Research ArticleGENETIC DISORDERS

Two tissue-resident progenitor lineages drive distinct phenotypes of heterotopic ossification

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Science Translational Medicine  23 Nov 2016:
Vol. 8, Issue 366, pp. 366ra163
DOI: 10.1126/scitranslmed.aaf1090

Boning up on stem cells

Fibrodysplasia ossificans progressiva is a rare genetic disorder of uncontrolled bone growth, which manifests as formation of bone in the muscles, ligaments, and joints triggered by inflammation or injury. Dey et al. used mouse models with disease-causing mutations in ACVR1 protein localized to specific types of progenitor cells in ligaments or in muscles to clarify the biology of this disease and determine which cells give rise to abnormal bone. In addition, the authors showed that the harmful manifestations of these mutations can be successfully controlled by a selective inhibitor of ACVR1.

Abstract

Fibrodysplasia ossificans progressiva (FOP), a congenital heterotopic ossification (HO) syndrome caused by gain-of-function mutations of bone morphogenetic protein (BMP) type I receptor ACVR1, manifests with progressive ossification of skeletal muscles, tendons, ligaments, and joints. In this disease, HO can occur in discrete flares, often triggered by injury or inflammation, or may progress incrementally without identified triggers. Mice harboring an Acvr1R206H knock-in allele recapitulate the phenotypic spectrum of FOP, including injury-responsive intramuscular HO and spontaneous articular, tendon, and ligament ossification. The cells that drive HO in these diverse tissues can be compartmentalized into two lineages: an Scx+ tendon-derived progenitor that mediates endochondral HO of ligaments and joints without exogenous injury, and a muscle-resident interstitial Mx1+ population that mediates intramuscular, injury-dependent endochondral HO. Expression of Acvr1R206H in either lineage confers aberrant gain of BMP signaling and chondrogenic differentiation in response to activin A and gives rise to mutation-expressing hypertrophic chondrocytes in HO lesions. Compared to Acvr1R206H, expression of the man-made, ligand-independent ACVR1Q207D mutation accelerates and increases the penetrance of all observed phenotypes, but does not abrogate the need for antecedent injury in muscle HO, demonstrating the need for an injury factor in addition to enhanced BMP signaling. Both injury-dependent intramuscular and spontaneous ligament HO in Acvr1R206H knock-in mice were effectively controlled by the selective ACVR1 inhibitor LDN-212854. Thus, diverse phenotypes of HO found in FOP are rooted in cell-autonomous effects of dysregulated ACVR1 signaling in nonoverlapping tissue-resident progenitor pools that may be addressed by systemic therapy or by modulating injury-mediated factors involved in their local recruitment.

INTRODUCTION

Heterotopic ossification (HO) broadly describes the formation of ectopic endochondral bone in muscles, tendons, ligaments, and other soft tissues. HO is a debilitating complication of fractures, joint replacement surgery, and other soft tissue trauma, suggesting a process of disordered injury repair. Fibrodysplasia ossificans progressiva (FOP) is a congenital HO syndrome in which individuals have minor skeletal abnormalities at birth but develop progressive HO during childhood and young adulthood, culminating in severe immobilization and reduced life expectancy because of restrictive lung disease and traumatic injuries (1). Progression may occur in episodic flares, which can follow accidental trauma, surgery, intramuscular immunization, inflammation, or viral prodromes. Recently, it has been recognized that a substantial portion of disease progression occurs gradually without known flares, antecedent injury, or triggers, but it is unclear if such activity is mechanistically distinct from flare-related episodes (2). FOP arises from gain-of-function mutations in bone morphogenetic protein (BMP) type I receptor ACVR1 (ALK2), with about 97% of individuals harboring a classic ACVR1R206H variant (36). Using genetically engineered mice harboring this variant, we recently found that ACVR1R206H drives HO in FOP by conferring aberrant activation of the BMP signaling pathway by activin ligands (7), a signaling defect also observed in mesenchymal stem cells derived from patient-derived induced pluripotent stem cells (iPSCs) (8). Because maladaptive BMP/activin/transforming growth factor–β (TGF-β) family ligand signaling may be a shared property of both genetic and acquired forms of HO (915), it has been suggested that FOP and HO might be mediated by common effector and progenitor cells. However, the identity and niche of these progenitors as well as their mechanistic relationship to either triggered or spontaneous HO have yet to be determined.

Previous approaches sought to identify cell populations contributing to HO lesions via immune histology or genetic marking techniques in animal models of HO caused by exogenous BMP ligands. These studies explored the role of diverse tissue-resident mesenchymal, vascular, circulating, hematopoietic, and bone marrow (BM)–derived populations, demonstrating their participation, but not identifying the populations that are sufficient to initiate this process and manifest the cell-autonomous effects of dysregulated BMP signaling (1622). Here, we used tissue-targeted expression of a ligand-responsive and a constitutively active ACVR1 mutant to identify two nonoverlapping tissue-resident populations that appear to be responsible for different anatomic phenotypes of FOP. The differential effects of these mutations reveal potentially distinct ligand-regulated and ligand-independent aspects of these phenotypes.

RESULTS

FOP exhibits diverse spatiotemporal phenotypes

FOP manifests with HO in multiple soft tissue compartments, including skeletal muscle, intra- and periarticular tissues, ligaments, fascia, and tendons, as demonstrated in radiographs obtained from individuals with classic FOP due to ACVR1R206H (Fig. 1, A to D). This spectrum of phenotypes manifests in distinct tissues with varying natural histories and functional consequences (2, 6, 23). Intramuscular HO in FOP is frequently preceded by local trauma or symptoms of myositis or swelling constituting a “flare,” and infiltrates the muscle to cause altered mechanics, pain, and reduced range of motion that progresses to immobilization (Fig. 1, A and B) (6, 24). HO may also affect peri- and intra-articular structures (Fig. 1, B and C), including ossification of articular cartilage, fascia, ligaments, and tendons, with direct impact on joint mobility, as well as exostosis or osteochondroma formation on long bones (Fig. 1D). Less is known about the triggers for bone formation in cartilage, tendon, and ligament tissues, including the role, if any, of injury, or of physiologic versus pathophysiologic mechanical loading. Moreover, a large proportion of disease progression in FOP occurs in the absence of known triggers or prodromes (2, 6, 25).

Fig. 1. The classic FOP-causing ACVR1R206H allele is associated with intramuscular, periarticular, ligament, and tendon ossification in human and mouse.

