cell culture. BMSC stem any their from expanded maintain greatly be BMSCs can and matrix, properties extracellular Using in expression Bmp2 activates VegfA marrow bone clonal Bmp2-cKO of of capacity (BMSCs) the in cells reduction stromal 90% a is There os capacity mature to reduced differentiate presents to and decreased is population MSC ntenme fCUFcoe dt o shown). not effect (data no clones had CFU-F rBmp2 assay of CFU clones. the number to MSC the plating Control-Het, on on initial the at Bmp2 added in of ng/ml) CFU-F effect (40 of threshold reduction a 30% suggesting significant a was Ss a lontdi h Bmp2-cKO the in noted also was MSCs, 2011). Jain, between and interactions (Carmeliet stable cells in endothelial important and be MSCs to reported is Ang-1 Bmp2 nvitro in notolssadlnst Ss4089 MSCs to links and osteoblasts in oto n Bmp2-cKO and control situ in Osteocalcin (B) quantified analysis. section for histomorphometric per evaluated regions were separate animal of per images 2–3 2–3 with and litters, sections to independent surface Three (osteoblast surface). OB.S/BS bone reduced with flat mostly sebat.I h Bmp2-cKO indicate the arrows In Blue osteoblasts. osteoclasts. and evaluation osteoblasts for of hematoxylin with counterstained staining sebat;Oy sects cl as 20 OB, bars: marrow; Scale bone osteocytes. BM, Ocy, vessel; osteoblasts; outlines blood line BV, dashed surface; Orange bone areas evaluated. different were of bone images per 2–3 and sections 2–3 genotype, xrsino sei,Cla n secli in Osteocalcin and Col1a1 Osterix, and analysis of histomorphometric expression and Histology 3. Fig. oto n Bmp2-cKO and control ob MCclue,an cultures, BMSC ebat nteBmp2-cKO the in teoblasts yrdzto nfmr f5-day-old of femurs in hybridization ob ob ob nml.Tolteswt each with litters Two animals. . A itlg ihTrap with Histology (A) ob a cells, ob -SMA Fg D et.There left). 6D, (Fig. Osterix oe,otolssare osteoblasts model, nvitro in nvitro in + MCcell BMSC , Col1a1 esr of measure m oform to , m. ob and Journal of Cell Science aiae yqTPRuigaTqa sa n FAM-labeled and shown). assay of not TaqMan (data induction a primers using VegfA qRT-PCR by The validated right). 7A, (Fig. hrceitcsog-ieo oe-obapaac fthe The of appearance cultures. honey-comb day or sponge-like 10 stain characteristic and with structures a mineral mineral-collagen 7 well-organized into differentiated for VonKossa/VanGieson BMSCs (bottom) the images images phase-contrast and shows S7A (top) Fig. material Supplementary a 88% high were at XC-marrowECM ( XC-marrowECM the replated of positive ng/ml on then 100 SMA were on with grown response and cells BMSCs cell the days 2 VegfA. tested The we type time, TX). 10 Over collagenase density. Antonio, with for San released grown LLC, (StemBioSystems were BMSCs eurdfor required endogenous the and osteoblasts, producing 4090 ef induces VegfA -SMA + MC ail ifrnit omtr mineral- mature to differentiate rapidly BMSCs ora fCl cec 2 (18) 126 Science Cell of Journal a -SMA Bmp2 a -SMA + RAepeso t4 4ad4 hours 48 and 24 4, at expression mRNA MCdifferentiation BMSC + hnaaye yFC Fg 7A). (Fig. FACS by analysed when ) Bmp2 Bmp2 RAwas mRNA a eeis gene -SMA a + - nrae eesi h Bp-rae utrs(supplementary cultures rBmp2-treated