Blood flow controls bone vascular function and osteogenesis

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1 RTICLE Reeived 8 Jan 16 epted 19 Ot 16 Published 6 De 16 DOI: 1.138/nomms1361 OPEN lood flow ontrols bone vasular funtion and osteogenesis Saravana K. Ramasamy 1,, njali P. Kusumbe 1,3, Maria Shiller 1, Dagmar Zeushner 4, M. Gabriele ixel 1, Carlo Milia 5, Jaba Gamrekelashvili 6, nne Limbourg 7, lexander Medvinsky 8, Massimo M. Santoro 5,9, Florian P. Limbourg 6 & Ralf H. dams 1 While blood vessels play important roles in bone homeostasis and repair, fundamental aspets of vasular funtion in the skeletal system remain poorly understood. Here we show that the long bone vasulature generates a peuliar flow pattern, whih is important for proper angiogenesis. Intravital imaging reveals that vessel growth in murine long bone involves the extension and anastomoti fusion of endothelial buds. Impaired blood flow leads to defetive angiogenesis and osteogenesis, and downregulation of Noth signalling in endothelial ells. In aged mie, skeletal blood flow and endothelial Noth ativity are also redued leading to dereased angiogenesis and osteogenesis, whih is reverted by geneti reativation of Noth. lood flow and angiogenesis in aged mie are also enhaned on administration of bisphosphonate, a lass of drugs frequently used for the treatment of osteoporosis. We propose that blood flow and endothelial Noth signalling are key fators ontrolling ageing proesses in the skeletal system. 1 Faulty of Mediine, Department of Tissue Morphogenesis, Max-Plank-Institute for Moleular iomediine and University of Münster, D Münster, Germany. Researh group Integrative Skeletal Physiology, Institute of Clinial Sienes, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W1 NN, UK. 3 Researh group Tissue and Tumor Miroenvironments, Kennedy Institute of Rheumatology, University of Oxford, Oxford OX3 7LY, UK. 4 Eletron Mirosopy Unit, Max-Plank-Institute for Moleular iomediine, D Münster, Germany. 5 VI Vesalius Researh Center, KU Leuven, 3 Leuven, elgium. 6 Department of Nephrology and Hypertension, Hannover Medial Shool, D-365 Hannover, Germany. 7 Department of Plasti and Reonstrutive Surgery, Hannover Medial Shool, D-365 Hannover, Germany. 8 Researh group Ontogeny of Haematopoieti Stem Cells, MRC Centre for Regenerative Mediine, University of Edinburgh, Edinburgh EH16 4UU, Sotland. 9 Moleular iotehnology Center, Department of Moleular iotehnology and Health Sienes, University of Torino, 116 Torino, Italy. Correspondene and requests for materials should be addressed to S.K.R. ( s.ramasamy@s.mr.a.uk) or to R.H.. ( ralf.adams@mpi-muenster.mpg.de). NTURE COMMUNICTIONS 7:1361 DOI: 1.138/nomms

2 RTICLE NTURE COMMUNICTIONS DOI: 1.138/nomms1361 Osteogenesis is ritial for the maintenane of a healthy and fully funtional skeletal system. Loss of bone mass is a major health issue assoiated with ageing and diseases suh as osteoporosis. During development of the mammalian skeletal system, bone formation is tightly oupled to angiogeni growth of blood vessels 1 3. In the embryo, mesenhymal ondensations express vasular endothelial growth fator (VEGF-), a master regulator of angiogenesis and ligand for the reeptor tyrosine kinase VEGFR (ref. 4). VEGF- ontrols growth plate morphogenesis, artilage remodelling, blood vessel invasion and ossifiation during skeletal development 5 7. ordingly, bone is a highly vasularized tissue ontaining an extensive vasular network of large vessels and apillaries. Reently, we have identified a distint apillary subtype alled type H, haraterized by high expression of the markers CD31 and Endomuin (Emn), whih ouples angiogenesis and osteogenesis in mie 8,9. Osteoprogenitors, bone forming mesenhymal ells identified by the expression of the transription fator Osterix, were seletively loalized in proximity to type H apillaries but were absent around diaphyseal type L vessels (expressing lower levels of CD31 and Emn). Type H endothelial ells (ECs) serete osteogeni fators and maintain Osterix þ osteoprogenitors, but this ruial vessel subtype delined in ageing animals, whih was aompanied by redued osteoprogenitor numbers and loss of bone mass 8,9. In the bone of ovarietomized mie, a model of osteoporosis, type H apillaries were also redued 1. The effet of VEGF-/VEGFR signalling in ECs is strongly linked to the Noth pathway. While VEGF- promotes EC sprouting and proliferation, these proesses are suppressed by Noth reeptors and the ligand delta-like 4 (Dll4) 11,1. ordingly, redued Dll4 expression or inhibition of Noth triggered exessive EC sprouting and hyperproliferation in animal models of developmental and tumour angiogenesis Surprisingly, the ativation of Noth was found to promote angiogenesis in the bone endothelium, whih involved the pararine (also termed angiorine ) release of signals by ECs that are required for hondroyte maturation, Sox9 expression and VEGF expression 9. In addition to moleular pathways, the behaviour of ECs is strongly ontrolled by physial parameters suh as blood flow, whih has roles in angiogenesis 17, vessel remodelling 18 and numerous vasular pathologies 19,. Haemodynamis is also oupled to the homeostasis of the skeletal system 1.Dereasedbloodflowwasfoundtobeassoiatedwith redued bone mass in elderly women. Similarly, hypertension in older men and women is assoiated with inreased bone mineral density 3. Case studies reveal that redued blood supply ause death of bone ells in the osteonerosis patients 4.dditionally,ative blood supply is essential for allus formation during frature healing and repair 5. Impaired blood vessel formation in fratures an result in delayed bone healing and regeneration 6. Thus, blood flow has been linked to bone repair and maintenane 7,buthardly anything is known about the moleular proesses oupling haemodynamis to bone EC funtion and osteogenesis. Here, we show that blood flow is ruial for the formation of type H apillaries and angiogeni growth of the vasulature in bone. Disrupted or pharmaologially redued blood flow results in defetive angiogenesis and osteogenesis, and downregulates Noth signalling in bone endothelium. We also find that redued blood flow and Noth ativity in the bone endothelium of aged mie affets angiogenesis and osteogenesis, whih is reverted by geneti approahes ativating Noth in ECs. The sum of our work