(A to D) Radiographic manifestations in patients with ACVR1R206H/+ genotype-confirmed classic FOP. (A) Female patient with intramuscular ossification (arrow) bridging the ischium and left femoral shaft, infiltrating the gluteus and hamstring. (B) Male patient with intramuscular ossification infiltrating the biceps (long arrow) and dense periarticular ossification surrounding the olecranon and coronoid fossae (short arrows). (C) T1 turbo spin echo magnetic resonance imaging of a female patient reveals ossification of tibiotalar, talonavicular, and naviculocalcaneal ligaments and the Achilles tendon insertion (arrows), resulting in permanent plantar flexion. (D) A male patient with osteochondroma (arrow) of the distal humerus. (E to H) Micro–computed tomography (CT) imaging of Rosa-CreERT2:Acvr1[R206H]FlEx/+ mice treated with tamoxifen for 10 to 12 weeks reveals diffuse axial and appendicular HO after 4 weeks, involving the paraspinal ligaments and Achilles tendons (E), periarticular ossification of the knees (F), sporadic intramuscular ossification infiltrating the hamstring and gastrocnemius, bridging ischium, femur, and tibia (G), and HO of the interscapular muscles (H) resulting from frequent handling for examination.

Global postnatal expression of Acvr1R206H in mice recapitulates human FOP phenotypes

Because germline expression of Acvr1R206H in knock-in mice results in prenatal lethality (19), a conditional-on Acvr1[R206H]FlEx knock-in allele (7) was expressed globally via the tamoxifen-inducible Rosa-CreERT2 transgene in 10- to 12-week-old mice, resulting in spontaneous ligamentous, tendon, periarticular, and intra-articular HO diffusely throughout the axial and appendicular skeleton within 8 to 12 weeks, mimicking the pattern seen in human disease (Fig. 1, E to H). Sporadic intramuscular HO was observed in some of the mice, with about 15% developing HO in hindlimb muscles (Fig. 1G), reminiscent of intramuscular disease in humans (Fig. 1, A and B), and infrascapular soft tissues (Fig. 1H), also a frequent site of flare-related activity in patients (2, 23), particularly when handled frequently for examination. After tamoxifen-induced global expression of the mutant allele, Acvr1[R206H]FlEx/+ mice demonstrated a spectrum of injury-mediated and spontaneous FOP phenotypes reminiscent of human disease.

Scx+ progenitor lineages are sufficient for spontaneous ligament, tendon, and joint HO

Seeking to determine the lineage(s) sufficient for the cell-autonomous effects of mutant ACVR1 in various tissues, we postulated that tendon-derived stem cells marked by the transcription factor Scleraxis (Scx) (26) and their analogous populations in ligaments and fascia might account for tendon and ligament HO. Expression of the conditional Acvr1R206H allele via the Scx-Cre transgene (Fig. 2A) resulted in spontaneous and progressive ligament, tendon, and periarticular ossification, affecting distal hindlimbs at the tibialis anterior and patellar ligaments, Achilles tendon, and knee joints (Fig. 2, A and B), as well as diffuse involvement of forelimb, shoulder, costochondral, and hip joints, and paraspinal ligaments (fig. S1), consistent with the pattern of expression of the Scx-GFP reporter allele. Radiographic HO in these mice was first evident between 8 and 18 weeks of age, and progressed over 52 weeks (Fig. 2A and fig. S2). Similar to human disease, severe HO was sporadic, with variable involvement of individual hindlimb joints, ligaments, and tendons (fig. S2). For example, the Achilles tendon had radiographic evidence of HO in 33% of limbs at 28 weeks, which increased to 83% at 52 weeks.

Fig. 2. Expression of mutant ACVR1R206H or ACVR1Q207D alleles in Scx-lineage tendon-resident progenitor cells results in spontaneous periarticular, ligament, and tendon ossification.

(A) Scx expression is localized to tibiotalar ligament and the patellar and Achilles tendons, as observed by ex vivo fluorescence in Scx-GFP transgenic mice. Spontaneous ossification (white arrows) of tibiotalar ligament, patellar tendon, and Achilles tendon progresses slowly in Scx-Cre:ACVR1R206H mice from 4 to 18 weeks, with rapid progression in Scx-Cre:ACVR1Q207D-Tg mice from 4 to 8 weeks. (B) Micro-CT of Scx-Cre:ACVR1R206H mice reveals distinct ossification of the tibiotalar ligament, Achilles tendon, and periarticular ossification, but no intramuscular ossification. (C) Scx-Cre:ACVR1Q207D-Tg:Rosa26-YFP mice injected with CTX (P21, gastrocnemius) develop typical tendon and ligament ossification, as seen by x-ray (P42) corresponding to areas of Scx expression via Scx+YFP+ fluorescence, but lack evidence of any intramuscular ossification (representative of five transgenic mice injected). (D) Cryosections of fibular head counterstained for CD45 (magenta) reveal Scx expression (green fluorescence) at ligamentous insertions (white arrow) but not in adjacent skeletal muscle (red arrow). Scale bar, 500 μm. (E) Sections of Achilles tendon reveal scattered nucleated Scx-expressing cells. Scale bar, 50 μm. The HO lesions infiltrating the (F and G) talonavicular ligament (scale bar, 50 μm), (H and I) talonavicular ligament in higher magnification (scale bar, 50 μm), and (J and K) patellar tendon (scale bar, 200 μm) (fluorescence imaging shown in upper panels; AR and AB staining shown in lower panels) in Scx-Cre:ACVR1Q207D-Tg;Rosa26-YFP mice reflect the contribution of Scx+YFP+ cells (white arrows, upper panels) to nearly all AB-stained hypertrophic chondrocytes and heterotopic cartilage (black arrows, lower panels) in these lesions but essentially no contribution to osteocytes or mineralized matrix (red arrows) stained with AR, with 4′,6-diamidino-2-phenylindole (DAPI) as a nuclear counterstain.

To test whether the ligand sensitivity of the Acvr1R206H allele might account for its variable penetrance, the ligand-independent constitutively active ACVR1Q207D transgene (a man-made mutation not seen in patients) was expressed in Scx+ lineages. Scx-Cre:ACVR1Q207D-Tg mice exhibited severe HO of the Achilles tendon, tibialis ligaments, knee, and costochondral joints, with 100% penetrance by 8 weeks (Fig. 2A). All anatomic phenotypes observed in Acvr1[R206H]FlEx/+ mice were also seen in ACVR1Q207D-Tg mice, albeit with greater penetrance, severity, and more rapid onset (figs. S1 and S2). These effects were restricted to tendons and ligaments, and direct muscle injury with cardiotoxin (CTX) injection did not cause muscle ossification or exacerbate tendon ossification (Fig. 2C). Scx-GFP fluorescence confirmed the presence of Scx+ cells at tendinous insertions of the patella, but not in adjoining medialis or gastrocnemius muscle or BM hematopoietic (CD45+) cells (Fig. 2, D and E).

Histology revealed replacement of ligamentous and tendon structures with nascent and mineralized chondrogenic matrix and BM (Fig. 2, F to K), reflecting endochondral ossification rather than the non-endochondral calcification seen in calcifying tendinopathies (27). The talus (Fig. 2, F to I), distal tibia, and patellar bones (Fig. 2, J and K) featured prominent exostoses contiguous with bridging ligaments, similar to those seen in human FOP disease (Fig. 1C). Yellow fluorescent protein (YFP) activity via the Rosa26-YFP reporter revealed an Scx+ lineage origin of 100% (164 of 164 nuclei counted) of Alcian Blue (AB)–stained hypertrophic chondrocytes in HO lesions, but no detectable contribution to osteocytes or mineralized cortex (0 of 64 nuclei counted) in HO lesions of the talonavicular, tibialis anterior, and patellar ligaments, suggesting that mutant ACVR1 induces the chondrogenic differentiation of Scx+ lineages to provide a substrate for endochondral bone.

