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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Bone Miner Res. Author manuscript; available in PMC Aug 1, 2013.
Published in final edited form as:
PMCID: PMC3556640
NIHMSID: NIHMS371133

An Acvr1 R206H knock-in mouse has fibrodysplasia ossificans progressiva

Abstract

Fibrodysplasia ossificans progressiva (FOP; MIM #135100) is a debilitating genetic disorder of dysregulated cellular differentiation characterized by malformation of the great toes during embryonic skeletal development and by progressive heterotopic endochondral ossification post-natally. Patients with these classic clinical features of FOP have the identical heterozygous single nucleotide substitution (c.617G>A; R206H) in the gene encoding ACVR1/ALK2, a bone morphogenetic protein (BMP) type I receptor. Gene targeting was used to develop a knock-in mouse model for FOP (Acvr1R206H/+). Radiographic analysis of Acvr1R206H/+ chimeric mice revealed that this mutation induced malformed first digits in the hind limbs and post-natal extra-skeletal bone formation, recapitulating the human disease. Histological analysis of murine lesions showed inflammatory infiltration and apoptosis of skeletal muscle followed by robust formation of heterotopic bone through an endochondral pathway, identical to that seen in patients. Progenitor cells of a Tie2+ lineage participated in each stage of endochondral osteogenesis. We further determined that both wild-type and mutant cells are present within the ectopic bone tissue, an unexpected finding that indicates that although the mutation is necessary to induce the bone formation process, the mutation is not required for progenitor cell contribution to bone and cartilage. This unique knock-in mouse model provides novel insight into the genetic regulation of heterotopic ossification and establishes the first direct in vivo evidence that the R206H mutation in ACVR1 causes FOP.

Keywords: ACVR1/Acvr1, ALK2, fibrodysplasia ossificans progressiva, FOP, heterotopic ossification, endochondral bone

Introduction

Heterotopic ossification (HO), the formation of ectopic (extra-skeletal) bone in soft tissues, is most commonly associated with acute tissue damage such as severe burns, spinal cord and head injuries, and high impact trauma including war-induced injuries.(1-5) Heterotopic ossification also occurs commonly in patients following total hip replacement surgery and is a complication of aging, occurring in association with atherosclerosis, valvular heart disease, and pressure ulcers. Heterotopic bone is qualitatively normal bone tissue that is induced to form at extra-skeletal sites. Little is known about the cellular events or molecular mechanisms that induce and promote heterotopic ossification.

In addition to trauma-induced heterotopic ossification, two rare inherited human diseases of heterotopic ossification, fibrodysplasia ossificans progressiva (FOP) and progressive osseous heteroplasia (POH), have also been described.(6) The genetic mutations that cause these disorders have been identified, providing the opportunity to understand the cellular and molecular regulation of bone formation.

Genetic analyses identified heterozygous mutations in ACVR1 (also known as ALK2), a bone morphogenetic protein (BMP) type I receptor, in all FOP patients.(7,8) FOP is diagnosed clinically on the basis of two defining, or classic, FOP features: congenital malformation of the great toes and progressive heterotopic endochondral ossification that develops in characteristic anatomic patterns.(6)

Spontaneous and episodic flare-ups (episodes of heterotopic ossification) in FOP typically begin during childhood, however flare-ups can also be activated by soft tissue trauma and injury. The subsequent extensive bone formation causes extra-articular ankylosis of the joints of the axial and appendicular skeleton leading to progressive immobility. In addition to post-natal soft tissue ossification, patients with FOP also have developmental skeletal malformations including characteristic great toe malformations and other common but variable skeletal features such as proximal medial tibial osteochondromas, cervical spine malformations, short, broad femoral necks, costo-vertebral malformations, scoliosis, and fusion of vertebrae and diarthrodial joints.(8,9)

All patients with classic FOP features share a single nucleotide substitution (c.617G>A) in the glycine-serine (GS) activation domain of the ACVR1/ALK2 receptor that replaces arginine with histidine in codon 206 (R206H). Protein modeling predicts that this amino acid substitution alters receptor signaling activity(10) and functional analyses demonstrate that the ACVR1 R206H mutation induces increased BMP signaling that is both ligand independent and BMP responsive.(11-14)

Signaling through bone morphogenetic proteins (BMPs) and their receptors is a key mechanism regulating chondrogenesis and endochondral bone formation.(15-18) BMP receptor signaling is essential for the mesenchymal cell condensations that precede skeletal element formation. BMP signaling also participates in proliferation, differentiation, and maturation of chondrocytes during the development of cartilage and bone. These effects of BMP signal transduction occur through both canonical SMAD signaling and non-canonical MAPK pathways.(16,19,20).