S7B). Fig. the material in levels increased yFC,a ecie bv.The above. described as FACS, by 7.Nrhr eeepeso nlssa a 0soshigh shows 10 day at analysis of expression levels Fig. gene material supplementary Northern mineral and in S7A. these quantified differentiation as form osteoblast Ghosh- minerals, cultures accelerates 2003; increased rBmp2 Control al., and et 1996). structures (Bonewald al., osteoblasts models et calvarial osteoblast Choudhury primary 2T3 in in observed and as structures mineral Bmp2 asfreauto fmnrlqaiyadqatt.A hw in 8 shown at As and quantity. and analysis, quality expression mineral gene of for were evaluation differentiation days, Cultures for 8 rBmp2. to days and ng/ml changed 1 100 at were minus or collected cultures plus the The day), the (0 confluent days, two. or medium be or to 2 density (Ad5–GFP) day high After at next replated CMV–GFP and (Ad5–Cre). collected are with CMV–Cre cells with Adenovirus-5 Adenovirus-5 either with a -SMA fx/fx Osterix + ie hs el r 80–90% are cells These mice. 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Allen, References S.E.H.; to at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.118596/-/DC1 online available material AR44728 Supplementary for PMC and in months. Health Deposited AR054616 12 of Y.M.]. after Institutes to release R01 ES071003-11 National and US R01DE020843 the numbers by supported [grant Data was work I.K. This and D.R. Funding V.D. and B.K., J.N. A.L., S.E.H., 3.6Col1a1- W.Y., mice: of J.G.-H., M.A.H., supply a-SMA-Cherry analysis: and J.N., Construction and testing: S.L. mechanical Cre and and D.Q. studies V.D., vascular J.R.E., Assistance J.F.M. uCT, and histology, Y.M. floxed R.M., with Bmp2 G.S., M.A.H., of D.G., production J.G.-H., S.E.H., and models: A.R., mouse Generation W.Y., S.E.H. and conduct: V.D Study X.-D.C., S.E.H. design: Study Mundy. R. contributions Author Gregory to dedicated is the study producing This on lab. work Mishina’s to Harris E. S. Bmp2 allowing for NIEHS thank We Acknowledgements Student’s analysis Statistical aclrzto a sesdwt hehl f20 hra oewsa a at was bone whereas 200, of threshold by a 600 2–5 of with at threshold periosteum assessed the was on Vascularization and marrow bone eete indit ifrn iecassadpotda oa lo esl ihna vessels within vessels blood blood total the range. as of plotted diameter diameter and given classes The size region. different the into metaphysis of out binned the shaft carried then were near was bone region manner, the vascular similar of periosteal middle a The the analyzed. in near were that region, tibias diaphysis trabecular and the the femurs in includes that volume plate a growth and the bone below just volume a region, metaphysis cKO lcdi 0 urs ni ed oebd ise eeebde nOCT- in then embedded at The were and sectioned Tissues and morphology. days PA) embed. 