Matrix Biology

Collagen I provides mechanical stability to organs and plays a critical role in several physiological processes such as wound healing and bone remodeling. Its ability to form fibrils depends on the proteolytic conversion of soluble procollagen I into mature protomers. However, the mechanistic aspects of this critical step have been difficult to analyze due to the lack of recombinant procollagen I suitable for structural and biochemical analyses. Here, we describe the successful production and purification of a heterotrimeric mini-procollagen I [1(I)22(I)] together with its homotrimeric counterpart [1(I)3] in HEK293-F cells. These tools were used to probe the specific role of the 2(I) chain both in procollagen I assembly and proteolytic maturation. While the presence of 2(I) in the heterotrimer does not strengthen trimer interfaces on its own, as revealed by cryo-electron microscopy, it provides a clear functional advantage to the procollagen substrate that is cleaved more efficiently by bone morphogenetic protein 1 (BMP-1) than the homotrimer. Furthermore, we demonstrate that the first cleavage event in the heterotrimer is at the level of the 2(I) chain, through a mechanism involving its C-telopeptide and a specific PRS motif close to the cleavage site. The preferential cleavage of 2(I) by BMP-1 can be partially corrected in the presence of PCPE-1 (procollagen C-proteinase enhancer 1) but the efficacy of the correction depends on its position on the procollagen trimer. Finally, we solved the high-resolution structure of the complex between the heterotrimer and PCPE-1 and proposed a new mechanism to explain the enhancing activity of PCPE-1.

Collagen biosynthesis is a complex multistep process, essential for functional tissue formation and repair. It is highly active during embryogenesis and wound healing, leading to physiological fibrogenesis, while its dysregulation induces pathologies affecting connective tissues, namely fibrosis. There are several key cellular microenvironmental factors regulating collagen biosynthesis, including vitamin C, oxygen tension and growth factors. Whilst the function of these distinct factors has previously been studied individually, how they operate together, synergistically or not, is not fully characterized. Using primary human dermal fibroblasts, we demonstrated that ascorbic acid (AA) and hypoxia both induced collagen I deposition when applied individually and had additive effects when combined together. Their effects were associated to the expected increase in collagen prolyl hydroxylation. However, AA induced a much stronger collagen secretion compared to hypoxia, while hypoxia leads to a higher collagen I hydroxylation. This observation challenged the common view of collagen prolyl hydroxylation being the central mechanism of AA-induced collagen secretion. Using proteomic analysis of the secretome, biochemical and cell imaging approaches, we further showed that increased CXCL12 extracellular levels and activation of its cognate CXCR4 receptor are at the core of AA-induced collagen intracellular trafficking and secretion. This mechanism is specific to AA and is not involved under hypoxia. The existence of distinct mechanisms underlying the effects of AA and hypoxia is further demonstrated by the different ultrastructure of collagen fibrils in the two conditions. Together, our results contribute to explain how these two triggers drive collagen biosynthesis and pave the way for potential pro- or anti-fibrotic strategies aimed at improving tissue repair.

TGF-{beta} signals through a conserved pathway yet produces diverse outcomes, pointing to regulatory mechanisms upstream of receptor engagement. A family of four latent TGF-{beta} binding proteins (LTBPs) tether pro-TGF-{beta}s and control their localization and activation, but how distinct LTBPs contribute to this regulation is unclear. Here we combine cryo-electron microscopy with functional assays to dissect LTBP-pro-TGF-{beta} interactions. We resolve the LTBP-1/pro-TGF-{beta}1 and LTBP-3/pro-TGF-{beta}3 complex structures, revealing a conserved yet plastic hydrophobic binding interface, and systematically map key residues across all four LTBP and three pro-TGF-{beta} subtypes that determine binding specificity and affinity. Unexpectedly, LTBP-2, previously thought incapable of TGF-{beta} binding, engages pro-TGF-{beta}1 through a covalent linkage via its 16th EGF-like domain, providing a mechanistic link between LTBP-2 function and TGF-{beta} signaling. Beyond tethering, LTBP-3 shifts pro-TGF-{beta}3 from spontaneous activation toward integrin dependence, establishing LTPBs as dynamic modulators that dictate not only where but also how TGF-{beta} is unleashed. Collectively, these findings establish that LTBPs are not redundant ECM scaffolds but a family of functionally distinct regulators that differentially encode TGF-{beta} spatial localization and activation, with implications for isoform-selective therapeutic strategies.