Initial investigations of the cellular events that lead to heterotopic ossification used in vivo BMP protein implants to induce bone formation.(21,22) More recently, transgenic mouse lines that express BMP4 under the control of the neuron-specific enolase promoter or that conditionally over-express constitutively active Acvr1/Alk2Q207D were shown to develop progressive heterotopic ossification within skeletal muscle.(23-26) However, none of these models fully reproduce the phenotype and progression of FOP and more faithful disease models are required to understand the molecular and cellular mechanisms that direct heterotopic bone formation as well as to serve as in vivo systems to test potential therapies.

In this study, we describe the development and characterization of an Acvr1 R206H (c.617G>A) knock-in mouse (Acvr1R206H/+). Although germline transmission of this mutation is perinatal lethal, mice that are chimeric for Acvr1R206H/+ cells recapitulate every clinical feature of patients with classic FOP including embryonic skeletal malformations and postnatal heterotopic endochondral bone formation. In addition, histological analyses of developing heterotopic ossification demonstrate the same progression of cellular events including inflammation-induced catabolism of connective tissues followed by a robust anabolic tissue replacement by cartilage and bone.

Material and Methods

Generation of R206H Acvr1 knock-in mice

Acvr1 sequences from a mouse C57BL/6 BAC library were inserted into the retroviral vector pL253 by a BAC recombineering strategy. Acvr1 codon 206 was engineered with the FOP mutation (CGC>CAC) in murine exon 5 and a neor marker gene under the regulation of the Pgk promoter(27) was added (Figure 1A). Correctly targeted homologous recombination was determined by BglII digestion and Southern analysis; a 14.4 kb fragment indicated the presence of the mutant allele and a 12.4 kb fragment corresponded to the endogenous allele (Figure 1B, C). From 300 G418 resistant ES colonies, we identified 16 positive ES clones showing homologous recombination; positive clones were karyotyped, verified by sequencing, and used for blastocyst injection into CD-1/BALB/c mice. Six different clones were injected to generate chimeras. The proportion of mutant cells in the resulting mutant/wild-type cell chimeric progeny was estimated by coat color; mutant cells from C57BL/6J ES cells generate black coat and wild-type cells from CD-1/BALB/c are white. Additional details are provided in Supplemental Information.

Figure 1
Generation of Acvr1R206H/+ knock-in mouse model for FOP

All animal studies were approved by the Institutional Animal Use and Care Committee of the University of Pennsylvania.

Imaging analyses

Micro-computed tomography (μCT)

Paraformaldehyde (PFA)-fixed whole mouse specimens were imaged using an eXplore Locus SP μCT specimen scanner (GE Healthcare) at the Small Animal Imaging Facility of the University of Pennsylvania. Volumetric data were acquired using the following parameters: 80 kVp and 80 μA X-ray tube voltage and current, 250 μm aluminum filter, 1.7 s integration time, 400 views at 0.5° increments, 2×2 detector bin mode, 4 averages, 1 hr scan time. Image data were reconstructed at a resolution of 40.5 μm isotropic voxels using a Feldkamp cone beam algorithm. The reconstructed 3D data were analyzed and volume rendered using OsiriX software (www.osirix-viewer.com).

