Decreased levels of Cx43 gap junctions result in ameloblast dysregulation and enamel hypoplasia in Gja1Jrt/+ mice

Coordinated differentiation of the ameloblast cell layer is essential to enamel matrix protein deposition and subsequent mineralization. It has been hypothesized that this process is governed by Cx43‐based gap junctional intercellular communication as oculodentodigital dysplasia (ODDD) patients harboring autosomal‐dominant mutations in Cx43 exhibit enamel defects typically resulting in early adulthood tooth loss. To assess the role of Cx43 in tooth development we employ a mouse model of ODDD that harbors a G60S Cx43 mutant, Gja1^Jrt/+, and appears to exhibit tooth abnormalities that mimic the human disease. We found that total Cx43 plaques at all stages of ameloblast differentiation, as well as within the supporting cell layers, were greatly reduced in Gja1^Jrt/+ incisors compared to wild‐type littermate controls. To characterize the Gja1^Jrt/+ mouse tooth phenotype, mice were sacrificed prior to tooth eruption (postnatal day 7), weaning (postnatal day 21), and adulthood (2 months postnatal). A severely disorganized Gja1^Jrt/+ mouse ameloblast layer and abnormal accumulation of amelogenin were observed at stages when the cells were active in secretion and mineralization. Differences in enamel thickness became more apparent after tooth eruption and incisor exposure to the oral cavity suggesting that enamel integrity is compromised, leading to rapid erosion. Additional analysis of incisors from mutant mice revealed that they were longer with a thicker dentin layer than their wild‐type littermates, which may reflect a mechanical stress response to the depleted enamel layer. Together, these data show that reduced levels of Cx43 gap junctions result in ameloblast dysregulation, enamel hypoplasia, and secondary tissue responses. J. Cell. Physiol. 223:601–609, 2010. © 2010 Wiley‐Liss, Inc.     

Bone morphogenetic protein signaling through ACVR1 and BMPR1A negatively regulates bone mass along with alterations in bone composition

Bone quantity and bone quality are important factors in determining the properties and the mechanical functions of bone. This study examined the effects of disrupting bone morphogenetic protein (BMP) signaling through BMP receptors on bone quantity and bone quality. More specifically, we disrupted two BMP receptors, Acvr1 and Bmpr1a, respectively, in Osterix-expressing osteogenic progenitor cells in mice. We examined the structural changes to the femora from 3-month old male and female conditional knockout (cKO) mice using micro-computed tomography (micro-CT) and histology, as well as compositional changes to both cortical and trabecular compartments of bone using Raman spectroscopy. We found that the deletion of Acvr1 and Bmpr1a, respectively, in an osteoblast-specific manner resulted in higher bone mass in the trabecular compartment. Disruption of Bmpr1a resulted in a more significantly increased bone mass in the trabecular compartment. We also found that these cKO mice showed lower mineral-to-matrix ratio, while tissue mineral density was lower in the cortical compartment. Collagen crosslink ratio was higher in both cortical and trabecular compartments of male cKO mice. Our study suggested that BMP signaling in osteoblast mediated by BMP receptors, namely ACVR1 and BMPR1A, is critical in regulating bone quantity and bone quality.     

High Bone Mass in Mice Lacking Cx37 Because of Defective Osteoclast Differentiation