7–10 Hatfield, to Sciences, for 8 ready Microscopy day) (Electron until #4583 every sucrose Tissue-Tek 30% (changed in EDTA placed 15% in demineralization eosrcino tcswscetdwt h uoiulz opnn of component AutoVisualize the with created MD). and Bethesda, (Olympus) The was Cybernetics, Viewer microscope. (Media 2.1 stacks 1000 AutoQuant FV10-ASW FV of a Olympus using confocal reconstruction analyzed a were using images tissue-tape visualized the on staining performed. also was rBmp2 ng/ml 40 without or with assays colony 50 of mM addition by the 10 evaluated with acid-2-phosphate, were induction colonies osteogenic CFU-OB after staining staining. VonKossa Violet Crystal using evaluated were ie ih4 aaomleyea 4 at paraformaldehyde 4% with fixed littermates, SACer rngncmice transgenic -SMA-Cherry m 190-196. epnet seprsstherapies. osteoporosis to response hcns,uigteKwmt aesse o rsraino oetissue bone of preservation for system tape Kawamoto the using thickness, m a ob SA–hryrpre rngncmdlwsue omntrvascular monitor to used was model transgenic reporter -SMA1–Cherry 4 n hi itraewl-yeadhtrzgu ieuigGaha Prism GraphPad using mice heterozygous and wild-type littermate their and ehdta losdtie iulzto ftemcoaclrsrcue in structures microvascular the of visualization detailed allows that method lxdmuemdlwt epo ebr fD Yuji Dr of members of help with model mouse floxed t m tssadAOAwr efre ocmaedt ewe Bmp2- between data compare to performed were ANOVA and -tests Tad3 lo eslmapping vessel blood 3D and CT a SACer;m2e or -SMA-Cherry;Bmp2Het nvivo in a -SMA m T h aclrmp eeidpnetyioae o the for isolated independently were maps vascular The CT. h rngn osse fa2kb 2 a of consisted transgene The . b nsitu in + gyeohsht n 0 Mdxmtaoe CFU dexamethasone. mM 100 and -glycerophosphate 21) yoi,HF n oedevelopment. bone and HIFs Hypoxia, (2010). population P , ob yrdzto,a es w needn itr were litters independent two least at hybridization, .5wscniee ssaitclsgiiac.For significance. statistical as considered was 0.05 ie fe ek fclue F- colonies CFU-F culture, of weeks 2 After mice. Bone nvivo in 20) eisem ilg,rglto,and regulation, biology, Periosteum: (2004). 35 a a SACer;T eecletdand collected were -SMA-Cherry;WT, 1003-1012. , -SMA-Cherry;Bmp2cKO ˚ m o – as olwdby followed days, 1–2 for C eouin(ai ta. 2009). al., et (David resolution m a hnosre fe DAPI after observed then was a SA( -SMA m ob Acta1 ascorbic- M n their and Bone gene) 47 , Journal of Cell Science onv .Y,Rmcada,R,Wszn,M . aaoo . Maidment, M., Yamamoto, N., M. Wosczyna, R., Ramachandran, Y., V. Lounev, hs-huhr,N,Coduy .G,Hri,M . ony . ud,G R., G. Mundy, J., Wozney, A., M. Harris, G., G. Choudhury, N., Ghosh-Choudhury, i,Y,Brnsn .D,Ja . oiu,S,Brn . err,N n Olsen, and N. Ferrara, R., Baron, S., Lotinun, S., Jia, D., A. Berendsen, and Y., M. Mina, Liu, C., A. Lichtler, S., M. Kronenberg, A., Braut, W., H. Woitge, F., Liu, T. R. Franceschi, and G. Zhao, C., Ge, Y., Li, Q., Yang, X., Zhao, T.-G., Kwon, hs-huhr,N,Wnl,J . op .A,Hri,M . ureo .L., D. Guerrero, A., M. Harris, A., B. Koop, J., J. Windle, N., C. Ghosh-Choudhury, M. Mullins, and A. J. Dutko, aazc . i . ag .P,Jag . aoh,K,Aas .J,Aguila, J., D. Adams, K., Lamothe, X., Jiang, L.