X-ray imaging

Whole-body radiographic images of PFA-fixed mice were performed with a prototype digital breast imaging system (Selenia Dimensions, Hologic, Bedford MA) located at the Hospital of the University of Pennsylvania. Both projection radiographic and tomosynthesis images were acquired. Radiographic images were acquired using a 2.0× geometric magnification at 25 kVp and 90 mAs with a 0.1 mm nominal focal spot and a W/Rh target/filter combination. Image processing by the manufacturer was restricted to flat-field corrections. The resultant linear projection images have a 35 μm pixel size in the plane of the mice. The images were analyzed using ImageJ software (http://rsbweb.nih.gov/ij/).

Histological analyses

Heterotopic lesions were dissected based on imaging analyses. Fixed tissues (4% PFA) were decalcified using Immunocal™ (Decal Chemical Corporation, Tallman, NY), embedded in paraffin, and sectioned serially at 7 microns. Control sections were from age-matched wild-type mice. Sections were stained with Harris Modified hematoxylin and eosin Y solution, safranin O, or alcian blue, and mast cells were detected by CEM staining (American MasterTech).

Deparaffinized sections were treated for antigen retrieval with 10 mM sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched with 3% hydrogen peroxide solution. Sections were blocked (Background Buster; American MasterTech) then incubated with primary antibody overnight at 4°C, followed by incubation with appropriate HRP linked secondary antibody and development of color using DAB (SuperPicTure™ Polymer, Invitrogen). Primary antibodies detected: phosphorylated-Smad1/5/8 and phosphorylated-p38-MAPK (Cell Signaling Technology); collagen II, collagen X, myeloperoxidase, and TGFβ (Abcam); proliferative cell nuclear antigen (PCNA) and CD45 (Santa Cruz); F4/80 (AbD Serotech); Tie2, Cleaved Caspase-3, and Neomycin phosphotransferase II (Millipore). Sections were counterstained by hematoxylin. TUNEL staining used the In Situ Cell Death Detection Kit (Roche).

Double immunohistochemical staining used the PicTure™-Double Staining Kit (Invitrogen). Processed sections were incubated overnight at 4°C with rabbit pSmad1/5/8 and mouse Neomycin phosphotransferase II (Abcam) antibodies, followed by incubation with alkaline phosphatase–conjugated anti-rabbit and HRP linked anti-mouse secondary antibodies. DAB (SuperPicTure™ Polymer, Invitrogen) was used for HRP color development (brown), followed by Vector Blue (Vector Laboratories) staining for alkaline phosphatase.

Skeletal muscle injury by cardiotoxin

Quadriceps muscle injury was induced with 100 μl of 10 μM cardiotoxin (Calbiochem, San Diego, California).(28) Contralateral injections of saline were used as controls. R206H chimeric mice developed immobility in the cardiotoxin-injected hind limb by 30 days post injection and were analyzed by X-ray, μCT, and histology (n=3).

Results

Gene targeting to generate an R206H Acvr1 allele

The ACVR1/ALK2 gene is highly conserved between human and mouse and identical at codon 206(29). Acvr1 from a mouse C57BL/6 BAC clone was modified to replace codon 206 in exon 5 with the FOP R206H mutation (CGC>CAC) and add a neor marker gene (Figure 1 and Supplemental Information). Following blastocyst injection of ES cells positive for homologous recombination, the resulting chimeric mice (Supplemental Figure 1) were bred with C57BL/6 or CD-1 mice but viable progeny with germline transmission of the mutant allele were not recovered (see Supplemental Information). Chimeras with estimated 70-90% mutant cells were used for phenotypic analysis of effects of the heterozygous Acvr1R206H knock-in allele.

Acvr1R206H/+ chimeric mice develop characteristic clinical features of classic FOP patients

In addition to post-natal heterotopic ossification, FOP patients with the ACVR1 R206H mutation have congenital malformation of the great toes (a shortened first metatarsal with a single or delta shaped proximal phalanx) (Figure 2A). Patients can be diagnosed with FOP solely on the basis of digit malformations, even before the appearance of pre-osseous soft tissue lesions.(30)

Figure 2
Acvr1R206H/+mice display classic FOP phenotypes

At birth, 13 of 27 Acvr1R206H/+ knock-in chimeric mice displayed shortened first digits in the hind limbs (Figure 2B and Supplemental Figure 2). The absence of the great toe malformation in some chimeric mice is expected due to variable distributions of mutant cells (also see Supplemental Information). Consistent with patients who have ACVR1 R206H mutations; first digit malformations were observed in hind limbs but not fore limbs. X-ray and μCT analyses revealed shortened or absent proximal and distal phalanges that were comparable to the unique and characteristic malformations in FOP patients (Figure 2B).