highlights entral roles of Noth signalling in bone endothelium and its regulation by blood flow, whih is relevant for age-related bone loss and, potentially, for therapeuti approahes aiming at the maintenane or restoration of bone mass. Results Vasular organization and flow pattern in bone. We investigated the arrangement of arteries, veins and apillaries in tibia to understand fundamental aspets of blood flow pattern in bone. Immunostaining showed that CD31 hi a-sm-overed Emn-negative arteries and distal arterioles seletively onneted to CD31 hi Emn hi apillaries (type H) in the metaphysis and endosteum, but not to diaphyseal sinusoidal (type L) vessels in atively growing (3-week-old) long bone (Fig. 1a, Supplementary Fig. 1a). This pattern indiates that arterial blood enters the long bone at its distal ends as well as at the inner surfae of ompat bone, and flows through type H apillaries into the highly branhed sinusoidal vasulature. From this intriate network, blood is drained by a large Emn þ CD31 lo a-sm-negative vein with a lumen size of 41 mm in the enter of the diaphysis (Fig. 1a e; Supplementary Fig. 1b). To measure blood veloity in type H and type L vessels of long bone, intravital imaging was performed after dextran injetion in living Flk1-GFP reporter mie (Supplementary Movies 1 and ). Consistent with the hierarhial organization of the bone vasulature, line-sanning analysis in living mie revealed that blood veloity in type H apillaries was muh higher (.98±.1 mm s 1 ) than in type L sinusoidal vessels (.16±4 mm s 1 ; Fig. 1f). Furthermore, blood veloity dropped further after eah vessel branh point in the metaphysis until it finally reahed the low veloity harateristi for diaphyseal type L vessels (Fig. 1g). ordingly, alulated shear rate is signifiantly higher for type H apillaries than in type L vessels (Supplementary Fig. 1). The observed differenes in blood flow were also in line with the higher transription of the flow-regulated genes Peam1, Nos3, Iam1 and Cdh5 in freshly isolated type H ECs than type L endothelium (Supplementary Fig. 1d). These findings raised the possibility that phenotypi differenes between type H and L vessels (Fig. 1h) might be linked to blood flow. lood flow regulates bone angiogenesis. To haraterize the role of haemodynami fores in the generation of type H vessels that mediate angiogenesis, we modulated blood flow in bone. We analysed the 3-week-old tibial vasulature after ligation of the femoral artery (Fig. a and Supplementary Fig. a), whih is the main vessel supplying blood to the lower limb 8,9. t 48h post surgery (hps), the inhibition of loal blood flow resulted in the strong redution of olumnar vessels and endothelial bud strutures in proximity of the growth plate (Fig. b and Supplementary Fig. b) without promoting endothelial apoptosis (Supplementary Fig. ). t an earlier time point after surgery (16 hps), profound anastomoses of buds ould be observed in tibial blood vessels near the growth plate (Supplementary Fig. d). is an inhibitor of the alpha 1-adrenergi reeptor that redues blood pressure via relaxation of vasular smooth musle and thereby inreases flow into peripheral apillaries 3. has been reently shown to redue bone formation in mie, whih has been linked to alpha 1-adrenoreeptor signalling in osteoblasts 31. We found that administration of for weeks led to signifiantly redued blood flow in bone (Fig. ), whih might result from the diversion of flow into other organs. The observed flow redution was aompanied by dereased abundane of endothelial buds near the growth plate and of the number of metaphyseal CD31 hi type H vessels (Fig. d f) suggesting dereased angiogenesis. nalysis of EdU þ ells on treatment showed dereased proliferation of ECs (Fig. g and Supplementary Fig. e). The expression of flow-regulated genes was signifiantly downregulated in freshly isolated bone ECs following treatment (Supplementary Fig. f). Similar phenotypi hanges in blood vessels were observed in NTURE COMMUNICTIONS 7:1361 DOI: 1.138/nomms1361

3 NTURE COMMUNICTIONS DOI: 1.138/nomms1361 RTICLE mie treated with another flow-reduing drug, Clonidine (Supplementary Fig. g,h). Together, these data indiate that angiogenesis in bone is impaired by alterations in blood flow. Mode of angiogenesis in bone. s pharmaologial and surgial impairments in blood flow led to profound morphologial hanges in type H vessels, we investigated the mode of blood a α SM/Emn/DPI b CD31/Emn CD31/Emn es es mp mp mp dp dp dp es V 3 μm 1 μm d α SM/Emn/DPI e f Type H Type L V 1 μm rtery Type L Type H Erythroyte veloity (mm s 1 ) Type H Type L g h CD31/DPI Flk1-GFP / Dextran Erythroyte veloity (mm s 1 ) ranh points mp Type H Type L dp μm Metaphysis Diaphysis Figure 1 lood flow dynamis in long bone. (a) Tile san image of tibial vasulature immunostained for Emn (red) and a-smooth musle atin (green). rteries overed by a-sm þ ells onnet (arrows) to metaphyseal (mp) type H vessels near growth plate (dashed lines, ). Sinusoidal type L vessels onnet to entral large vein (yellow arrowhead). (b,) Confoal images of 4-week-old metaphysis near growth plate () (b, top panels) or diaphysis (dp) (, top). CD31 þ (green) Emn- (red) arteries terminate in CD31 þ Emn þ type H vessels in the metaphysis (mp) and endosteum (es) but not in type L vessels in diaphysis (dp). lue arrows in Emn-stained (red) overview images (bottom panels) illustrate blood flow diretion from metaphyseal vessel olumns (b) and endosteum () into the adjaent sinusoidal network and veins. (d) Maximum intensity projetion of transversal setions of 4-week-old tibia immunostained for a-sm (green) and Emn (red). Sinusoidal type L vessels (arrowheads) onnet to a large entral vein (v). Multiple smooth musle-overed CD31 þ Emn-arteries ross the diaphysis. Dashed lines mark ompat bone. (e) Diagram of arterial (green arrows), type H (red arrows) and sinusoidal/venous flow (blue arrows) in murine long bone. (f) Graph showing blood veloities alulated from line sanning of type H and type L vessels after Dextran (,, Da) injetion. Data represent mean±s.d. (n ¼ 5 biologial repliates). P values, two-tailed unpaired t test. (g) Overview image and representative line sans showing blood veloities in -week-old long bone. Note dereasing veloity of erythroytes at eah branh point (represented as 1,, 3, 4 ) at the interfae between olumnar type H (right) and diaphyseal type L vessels (left). Data represent mean±s.d. (n ¼ 5 biologial repliates). (h) Confoal images of CD31 (green) expression in the metaphysis and diaphysis of 4-week-old tibia. Note differenes in CD31 levels. Sale bars are as indiated in the respetive images. DPI (blue) is used for ounterstaining of nulei. NTURE COMMUNICTIONS 7:1361 DOI: 1.138/nomms