Muscle-resident Mx1+ progenitor lineages are sufficient for intramuscular HO

It was previously shown that the BM-derived Mx1+ lineages serve as a reservoir of osteoprogenitors in fracture healing (28). After injections with polyinosinic-polycytidylic acid (pIpC) at ages postnatal day 7 (P7) to P21, Mx1-Cre mice exhibited high frequency of recombination via the Rosa26-mTmG and Rosa26-YFP reporter alleles in microvascular endothelial cells (ECs) costaining with von Willebrand factor (vWF), in >90% of CD45+ BM cells, and in 30 to 50% of muscle interstitial cells located outside of laminin-stained myofibers (Fig. 3, A to D, and figs. S3 and S4). Mx1-Cre:Acvr1[R206H]FlEx/+ mice did not exhibit spontaneous HO, but when subjected to CTX-induced muscle injury at P21, intramuscular HO that spared tendons and ligaments was observed (Fig. 3E and fig. S3A). As noted in human disease (Fig. 1A), intramuscular HO in these mice resulted in fused long bones and bridged joints (Fig. 3F), restricting hindlimb mobility. Thus, in contrast to the Scx+ tendon and ligament phenotype, expression of mutant ACVR1 in the Mx1+ population resulted in a nonoverlapping intramuscular HO phenotype that required extrinsic injury to manifest. Similar to Scx-Cre, the HO seen in Mx1-Cre:Acvr1[R206H]FlEx/+ mice was sporadic and incompletely penetrant, with 30 to 50% of injured hindlimbs developing severe HO at 60 days (fig. S3).

Fig. 3. Expression of mutant ACVR1R206H or ACVR1Q207D alleles in Mx1-lineage results in injury-dependent intramuscular HO.

(A) Treatment of Mx1-Cre:Rosa26-mTmG (mTmG) mice with pIpC activates green fluorescent protein (GFP) expression within skeletal muscle interstitial cells (red, mTomato; green, mGFP; top and middle panels, whole-mount fluorescence; lower panel, cryosection; scale bar, 50 μm). (B) Gastrocnemius muscles of pIpC-treated Mx1-Cre:Rosa26-YFP mice reveal a subset of Mx1+ lineage (YFP, green) cells in the interstitium and microvascular endothelium costaining with DAPI (blue) and vWF (red). The same finding is shown at higher magnification in inset panels (top panel, scale bar, 50 μm; bottom panel, scale bar, 10 μm). Mx1+ cells were located consistently outside of myofiber basement membranes (C), based on laminin staining (magenta; top panel, scale bar, 50 μm; bottom panel, scale bar, 25 μm), and they accounted for a high percentage of BM cells based on costaining with CD45 (D) (magenta; top panel, scale bar, 200 μm; bottom panel, scale bar, 100 μm). (E) Treatment of Mx1-Cre:Acvr1[R206H]FlEx/+:Rosa26-YFP and Mx1-Cre:ACVR1Q207D-Tg:Rosa26-YFP mice with pIpC results in injury-dependent ossification of hindlimb muscles after CTX-mediated injury, with YFP-marked intramuscular bone. (F) Micro-CT imaging of Mx1-Cre:Acvr1[R206H]FlEx/+ mice treated with pIpC and CTX reveals ossification infiltrating hamstring and gastrocnemius, fusing ischium and tibia without articular involvement. (G) Intramuscular HO and heterotopic BM in Mx1-Cre:ACVR1Q207D-Tg:Rosa26-mTmG mice are derived from GFP-marked Mx1+ cells, with detail of lesion structure shown in (G) (scale bar, 1 mm) and at higher magnification (H) (scale bar, 100 μm; higher-magnification panels on right), revealing a mutant Mx1+ origin based on green fluorescence (white arrows) for nearly 100% of AB-stained hypertrophic chondrocytes (black arrows) and an Mx1+ origin for a portion of periosteal cells (red arrows), in contrast to no contribution to AR-stained mineralized matrix or associated osteocytes.

To test whether the ligand sensitivity of the Acvr1R206H allele might account for the need for extrinsic injury or variable penetrance, Mx1-Cre was used to express the constitutively active ACVR1Q207D transgene. Surprisingly, no spontaneous HO occurred when ACVR1Q207D was expressed via Mx1-Cre after pIpC injections (fig. S3). After CTX-induced injury, however, severe HO occurred in 100% of treated mice by 60 days. Although ligand-independent activity of ACVR1Q207D resulted in more highly penetrant HO than ligand-sensitive Acvr1R206H in Scx+ or Mx1+ populations, the requirement for antecedent injury for intramuscular HO with both mutants suggested that enhanced receptor signaling is necessary but not sufficient for intramuscular HO.

Immunochemical staining revealed that ossified lesions contain hypertrophic chondrocytes, marrow, cartilage, and heterotopic growth plates, consistent with an endochondral process (Fig. 3, G and H). Sequential fluorescence imaging and AB/AR (Alizarin Red) staining of sections revealed that nearly 100% of the hypertrophic chondrocytes of intramuscular heterotopic lesions (202 of 204 nuclei counted) were derived from Mx1+ lineage YFP–marked tissues (Fig. 3H), whereas few heterotopic osteocytes (1 of 24 nuclei counted) and only occasional periosteal cells were derived from YFP-marked lineages.

Because Mx1-Cre marked muscle interstitial, BM, and microvascular endothelial lineages (Fig. 3B), we performed additional studies to determine the subcompartment(s) responsible for this phenotype. Given the high frequency of recombination in the BM, including CD45+ cells (Fig. 3D and fig. S4), and the previous observation that BM Mx1+ cells are an important osteoprogenitor lineage (28), the role of the BM was tested by reciprocal BM transplants between wild-type (WT) and mutant reporter mice (Mx1-Cre:ACVR1Q207D-Tg:Rosa26-YFP). We used mutant mice with the highly penetrant ACVR1Q207D allele rather than the incompletely penetrant Acvr1[R206H]FlEx, as detecting a negative result with a power of 95% and an α = 0.05 would require at least 30 transplants based on a penetrance of 30% for Acvr1[R206H]FlEx (figs. S2 and S3). When mutant reporter donor or recipient mice were injected with pIpC from P7 to P21, consistently high-frequency (>90%) recombination of BM was observed based on reporter fluorescence (Fig. 4A). When neonatal WT mice that had been conditioned prenatally with busulfan were injected at P2 with BM obtained from mutant reporter mice previously treated with pIpC (P7 to P21), replacement of BM with 78.2 ± 15.9% (n = 5) YFP+ cells was observed by P21, approaching frequencies seen in donor mice (Fig. 4, B and C). Despite high-efficiency engraftment with mutant ACVR1 marrow, no HO was observed with or without intramuscular CTX treatment in any of these mice (zero of five). Conversely, when mutant reporter mice conditioned prenatally with busulfan were injected at P2 with BM obtained from WT mice at P21, the frequency of YFP+ cells in BM after pIpC was markedly decreased (0.5 to 5%, n = 11), consistent with near total replacement of marrow with WT cells (Fig. 4D), yet no attenuation of HO was observed after CTX treatment in any of these mice. These results suggested that cell-autonomous effects of mutant ACVR1 are not mediated by transplantable BM lineages.