Movement and activity of the mice appeared normal during the first several weeks after birth. However, by 6-8 weeks of age, most chimeras with a high proportion of mutant cells displayed severe physical disability evidenced by soft tissue swelling, ankylosed joints, limited mobility, and difficulty in movement. μCT and X-ray analyses of five Acvr1R206H/+ chimeras revealed extensive heterotopic ossification in skeletal muscle causing ankylosis of major joints of the axial and appendicular skeleton, as is often observed in FOP patients (Figure 2C, Table 1).

Table 1
Comparison of FOP clinical features with Acvr1R206H/+ chimeric mice.

Other common but more variable FOP features(8) were observed in the chimeric mice including fusion of the posterior facet joints of the subaxial cervical vertebrae (Figure 2D) and variable rib fusions and costovertebral malformations with secondary scoliosis (Figure 2E). Osteochondromas, cartilage-capped bony out-growths that typically form in metaphyseal regions, occur in most patients with FOP(31) and in Acvr1R206H/+ chimeras (Figure 2F), most commonly at the proximal medial tibia, but also in other bones, notably the humerus and femur. Chimeras with lower percentages of mutant cells (based on coat color) were also examined and found to display subsets of the FOP-associated phenotypes (Supplemental Information and Supplemental Figure 1).

Distinct cellular events are associated with heterotopic endochondral ossification

Cellular events associated with FOP lesion progression have been defined through histological analyses that demonstrated formation of heterotopic ossification through a complex process that involves an initial cell and tissue catabolic phase followed by an anabolic phase.(22,32) Destruction of connective tissues, such as skeletal muscle, is accompanied by a robust inflammatory response. This phase is followed by proliferation of fibroblast-like cells that are subsequently replaced through chondrogenesis, angiogenesis, and osteogenesis to form endochondral bone tissue with mature marrow elements. Histological analyses showed these same stages of tissue metamorphosis in the Acvr1R206H/+ mice (Figure 3).

Figure 3
Histological analysis of cellular events during heterotopic bone formation in Acvr1R206H/+ knock-in chimeric mice

Apoptosis of connective tissue and abundant immune cell infiltration are initial events in lesion formation

Early evidence of skeletal muscle degeneration in Acvr1R206H/+ mouse lesions was indicated by loss of peripheral nuclei and many myofibers with central nuclei (Figure 3A, B). These degenerating cells were positively labeled by nuclear DNA fragmentation (TUNEL) assays (Figure 3A) and activated caspase-3 (Figure 3B). Although some necrosis of the degenerating muscle tissue cannot be excluded, the data demonstrate that apoptosis occurs at the earliest stages of HO lesion formation and that mature muscle cells and other connective tissue cells are actively lost through apoptosis.

A strong inflammatory response was also observed within the degenerating tissues. Tissues containing enucleated cells (dead) and ghost bodies were infiltrated with CD45+ lymphocytes (Figure 3C). Large numbers of polymorphonuclear cells that were positive for the neutrophil marker myeloperoxidase surrounded the dead and degenerating myofibers and indicate neutrophil participation in scavenging degrading cells/tissues (Figure 3D).

Fibroproliferation follows the inflammatory response and skeletal muscle apoptosis

Apoptosis of skeletal muscle is accompanied by expanding islands of spindle-shaped, nucleated fibroblastic cells, indicated by proliferative cell nuclear antigen (PCNA) staining (Figure 3E), as tissue is cleared of dead muscle cells and the lesion transitions from a catabolic to an anabolic phase. Such tissue fibrosis has been associated with secreted growth factors and collagens that provide a tissue environment that supports tissue remodeling and replacement.(33,34)

Activated macrophages and cells of monocyte origin are present within degenerating muscle tissue and in regions of newly forming fibroproliferative cells (Figure 3F) suggesting an active role of macrophages in this tissue remodeling. Activated, granular mast cells (Figure 3G) were present at every stage of lesion formation with the most pronounced presence during the highly vascular fibroproliferative stage, as well as at sites initiating chondrogenesis, identical to that seen in human lesions.(35) Control skeletal muscle from wild-type mice showed only rare scattered mast cells which were non-granular and smaller in size than those in the Acvr1 R206H-induced lesions.