4 RTICLE NTURE COMMUNICTIONS DOI: 1.138/nomms1361 a b CD31/Emn Femoral artery ligation Ligation Contralateral 48 hps Ipsilateral μm Contralateral Ipsilateral Inset Inset 1.5 P=14 d Fold hange in blood flow 1..5 Emn Insets e CD31 3 μm f Fold hange type H g EdU+ ells per 1 um metaphysis vessel area Figure Changes in blood flow impair bone angiogenesis. (a) Shemati representation of unilateral femoral artery ligation in 3-week-old mie. Results ompare operated (ipsilateral) legs with ontralateral ontrols. (b) Representative onfoal images showing CD31 (green) and Emn (red) immunostaining of tibia from ligated and ontralateral side at 48 h post surgery (hps). Note deline in vasular front strutures, buds and olumns on ligation. Insets show vessel buds (blue arrowheads) on the ontralateral side and appearane of pointed, sprout-like strutures (white arrowheads) after ligation. () nalysis of blood flow in -treated tibia ompared with saline-treated ontrol. Data represent mean±s.d. (n ¼ 4 biologial repliates). P values, two-tailed unpaired t test. (d,e) Confoal images of 4-week-old -treated and ontrol tibial metaphysis immunostained for Emn (red; d) or CD31 (green; e). Insets in d show vessel buds (blue arrowheads), whih appeared ollapsed (white arrowheads) after treatment. CD31 (green) expression was dereased in -treated animals (e). (f) Flow ytometri quantifiation of tibial type H ECs after treatment with or ontrol (saline). (g) Quantitation of EdU þ ECs present in the metaphysis of -treated and ontrol mie. Data represent mean±s.e.m. (n ¼ 4 biologial repliates). P values, two-tailed unpaired t test. vessel growth in postnatal bone. While the olumnar organization is a harateristi feature of metaphyseal type H vessels, these olumns were interonneted by distal, loop-like arhes. ud-shaped protrusions emerging from those arhes were loalized in lose proximity of hypertrophi growth plate hondroytes (Fig. 3a). Their juxtaposition to hypertrophi hondroytes, a known soure of VEGF- (ref. 5), suggested that buds represent the leading edge of the growing vasulature, whih would make them funtionally equivalent to endothelial tip ells and sprouts observed in other organs 3,33. ordingly, the length of olumns and the abundane of buds inreased during postnatal bone growth (Supplementary Fig. 3a ). In adult (1-week-old) long bone, arhes remained as the most prominent distal vessel struture, while the abundane of buds was strongly dereased (Fig. 3b; Supplementary Fig. 3b). In line with the previously reported redution of type H vasulature in long bone of aged mie 8, CD31 hi vessel arhes in 8-week-old mie were sparse and spanned longer distanes than in adults, while buds were almost ompletely absent (Fig. 3b). To gain insight into the dynamis of blood vessel formation in postnatal long bone, we developed an intravital live imaging tehnique using the endothelium-speifi Flk1-GFP reporter mie to monitor blood vessel growth in long bone. The observation of green fluoresent protein (GFP) þ ECs next to the growth plate demonstrated the emergene of buds from arhes (Fig. 3 and Supplementary Movie 3). Stati imaging and ultrastrutural analysis by sanning eletron mirosopy indiated that lumenized buds arried short filopodia, whih onneted to the surrounding hondroyte matrix (Fig. 3d,e and Supplementary Fig. 4a). Other buds laked visible filopodia and were found in proximity of intat hondroytes (Fig. 3e and Supplementary Fig. 4b d), suggesting that they were not atively extending at the time of analysis. Mosai fluoresent labelling of ECs by tamoxifen-indued, EC-speifi reombination of R6-Confetti reporter mie indiated that buds were omposed of multiple ells enlosing a luminal spae (Fig. 3f and Supplementary Fig. 4e,f). Dynami and stati imaging approahes also established that new arhes were formed through the anastomoses of two neighbouring bud strutures (Fig. 3g i and Supplementary Movie 4). Thus, arhes and buds are dynami strutures that are formed during ative angiogenesis and mediate vessel growth in long bone. Due to redued or absent endothelial budding after 4 NTURE COMMUNICTIONS 7:1361 DOI: 1.138/nomms1361

5 NTURE COMMUNICTIONS DOI: 1.138/nomms1361 RTICLE a CD31 Ch b CD31/Emn 4 weeks 1 weeks 8 weeks Ch Ch Ch Flk1-GFP e Still images from supplementary video. 3 h 1 h h 3 h 5.3 h 5.6 h 6 h 7 h Cdh5-CreERT X R6-mTmG f d SEM Ch V Cdh5-CreERT X R6-Confetti V 1 μm g DIC GFP Merge 5 μm 7 dpi 1 dpi h hondroytes buds arhes CD31/Ter119 i Still images from supplementary video. 4 μm Flk1-GFP min 3 min 5 min 6 min 7 min 8 min 9 min Figure 3 lood vessel growth in bone. (a) Maximum intensity projetion of metaphyseal vessel strutures (CD31, red): distal, loop-like arhes () and bud-shaped protrusions () present in proximity of hypertrophi hondroytes (Ch). (b) Representative onfoal images of tibia setions from 4, 1 and 8-week-old mie immunostained for Emn (red) and CD31 (green). CD31 hi Emn hi ECs form olumns, distal arhes () and buds () at the vasular growth front, whih were abundant at 4 weeks but delined during ageing (1 and 8 weeks). rrowheads indiate Emn- arterioles. Ch, hondroytes. () Still images of the indiated time points from a 1 h time lapse movie of 1-day-old Flk1-GFP metatarsal. rrow indiates bud emerging from arh (). (d) SEM images of blood vessel (V) next to hondroytes (Ch) in 4-week-old tibia with distal arh () and bud (; arrowhead). Right panel shows higher magnifiation of inset with bud emerging from arh. (e) Merged onfoal and differential interferene ontrast (DIC) image showing distal vessel buds () genetially labelled by GFP (green). rrow indiates a filopodia-bearing endothelial bud displaing an apoptoti hondroyte, arrowhead points at filopodiafree bud next to intat hondroyte. (f) 4-week-old Cdh5-CreERT R6-Confetti double transgenis at 7 and 1 days post injetion (dpi) showing high degree of mosaiism in endothelial olumns, arhes and buds. (g) Shemati illustration of vessel growth in long bone. Invading vessels buds () displae apoptoti growth plate hondroytes () and, through anastomosis, generate new arhes (), from whih new buds an emerge. (h) Representative onfoal images showing ontat formation and anastomosis (arrows) of CD31 þ (green) vessel buds in 4-week-old tibia. Note red presene of Ter-119 þ RCs (red) in buds and forming vessel arhes. (i) Still images (time indiated) from movie showing anastomosis (arrows) of buds (arrowheads in left image) in 1-day-old Flk1-GFP (green) metatarsal. rrows show the disappearane of juntion between anastomosing buds at later time points. the ompletion of postnatal bone growth, vessel arhes remain as the predominant distal vasular struture in adult and aged long bone (Fig. 3b). These results also indiate that the defetive angiogenesis observed under flow-modulated onditions is due to defetive bud formation and anastomosis at the vasular front (Fig. b and Supplementary Figs b and 4g). Flow-mediated oupling of angiogenesis and osteogenesis. s angiogenesis in bone is tightly oupled with osteogenesis, we investigated whether modulation of blood flow would affet bone formation. oth femoral artery ligation and treatment led to signifiant redutions in Osterix-expressing osteoprogenitors (Fig. 4a and Supplementary Fig. 5a,b). NTURE COMMUNICTIONS 7:1361 DOI: 1.138/nomms

6 RTICLE NTURE COMMUNICTIONS DOI: 1.138/nomms1361 Moreover, miro-ct analysis onfirmed that administration led to loss of mineralized bone (Fig. 4d,e and Supplementary Fig. 5). This is likely to reflet altered bone formation rates, as osteolast numbers were not signifiantly hanged (Supplementary Fig. 5d). Defetive blood flow also led to redued expression of pro-osteogeni fators in freshly sorted bone ECs (Fig. 4f). Next, we investigated the moleular link between blood flow and angiogenesis in bone. Peam1/CD31 is highly expressed by ECs in type H arh and bud strutures (Fig. 1h) and was shown to have mehanosensory ativity 34,35. To evaluate a potential involvement of CD31 in the generation of speifi vessel subtypes, we analysed the vasulature in long bone of Peam1 knokout mie 36. However, in spite of absent CD31 expression, these mutants did not show defets in bone angiogenesis (Supplementary Fig. 6a). Likewise, Osterix-expressing ells were unaffeted in Peam1-defiient mie (Supplementary Fig. 6b) indiating that this moleule is dispensable for physiologial bone angiogenesis and osteogenesis. Flow modulates endothelial Noth signalling. Noth signalling in bone endothelium promotes blood vessel growth and has been shown to ouple angiogenesis and osteogenesis 9. Immunostaining showed that Dll4, the ritial Noth ligand in bone ECs, was highly expressed on the endothelium of arteries and distal type H buds, whih was independent of PECM1/CD31 expression (Supplementary Fig. 6). The transription fator RP-Jk is an essential regulator of Noth-indued gene expression inside and outside the vasulature 37 39, and Cdh5(PC)-CreERT-mediated a Osx/Emn Contralateral Ipsilateral b % Osx+ ells Contralateral Ipsilateral d Osx/Emn miro-ct 1 μm e V/TV f 6 4 Rel. fold mrn levels P=5 P=477 Trabeular thikness (mm) P= Sorted bone ECs P=35 P=68 P=93 Trabeular N. (mm 1 ) 3 1 P=.139 P=.9313 Tgfb1 Tgfb Tgfb3 Wnt1b Pdgfb mp mp4 Fgf1 Figure 4 Flow-mediated angiogenesis is oupled to osteogenesis. (a) Maximum intensity projetions of Emn (red) and Osterix (Osx, green) immunostaining in ontralateral (ontrol) and ipsilateral (ligated) tibia at 48 hps after femoral artery ligation. (b) Quantitation of Osx þ ells in ontralateral and ipsilateral tibia after femoral artery ligation. Data represent mean±s.e.m (n ¼ 5 mie in three independent experiments). P values, two-tailed unpaired t test. () Confoal images of and ontrol tibial setions immunostained for Emn (red) and Osterix (Osx, green). (d) 3D rendering showing miro-ct analysis of mineralized regions in the metaphysis of ontrol and -treated tibiae. (e) Histomorphometrial data derived from miro-ct sans. Note redued bone parameters after treatment. (f) RT-qPCR for the expression of known pro-osteogeni fators suh as Tgfb1, Tgfb, Tgfb3, Wnt1b, Pdgfb, mp, mp4 and Fgf1 (normalized to tb expression) in sorted bone ECs from ontrol and -treated mie. Data represent mean±s.d. (n ¼ 8 biologial repliates). P value, two-tailed unpaired t test. 6 NTURE COMMUNICTIONS 7:1361 DOI: 1.138/nomms1361

7 NTURE COMMUNICTIONS DOI: 1.138/nomms1361 RTICLE inativation of the Rbpj gene led to defetive growth and severe dilation of metaphyseal vessels in the resulting Rbpj idec mutants 9. Consistent with these morphologial alterations, transript analysis of sorted Rbpj idec bone ECs revealed a downregulation of the flow-modulated genes Klf, Nos3 and Peam1 relative to ontrol littermates (Supplementary Fig. 7a). Conversely, EC-speifi and induible (Cdh5(PC)-CreERT- ontrolled) overexpression of the ative Noth1 intraellular domain (NICD), whih was reently shown to enhane artery formation in long bone 4, led to the upregulation of Klf, Nos3 and Peam1 in ECs (Supplementary Fig. 7a). Thus, Noth signalling ontrols the expression of flow-ontrolled genes, whih presumably reflets morphologial alterations in the bone vasulature. Other results indiate that the expression of Noth pathway omponents and Noth signalling in ECs are regulated by flow. treatment led to a strong redution of Dll4 expression by bone ECs at the transript and protein level (Fig. 5a,b). Likewise, mrns for several Noth pathway target genes were signifiantly downregulated in freshly isolated bone ECs from -treated animals (Fig. 5b). Tibial ECs from femoral artery ligation experiments (16 hps) also showed redued expression of Dll4 (Supplementary Fig. 7b) and other Noth downstream target genes (Fig. 5). Conversely, when mie were treated for 48 h with angiotensin II to inrease blood flow, sorted bone EC showed inreased expression of Noth target genes (Supplementary Fig. 7). The exposure of ultured ECs to laminar flow inreased the level of NICD protein and upregulated the expression of known Noth target genes (Supplementary Fig. 7d,e). Together, these data indiate that endothelial Noth signalling is ontrolled by blood flow. lood flow in aged mie bones. Skeletal ageing is assoiated with dereased bone formation and loss of type H vasulature 8. Gene expression analysis in freshly isolated bone ECs from aged mie revealed that transript levels for numerous