Fig. 4. Mx1-lineage muscle interstitial but not BM cells are sufficient for intramuscular HO.

(A to D) Reciprocal BM transplant experiments demonstrate that medullary expression of mutant ACVR1 is dispensable for HO. (A) Control Mx1-Cre:Rosa26-YFP or mutant Mx1-Cre:ACVR1Q207D-Tg:Rosa26-YFP mice treated with pIpC exhibited >90% YFP labeling of marrow cells. Control or mutant mice were preconditioned antenatally with busulfan (E16, maternal intraperitoneal injection) and engrafted at P2 with total BM cells (5 × 105 cells, intraperitoneally) harvested at P21 from WT or mutant donor mice previously treated with pIpC (P7 to P21). In contrast to WT mice engrafted with WT marrow (B), WT mice engrafted with mutant marrow (C) exhibited a high percentage of YFP+ BM comparable to mutant donor mice (A) by flow cytometry (lower panel, representative flow plot shown; 78.2 ± 15.9%; n = 5) and ex vivo fluorescence (white arrows) but did not exhibit injury-dependent ossification after CTX, despite the presence of infiltrating YFP+ cells due to CTX-induced inflammation (red arrow). Conversely, Mx1-Cre:ACVR1Q207D-Tg:Rosa26-YFP mice engrafted with WT marrow (D) exhibited very low frequencies of residual YFP+ BM by flow cytometry (bottom panel; 0.5 to 5%; n = 11) but exhibited robust CTX-induced intramuscular ossification with YFP+ lesions (white arrows, top and middle panels). (E to I) Mx1+ lineage muscle interstitial cell engraftment studies demonstrate that Mx1+ lineage interstitial cells are sufficient for injury-dependent intramuscular HO. Mx1+YFP+ (5 × 105) cells sorted from the muscles of P21 control Mx1-Cre:Rosa26-YFP or mutant Mx1-Cre:ACVR1Q207D-Tg:Rosa26-YFP mice previously treated with pIpC (P7 to P19) were transplanted into gastrocnemius muscles of Dmdmdx-5cv:Rag1null (Mdx−/−) mice (P21) in Matrigel (E to F). In comparison to Mdx−/− control mice injected with Matrigel only (G, left), Mdx−/− mice injected with WT Mx1+YFP+ cells exhibited engraftment after 6 weeks based on YFP fluorescence but no HO with or without injury (G, middle panel; no lesions seen in five treated mice), whereas Mdx−/− mice injected with mutant Mx1+YFP+ cells exhibited engraftment and developed intramuscular ossification after CTX treatment (G, right panel; lesions seen in three of five mice treated). (H) Histological analysis of mice injected with WT Mx1+YFP+ control cells demonstrated engraftment of YFP+ cells interspersed in gastrocnemius and popliteal fossa (left panel, scale bar, 2 mm), all of which stain with ORO (inset right panels, scale bar, 200 μm). (I) Mice engrafted with ACVR1Q207D Mx1+YFP+ cells demonstrated engraftment of YFP+ cells throughout HO lesions of the gastrocnemius, with mineralization evident by AR, and formation of ectopic cartilage demonstrated by AB (upper panels and lower left panel, scale bar, 2 mm). At higher magnification, YFP fluorescence was observed to colocalize with mineralized areas stained by AR, whereas no YFP fluorescence was seen in heterotopic marrow, shown by fluorescence and DAPI counterstaining (inset panels at lower right, scale bar, 200 μm).

To test the sufficiency of the interstitial muscle cell population, muscle-resident Mx1+ lineages were marked by pIpC treatment (P7 to P21) in Mx1-Cre:Rosa26-YFP (control) or Mx1-Cre: ACVR1Q207D-Tg: Rosa26-YFP (mutant) mice, dispersed by mechanical and enzymatic dissociation and isolated by fluorescence-activated cell sorting (FACS), and then injected into the muscles of Dmdmdx-5cv:Rag1−/− recipients (Fig. 4, E and F). Recipient mice injected with labeled control cells demonstrated engraftment of YFP+ cells in muscle but did not demonstrate HO with or without CTX injury (zero of five mice tested; Fig. 4G). Rather, engrafted YFP+ cells were observed to stain with Oil Red O (ORO), consistent with an adipogenic fate after injury (Fig. 4H). In contrast, recipients engrafted with mutant reporter cells reliably demonstrated HO after CTX injury (three of five mice tested). The histological examination of engrafted mutant cells revealed colocalization with areas of mineralization and chondrogenic differentiation stained by AR/AB as well as the absence of YFP+ cells in heterotopic marrow (Fig. 4I). These findings suggested that expression of mutant ACVR1 in Mx1+ muscle-resident interstitial lineages mediates HO by skewing a normally adipogenic population toward an endochondral fate, whereas expression of mutant ACVR1 in the BM is dispensable for HO.

Endothelial, BM, pericyte, and smooth muscle expression of mutant ACVR1 are insufficient for HO

To corroborate results obtained by cell transplantation studies, the contribution of various lineages was tested directly by mating a panel of lineage-specific Cre strains against conditional ACVR1 mutant mice. As in cell transplantation experiments, mice expressing the highly penetrant ACVR1Q207D-Tg were used for these studies, because detecting a negative result with 95% power and an α = 0.05 for each lineage-targeting experiment would require observations from at least 30 compound heterozygous mice based on a 30% penetrance of Acvr1R206H (figs. S2 and S3). Thus, conditional ACVR1Q207D-Tg mice expressing the Rosa-YFP reporter allele to confirm lineage-specific recombination (29, 30) were mated with Myf6-Cre, SM22α-Cre, Vav1-Cre, Cadh5-CreERT2, Cspg4-CreERT2, and Pax7-Cre-ERT2 strains to target expression of ACVR1Q207D to skeletal myofiber, smooth muscle, BM, vascular endothelial, pericyte, and satellite cell compartments, respectively (Table 1). These matings yielded viable progeny in Mendelian ratios. However, activation of mutant ACVR1 expression during development or after tamoxifen injection (P7 to P21) failed to generate spontaneous or CTX injury–induced HO by x-ray (Table 1) within 90 days, despite confirmation of efficient recombination in target tissues.

Table 1. Cre recombinase–expressing mouse strains used for targeting mutant ACVR1.