Connexin (Cx) proteins are essential for cell differentiation, function, and survival in all tissues with Cx43 being the most studied in bone. We now report that Cx37, another member of the connexin family of proteins, is expressed in osteoclasts, osteoblasts, and osteocytes. Mice with global deletion of Cx37 (Cx37^−/−) exhibit higher bone mineral density, cancellous bone volume, and mechanical strength compared with wild type littermates. Osteoclast number and surface are significantly lower in bone of Cx37^−/− mice. In contrast, osteoblast number and surface and bone formation rate in bones from Cx37^−/− mice are unchanged. Moreover, markers of osteoblast activity ex vivo and in vivo are similar to those of Cx37^+/+ littermates. sRANKL/M-CSF treatment of nonadherent Cx37^−/− bone marrow cells rendered a 5-fold lower level of osteoclast differentiation compared with Cx37^+/+ cell cultures. Further, Cx37^−/− osteoclasts are smaller and have fewer nuclei per cell. Expression of RANK, TRAP, cathepsin K, calcitonin receptor, matrix metalloproteinase 9, NFATc1, DC-STAMP, ATP6v0d1, and CD44, markers of osteoclast number, fusion, or activity, is lower in Cx37^−/− osteoclasts compared with controls. In addition, nonadherent bone marrow cells from Cx37^−/− mice exhibit higher levels of markers for osteoclast precursors, suggesting altered osteoclast differentiation. The reduction of osteoclast differentiation is associated with activation of Notch signaling. We conclude that Cx37 is required for osteoclast differentiation and fusion, and its absence leads to arrested osteoclast maturation and high bone mass in mice. These findings demonstrate a previously unrecognized role of Cx37 in bone homeostasis that is not compensated for by Cx43 in vivo.     

Development of Mice with Osteoblast-Specific Connexin43 Gene Deletion

Genetic deficiency of Cx43 in vivo causes skeletal developmental defects, osteoblast dysfunction and perinatal lethality. To determine the role of Cx43 in the adult skeleton, we developed two models of osteoblast-specific Cx43 gene deletion using Cre mediated replacement of a “floxed” Cx43 allele with a LacZ reporter gene. Cre recombinase expression in osteoblasts was driven by either the osteocalcin OG2 promoter or the 2.3 kb fragment of the Colα1(I) promoter. Homozygous Cx43^fl/flmice, in which the Cx43 coding region is flanked by two loxP sites, were crossed with Cre expressing mice in a heterozygous Cx43-null background [Cx43^±; Colα1(I)-Cre or Cx43^±; OG2-Cre]. Cx43 gene ablation was demonstrated in tissues by selective X-gal staining of cells lining the endosteal surface, and in cultured osteoblastic cells from calvaria using different approaches. Although no LacZ expression was observed in proliferating calvaria cells, before osteoblast differentiation begins, post-proliferative cells isolated from conditional knockout mice [Cx43^fl/?; Colα1(I)-Cre or Cx43^fl/?; OG2-Cre] developed strong LacZ expression as they differentiated, in parallel to a progressive disappearance of Cx43 mRNA and protein abundance relative to controls. Selective Cre mediated Cx43 gene inactivation in bone forming cells will be useful to determine the role of Cx43 in adult skeletal homeostasis and overcome the perinatal lethality of the conventional null model.     

Enhanced periosteal and endocortical responses to axial tibial compression loading in conditional connexin43 deficient mice

The gap junction protein, connexin43 (Cx43) is involved in mechanotransduction in bone. Recent studies using in vivo models of conditional Cx43 gene (Gja1) deletion in the osteogenic linage have generated inconsistent results, with Gja1 ablation resulting in either attenuated or enhanced response to mechanical load, depending upon the skeletal site examined or the type of load applied. To gain further insights on Cx43 and mechanotransduction, we examined bone formation response at both endocortical and periosteal surfaces in 2-month-old mice with conditional Gja1 ablation driven by the Dermo1 promoter (cKO). Relative to wild type (WT) littermates, it requires a larger amount of compressive force to generate the same periosteal strain in cKO mice. Importantly, cKO mice activate periosteal bone formation at a lower strain level than do WT mice, suggesting an increased sensitivity to mechanical load in Cx43 deficiency. Consistently, trabecular bone mass also increases in mutant mice upon load, while it decreases in WT. On the other hand, bone formation actually decreases on the endocortical surface in WT mice upon application of axial mechanical load, and this response is also accentuated in cKO mice. These changes are associated with increase of Cox-2 in both genotypes and further decrease of Sost mRNA in cKO relative to WT bones. Thus, the response of bone forming cells to mechanical load differs between trabecular and cortical components, and remarkably between endocortical and periosteal envelopes. Cx43 deficiency enhances both the periosteal and endocortical response to mechanical load applied as axial compression in growing mice.     