Noth target genes were strongly downregulated (Fig. 5d,e). Measurements using radiolabelled mirospheres in rats have previously shown that blood flow in aged bone is dereased relative to young adult animals 41. Our own analysis also indiated a remarkable deline in blood flow in long bone of aged mie relative to young animals (Fig. 5f). These data suggest that the redutions in both Dll4 expression and Noth signalling in ECs of the aged bone vasulature might be indued or enhaned by hanges in blood flow. To further investigate the link between endothelial Noth ativity, blood flow and organization of the bone vasulature, was administered to transgeni mie overexpressing ative NICD of Noth1 (NICD ioe-ec ) 4 in ECs. Remarkably, bud strutures at the vasular growth front were preserved in Proazosin-treated NICD ioe-ec gain-of-funtion mutants (Fig. 5g and Supplementary Fig. 7f). Thus, Noth ativation in bone ECs was suffiient to overome defets aused by administration of. Next, we addressed whether EC-speifi Noth ativation would be also suffiient to overome age-related hanges in the bone vasulature. For this purpose, NICD ioe-ec mutants at the age of weeks reeived injetions of tamoxifen before analysis 4 days later (Fig. 5h). This approah led to the reappearane of type H vessels and formation of Dll4-positive endothelial buds in the NICD ioe-ec but not in the ontrol metaphyseal region (Fig. 5i,j and Supplementary Fig. 8a,b). seond strategy involved EC-speifi inativation of the gene enoding Fbxw7 (Fbxw7 idec ) 43, whih mediates the polyubiquitination and proteasomal degradation of ative Noth. Tamoxifen treatment of aged Fbxw7 idec mie also lead to the reappearane of CD31 hi apillaries together with Dll4-positive vessels buds, whih were absent in ontrol littermates (Fig. 5h and Supplementary Fig. 8). Changes in the vasulature of aged NICD ioe-ec and Fbxw7 idec mutants were aompanied by signifiant inreases in Osterix-expressing osteoprogenitors (Fig. 5k; Supplementary Fig. 8d,e) and enhaned bone formation, as seen by miro-ct analysis (Supplementary Fig. 9a d). The formation of new bone also involved enhaned bone remodelling indiated by the inreased abundane of osteolasts (Supplementary Fig. 9e). Thus, EC-speifi Noth-mediated reativation of type H vessels in aged mie an promote both angiogenesis and osteogenesis leading to improved bone mass. isphosphonate inreases bone blood flow and angiogenesis. isphosphonates are ommonly used mediations for osteoporosis treatment. While the inhibition of hydroxyapatite breakdown by osteolasts is the main mehanism of ation for bisphosphonates, it has been proposed that this lass of antiresorptive drugs also ats on other ell types and inhibits apoptosis of osteoblasts and osteoytes 44,45. s age-related hanges in bone mass in mie strongly orrelate with alterations in the abundane of CD31 hi vessels in bone 8, blood flow, EC proliferation and the presene of endothelial buds (Fig. 6a), we tested whether bisphosphonate treatment would affet any of these vasular parameters. Indeed, administration of lendronate to aged mie ( mg kg 1 ) twie a week for 6 weeks led to substantial inreases in type H vessels and Osterix þ osteoprogenitors relative to ontrol animals (Fig. 6b,). lendronate also indued the reappearane of vessel buds in the metaphysis and expression of the Noth ligand Dll4 (Fig. 6d). Expression analysis of freshly sorted ECs from aged bone showed that lendronate signifiantly inreased transript levels of several Noth target genes (Fig. 6e), whereas suh hanges were not indued by lendronate in ultured primary bone ECs (Supplementary Fig. 9f). In addition, lendronate treatment led to signifiantly inreased blood flow (Fig. 6f). To understand whether the inrease in blood flow on lendronate treatment preedes or sueeds new blood vessel formation, we measured flow in the bone vasulature after short treatment with bisphosphonate. Following three injetions of lendronate every 1 h, 3-week-old mie showed signifiantly inreased perfusion of the bone vasulature at 3 min after last injetion (Fig. 6g). Taken together, these data argue that bisphosphonate treatment an trigger a omplex response in bone, whih involves enhanements in blood flow, ativation of endothelial Noth signalling and vessel growth. Disussion In addition to their onventional role as a onduit system for blood irulation, there is inreasing evidene that vasular ells generate speialized miroenvironments supporting stem and progenitor ells. In bone, the loal vasulature provides nihes for haematopoieti stem ells and thereby regulates haematopoiesis Likewise, bone formation and frature healing are ontrolled by blood vessels and EC-derived moleular signals 8,9,5,51. The osteogenesis-promoting effet of the bone vasulature in physiologial settings was reently attributed to the type H apillary subset, whih is haraterized by high expression of the ell adhesion protein CD31/PECM1 and assoiation with perivasular Osterix-positive ells 8,9. Indiating potential roles in disease and therapy, CD31 hi Emn hi apillaries were dereased in ovarietomy-indued osteoporoti mie, whereas the administration of athepsin K inhibitor, whih impairs bone resorption, led to an inrease in CD31 hi Emn hi vasulature 1. NTURE COMMUNICTIONS 7:1361 DOI: 1.138/nomms

8 RTICLE NTURE COMMUNICTIONS DOI: 1.138/nomms1361 one growth is ontrolled by geneti programs and physial fators suh as mehanial loading 5,53. Chondrogenesis, hondroyte differentiation, VEGF expression in the growth plate and angiogenesis are also influened by mehanial signals 5,54,55. Our urrent study unravels the ellular basis of angiogeni vessel growth in the metaphysis by type H apillaries in diret proximity of growth plate hondroytes with live imaging, whih revealed that new anastomoti onnetions (arhes) are formed by fusion of multiellular vessel buds. lood flow is required for the formation and anastomoti fusion a DPI/Dll4 b Rel. fold mrn levels Sorted bone ECs P=5 P= P=.6 P=4 P=.7 Dll4 Hes1 Hes5 Hey1 Hey Jag1 Rel. fold mrn expression P=135 P=7 P=.1 P=119 P=14 Dll4 Hes1 Sorted bone ECs Contralateral Ipsilateral Hes5 Hey1 Hey Jag1 d Rel. fold mrn levels 3 1 g Sorted bone ECs P=4 P=3 P=1 P=4 P= Dll4 Hes1 Hes5 Hey1 Hey Jag1 Young Old CD31/Emn e Young Old Dll4/Emn Insets f Fold hange in blood flow Young ( weeks) Old (8 weeks) NICD ioe-ec NICD ioe-ec NICD ioe-ec NICD ioe-ec h / NICD ioe-ec / Fbxw7 iδec d1 d5 Tamoxifen-1 (i.p) ged mie (>1 yr) Rest 16 days d6 d1 Tamoxifen- (i.p) Rest 16 days nalysis i ged tibia CD31/Emn j uds per mm metaphysis 1 μm NICD ioe-ec NICD k ged tibia DPI/ Osx 1 μm NICD ioe-ec 8 NTURE COMMUNICTIONS 7:1361 DOI: 1.138/nomms1361