Various tissue promoter-specific Cre-expressing mouse strains were mated with homozygous ACVR1Q207D-Tg/Q207D-Tg mice and tested for the capacity to generate live compound transgenic or compound knock-in and transgenic offspring. No live compound mutant mice were produced as a result of mating ACVR1Q207D-Tg/Q207D-Tg mice with Pax7-Cre, SM-MHC-Cre, or Tie2-Cre strains among the stated number of live births per mating strategy. In matings with Myf6-Cre, SM22α-Cre, and Vav1-Cre, compound mutant mice were produced in expected Mendelian ratios, but no spontaneous HO was seen in a minimum of 10 live compound mutants observed for up to 16 weeks of age, nor did any HO occur with intramuscular CTX injection at P10 to P14 in a minimum of 5 injected compound mutants. When offspring of ACVR1Q207D-Tg/Q207D-Tg and Pax7-CreERT2 or Cadh5-CreERT2, Cspg4-CreERT2 matings were injected with tamoxifen (50 mg/kg, intraperitoneally, daily, P14 to P21), high-efficiency recombination was observed among sublaminar satellite cells and vascular ECs, respectively. However, no spontaneous or CTX-induced HO was observed among a minimum of 10 compound mutant mice after tamoxifen injection and observation up to 16 weeks of age.

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Acvr1R206H confers activin A–induced SMAD1/5/8 activation and endochondral differentiation to Mx1+ and Scx+ lineages

Similar to previous observations (7, 8), expression of Acvr1R206H in myofibroblasts did not increase basal activation of BMP receptor–associated SMAD1/5/8 but conferred an ability to activate SMAD1/5/8 in response to activin A, a property not seen in WT cells, and moderately enhanced sensitivity to BMP4 (fig. S5A), whereas activation of SMAD3 by activin A was essentially unchanged in mutant versus WT cells. Similarly, Mx1+YFP+cells isolated from the skeletal muscle interstitium of pIpC-activated Mx1-Cre:Acvr1[R206H]FlEx/+:Rosa26-YFP mice did not exhibit baseline changes in alkaline phosphatase activity compared to WT Mx1+ cells (Fig. 5A) but exhibited markedly enhanced sensitivity to ligand-induced differentiation in response to activin A and BMP4, but not BMP6 (Fig. 5, B to D). Consistent with these observations, the baseline expression of a panel of endochondral genes was not altered with expression of Acvr1R206H; however, challenge with activin A induced expression of the endochondral genes Fmod and Col1a2 (fig. S5, B and C). Consistent with the earlier finding that activin A is a critical mediator of HO in Acvr1[R206H]FlEx/+ mice (7), these findings demonstrate that Acvr1R206H confers aberrant activin A–mediated signaling as well as osteogenic and chondrogenic differentiation in muscle-resident Mx1+ lineages.

Fig. 5. Expression of ACVR1R206H in Mx1 and Scx-lineage cells modifies their osteochondrogenic differentiation potential in a ligand-dependent manner.

(A) Spontaneous (n = 8) and (B to D) ligand-induced alkaline phosphatase expression in freshly isolated interstitial Mx1+ACVR1R206H and Mx1+ control cells obtained from quiescent, noninjured muscles after culture in the presence or absence of ligand for 4 days (n = 4). (B) †P = 0.006, **P = 0.01, *P = 0.03; (C) *P = 0.02, **P = 2 × 10−6. (E) Spontaneous (n = 4) and (F to H) ligand-induced alkaline phosphatase expression in freshly isolated YFP+ cells from 2-week-old Scx-Cre:ACVR1R206H-Tg:Rosa26-YFP (Scx+ACVR1R206H) and Scx-Cre:Rosa26-YFP (Scx+WT) mice after culture in the presence or absence of ligand for 4 days (n = 4). *P = 1.3 × 10−6, **P = 0.0001. Expression of BMP and TGF-β transcriptional targets and endochondral genes in Scx+ACVR1R206H and Scx+WT cells without (I) and with (J) activin A (1 nM) treatment for 48 hours (n = 4). (I) *P = 0.02; (J) *P = 0.02, **P = 0.003, ***P = 0.02. Unpaired Student’s t test was used for statistical analysis.

Similar to Mx1+ cells, Acvr1R206H Scx+ cells did not exhibit enhanced expression of alkaline phosphatase in the absence of exogenous ligand (Fig. 5E) but acquired sensitivity to activin A, which induced the differentiation of mutant but not WT Scx+ cells (Fig. 5F). In contrast to Mx1+ cells, mutant Scx+ cells did not exhibit enhanced differentiation in response to BMP4 or BMP6 (Fig. 5, G and H). In the absence of exogenous ligand, Acvr1R206H did not induce the activation of endochondral genes (Fig. 5I) but decreased the expression of Col1A2, Col2A1a, and Fmod, apparently modulating tendon phenotype. Treatment with activin A induced the expression of Id3, Sox9, and Col2A1a in ACVR1R206H compared to WT Scx+ cells, consistent with activin-mediated chondrogenic differentiation (Fig. 5J).

To analyze subpopulations of Scx+ lineages that might contribute to tissue-specific HO, we combined multiparameter FACS using surface lineage markers with Scx-regulated expression of Rosa26-YFP (fig. S6A). Total Scx+YFP+ cells accounted for ≤5% of tendon-derived mononuclear cells (fig. S6A). Acvr1R206H/+, but not WT Scx+, cells exhibited enhanced osteogenic and chondrogenic differentiation in response to activin A, but exhibited decreased adipogenic differentiation in the presence or absence of exogenous ligands (fig. S6, B to D). These results suggested that Acvr1R206H exerts ligand-dependent and possibly ligand-independent effects on the fate of Scx+ fibroadipogenic progenitor subpopulations. To test the impact of enhanced, ligand-independent ACVR1 signaling on Scx+ fibroadipogenic progenitors with a highly penetrant mutation, platelet-derived growth factor receptor α–positive (PDGFRα+) and PDGFRα fractions of Scx+ cells were fractionated from tendons of Scx-Cre:ACVR1Q207D-Tg:Rosa26-YFP mice and cultured in specific osteogenic, chondrogenic, and adipogenic media (fig. S6, E to G). Constitutive activation of ACVR1 signaling in these lineages revealed markedly enhanced osteogenic and chondrogenic potential among the PDGFRα+ subset, compared to PDGFRα or unfractionated Scx+ cells, suggesting an important role of this fibroadipogenic progenitor population in mediating the effects of mutant ACVR1.