In addition to abundant monocytes/macrophages and activated mast cells, fibroproliferative regions were angiogenic, detected by staining for alpha smooth muscle actin (Figure 3H) and von Willebrand factor, throughout this transition from a catabolic lesion to an intensely anabolic lesion.

Extra-skeletal bone formation occurs by an endochondral process

The robust fibroproliferative response in developing lesions was followed by chondrogenesis as detected by cell morphology and safranin O staining (Figure 3I). These areas of newly formed cartilage express characteristic markers of early chondrocyte differentiation (collagen II; Figure 3J) and maturation (collagen X; Figure 3K) with the characteristic changes in cell morphology from early to late stage chondrogenesis and hypertrophy, chondrocyte apoptosis, and replacement by osteoblasts. Subsequent bone formation is coordinated with angiogenesis, the appearance of bone marrow, and mature bone formation as in FOP heterotopic ossification (Figure 3L).

Skeletal muscle injury in Acvr1R206H/+ chimeric mice triggers heterotopic ossification

Although HO lesions can form spontaneously in FOP patients, tissue injury can also induce HO.(9) In order to determine whether the Acvr1R206H/+ knock-in mouse similarly responds to tissue trauma by forming heterotopic bone, we used an established skeletal muscle injury model of intramuscular injection of cardiotoxin.(28,36) By 6 weeks post cardiotoxin injection, Acvr1R206H/+ mice showed progressive immobility in response to cardiotoxin-induced injury, and X-ray (Figure 4A) and μCT analysis revealed substantial heterotopic ossification at the site of cardiotoxin injection and in the surrounding soft tissues by 6 weeks. Histological analysis (Figure 4B) demonstrated endochondral bone formation with all the previously described stages of HO lesion formation, including apoptotic muscle tissue degeneration, mononuclear infiltration, inflammation with acute fibroproliferative response, chondrogenesis, and bone formation with marrow elements (Figure 4B). No heterotopic ossification or earlier stages of lesion formation were observed in PBS-treated contra-lateral limbs of Acvr1R206H/+ knock-in (Figure 4A) or in wild-type mice. Although relatively minor injury by PBS injection is not sufficient to stimulate heterotopic ossification, our results demonstrate that more severe connective tissue injury in the context of the R206H mutation triggers a severe inflammatory response that is followed by development of heterotopic ossification.

Figure 4
Skeletal muscle injury induces heterotopic ossification in Acvr1R206H/+ mice

Tie2+ progenitor cells contribute to heterotopic ossification

Lineage tracing studies in mouse models of heterotopic ossification previously demonstrated that Tek/Tie2+ lineage cells are a progenitor cell population recruited to differentiate to cartilage and bone cells in HO lesions.(28,37,38) Consistent with these studies, abundant Tie2+ cells were detected in regions of skeletal muscle tissue degradation and fibroproliferation in Acvr1R206H/+ lesions (Figure 5A). At later stages, fibroproliferative cells and many newly formed chondrocytes were Tie2+ (Figure 5A). By contrast, Tie2+ cells were not observed in skeletal muscle tissue from wild-type mice (Figure 5A), except in vessels. These data indicate that a Tie2+ cell lineage contributes to the progenitor cells that form all stages of R206H Acvr1-induced heterotopic endochondral bone.