9 NTURE COMMUNICTIONS DOI: 1.138/nomms1361 RTICLE of buds in the vasular front, type H vessel formation and angiogenesis in bone. Thus, CD31 hi Emn hi apillaries are not only onneting diretly to distal arterioles and are therefore perfused at higher veloity than the highly branhed sinusoidal network, but blood flow also regulates fundamental properties of the bone vasulature and its ability to promote osteogenesis. The relationship between blood pressure, flow in the skeletal system and bone mass in the linial ontext is omplex and linked to numerous fators suh as nutrient delivery, the regulation of loal metabolism or the influx of alium and phosphate 1,7. Dereased blood flow in bone has been reported in pathologial onditions suh as osteoporosis and osteonerosis 4. Likewise, ageing is not only assoiated with bone loss but also with redued blood flow in the skeletal system 41. Our new findings link these flow and ageing-related hanges to redued Noth ativity in ECs. While endothelial Noth signalling is known to suppress vessel growth in a wide range of organs and model organisms 13,56,57, the pathway ats as a strong stimulator of angiogenesis and thereby osteogenesis in the skeletal system 9. The expression of Noth pathway genes and bone formation were strongly redued by treatments disturbing normal blood flow or in aged animals, whereas the reativation of Noth signalling in ECs was suffiient to restore loal angiogenesis and bone formation (Fig. 7). number of reports addressing the relationship between flow and Noth signalling suggest omplex and, potentially, ontextdependent interations. Noth pathway genes are highly expressed by arterial ECs, whih are exposed to higher flow rates and shear stress, whereas venous expression is low or undetetable 58,59. This is onsistent with numerous studies reporting Noth ativation in blood vessel ECs in response to flow and laminar shear stress 6 6. In ontrast, loss of blood flow led to inreased vasular Noth signalling in the vasulature of zebrafish embryos 63. Noth failitates vessel onstrition during the regression (pruning) of apillaries 64, but Noth ativity is also required for nitri oxide indution and vasodilation in response to ishaemia in the adult vasulature 65. Furthermore, Noth ativation is a hallmark of arteriovenous malformations and onstitutive ativation of Noth4 has been shown to indue the enlargement of apillary vessels into arteriovenous shunts 66. We propose that Noth ontrols blood flow and expression of flow-ontrolled genes in the bone endothelium presumably by ontrolling vasular morphology, angiogeni growth and the formation of arteries/arterioles in bone. Whether suh effets might also involve the release of vasular autooids (that is, loal ating fators) suh as nitri oxide, whih was shown to redue bone resorption 67, or pro-anaboli moleules that promote bone formation, suh as alitonin gene-related peptide 68, remains to be investigated. Noth signalling also has important ell-autonomous roles in the osteoblast lineage and preserves mesenhymal progenitors by preventing exessive differentiation of these ells 69,7. Noth ativation in mature osteoytes triggers a strong skeletal anaboli response in adult mie, whih is suffiient to resue age-assoiated and ovarietomy-indued bone loss 71. s osteoyte proesses physially interat with the vasular endothelium 7, it would be worthwhile to investigate whether different ell types ommuniate through diret Noth-ligand interations. Our findings also support that Noth ativity and pathway gene expression in ECs depend on flow, whih is therefore ritial for the formation of type H apillaries, the formation of endothelial buds and thereby the expansion of the growing vasulature, and Noth-mediated oupling of angiogenesis and osteogenesis. ordingly, redued blood flow in the ageing organism might be a ause of redued endothelial Noth ativity, whih, in turn, might ontribute to age-related bone loss. Supporting this onept, the reativation of Noth signalling in the ageing vasulature was suffiient to indue loal growth of type H apillaries, whih was aompanied by the expansion of vessel-assoiated Osterix þ osteoprogenitors and formation of mineralized bone. Remarkably, treatment of mie with bisphosphonate, the lass of drugs that is ommonly used in humans to prevent the loss of bone mass, also led to the indution of endothelial Noth expression, the emergene of CD31 hi Emn hi apillaries and inreased blood flow. While it remains unlear whether the effet of lendronate on bone ECs is diret or mediated by hanges in other ell types, our findings nevertheless indiate that properties of the loal vasulature might play a far more entral role in the regulation of bone mass than previously appreiated. Methods ge groups and genetially modified mie. C57L/6 J males were used for all experiments involving wild-type mie and pharmaologial treatments, whereas both males and females animals were analysed in the geneti experiments. Mie at the age of 4 weeks and 453 weeks were hosen for young and aged groups, respetively, unless speified otherwise. For treatment, C57L/6 J mie were given.5 mg kg 1 body weight dose every seond day from P1 to P before they were killed and analysed at P4. For lendronate treatment in aged mie, 75-week-old C57L/6 J mie were given mg kg 1 body weight dose twie a week for 5 weeks and killed on the 6th week. Mosai multi-olour labelling of ECs was ahieved by ombining R6R-Confetti reporter 73, whih an express four different fluoresent proteins in a stohasti and Cre-dependent fashion, with Cdh5(PC)- CreERT transgenis 8,74 expressing tamoxifen-induible CreERT speifially in ECs. 4-Hydroxy tamoxifen was administered (1 mg) by five onseutive, daily injetions, whih was followed by analysis of the bone vasulature at 7 or 1 days after the last injetion, as indiated. For Fbxw7 deletion in the vasulature of aged mie, mie arrying loxp-flanked Fbxw7 gene (Fbxw7 floxed ) 43 were interbred with the Cdh5(PC)-CreERT line. Fbxw7 floxed/floxed Cdh5(PC)-CreERT T/ þ males were interbred with Fbxw7 floxed/floxed females