A similar analysis of Mx1+ subpopulations was performed. Because muscle-resident Mx1+YFP+ cells accounted for a sizable (30 to 50%) fraction of mononuclear muscle interstitial cells (fig. S7A), these cells were fractionated further for analysis. WT Mx1+YFP+ cells lacking CD31 and CD45 (Lin) and enriched for Sca1+ demonstrated spontaneous and high-frequency adipogenic differentiation, which was markedly diminished in Acvr1R206H/+ cells (fig. S7B), suggesting that Acvr1R206H alters the potential of a fibroadipogenic Mx1+ subset. To test the impact of enhanced, ligand-independent ACVR1 signaling on Mx1+ subpopulations with a highly penetrant mutation, interstitial muscle cells were fractionated from hindlimbs of Mx1-Cre:ACVR1Q207D-Tg:Rosa26-YFP mice. Among Sca1+ cells, the enhanced osteogenic and chondrogenic potential of these mutant cells was enriched in CD31 and CD45 fractions (fig. S7C). Similarly, when Mx1+ cells expressing ACVR1Q207D were compared to WT cells, Sca1+, Lin, CD31, and CD45 fractions of mutant cells exhibited enhanced osteogenic and chondrogenic potential compared to WT (fig. S7D).

ACVR1 inhibition prevents intramuscular and joint HO in Acvr1[R206H]FlEx/+ mice

A selective, ACVR1-biased BMP type I receptor inhibitor, LDN-212854, has been shown to inhibit intramuscular HO in the adenovirus Cre-triggered ACVR1Q207D-Tg mouse model (14). This ACVR1-selective inhibitor was tested in the Rosa26-CreERT2:Acvr1[R206H]FlEx/+ mouse to determine whether spontaneous tendon and ligament HO respond differently to ACVR1 inhibition than do injury-induced intramuscular HO and whether ACVR1 inhibition is equally effective in an activin ligand–mediated model (7) compared to the previously tested ligand-independent model. Administration of LDN-212854 essentially abrogated spontaneous joint and ligamentous HO as well as the sporadic and handling-induced intramuscular HO seen in mice when administered for 4 weeks after tamoxifen administration (Fig. 6). Together, these data demonstrate that selective inhibition of ACVR1 in vivo is sufficient to abrogate the cell-autonomous and activin-mediated effects of mutant ACVR1 in tendon-derived and muscle-resident interstitial populations when administered prophylactically.

Fig. 6. Treatment with an ACVR1-selective kinase inhibitor prevents HO in global knock-in Acvr1R206H mice in vivo.

Adult Rosa-CreERT2:Acvr1[R206H]FlEx/+ mice were administered tamoxifen (40 mg/kg per day, intraperitoneally × 8 days) while simultaneously beginning a 4-week course of either vehicle or ACVR1-selective inhibitor LDN-212854 (3 mg/kg, subcutaneously, twice daily). After 4 weeks, vehicle-treated animals developed spontaneous joint and ligamentous HO, as well as prominent interscapular HO at sites of handling and examination (red arrows, top panels), whereas animals treated with LDN-212854 demonstrated near-complete inhibition of joint, ligamentous, and interscapular HO (red arrow, middle panel), shown quantitatively as a marked reduction in HO volume with LDN-212854 treatment (lower panel, **P = 0.0017 versus vehicle-treated controls).

DISCUSSION

We identified two distinct tissue-resident progenitor lineages that drive muscle versus tendon and ligament HO: an Mx1+ interstitial lineage in muscle that gives rise to injury-dependent intramuscular HO and an Scx+ lineage that gives rise to apparently spontaneous HO of tendons and ligaments. Expression of mutant ACVR1 in either of these lineages in vivo exerted chondrogenic effects, and the cartilage and hypertrophic chondrocytes of heterotopic bone lesions were entirely derived from mutant cells. Expression of Acvr1R206H in these lineages also conferred aberrant activin A–mediated endochondral differentiation, consistent with previous observations in surrogate cell lineages and in vivo in conditional knock-in mice (7, 8). Our findings are consistent with earlier studies demonstrating chondrogenic effects of gain-of-function ACVR1 mutants, shown in embryonic fibroblasts from Acvr1R206H/+ knock-in mice, micromass cultures induced to express ACVR1R206H, and developing chick limb buds induced to express ACVR1Q207D (3133). These data suggest that gain-of-function ACVR1 mutations promote ectopic chondrogenesis as a substrate for heterotopic endochondral ossification, which we now confirm as the mechanism of action within two tissue-resident mesenchymal progenitor populations.

In contrast to previous approaches that analyzed various cell populations contributing to HO lesions by immunohistochemistry and lineage tracing, the current study identified cell lineages that are sufficient to initiate HO, resulting from the cell-autonomous effects of ACVR1 mutations. Previous studies implicated Glast-expressing muscle interstitial and perivascular lineages, CD56+PDGFRα+ muscle interstitial cells, Tie2-expressing endothelial-related cells, and marrow-derived Col1+CD45+ osteoprogenitors as potential contributors to HO lesions in humans or HO lesions induced by injections of recombinant BMP ligands or overexpression of BMP transgenes in mice (1622). These previous studies demonstrated that a wide variety of lineages may be recruited to HO lesions but do not identify the lineages sufficient to initiate HO. Consistent with this notion, chimeric Acvr1R206H knock-in mice obtained by blastocyst injection develop HO lesions that incorporate both mutation-positive and mutation-negative cells (19). The current study similarly demonstrates heterogeneity of mutation-positive and mutation-negative cells in lesions but goes further to show that mutant ACVR1 initiates this process by endochondral ossification in two anatomically distinct progenitor lineages.

An earlier study examining lesions from a BM-chimeric FOP patient engrafted with WT BM as treatment for aplastic anemia and HO lesions in BM-chimeric mice revealed the participation of donor-derived hematopoietic cells in early preosseous lesions but not in mature heterotopic bone (34). Despite complete engraftment of the FOP patient with WT BM, FOP disease continued to progress, indicating that expression of mutant ACVR1 in transplantable BM cells is dispensable. Here, we used reciprocal BM transplantation as well as hematopoietic and vascular-targeted expression via the Vav1 promoter to eliminate hematopoietic and other transplantable BM-derived lineages as necessary or sufficient sites of mutant ACVR1 signaling in HO. The possibility of a cell-autonomous BM effect was important to evaluate because the Mx1-Cre knock-in allele, introduced at the interferon-responsive myxovirus resistance locus, has been used to mark hematopoietic BM-derived cells (35), a subset of which function as primitive osteoprogenitors that migrate to sites of injury to mediate fracture repair (28). The BM-derived Mx1+LinSca1+PDGFRα+ cells in that study had a similar surface phenotype to the muscle-localized Mx1+ cells in the current study; however, we showed that the muscle interstitial Mx1+ population functions independently of the BM-derived population and can engraft in WT muscle to initiate HO.