Matrix metalloproteinase 13 (MMP13) is a zinc-dependent protease that plays key roles in extracellular matrix remodeling. Like several other MMPs, MMP13 has been shown to interact with heparan sulfate (HS), a highly sulfated glycosaminoglycan found at the cell surface and in the extracellular matrix, but the significance of the interaction remains unknown. Here we report that while zymogen and mature forms of MMP13 both bind HS with high affinity, their interactions with HS display markedly different characteristics in terms of preferred HS structure and binding kinetics. By structure-guided mutagenesis, we identified a large HS-binding site of MMP13 consists of 10 residues in the hemopexin domain, 3 residues in the catalytic domain, and 2 residues in the linker region. While these basic residues participate in binding to both zymogen and mature forms of MMP13, the relative contribution of many residues differs substantially between the two forms, which likely contributes to their distinct HS-binding characteristics. Binding of HS to mature MMP13 resulted in selective inhibition of the collagenase activity of MMP13 in a length- and sulfation-dependent manner, but the binding had no effect on degradation of non-collagen substrates. Mechanistically, the inhibitory effect of HS likely results from reduced interdomain flexibility after binding of HS, and/or HS-induced dimerization of MMP13. In sum, our study establishes HS as a multifaceted regulator of MMP13 activity, and discovers that the HS-binding site of MMP13 is a novel exosite that can be targeted to inhibits its collagenase activity.

Fibrosis severity is routinely inferred from collagen abundance, although whether collagen quantity determines pathological fibrosis remains unclear. In Duchenne muscular dystrophy (DMD), chronic muscle injury and inflammation drive extracellular matrix accumulation, making these processes difficult to disentangle. We exploit sarcospan overexpression in mdx mice, a model of DMD (mdxTG), which improves membrane integrity and muscle function despite persistent matrix remodeling. mdxTG muscle accumulates more collagen than mdx yet lacks its dense macrophage-rich scars. Matrisome proteomics and spatial transcriptomics reveal compositionally and spatially distinct matrix states, while decellularized mdxTG matrix protects myotubes from membrane damage relative to mdx matrix. Despite these differences, both dystrophic matrices remain stiff and induce nuclear YAP in fibro-adipogenic progenitors. Verteporfin suppresses collagen production and reduces fibrosis in vivo, while nuclear YAP is increased in FAPs from patients with DMD. Thus, collagen abundance alone does not define pathological fibrosis; matrix organization, biological activity, and mechanosignaling distinguish functionally distinct fibrotic states.

Tendon injuries often result in fibrosis, compromising function and predisposing to re-injury. Here, we used a full-width, non-repair Achilles tendon transection model in young (3 weeks old) and adult (18-20 weeks) rats to elucidate the cellular mechanisms governing regenerative versus fibrotic healing. Functional and biomechanical analyses revealed that young tendons recovered motion and load-bearing capacity more rapidly but exhibited more fibrotic early healing. Single-nuclei RNA sequencing identified seven major cell populations within the connective tissue compartment. Adult tendons maintained a "synthetic fibroblast" population marked by upregulated ECM synthesis, reduced stress-related gene expression, whereas young tendons favored expansion of Cxcl12/Lrp6/Gas6 fibrotic fibroblasts linked to oxidative and pro-angiogenic signaling. The young group showed sustained activation of Nox4-Gas6 pathways driving a self-reinforcing fibrotic circuit. These findings define a fibroblast lineage bifurcation that dictates oxidative stress signaling as a key regulator of fibrotic remodeling, highlighting potential therapeutic targets to promote regenerative tendon repair.

PCPE-2 (procollagen C-proteinase enhancer-2) is an extracellular glycoprotein playing dual functions in the regulation of BMP-1/tolloid-like proteinases. Like its homologue PCPE-1, PCPE-2 can enhance the proteolytic maturation of fibrillar procollagens, but it also acts as a potent and specific inhibitor of BMP-1 through formation of a high-affinity complex with the protease. Here, we investigated the molecular basis of this inhibitory interaction using complementary biochemical, biophysical and structural approaches. We show that the two CUB domains of PCPE-2 must be covalently linked for efficient BMP-1 binding and inhibition, consistent with cooperative engagement of the protease. Cryo-electron microscopy combined with density-guided structural modelling and hydrogen/deuterium-exchange mass spectrometry supports a bipartite interaction complex in which the CUB1 domain of PCPE-2 engages the catalytic domain of BMP-1, while the CUB2 domains of both proteins interact together. The interaction involves the calcium-binding surfaces of the PCPE-2 CUB domains that also participate in procollagen recognition, providing a structural framework for understanding the mutually exclusive interactions of PCPE-2 with BMP-1 and procollagen. Allosteric inhibition is supported by the binding of PCPE-2 CUB1 opposite to the active site cleft, leading to the shielding of the residues surrounding the S1-prime pocket, as observed by HDX-MS. Together, these findings define the architecture of the inhibitory PCPE-2/BMP-1 complex and provide the molecular basis for understanding the distinct regulatory activities of the two procollagen C-proteinase enhancers.