-P., Wang, H., Li, Z., D., Kalajzic, Z. Mason, N., S. Stapleton, L., J. Fitch, C., Wan, S., Z. Kronenberg, Al-Aql, H., A., Li, K. L., Jacobsen, Wang, D., Repic, G., B. Matthews, S., Pejda, D., Grcevic, ekr,M .L,vnBzojn .L,vndrHrt . ogna,J,vnDer van J., Hoogendam, G., Horst, der van L., R. Bezooijen, van L., S. M. M. P. Deckers, Rowe, and M. A. Hedge, A., Martin, V., David, lhkHirc,J,Go . ag . ars .A,Lcte,A,Kem . Zhang, B., Kream, A., Lichtler, A., M. Harris, W., Yang, D., Guo, J., Gluhak-Heinrich, rsn . a,S,Csela . hn .W,Crel,M,Pr,T . Andriolo, S., T. Park, M., Corselli, W., C. Chen, L., Casteilla, S., L. Yap, R. M., Crisan, Jilka, and C. S. Manolagas, Q., J. Feng, V., Dusevich, D., X. Chen, B. D. Burr, F. J. Martin, and J. Selever, E., Klysik, Y., Bai, J., Wang, M., Bonilla-Claudio, P., N. Camacho, L., S. Dallas, R., M. Dallas, J., Rosser, E., S. Harris, F., L. Bonewald, M. Riminucci, and I. Saggio, G., P. Robey, P., Bianco, lhkHirc,J,Yn,W,Hri,M .adHri,S E. S. Harris, and A. M. Harris, W., Yang, J., Gluhak-Heinrich, amle,P n an .K. R. Jain, and P. Carmeliet, and F. T. Linsenmayer, L., M. Crochiere, K., J. Kubilus, A., Bandyopadhyay, J. C. Tabin, and V. Rosen, D., B. Harfe, K., Cox, K., Tsuji, A., Bandyopadhyay, hs-huhr,N,Abu,S . ihmr,R,Clse . Mahimainathan, A., Celeste, R., Nishimura, L., S. Abboud, N., Ghosh-Choudhury, saia,A,Jms .W,Zr,J . oa,R,Crel,M,Pn . in,P., Liang, A., Pan, M., Corselli, R., Goyal, N., J. Zara, W., A. James, A., Askarinam, .D,Soe .M,Gae,D . odae,D .adKpa,F S. F. Kaplan, and J. D. skeletogenesis. heterotopic Am. Goldhamer, to Surg. L., contribute Joint D. that cells Glaser, progenitor M., of Identification E. Shore, D., A. bod .L n ars .E. S. Harris, and L. S. Abboud, dpct differentiation. adipocyte R. B. tissues. skeletal murine in transgenes E. B. HIF-1 Kream, and Runx2 expression. between gene factor interactions growth endothelial functional and Physical ony .M,Mny .R n ars .E. 2-T-an S. Harris, BMP and from R. Endocrinology G. derived Mundy, osteoblasts M., J. Wozney, F eotrt dniya sepoeio population. osteoprogenitor an I. and identify Kalajzic, to and VEGFR1 reporter W. GFP both D. al. Rowe, on L., et H. F. dependent E. is Morgan, L., osteogenesis T. signaling. Clemens, distraction VEGFR2 R., during S. al. Gilbert, formation et M., J. R. Cole, D. Adams, P., cells. Maye, progenitor mesenchymal X., identifies Jiang, S., M. Cell factor growth endothelial vascular A. osteoblast-derived through Lo angiogenesis and stimulate E. S. Papapoulos, C., Bent, . eg .Q,Sih .C,Dco,P tal. cytodifferentiation. et tooth postnatal P. Dechow, in BMP4 C., of L. action Smith, Q., J. Feng, J., hshgyorti MP)i e oernlhroeadvascularization and hormone renal bone new a al. modulator. is et (MEPE) L. phosphoglycoprotein organs. Zhang, human B., multiple in Zheng, cells stem B., mesenchymal Sun, G., facial control to program transcriptional dose-dependent development. a skeletal regulates signaling Bmp formation. bone represents vitro in mineralization that A. Boskey, and B. Boyan, nature, disease. their skeletal of discussion incurable critical in 1066. a significance cells): stem and (skeletal identity, marrow bone human in cells nst yrdzto ihehne estvt nsf,bn n et iseusing tissue teeth and bone soft, in probes. sensitivity RNA enhanced tagged with Digoxigenin hybridization situ in eie eecya rgntrclsadpeet hi ifrnito into differentiation their prevents marrow- and of expansion cells facilitates progenitor cells osteoblasts. marrow mesenchymal bone by derived made matrix Extracellular angiogenesis. of Bone the in expression gene regulating in role their chondrocytes. and underlying periosteum and and patterning J. limb perichondrium in C. 