In addition to marking a subset of BM, Vav1-Cre and Mx1-Cre also mark microvascular endothelium (28), as confirmed in the present study. We excluded the possibility that mature ECs are sufficient to account for the Mx1 phenotype using both Cadh5-CreERT2 and Vav1-Cre mice but have not ruled out the possibility that postnatal administration of pIpC to Mx1-Cre mice could have marked Cadh5 immature EC progenitors that might contribute. After a report that Tie2-lineage cells may contribute to HO (22), a recent study demonstrated that a muscle-resident, Tie2-lineage, LinSca1+PDGFRα+ population contributes to BMP-induced heterotopic bone (21). It is likely that the interstitial Mx1+LinSca1+PDGFRα+ population identified in the current study overlaps substantially with these nonendothelial Tie2-lineage interstitial cells; however, the present study extends these concepts by showing that this population is sufficient to initiate HO in vivo through the chondrogenic effects of mutant ACVR1, an activin A–mediated gain of function that appears to be absent in FOP mutation–expressing iPSC-derived ECs (36), that mature Cadh5-lineage ECs cannot initiate HO, and that muscle-resident CD31+ cells do not manifest the chondrogenic or osteogenic effects of mutant ACVR1.

Our observations that Mx1+ LinSca1+PDGFRα+ interstitial cells exhibit markedly enhanced chondrogenic potential and decreased adipogenic potential with mutant ACVR1 and that, in muscle engraftment studies, the default adipogenic fate of WT Mx1+ interstitial cells was modified by mutant ACVR1 to form bone led us to postulate that Mx1+ HO progenitors represent “reprogrammed” fibroadipogenic progenitor cells. Fibroadipogenic progenitors, defined as muscle-resident LinSca1+PDGFRα+ cells, play physiologic roles in muscle injury repair and tissue homeostasis, contribute to fatty infiltration and fibrosis of muscles in settings of injury or myopathy, and may contribute to HO (18, 3739). These cells may overlap with PW1+ muscle interstitial cells, which express PDGFRα and Sca1 early in ontogeny and also exhibit fibro-adipogenic potential (40).

Although Scx-expressing lineages have been implicated in the normal development and maintenance of tendon and ligament tissues (26, 41), less is known about their contribution to disease or the impact of BMP and TGF-β family signaling upon their function. Scx lineage tendon–derived progenitors lack hematopoietic lineage markers and express Sca1 (42), consistent with current findings. The significance of PDGFRα expression in ligament and tendon-derived progenitor cells in relation to regenerative, fibroadipogenic, or osteogenic function was previously unexplored. We found that Scx+ cells and particularly the PDGFRα+ subfraction exhibited enhanced chondrogenic potential with expression of mutant ACVR1 even in the absence of ligand, consistent with the spontaneous HO of tendons and ligaments observed in two mutant ACVR1 mouse strains when activated by Scx-Cre. When constitutively active ACVR1Q207D was expressed in Scx+ lineages, these same phenotypes occurred with greater penetrance, suggesting that activin expression may be a regulator of HO in those tissues, requiring a threshold of expression for penetrance. It is possible that injury-induced factors required to activate Mx1+-derived lineages are constitutively produced in the milieu of mechanosensitive tendon and ligament tissues, thereby eliminating the need for experimentally induced injury. Because Scx-derived tissues function in force transmission and are regulated by signals mediated by physiologic and pathophysiologic loading (41), Scx+ progenitors may be triggered by mechanical loading in the presence of mutant ACVR1 to undergo osteogenic and chondrogenic differentiation, yielding spontaneous endochondral lesions in the absence of known extrinsic stimuli.

ACVR1Q207D causes constitutive ligand-independent activation of BMP signaling, whereas Acvr1R206H confers enhanced sensitivity to various ligands including activin A (5, 33, 4345). Expression of the knock-in Acvr1R206H allele in Mx1+ and Scx+ lineages sensitized cells to activin A–induced SMAD1/5/8 signaling and chondrogenic differentiation not seen in WT cells, extending our recent findings (7). Activin A and related ligands are induced by injury in muscle and other tissues, suggesting a possible contributing mechanism for HO after muscle injury in mice expressing mutant ACVR1 via Mx1-Cre. Because activin ligands are effectors of muscle homeostasis and regenerative responses after injury (46, 47), the functions gained by activin A in mutant Mx1+ are potential mechanisms for their contribution to HO. The persistent need for muscle injury with ligand-insensitive ACVR1Q207D Mx1+ cells suggests, however, that ligand-independent injury-mediated factors may be required to prime an endochondral differentiation program.

A limitation of this study is that to overcome embryonic lethality caused by global expression of mutant ACVR1 in mice (19), tissue-specific and/or postnatal expression of mutant ACVR1 alleles was driven by various Cre mouse strains. Postnatal expression of ACVR1 did not replicate some developmental phenotypes associated with FOP, including skeletal malformations such as hallux valgus and osteochondroma. Moreover, none of the Cre-targeting strategies, including Rosa26-CreERT2, would reveal the impact of expressing this mutation in all cells from conception as occurs in affected humans. In Scx-Cre mice, mutant ACVR1 was expressed developmentally in tendon-related lineages, whereas in Mx1-Cre mice expression was activated by pIpC injection only after birth, making functional comparisons of these lineages not strictly equivalent. In certain experiments, we used a constitutively active, ligand-independent ACVR1Q207D mutant allele, which is not meant to represent human FOP disease, but was used here to increase sensitivity for sporadic phenotypes in tissue targeting and cell transplantation experiments, as well as to demonstrate potential ligand-dependent phenotypes based on comparisons with Acvr1R206H. The observation that intramuscular HO occurs sporadically without intentional injury in a minority (15%) of Acvr1[R206H]FlEx mutant mice driven by Rosa26-CreERT2 but occurs in Mx1-Cre driven ACVR1Q207D or Acvr1R206H only with muscle injury suggests that Rosa-CreERT2, being nearly universal in its expression, may affect a wider array of muscle-associated progenitors or target the same populations with greater efficiency than Mx1-Cre. Although expression of Acvr1R206H in Mx1+ and Scx+ lineages together accounts for much of the phenotypic spectrum of FOP in humans, the identification of these lineages does not rule out a distinct or redundant contribution of other lineages within these or other tissue compartments. One might extrapolate from these results that nongenetic HO may derive from similar lineages, to the extent that FOP and acquired HO share biological mechanisms; however, this concept will need to be tested rigorously, because Acvr1R206H progenitor cells and mice exhibit activin gain of function not seen in acquired forms of HO, and as a consequence, the cellular progenitors and signals that recruit HO in each disorder could be distinct.

The identification of two distinct HO-driving progenitors has implications for the development of therapy. Local therapies neutralizing a single population in a given tissue are unlikely to provide satisfactory treatment for these syndromes, whereas systemic strategies targeting shared phenotypes, signaling, recruitment, or function of multiple tissue-resident progenitors are more likely to be effective—perhaps via the common marker PDGFRα or its ligands. Strategies to neutralize activin A could be effective in FOP, based on the gained function of activin A in these two distinct lineages and our previous findings in the Acvr1R206H model (7), but the impact of this therapy on the constitutive functions of activin in these tissues will need to be considered, and the contribution of muscle injury appears to be not only independent of ligand but also independent of ACVR1 signaling. The distinction raised here between injury-induced and injury-independent phenotypes has implications for trial design, because recent clinical trials for the treatment of FOP have been designed to intercept flare-associated events (48), yet controlling a major portion of disease progression that occurs in the absence of flares or injury (2) might require chronic therapy. In the absence of injury or acute flares, and particularly in tendons, ligaments, and fascia, hypotheses regarding the underlying trigger(s) such as ligands, inflammation, force transduction, and pathophysiologic stress will need to be tested directly and are likely to reveal how tissue homeostasis and regeneration are regulated in these tissues.