Osteoblast connexin43 modulates skeletal architecture by regulating both arms of bone remodeling

Connexin43 (Cx43) has an important role in skeletal homeostasis, and Cx43 gene (Gja1) mutations have been linked to oculodentodigital dysplasia (ODDD), a human disorder characterized by prominent skeletal abnormalities. To determine the function of Cx43 at early steps of osteogenesis and its role in the ODDD skeletal phenotype, we have used the Dermo1 promoter to drive Gja1 ablation or induce an ODDD mutation in the chondro-osteogenic linage. Both Gja1 null and ODDD mutant mice develop age-related osteopenia, primarily due to a progressive enlargement of the medullary cavity and cortical thinning. This phenotype is the consequence of a high bone turnover state, with increased endocortical osteoclast-mediated bone resorption and increased periosteal bone apposition. Increased bone resorption is a noncell autonomous defect, caused by exuberant stimulation of osteoclastogenesis by Cx43-deficient bone marrow stromal cells, via decreased Opg production. The latter is part of a broad defect in osteoblast differentiation and function, which also results in abnormal structural and material properties of bone leading to decreased resistance to mechanical load. Thus Cx43 in osteogenic cells is a critical regulator of both arms of the bone remodeling cycle, its absence causing structural changes remindful of aged or disused bone.     

Transgenic Overexpression of Ephrin B1 in Bone Cells Promotes Bone Formation and an Anabolic Response to Mechanical Loading in Mice

To test if ephrin B1 overexpression enhances bone mass, we generated transgenic mice overexpressing ephrin B1 under the control of a 3.6 kb rat collagen 1A1 promoter (Col3.6-Tg^efnb1). Col3.6-Tg^efnb1 mice express 6-, 12- and 14-fold greater levels of full-length ephrin B1 protein in bone marrow stromal cells, calvarial osteoblasts, and osteoclasts, respectively. The long bones of both genders of Col3.6-Tg^efnb1 mice have increased trabecular bone volume, trabecular number, and trabecular thickness and decreased trabecular separation. Enhanced bone formation and decreased bone resorption contributed to this increase in trabecular bone mass in Col3.6-Tg^efnb1 mice. Consistent with these findings, our in vitro studies showed that overexpression of ephrin B1 increased osteoblast differentiation and mineralization, osterix and collagen 1A1 expression in bone marrow stromal cells. Interaction of ephrin B1 with soluble clustered EphB2-Fc decreased osteoclast precursor differentiation into multinucleated cells. Furthermore, we demonstrated that the mechanical loading-induced increase in EphB2 expression and newly formed bone were significantly greater in the Col3.6-Tg^efnb1 mice than in WT littermate controls. Our findings that overexpression of ephrin B1 in bone cells enhances bone mass and promotes a skeletal anabolic response to mechanical loading suggest that manipulation of ephrin B1 actions in bone may provide a means to sensitize the skeleton to mechanical strain to stimulate new bone formation.     