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PLoS 137 20) dniiaino nqemlclrsboan nthe in subdomains molecular unique of Identification (2008). 91 Nature 143 652-663. , 331-339. , 19 .Bn ie.Res. Miner. Bone J. Development 1545-1553. , 1386-1397. , 473 .Ci.Invest. Clin. J. e.Biol. Dev. 20) eurmn fBP2idcdphosphatidylinositol BMP-2-induced of Requirement (2002). 20) o os tiigaoei o ufcett confirm to sufficient not is alone staining Kossa von (2003). 150 298-307. , 2 21) oeua ehnssadciia applications clinical and mechanisms Molecular (2011). e216. , 4012-4023. , 22 21) npht M inln ndevelopment. in signaling BMP SnapShot: (2011). ice.Med. Biochem. wk .W. C. ¨wik, 1943-1956. , 139 20) uoeuaino os M- gene BMP-2 mouse of Autoregulation (2001). 321 .Bo.Chem. Biol. J. n.J e.Biol. Dev. J. Int. 20) s fa lh-mohmsl actin muscle alpha-smooth an of Use (2008). 709-719. , 162-174. , 23 tmCells Stem 122 .Cl.Biochem. Cell. J. 596-609. , 3101-3113. , ie xrsigtasei mice. transgenic expressing tigen 21) ua eiaclrse cells stem perivascular Human (2013). 20) oemrhgntcproteins morphogenetic Bone (2002). 21) e oe n ehns of mechanism and roles New (2010). 20) eiaclroii for origin perivascular A (2008). 18 277 elSe Cell Stem Cell 59-80. , 30 Bone 21) nvv aemapping fate vivo In (2012). acf iseInt. Tissue Calcif. 19) motlzdmurine Immortalized (1996). 21) ‘eecya’ stem ‘‘Mesenchymal’’ (2010). 187-196. , 33361-33368. , 48 u.Gn Ther. Gene Hum. 20) arxextracellular Matrix (2009). Bone 645-653. , 46 112 ice.Bohs Res. Biophys. Biochem. 1533-1545. , 43 3582-3593. , 20) Quantitative (2008). 501-510. , a 3 nuevascular induce 301-313. , 20) Bone (2008). 72 537-547. , 21 .Bone J. 1057- , (2009). (2007). (2011). (2012). (2006). as . amle,G n ciai E. Schipani, and G. Carmeliet, C., Maes, as . oaah,T,Slg .K,Trees . oh .I,Mce,S., Mackem, I., S. Roth, S., Torrekens, K., M. Selig, T., Kobayashi, C., Maes, as . oses . atnoa . rgt . ongahs . tcmn,I., Stockmans, L., Coenegrachts, B., Drogat, S., Bartunkova, S., Goossens, C., Maes, ciai . as . amle,G n eez,G L. G. Semenza, and G. Carmeliet, C., Maes, E., M. Schipani, H. Kronenberg, and E. Schipani, trasotr . air .B,Kn,A,Rlso,O,Lre,H,Bjarnadottir, H., Larsen, O., Rolfsson, A., Kong, B., J. Cazier, U., Styrkarsdottir, and E. S. Harris, M., Ray, A., M. Harris, P. D., Guo, G., Knaus, Scott, T., Castranio, and P., A. Singh, C. Hiepen, J., E., Kopf, S. C., Harris, D., Sieber, Tang, W., Hou, H., Jin, M., Wang, R., Xie, M., Zhang, B., Shu, Saggio, S., Piersanti, S., Cesare, Di S., Michienzi, A., Funari, B., Sacchetti, ya,J . yc,C . ere,D . holy . ’un,E . ai,C A., C. Patil, C., E. O’Quinn, S., Thiolloy, S., D. Perrien, C., C. Lynch, S., J. Nyman, ak . pne,J . o,B . oaah,T,Fjsk,J,Cees .L,Lin, L., T. Clemens, J., Fujisaki, T., Kobayashi, I., B. Koh, A., J. Spencer, D., Park, ikl W. Nickel, enti,R S. R. Weinstein, Rosen, and J. C. Tabin, D., Wang,Q.,Huang,C.,Xue,M.andZhang,X. B. Harfe, A., Bandyopadhyay, K., Cox, K., Tsuji, on .M,Bue,A .adCpch,M R. M. Capecchi, and M. A. Boulet, M., A. Moon, ol .R,Twe,D .adSla .J. M. A., Silva, and C. A. O’Brien, D. Towler, M., R., Almeida, G. Wohl, Y., Wang, Q., Liu, C., Wan, S., R. Weinstein, K. Miyazono, and