MATERIALS AND METHODS

Study design

This study was designed to ascertain whether the tissue-specific expression of hyperactive mutant ACVR1 alleles targeted to specific progenitor compartments could replicate tissue-specific phenotypes of FOP. These studies used a conditional knock-in mouse expressing the FOP-causing Acvr1R206H allele (7), as well as a previously described conditional transgenic mouse expressing the constitutively active ACVR1Q207D mutant allele (15). Comparison of the effects of the ACVR1Q207D transgene and Acvr1R206H allele was made to discern differences in ligand-mediated signaling and biochemical activity between these mutants (5, 33, 44, 45). For transplantation experiments, Cre-mediated expression, and identification of functionally relevant subcompartments, we used the ACVR1Q207D-Tg strain for its high sensitivity for in vivo and cellular phenotypes, whereas the Acvr1R206H allele was used to confirm that these findings were not artifacts resulting from ectopic expression or the high level of signaling activation mediated by ACVR1Q207D. Tissue-specific expression and cell engraftment experiments were assessed by gross anatomical and x-ray phenotyping for evidence of joint immobility and HO lesions, and inspection of histochemical staining by observers blinded with respect to genotype and treatment. Mice were assigned to different experimental groups based on their genotypes: the expression of mutant ACVR1 knock-in or transgenic alleles, various Cre deleter alleles, and the Rosa26-YFP reporter. Animals expressing all three alleles were assigned to the mutant (test) group, and those expressing only Cre and Rosa26-YFP were assigned to the WT (control) group. In Mx1-Cre experiments, pIpC was administered to all mice regardless of genotype. Observations in vivo were confirmed at a minimum in 10 compound transgenic or knock-in mice of a given genotype and treatment condition, or in at least 5 BM-transplanted or muscle-engrafted mice, unless otherwise noted in individual experiments. All other assays, including cell differentiation and flow cytometric measurements, were performed with a minimum of three experimental or biological replicates, unless otherwise noted, and data are represented as means ± SEM based on replicate measurements.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/8/366/366ra163/DC1

Materials and Methods

Fig. S1. Micro-CT imaging of Scx-Cre:Acvr1[R206H]FlEx/+, Scx-Cre:ACVR1Q207D-Tg, and Scx-Cre mice.

Fig. S2. Natural history study of Scx-Cre:Acvr1[R206H]FlEx/+ and Scx-Cre:ACVR1Q207D-Tg mice.

Fig. S3. Natural history study of Mx1-Cre:Acvr1[R206H]FlEx/+ and Mx1-Cre:ACVR1Q207D-Tg mice.

Fig. S4. Flow cytometric profile and gating strategy for isolation of Mx1+ lineage cells.

Fig. S5. Activin A elicits SMAD1/5/8 activation in Acvr1R206H but not WT myofibroblasts.

Fig. S6. Impact of Acvr1R206H and ACVR1Q207D upon differentiation of Scx+ cells.

Fig. S7. The impact of Acvr1R206H and ACVR1Q207D upon the differentiation potential of Mx1+ subpopulations.

Table S1. qRT-PCR primer sequences.

Table S2. Original data for Figs. 5 and 6 and figs. S5 to S7 (provided as an Excel file).

References (5759)

REFERENCES AND NOTES

  1. Acknowledgments: We thank Y. Mishina, E. Gussoni, and A. Aliprantis for providing critical reagents and experimental advice. We thank G. Buruzula from the Flow Core Facility, Joslin Diabetes Center, for technical assistance in FACS experiments; H. Nakajima for technical assistance with cryosectioning and microscopy; and D. Xia for assistance in x-ray analysis of mouse phenotypes. Cre-inducible constitutively active ACVR1Q207D transgenic mice were gifts from Y. Mishina. C57BL/6 Dmdmdx-5cv:Rag1null mice were gifts from E. Gussoni. SM22α-Cre transgenic mice were provided by J. Lepore, Scx-Cre and Scx-GFP mice were gifts from C. Tabin, and VE-Cadherin-CreERT2 mice were provided by L. Iruela-Arispe. Funding: This work was supported by the US NIH [HL079943 (to P.B.Y.), AR057374 (to P.B.Y.), DK036836 (to A.J.W.), and HL100402 (to A.J.W.)], Brigham and Women’s Hospital Cardiovascular Division T32 [HL007604 (to D.D.)], National Institute of Arthritis and Musculoskeletal and Skin Diseases P30 Center for Skeletal Research Core (AR066261), Department of Defense [MR140072 (to P.B.Y.)], Harvard Stem Cell Institute Seed Award (to P.B.Y.), International FOP Association Competitive Grant Award (to P.B.Y. and Y.S.), Harvard Stem Cell Institute funding for the Joslin Diabetes Center Flow Core Facility, Pulmonary Hypertension Association Clinician Scientist Career Development Award (to P.B.Y.), Leducq Foundation Transatlantic Network of Excellence Award (to P.B.Y.), Howard Hughes Medical Institute Early Career Physician-Scientist Award (to P.B.Y.), and Massachusetts Technology Transfer Award (to P.B.Y.). Author contributions: D.D., J.B., S.J.H., K.A.A., L.H., J.E., A.J.V., Y.S., A.H.M., and P.B.Y. designed experiments, performed experiments, and analyzed data. A.L. synthesized drug compounds. E.M.W.E. and A.v.S. provided clinical data. D.D., J.B., S.J.H., M.B.D., C.K., A.J.W., A.N.E., and P.B.Y. provided methodological and conceptual input. D.D., J.B., S.J.H., J.E., A.J.W., A.N.E., and P.B.Y. wrote and revised the manuscript. Competing interests: A.N.E., S.J.H., and L.H. are employees of Regeneron Pharmaceuticals Inc. and hold stock in the company. A.H.M. and P.B.Y. are inventors on patent applications PCT/US2014/020360 and PCT/US2014/026042 submitted by Partners Healthcare System Inc., covering small molecules that inhibit BMP receptor signaling for the treatment of FOP. S.J.H. and A.N.E. are inventors on patent applications US2014/0283158 and 20160075772 submitted by Regeneron Pharmaceuticals Inc. that cover the invention of the Acvr1[R206H]FlEx mouse model and the inhibition of activin ligands and activin receptors for the treatment of FOP, respectively. Data and materials availability: All noncommercially available materials described in this study may be obtained with a materials transfer agreement (MTA). The Acvr1[R206H]FlEx/+ knock-in mouse may be obtained from A. Economides at Regeneron Pharmaceutical through MTA. Original data for Figs. 5 and 6 and figs. S5 to S7 are provided in table S2.
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