to generate litters with Fbxw7 floxed/floxed Cdh5(PC)-CreERT T/ þ (Fbxw7 idec ) mutants and Cre negative Fbxw7 floxed/floxed (ontrol) littermates. To indue Cre-mediated gene inativation, Figure 5 Flow positively regulates endothelial Noth signalling in bone. (a) Dll4 (green) immunostaining of -treated and ontrol tibia setions. (b,) qpcr analysis of Dll4, Hes1, Hes5, Hey1, Hey and Jag1 expression (normalized to tb) in ECs sorted from -treated or ontrol long bone (b) and from ipsilateral (operated) and ontralateral sides of femoral artery ligated (16hps) limbs of mie (). Data represent mean±s.d. (n ¼ 5 biologial repliates). P values, two-tailed unpaired t test. (d) qpcr analysis of Dll4, Hes1, Hes5, Hey1, Hey and Jag1 expression (normalized to tb) in ECs sorted from 4 (young) and 85-week-old (old) long bone. Data represent mean±s.d. (n ¼ 5 biologial repliates). P values, two-tailed unpaired t test. (e) Representative onfoal images showing the metaphysis region near the growth plate (, dashed line) in young (4 week) and aged (85 week) tibia immunostained for Emn (red) and Dll4 (green). (f) lood flow measurements in young and old mie show redued flow upon ageing. Data represent mean±s.d. (n ¼ 5 biologial repliates). P values, two-tailed unpaired t test. (g) Maximum intensity projetions of tibial setions from NICD ioe-ec (Noth gain-of-funtion) mie and littermate ontrols treated with saline (ontrol) and, immunostained for CD31 (green) and Emn (red). lue arrowheads indiate buds and arhes, white arrowheads mark defetive buds in the vasular front. (h) Experimental sheme of tamoxifen administration to aged transgeni mie for EC-speifi ativation of Noth signalling. (i) Confoal images of aged NICD ioe-ec and littermate ontrol tibiae immunostained for CD31 (green) and Emn (red). Note inrease in CD31 þ vessels and buds (arrows) near NICD ioe-ec growth plate (, dashed blue line). (j) Quantifiation of bud strutures in the vasular front of aged NICD ioe-ec and littermate ontrol tibiae. Data represent mean±s.d. n ¼ 4 in three independent experiments. P values, two-tailed unpaired t test. (k) Confoal images of the metaphyseal region of aged NICD ioe-ec and littermate ontrol tibiae immunostained for Osterix (Osx, white). Nulei, DPI (blue). NTURE COMMUNICTIONS 7:1361 DOI: 1.138/nomms

10 RTICLE NTURE COMMUNICTIONS DOI: 1.138/nomms1361 a b lendronate Fold hange EdU uds Flow CD31/Emn ge (weeks) ged: 8 weeks ged: 8 weeks 3 μm lendronate Inset d Dll4/DPI CD31/Emn/Osx Osx/DPI 1 μm Rel. fold mrn expression 8 lendronate 6 4 P= P=3 lendronate e f g Dll4 Hes1 Hes5 Hey1 Jag1 Fold hange in blood flow 3 1 P=86 lendronate Fold hange in blood flow P=71 lendronate Figure 6 lendronate stimulates endothelial Noth signalling and flow in bone. (a) Graph showing age-dependent hanges in EdU þ ECs and bud strutures in distal tibia. lood flow measurements in the same age groups indiate a similar pattern. Data represent mean±s.d. (n ¼ 5 biologial repliates). P values, two-tailed unpaired t test. (b) Tile san images of tibia from ontrol and lendronate-treated 8-week-old mie. Setions were immunostained for CD31 (green) and Emn (red). Note appearane of CD31 þ blood vessels (arrows) in lendronate-treated metaphysis. () Vasular front in setions of ontrol and lendronate-treated aged tibia immunostained for CD31 (green), Emn (red) and Osterix (Osx, white). Nulei, DPI (blue). Inset shows CD31 þ blood vessels in lose proximity to osteoblasts. (d) Confoal images showing metaphysis of aged mie after lendronate administration. Immunostaining shows endothelial Dll4 (green). Nulei, DPI (blue). (e) qpcr analysis of Dll4, Hes1, Hes5, Hey1 and Jag1 transripts expression in ECs sorted from ontrol and lendronate-treated 8-week-old long bone. Data represent mean±s.d. (n ¼ 6 biologial repliates). P values, two-tailed unpaired t test. (f) Inreased blood flow in tibia of lendronate-treated aged (8-week-old) mie. Data represent mean±s.d. (n ¼ 6 biologial repliates). P values, two-tailed unpaired t test. (g) lood flow measurements in tibia after short-term treatment with lendronate. Data represent mean±s.d. (n ¼ 6 biologial repliates). P values, two-tailed unpaired t test. aged (41 year) mutant and ontrol mie were injeted with 1, mg tamoxifen (Sigma, T5648) intraperitoneally every day for 5 days. ll mie were rested for 16 days before subjeting them to a seond round of 5 tamoxifen injetions. fter another 16-day resting period, mie were analysed by olleting femur and tibia after euthanasia. For EC-speifi overexpression of the ative NICD, Gt(ROS)6Sor tm1(noth1)dam/j mie 4 arrying a Cre-induible transgene for NICD overexpression and Cdh5(PC)-CreERT transgenis were interbred. Tamoxifen administration (see injetion shedule for aged mie above) was used to generate CreERT-positive (NICD ioe-ec ) Noth gain-of-funtion mutants and orresponding Cre-negative ontrol littermates. Experiments involving animals were performed following protools approved by loal animal welfare boards and offiial institutions (LVES Lower Saxony, LNUV North Rhine Westphalia). Femoral artery ligation. Male 3-week-old C57L/6 J mie were subjeted to permanent femoral artery ligation 8. Mie were anaesthetized by intraperitoneal injetion of ketamine and xylazine before surgery. The femoral artery was ligated distal to the origin of the deep femoral artery and proximal to the popliteal artery. lood flow measurements in mouse feet were performed on 37 C heated pads before and immediately after surgery and on post-operative 16 and 48 h using a 1 NTURE COMMUNICTIONS 7:1361 DOI: 1.138/nomms1361