T. Watabe, L., Gerstenfeld, S., Kakar, K., Cox, D., B. Harfe, A., Bandyopadhyay, K., Tsuji, eii . hr,E . onv .Y,Kpa,F . alr,R n Olsen, and R. Kalluri, S., F. Kaplan, Y., V. Lounev, M., E. Shore, D., Medici, ag . ag . i . eCobuge . io . io .adZhang, and G. Xiao, H., Jiao, B., Crombrugghe, de Y., Li, F., Yang, W., Tang, eii .adOsn .R. B. Olsen, and D. Medici, J. M. and McLeod, A. T. Einhorn, P., D. Mortlock, F., E. Morgan, E., D. Hogan, H., Matsubara, J. M. Silva, and M. D. Ornitz, A., J. McKenzie, J., G. Schmid, D., M. Martinez, eeomn,rgnrto n disease. and regeneration development, aueotolss oeit eeoigadfatrdbnsaogwt invading with along bones fractured and developing vessels. M. into blood H. move osteoblasts, Kronenberg, mature and G. Carmeliet, kltlVG nacsbt-aei ciiyadrslsi xesvl ossified excessively al. in results et and M. activity Naessens, beta-catenin K., bones. enhances Haigh, VEGF O., skeletal Nyabi, K., Moermans, 20) ikg fotoooi ocrmsm 01 n soito oBMP2. to association and 20p12 chromosome al. to et C. osteoporosis Christiansen, Y., of Bagger, S., Linkage M. (2003). Sigurdardottir, D., V. Johannsdottir, E., development. embryonic mouse on 2 protein Y. Mishina, development. bone endochondral during Sci. al. Cell maturation et J. and J. proliferation R. chondrocyte O’Keefe, Y., Mishina, VEGF. and HIFs 1347-1353. by coupling osteogenesis-angiogenesis al. hematopoietic StemBook et a M. organize Riminucci, can sinusoids G., marrow P. bone microenvironment. Robey, in S., osteoprogenitors Ferrari, renewing E., Tagliafico, I., ifrnilefcsbtentels fMP2adMP9o tutrland structural R. on G. MMP-9 Mundy, and and MMP-2 A. of loss Mahadevan-Jansen, the M., between G. effects Pharr, Differential X., Bi, eetrsignaling. receptor regeneration. and T. maintenance Cell bone Stem D. in Cell Scadden, participants fate-restricted and dynamic, M. are MSCs H. Kronenberg, P., C. bone. of properties tissue-level ag rtisadptnilepr routes. export potential and proteins cargo .Bn on ug Am. Surg. Joint Bone J. V. odtoa uat fFgf4. of mutants conditional hsesn . oesn .K,Bse,A . lmn,T .e al. et L. T. hydration, Clemens, mice. vascularity, aged L., in angiogenesis, A. strength skeletal Boskey, and decrease K., glucocorticoids P. 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PLoS 1604-1612. , 21) rncitoa euaino aclrEdteilGot atr(VEGF) Factor Growth Endothelial Vascular of regulation Transcriptional (2012). 20) M4i ipnal o kltgnssadfatr-eln ntelimb. the in fracture-healing and skeletogenesis for dispensable is BMP4 (2008). 1671-1678. , Bmp2 21) ovrino aclredteilclsit utptn tmlk cells. stem-like multipotent into cells endothelial vascular of Conversion (2010). MOJ. EMBO 38 20) h ytr fnnlsia rti erto.Acretve on view current A secretion. protein nonclassical of mystery The (2003). 1424-1429. , abig,M:HradSe elInstitute. Cell Stem Harvard MA: Cambridge, . 16 1 124 20) nlecso eue xrsino aenlbn morphogenetic bone maternal of expression reduced of Influences (2008). e69. , 1400-1406. , 18) ifrnilsann fcriaeadbn nwoemouse whole in bone and cartilage of staining Differential (1980). e.Cell Dev. notolssadlnst Ss4097 MSCs to links and osteoblasts in 21) lccriod,otoye,adseea rglt:terl of role the fragility: skeletal and osteocytes, Glucocorticoids, (2010). 