Figure 5
Cells participating in heterotopic lesion formation

Acvr1 R206H is not required by progenitor cells that form ectopic cartilage and bone

Given that both Acvr1 R206H mutant and wild-type cells are present in the Acvr1R206H/+ chimeric mice, we investigated the contributions of both cell types to ectopic endochondral bone formation. Mutant Acvr1R206H/+ cells were detected by their expression of neomycin phosphotransferase II (neo) (Figure 1A). Both neo positive and negative cells were present in fibroproliferative areas of the developing ectopic lesions (Figure 5B) as well as in heterotopic bone, indicating that both mutant and wild-type progenitor cells participate in this process (Figure 5B). Both neo+ and neo- cells were also detected in histologic sections of malformed toes (Supplemental Figure 1).

Areas of heterotopic ossification showing tissue degradation, fibroproliferative, and endochondral ossification stages indicated that neo+ cells were present in greater numbers than neo- cells (Figure 5B, C), and cell counts for fibroproliferative and endochondral stages show that both contain ~65% neo+ cells (Supplemental Information and Supplemental Figure 3). To investigate whether BMP signaling is activated in neo expressing cells, as expected in the presence of the Acvr1R206H mutation, we conducted double immunohistochemical staining for neo and phosphorylated Smad1/5/8 (pSmad1/5/8) (Figure 5C). At both fibroproliferative and endochondral stages, ~80% of neo+ cells were also positive for pSmad1/5/8 (Supplemental Figure 3C, G) supporting a strong correlation of BMP signaling with the Acvr1 mutation. A small population of neo-/pSmad1/5/8+ cells (~17% of total cells) may represent wild-type cells that are recruited to heterotopic ossification, a process that involves activation of BMP signaling.

Discussion

Animal models of human genetic disease are vital for validating the exact genetic cause of a condition, for understanding the cellular and molecular mechanisms of disease pathology, and for developing translational strategies to prevent and treat affected individuals. The described chimeric knock-in animal model of the rare and disabling human genetic disorder fibrodysplasia ossificans progressiva (FOP) displays all of the embryonic and post-natal features of FOP that are present in the human condition. These mice validate that the recurrent mildly activating mutation of the BMP type I receptor ACVR1/ALK2 (c.617G>A; R206H) that occurs in all individuals with classic clinical features of the disease(6) is the direct genetic cause of FOP and of all of its resulting pathology.

Along with progressive heterotopic endochondral ossification, malformation of the great toes is a hallmark of classic FOP. Acvr1R206H/+ murine knock-in chimeras had malformations of hind limb first digits, nearly identical to those seen in patients with classic FOP. No malformations were seen in the fore limb digits of any of the chimeras. Acvr1R206H/+ chimeras also showed the full spectrum of congenital malformations observed in patients with FOP: fusion of subaxial cervical facet joints, costovertebral malformations, and osteochondromas of the proximal tibias and scattered other sites. Importantly, the knock-in mice also developed spontaneous and injury-induced FOP lesions that differentiated into mature heterotopic endochondral bone as described in patients with FOP. The study reveals that heterotopic ossification in FOP is not simply a process of ectopic bone formation within skeletal muscle. Rather the original skeletal muscle tissue is replaced with heterotopic bone in a complex multi-stage process characterized by an inflammatory and apoptotic catabolic phase followed by an anabolic endochondral phase. These findings establish that heterozygous substitution of c.617G>A in Acvr1 causes all of the congenital and post-natal features of FOP, and further establishes the first knock-in mouse model of classic human FOP.

This work sheds light on many important questions about the cellular targets of the FOP mutation, including those that could only be addressed in viable chimeras exhibiting the classic FOP phenotype. This mouse model is consistent with recent findings that cells of Tie2+ origin differentiate to form mature heterotopic bone through an endochondral pathway.(28,37,38) Importantly, both wild-type and mutant Tie2+ mesenchymal progenitor cells comprise much of the early anabolic fibroproliferative lesion in the chimeric mice and are capable of differentiating to heterotopic bone. This unexpected finding strongly suggests that the Acvr1R206H mutation is not required in precursor cells and that once formed, wild-type precursor cells can receive instructive signals in a cell non-autonomous manner to guide their differentiation through an endochondral pathway. We confirmed that the majority of mutant cells within lesions, as well as some wild-type cells, show activated BMP signaling, supporting their differentiation along a chondro/osseous pathway. This finding supports the physiological importance of designing preventions and treatments that target both cell autonomous and cell non-autonomous responses to BMP signaling.