Cataracts and Microphthalmia Caused by a Gja8 Mutation in Extracellular Loop 2

The mouse semi-dominant Nm2249 mutation displays variable cataracts in heterozygous mice and smaller lenses with severe cataracts in homozygous mice. This mutation is caused by a Gja8^R205G point mutation in the second extracellular loop of the Cx50 (or α8 connexin) protein. Immunohistological data reveal that Cx50-R205G mutant proteins and endogenous wild-type Cx46 (or α3 connexin) proteins form diffuse tiny spots rather than typical punctate signals of normal gap junctions in the lens. The level of phosphorylated Cx46 proteins is decreased in Gja8^R205G/R205G mutant lenses. Genetic analysis reveals that the Cx50-R205G mutation needs the presence of wild-type Cx46 to disrupt lens peripheral fibers and epithelial cells. Electrophysiological data in Xenopus oocytes reveal that Cx50-R205G mutant proteins block channel function of gap junctions composed of wild-type Cx50, but only affect the gating of wild-type Cx46 channels. Both genetic and electrophysiological results suggest that Cx50-R205G mutant proteins alone are unable to form functional channels. These findings imply that the Gja8^R205G mutation differentially impairs the functions of Cx50 and Cx46 to cause cataracts, small lenses and microphthalmia. The Gja8^R205G mutation occurs at the same conserved residue as the human GJA8^R198W mutation. This work provides molecular insights to understand the cataract and microphthalmia/microcornea phenotype caused by Gja8 mutations in mice and humans.     

Mechanical Loading Synergistically Increases Trabecular Bone Volume and Improves Mechanical Properties in the Mouse when BMP Signaling Is Specifically Ablated in Osteoblasts

Bone homeostasis is affected by several factors, particularly mechanical loading and growth factor signaling pathways. There is overwhelming evidence to validate the importance of these signaling pathways, however, whether these signals work synergistically or independently to contribute to proper bone maintenance is poorly understood. Weight-bearing exercise increases mechanical load on the skeletal system and can improves bone quality. We previously reported that conditional knockout (cKO) of Bmpr1a, which encodes one of the type 1 receptors for Bone Morphogenetic Proteins (BMPs), in an osteoblast-specific manner increased trabecular bone mass by suppressing osteoclastogenesis. The cKO bones also showed increased cortical porosity, which is expected to impair bone mechanical properties. Here, we evaluated the impact of weight-bearing exercise on the cKO bone phenotype to understand interactions between mechanical loading and BMP signaling through BMPR1A. Male mice with disruption of Bmpr1a induced at 9 weeks of age, exercised 5 days per week on a motor-driven treadmill from 11 to 16 weeks of age. Trabecular bone volume in cKO tibia was further increased by exercise, whereas exercise did not affect the trabecular bone in the control genotype group. This finding was supported by decreased levels of osteoclasts in the cKO tibiae. The cortical porosity in the cKO bones showed a marginally significant decrease with exercise and approached normal levels. Exercise increased ductility and toughness in the cKO bones. Taken together, reduction in BMPR1A signaling may sensitize osteoblasts for mechanical loading to improve bone mechanical properties.     

Loss-of-function of ACVR1 in osteoblasts increases bone mass and activates canonical Wnt signaling through suppression of Wnt inhibitors SOST and DKK1

BMPs (Bone morphogenetic proteins) such as BMP2 and BMP7 have been used about one decade as bone anabolic agents in orthopaedics. The BMP receptor ACVR1, which is a key receptor of BMP7, is expressed in bone. The pathological role of ACVR1 in humans has been reported: a point mutation in ACVR1 can cause fibrodysplasia ossificans progressiva (FOP) in which ectopic ossification occurs in skeletal muscles and deep connective tissues. The physiological function of ACVR1 in bone, however, is totally unknown. The purpose of this study is to investigate the endogenous role of ACVR1 in osteoblasts, one of the most dominant cell-types in bone. We generated Acvr1-null mice in an osteoblast-specific manner using an inducible Cre-loxP system. Surprisingly, we found that bone mass was increased in the Acvr1-null mice. Interestingly, canonical Wnt signaling was increased and expression levels of Wnt inhibitors Sost and Dkk1 were both suppressed in the null bones during the developmental stages. In addition, we confirmed that expression levels of both Sost and Dkk1 were upregulated by BMP7 dose-dependently in vitro. These results suggest that the Acvr1-deficiency can increase bone mass by activating Wnt signaling in which both Sost and Dkk1 expression levels are diminished. This study leads to a new concept of the BMP7-ACVR1-SOST/DKK1 axis in osteoblasts, in which BMP7 signaling through ACVR1 can reduce Wnt signaling via SOST/DKK1 and then inhibits osteogenesis. Although this concept is beyond the current known function of BMP7, it can explain the varied outcomes of BMP7 treatment. We believe BMP signaling can exhibit multifaceted effects by context and cell type.     