10 3428-3440. , 29 Bone 259-272. , yoieGot atrRev. Factor Growth Cytokine Cell 424-441. , 21) aclrtsusaeapiaysuc fBMP2 of source primary a are tissues Vascular (2012). 46 131 .Bn ie.Res. Miner. Bone J. 0Spl 1 Suppl. 90 19 564-570. , 21) h oeo nohla-eecya rniinin transition endothelial-mesenchymal of role The (2012). gn Cell Aging 329-344. , 324-336. , elRes. Cell 20) oe fTFbt aiysgaigi tmcell stem in signaling family TGF-beta of Roles (2009). Development .Bn ie.Res. Miner. Bone J. si seta o oehealing. bone for essential is ls 14-18. , 21) M2 u o M4 scuilfor crucial is BMP4, not but BMP2, (2011). 19 9 20) dl eecya tmcls In cells. stem mesenchymal Adult (2008). a.Rv Rheumatol Rev. Nat. 147-161. , 103-115. , Teratology 21) yoi-rvnptwy nbone in pathways Hypoxia-driven (2012). 127 u.J Biochem. J. Eur. 21b.Otols rcros u not but precursors, Osteoblast (2010b). e Dev. Sex 27 20) tesfatr eln:fatigue healing: fracture Stress (2009). 989-996. , 1619-1622. , 20) omllm eeomn in development limb Normal (2000). 21) xrsino endogenous of Expression (2011). 20) M2atvt,although activity, BMP2 (2006). 20 20) eetavne nBMP in advances Recent (2009). 21) noeosbn marrow bone Endogenous (2012). 343-355. , 26 22 2 1252-1260. , 134-141. , 299-301. , .Bn ie.Res. Miner. Bone J. 270 8 20) euainof Regulation (2009). 358-366. , 2109-2119. , 21a.Increased (2010a). Bone .Bo.Chem. Biol. J. 20) Self- (2007). Bone 48 (2011). (2010). (2010). ,524- Bone Nat. 51 24 , , Journal of Cell Science 4098 hn,H n rde,A. Bradley, and H. Lyons, Zhang, and R. R. Behringer, Y., Mishina, I., Yoshii, A., D. Gluhak-Heinrich, Ovchinnikov, S., and B. E. Yoon, S. Harris, Y., Mishina, Y., Cui, A., M. Harris, W., Yang, eet nano/hro n ada development. cardiac and amnion/chorion in defects vivo. in chondrogenesis M. K. Res. Dent. J. J. repair. fracture of portion intramembranous 320-330. the mimic that genes angiogenic 21) m2i eurdfrootbatdfeetainadpl vasculogenesis. pulp and differentiation odontoblast for required is Bmp2 (2012). 20) mraadBp1 aeoelpigfntosadaeesnilfor essential are and functions overlapping have Bmpr1b and Bmpr1a (2005). ora fCl cec 2 (18) 126 Science Cell of Journal 91 58-64. , rc al cd c.USA Sci. Acad. Natl. Proc. 19) iedfcetfrBP r oval n have and nonviable are BMP2 for deficient Mice (1996). 102 Development 5062-5067. , 122 2977-2986. , Bone 44 , ho . ars .E,Hr,D,Gn,Z,Nsiua . ud,G .adChen, and R. G. Mundy, R., Nishimura, Z., Geng, D., Horn, E., S. Harris, M., Zhao, hn,R,Oaoi .O,Hri,S . hn . so . eg .W n Zhao, and H.-W. Deng, C., Tsao, D., Chen, M., E., S. Wan, Harris, G., O., B. J. Oyajobi, Chen, R., Zhang, Y., Wang, W., Shi, C., Wan, X., Wu, T., Qiu, F., Zhang, uiepsntlbn formation. bone postnatal murine D. xrsini osteoblasts. in expression M. differentiation. osteoblast Res. and X. Miner. angiogenesis Bone promoting Cao, J. by formation and bone L. stimulates T. Clemens, 20) oemrhgntcpoenrcpo inln sncsayfrnormal for necessary is signaling receptor protein morphogenetic Bone (2002). 21) Wnt/ (2013). b ctnnsgaigatvtsbn opoeei rti 2 protein morphogenetic bone activates signaling -catenin 24 1224-1233. , Bone 52 20) utie M inln nosteoblasts in signaling BMP Sustained (2009). .Cl Biol. Cell J. 145-156. , 157 1049-1060. ,