In addition to the post-natal cellular events and targets of Acvr1R206H activity that lead to heterotopic ossification, this work also provides important insight into the pre-natal developmental targets of the FOP mutation. As in patients with FOP, the Acvr1R206H/+ chimeric mice develop malformations and subsequent ankylosis in a wide array of small joints of the axial and appendicular skeleton including, but not limited to, the great toes, the intervertebral joints, and the costo-vertebral joints, identical to those seen in individuals affected with FOP. These findings suggest that articular chondrocytes or pre-chondrocytes have a lower threshold and higher sensitivity for the activation of BMP signaling caused by Acvr1R206H compared to other cells of the developing skeleton. This observation is supported by earlier findings on the sensitivity of diarthrodial joint development to BMP morphogenetic gradients.(15,39-42) Further, the presence of widespread osteochondromas underscores that cells of the perichondrium are highly sensitive to the direct effects of BMP signaling and its interacting pathways.

Given that patients with the ACVR1 R206H mutation have relatively few major effects on development, the severe consequences of this mutation that lead to perinatal lethality upon germline transmission in our knock-in mouse model were unexpected. The precise defects leading to early lethality remain under investigation, however this mouse model was developed in an isogenic C57BL/6 background; it is possible that the Acvr1R206H mutation would provoke a less severe phenotype in an alternate genetic context. The extensive genomic heterozygosity in humans may support viability, as well as explaining the variations in disease severity that we observe among patients with the R206H mutation. Alternatively, since BMP signaling has been implicated in the development and function of many tissues and cells including germ cells, early mouse embryonic development may be more sensitive to perturbations in the level of BMP signaling and/or the expression pattern of Alk2 may be different in mice compared to humans.

Many additional questions remain unanswered by this work including the cause of the extremely robust inflammatory infiltration that occurs in early spontaneous FOP lesions, whether the ACVR1 mutation in FOP influences the immunosuppressive phenotype that has been associated with apoptosis,(43,44) the basis for the distinct anatomic progression of lesions, and the identity of the factors that direct the episodic progression of the disease. This FOP chimeric knock-in mouse model is novel and is rare among animal models by its recapitulation of all of the features of a complex human disease with complete fidelity, and thus provides a valuable tool to address important physiological questions and therapeutic strategies that can be applied to treat heterotopic ossification.

Supplementary Material

Supp Material

Acknowledgments

We thank Dr. Tobias Raabe (Gene Targeting Core) and Dr. Jean Richa (Transgenic Mouse Core) of the Perelman School of Medicine at the University of Pennsylvania. Sincere thanks to Dr. Vitali Lounev, Kevin Egan, Dr. Robert J Pignolo, and Dr. Kurt Hankenson for experimental assistance and/or discussions, and to the members of the Shore/Kaplan research group. This work was supported in part by the International Fibrodysplasia Ossificans Progressiva Association, the Center for Research in FOP and Related Disorders, the Ian Cali Endowment for FOP Research, the Whitney Weldon Endowment for FOP Research, the Isaac & Rose Nassau Professorship of Orthopaedic Molecular Medicine, the Rita Allen Foundation, the Penn Center for Musculoskeletal Disorders, and the National Institutes of Health (NIH R01-AR41916).

Footnotes

Author Contributions: Study design: EMS and SAC Study conduct: EMS Data collection: SAC, DZ, ALC, MRC, and RJC Data analysis: SAC, ALC, MRC, FSK, and EMS Data interpretation: SAC, ACW, ADAM, FSK, and EMS Drafting manuscript: SAC and EMS Revising manuscript content: DZ, ALC, MRC, RJC, ACW, ADAM, and FSK Approving final version of manuscript: SAC, DZ, ALC, MRC, RJC, ACW, ADAM, FSK, and EMS EMS takes responsibility for the integrity of the data analysis.

Disclosure Statement: ADAM received grant support from Hologic Inc. (Bedford, MA). All the other authors state that they have no conflicts of interest.

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