Characteristics of the Cfu-S Population In Mice Carrying the SlJ Allele

Abstract Abstract. A tentative characterization of haemopoietic stem cells with respect to their organ distribution, seeding fraction and colony formation in the spleen, radiosen-sitivity and humoral regulation was attempted in mice heterozygous for the mutant allele Sl^J and in their normal littermates. Sl^J/+ mice were characterized by a deficient CFU-s content of the blood and spleen and had slightly lower femoral CFU-s numbers. This CFU-s distribution could not be explained by differences in seeding efficiency ‘f’ between CFU-s of Sl^J/+ and +/+ origin in lethally irradiated recipients used in the CFU-s assay. the seeding fraction of CFU-s of +/+ origin did not differ in +/+ and Sl^J/+ recipients. However, in irradiated SI^J/+ recipient mice a 30% decrease was observed in the number of the colonies derived from splenic and femoral CFU-s of both +/+ and Sl^J/+ origin. the serum level of SHSF (splenic haemopoiesis stimulating factor) was decreased in Sl^J/+ mice, but significantly increased in Sl/Sl^d mice, as compared to their respective normal +/+ littermates. Endogenous colony formation in Sl^J/+ spleens was deficient in comparison to that observed in +/+ spleens, and distinct sex differences were observed. However, mutant and normal CFU-s from spleen and bone marrow had a similar survival following in-vitro y irradiation. Femurs and spleens of both Sl^J/+ and +/+ origin were implanted into both Sl^J/+ and +/+ hosts. Six weeks later the Sl^J/+ grafts contained less CFU-s than the +/+ grafts. These data show that the splenic stroma of Sl^J/+ mice is not defective in its capacity to lodge injected CFU-s but is deficient in its ability to maintain CFU-s under ‘steady-state’ conditions and stimulate their colony formation in a ‘perturbed state’. Some of the characteristics of Sl^J/+ mice segregate them from Sl/Sl^d mice, i.e. a deficient splenic CFU-s content, normal seeding fractions ‘f’ of CFU-s from spleen and bone marrow in the presence of an almost compensated anemia, and decreased serum levels of SHSF. the study of the Sl^J trait may be a useful extension of the current Sl/Sl^d model for exploration of hereditary defects in haematopoietic stroma.     

Runx1 is a central regulator of osteogenesis for bone homeostasis by orchestrating BMP and WNT signaling pathways

Runx1 is highly expressed in osteoblasts, however, its function in osteogenesis is unclear. We generated mesenchymal progenitor-specific (Runx1^f/fTwist2-Cre) and osteoblast-specific (Runx1^f/fCol1α1-Cre) conditional knockout (Runx1 CKO) mice. The mutant CKO mice with normal skeletal development displayed a severe osteoporosis phenotype at postnatal and adult stages. Runx1 CKO resulted in decreased osteogenesis and increased adipogenesis. RNA-sequencing analysis, Western blot, and qPCR validation of Runx1 CKO samples showed that Runx1 regulates BMP signaling pathway and Wnt/β-catenin signaling pathway. ChIP assay revealed direct binding of Runx1 to the promoter regions of Bmp7, Alk3, and Atf4, and promoter mapping demonstrated that Runx1 upregulates their promoter activity through the binding regions. Bmp7 overexpression rescued Alk3, Runx2, and Atf4 expression in Runx1-deficient BMSCs. Runx2 expression was decreased while Runx1 was not changed in Alk3 deficient osteoblasts. Atf4 overexpression in Runx1-deficient BMSCs did not rescue expression of Runx1, Bmp7, and Alk3. Smad1/5/8 activity was vitally reduced in Runx1 CKO cells, indicating Runx1 positively regulates the Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 signaling pathway. Notably, Runx1 overexpression in Runx2^-/- osteoblasts rescued expression of Atf4, OCN, and ALP to compensate Runx2 function. Runx1 CKO mice at various osteoblast differentiation stages reduced Wnt signaling and caused high expression of C/ebpα and Pparγ and largely increased adipogenesis. Co-culture of Runx1-deficient and wild-type cells demonstrated that Runx1 regulates osteoblast−adipocyte lineage commitment both cell-autonomously and non-autonomously. Notably, Runx1 overexpression rescued bone loss in OVX-induced osteoporosis. This study focused on the role of Runx1 in different cell populations with regards to BMP and Wnt signaling pathways and in the interacting network underlying bone homeostasis as well as adipogenesis, and has provided new insight and advancement of knowledge in skeletal development. Collectively, Runx1 maintains adult bone homeostasis from bone loss though up-regulating Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 and WNT/β-Catenin signaling pathways, and targeting Runx1 potentially leads to novel therapeutics for osteoporosis.     

Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging

Skeletal aging results in apoptosis of osteocytes, cells embedded in bone that control the generation/function of bone forming and resorbing cells. Aging also decreases connexin43 (Cx43) expression in bone; and osteocytic Cx43 deletion partially mimics the skeletal phenotype of old mice. Particularly, aging and Cx43 deletion increase osteocyte apoptosis, and osteoclast number and bone resorption on endocortical bone surfaces. We examined herein the molecular signaling events responsible for osteocyte apoptosis and osteoclast recruitment triggered by aging and Cx43 deficiency. Cx43‐silenced MLO‐Y4 osteocytic (Cx43^def) cells undergo spontaneous cell death in culture through caspase‐3 activation and exhibit increased levels of apoptosis‐related genes, and only transfection of Cx43 constructs able to form gap junction channels reverses Cx43^def cell death. Cx43^def cells and bones from old mice exhibit reduced levels of the pro‐survival microRNA miR21 and, consistently, increased levels of the miR21 target phosphatase and tensin homolog (PTEN) and reduced phosphorylated Akt, whereas PTEN inhibition reduces Cx43^def cell apoptosis. miR21 reduction is sufficient to induce apoptosis of Cx43‐expressing cells and miR21 deletion in miR21^fl/fl bones increases apoptosis‐related gene expression, whereas a miR21 mimic prevents Cx43^def cell apoptosis, demonstrating that miR21 lies downstream of Cx43. Cx43^def cells release more osteoclastogenic cytokines [receptor activator of NFκB ligand (RANKL)/high‐mobility group box‐1 (HMGB1)], and caspase‐3 inhibition prevents RANKL/HMGB1 release and the increased osteoclastogenesis induced by conditioned media from Cx43^def cells, which is blocked by antagonizing HMGB1‐RAGE interaction. These findings identify a novel Cx43/miR21/HMGB1/RANKL pathway involved in preventing osteocyte apoptosis that also controls osteoclast formation/recruitment and is impaired with aging.     

BMP3 suppresses osteoblast differentiation of bone marrow stromal cells via interaction with Acvr2b

Enhancing bone morphogenetic protein (BMP) signaling increases bone formation in a variety of settings that target bone repair. However, the role of BMP in the maintenance of adult bone mass is not well understood. Targeted disruption of BMP3 in mice results in increased trabecular bone formation, whereas transgenic overexpression of BMP3 in skeletal cells leads to spontaneous fracture, consistent with BMP3 having a negative role in bone mass regulation. Here we investigate the importance of BMP3 as a mediator of BMP signaling in the adult skeleton. We find that osteoblasts (OBL) and osteocytes are the source of BMP3 in adult bone. Using in vitro cultures of primary bone marrow stromal cells, we show that overexpression of BMP3 suppresses OBL differentiation, whereas loss of BMP3 increases colony-forming unit fibroblasts and colony-forming unit OBL. The ability of BMP3 to affect OBL differentiation is due to its interaction with activin receptor type 2b (Acvr2b) because knockdown of endogenous Acvr2b in bone marrow stromal cells reduces the suppressive effect of BMP3 on OBL differentiation. These findings best fit a model in which BMP3, produced by mature bone cells, acts to reduce BMP signaling through Acvr2b in skeletal progenitor cells, limiting their differentiation to mature OBL. Our data further support the idea that endogenous BMPs have a physiological role in regulating adult bone mass.     