11 NTURE COMMUNICTIONS DOI: 1.138/nomms1361 RTICLE Postnatal development geing Reativation of endothelial noth in aged mie Higher blood flow Higher endothelial noth rtery Lower blood flow Lower endothelial noth rtery Metaphysis ngiogenesis Osteogenesis ngiogenesis Osteogenesis ngiogenesis Osteogenesis rtery es Diaphysis Vein Lower blood flow Lower endothelial noth Vein Vein Figure 7 Shemati representation of key findings. Postnatal angiogenesis in bone involves the formation of arh and bud strutures. lood flow is higher in metaphyseal type H vessels, whih orrelates with higher expression and ativity of the Noth pathway. Redution of flow impaired endothelial Noth ativity, bone angiogenesis and osteogenesis. In aged mie, type H apillaries, vessel buds, endothelial Noth signalling and osteogenesis were low, all of whih were reativated by indution of EC-speifi Noth ativity leading to signifiant inreases in osteoprogenitors and mineralized bone. laser Doppler perfusion imager (PIM II, Perimed, Sweden). nimals were euthanized at 16 or 48 hps and tibias were olleted for analysis. Immunohistohemistry. Freshly disseted tibiae were olleted from wild type, mutant mie and their ontrol littermates. one were proessed and imaged as reported earlier 8,9. The following primary antibodies were used: Emn (s-65495, Santa Cruz, diluted 1:1), Peam1 onjugated to lexa Fluor488 (F368G, R&D Systems, 1:1), Peam1 (55337, D Pharmingen, 1:1), Osterix (s-536-r, Santa Cruz, 1:), alphasm-cy3 (C6198, Sigma, 1:1), Calitonin reeptor (ab114, bam, 1:75), Dll4 (F1389, R&D Systems, 1:1), iotin-onjugated CD45 (55377, eton Dikinson, 1:1) and Ter-119 (M115, R&D Systems, 1:1). fter setions had been washed with PS for three times, the following seondary antibodies were used: nti-rat lexa Fluor 594 (Invitrogen; 19, 1:4), anti-rabbit lexa Fluor 546 (Invitrogen; 1135, 1:4), anti-goat lexa Fluor 488 (Invitrogen; 1155, 1:4) and streptavidin lexa Fluor 488 (Invitrogen; S113, 1:4). Immunofluoresent stainings were analysed at high resolution with a Zeiss laser sanning onfoal mirosope, LSM78. Proliferating ells were labelled by injeting mie intraperitoneally with EdU (Invitrogen) 3 h before euthanasia. Mie were killed; bones were olleted and proessed following the protool mentioned before. For immunostaining EdU using Clik-iT hemistry, manufaturer s protools were followed. Image aquisition and analysis. Z-staks of images were proessed and 3D reonstruted with the Imaris software (version 7., itplane). Imaris, Image J, Zen (Carl Zeiss), Photoshop and Illustrator (dobe) softwares were used for image and movie proessing in ompliane with Nature s guide for digital images. For all quantifiations, a region of 3 4 mm from growth plate towards the audal region was seleted in the metaphysis. ll quantifiations were done with Image J software on high-resolution onfoal images. Quantitative RT PCR. For transript analysis, fresh bone samples were rushed and digested with Collagenase to prepare a single-ell suspension. Pure ECs were sorted by flow ytometry using a FCSria (D iosienes) diretly into the lysis buffer of RNeasy Mini Kit (Qiagen). Total RN was isolated following the manufaturer s protool and, subsequently, a total of 1 5 ng RN was used to generate DN with the isript DN Synthesis System (io-rad). Further, quantitative PCR (qpcr) was performed using TaqMan gene expression assays on an I PRISM 79HT Sequene Detetion System. TaqMan Gene Expression Master Mix (pplied iosystems) was used to perform qpcr for the FMonjugated TaqMan probes Peam1, Nos3, Dll4, Jag1, Hes1, Hey1, Hey, Hes5, nt, Cdh5, Sp7, glap, Tgfb1, Tgfb, Tgfb3, mp, mp4, Fgf1, Pdgfb and Wnt1b ommerially available at Life Tehnologies (Thermo Fisher Sientifi). ll the gene expression assays were normalized using endogenous VIC-onjugated tb probes. For the analysis of galp and Sp7 expression, RN was isolated from whole-bone samples. Freshly disseted bones were immediately flushed (to remove haematopoieti ells), rushed and lysed in lysis buffer for 15 min before proeeding for RN isolation, DN preparation and qpcr analysis as desribed above. one blood flow measurements. lood flow measurements were performed in tibia of mie following a previously published protool 75. riefly, anaesthetized mie were given intraardia injetion of fluoresent mirospheres (Invitrogen FluoSpheres 15 mm, at. no. F884). Mie were killed after 1 min and bones (tibia, femur and sternum) were olleted immediately. fter dealifying the bones for 4 48 h, they were subjeted to ethanoli lysis for 1 days in a shaker inubator at 37 C, 75 r.p.m. and proteted from light. Then the samples were run through filtration unit to ollet mirospheres in a nylon membrane. Finally, spheres were olleted in 1 ml of Cellosolve aetate and fluoresene intensity is quantified using miroplate reader. Intravital imaging. 1-day-old Flk1-GFP knok-in mie were anaesthetised using ketamine and xylazine. Following the areful dissetion of skin and musle overing the metatarsals, mie were plaed on stage of a Zeiss LSM78 laser sanning onfoal mirosope. Depending on the region of interest, loation of metaphysis or diaphysis was seleted for further imaging. fter 1 h, the animals were ontinuously provided with isoflurane and oxygen ombinations. lood vessel growth was monitored for 6 1 h ontinuously with every 5 1 min sanning depending on experimental requirements. Mie were euthanized with an overdose of anaesthetis after the experiment. ll the images were analysed using ZEN 1 and Imaris software platforms. Quantifiation of blood flow speed and shear rate. Movies of red blood ell (RC) movement in the bone vasulature were aptured in the anaesthetized Flk1-GFP mie after dextran (, kda) injetion using LSM78 onfoal mirosoope with the san speed of 1 frames per seond. Line san analysis was performed to trae the displaement of RCs and alulate the speed of erythroytes based on the distane RCs displaed per time unit ( ¼ number of frames 1/1 s). Using this blood flow speed, shear rate was alulated onsidering blood vessels as straight ylinders and blood as an ideal Newtonian fluid with onstant visosity. Under these onditions, shear rate (g) experiened by ECs lining a blood vessel is 8v/d where v is the blood speed and d is the diameter of the blood vessel. Flow ytometry. Freshly isolated bone samples were leaned thoroughly to remove the adherent musles and then rushed in ie-old PS with a mortar and pestle. Whole bone marrow was digested with Collagenase at 37 C for min. fter washing and filtering, single-ell suspensions were subjeted to immunostaining. Cells were stained with Emn (s-65495, Santa Cruz, 1:1) antibody for 45 min. fter washing in PS, ells were stained with anti-rat PC (Jakson laboratories, , 1:5) and CD31-onjugated with lexa Fluor 488 (R&Dsystems, F368G, 1:5) for 45 min. fter washing, data were aquired and analysed on D FCSVantage ytometer (D iosienes). Miro-CT analysis and histomorphometry. Tibiae were olleted from mutants and their littermate ontrols, or from treated mie and sham ontrols. ones were leaned thoroughly to remove surrounding soft tissues and fixed in 4% NTURE COMMUNICTIONS 7:1361 DOI: 1.138/nomms