Histone Deacetylase 3 Depletion in Osteo/Chondroprogenitor Cells Decreases Bone Density and Increases Marrow Fat

Histone deacetylase (Hdac)3 is a nuclear enzyme that contributes to epigenetic programming and is required for embryonic development. To determine the role of Hdac3 in bone formation, we crossed mice harboring loxP sites around exon 7 of Hdac3 with mice expressing Cre recombinase under the control of the osterix promoter. The resulting Hdac3 conditional knockout (CKO) mice were runted and had severe deficits in intramembranous and endochondral bone formation. Calvarial bones were significantly thinner and trabecular bone volume in the distal femur was decreased 75% in the Hdac3 CKO mice due to a substantial reduction in trabecular number. Hdac3-CKO mice had fewer osteoblasts and more bone marrow adipocytes as a proportion of tissue area than their wildtype or heterozygous littermates. Bone formation rates were depressed in both the cortical and trabecular regions of Hdac3 CKO femurs. Microarray analyses revealed that numerous developmental signaling pathways were affected by Hdac3-deficiency. Thus, Hdac3 depletion in osterix-expressing progenitor cells interferes with bone formation and promotes bone marrow adipocyte differentiation. These results demonstrate that Hdac3 inhibition is detrimental to skeletal health.     

Heterotopic ossification in mice overexpressing Bmp2 in Tie2+ lineages

Bone morphogenetic protein (Bmp) signaling is critical for organismal development and homeostasis. To elucidate Bmp2 function in the vascular/hematopoietic lineages we generated a new transgenic mouse line in which ectopic Bmp2 expression is controlled by the Tie2 promoter. Tie2^CRE/+;Bmp2^tg/tg mice develop aortic valve dysfunction postnatally, accompanied by pre-calcific lesion formation in valve leaflets. Remarkably, Tie2^CRE/+;Bmp2^tg/tg mice develop extensive soft tissue bone formation typical of acquired forms of heterotopic ossification (HO) and genetic bone disorders, such as Fibrodysplasia Ossificans Progressiva (FOP). Ectopic ossification in Tie2^CRE/+;Bmp2^tg/tg transgenic animals is accompanied by increased bone marrow hematopoietic, fibroblast and osteoblast precursors and circulating pro-inflammatory cells. Transplanting wild-type bone marrow hematopoietic stem cells into lethally irradiated Tie2^CRE/+;Bmp2^tg/tg mice significantly delays HO onset but does not prevent it. Moreover, transplanting Bmp2-transgenic bone marrow into wild-type recipients does not result in HO, but hematopoietic progenitors contribute to inflammation and ectopic bone marrow colonization rather than to endochondral ossification. Conversely, aberrant Bmp2 signaling activity is associated with fibroblast accumulation, skeletal muscle fiber damage, and expansion of a Tie2+ fibro-adipogenic precursor cell population, suggesting that ectopic bone derives from a skeletal muscle resident osteoprogenitor cell origin. Thus, Tie2^CRE/+;Bmp2^tg/tg mice recapitulate HO pathophysiology, and might represent a useful model to investigate therapies seeking to mitigate disorders associated with aberrant extra-skeletal bone formation.Subject terms: Mechanisms of disease, Central control of bone remodelling, Haematopoietic stem cells