Extracellular matrix (ECM) remodeling and angiogenesis are essential processes underlying endometrial decidualization during embryo implantation. Fibrillar collagens, the major structural components of the ECM, form the architectural framework of the decidua and undergo dynamic reorganization during this process. However, the functional role of type V collagen--a key regulator of collagen fibrillogenesis-- remains undefined. Here, we demonstrate that Col5a1 is highly expressed in decidual stromal and endothelial cells of the mouse uterus. Conditional deletion of Col5a1 leads to progressive uterine hemorrhage beginning at gestation day 8, culminating in complete embryo resorption and pregnancy loss by day 12. Col5a1-deficient decidua exhibits severe structural distortion, marked disorganization of fibrillar collagen, shallow and misdirected embryo invasion, and profound disruption of decidual angiogenesis and vascular network formation. Transcriptomic profiling further reveals distinct gene expression signatures and signaling pathways regulated by COL5A1 in the decidua. Collectively, these findings identify type V collagen as a critical ECM regulator required for maintaining decidual integrity, supporting angiogenesis, and ensuring successful pregnancy.

Nasal septal deviation affects approximiately 20% of newborns globally and 80% individuals in the United States. GWAS have linked PRDM16, a histone methyltransferase, to craniofacial abnormalities, yet its role in nasal septum development remains poorly understood. Global Prdm16 knockout mice (Prdm16-/-) exhibit severe craniofacial defects resembling Pierre Robin Syndrome but are neonatally lethal, precluding their potential applications for postnatal study. To address this, we generated an osteochondral lineage-specific, Prdm16 conditional knockout (Col2a1-Cre; Prdm16flox/flox; cKO) mouse model. Both sexes of cKO mice display significantly shorter nasal bone length, with a sex-dependent increase in nasal bone volume fraction of 12 wk old males. Nasal septal deviation is detectable as early as postnatal day 15 and progresses with age. Single-cell RNA sequencing (scRNA-seq) of 4 wk old nasal septal cartilage revealed a marked shift in chondrocyte composition: Mgp+ chondrocytes were substantially reduced, while Col10a1+/Serpina3n+hypertrophic chondrocytes were dramatically increased, indicating PRDM16 regulates chondrocyte phenotypes. Spatial transcriptomics localized Mgp+ chondrocytes and Col1a1high/Col3a1+fibrotic cells to the septal cartilage-bone interface (the site of deviation in cKO mice). Intercellular communication analyses revealed a switch in dominant sender cells from the fibrotic population in WT to Mgp+chondrocytes in cKO. MultiNicheNet bioinformatic analyses identified elevated TGF{beta}2 signaling at the nasal septal deviation site. Specifically, TGF{beta}2 secreted by Mgp+ chondrocytes was predicted to promote Col1a1/Col1a2 expression, resulting in fibrotic extracellular matrix (ECM) deposition and osteogenesis; consistent with elevated RUNX2 in cKO mice. TGF{beta}2 immunohistochemical staining confirmed increased TGF{beta}2 cells in the fibrous ECM and apical nasal cartilage of cKO, but not WT mice. Loss of PRDM16 also increased chondrocyte apoptosis at 4 and 12 wks of age. These findings demonstrate that loss of PRDM16 drives hypertrophic and fibrotic remodeling of nasal septal cartilage through dysregulation of TGF{beta}2 signaling, establishing a mechanistic basis for nasal septal deviation.

Sex differences in hair growth are clinically evident, but sex-dependent regulation of physiological hair cycling and injury-induced hair regeneration remains incompletely understood. We compared physiological dorsal hair-cycle progression and adhesive material-induced localized hair regeneration in male and female C3H/He mice. Males entered the second and third anagen phases earlier than females, indicating longer telogen phases in females. In contrast, localized hair regrowth after application and removal of a cyanoacrylate adhesive material appeared earlier in females. Ovariectomy induced widespread telogen-to-anagen transition and therefore did not permit isolation of ovarian-hormone effects on the localized response. RNA sequencing of intact dorsal skin identified sex-dependent baseline expression profiles involving inflammation, wound response, and tissue repair. Independent time-course quantitative PCR further demonstrated sex-dependent expression of inflammatory and reparative genes after adhesive material application. Local clodronate liposome administration alone induced delayed perifocal hair growth. When combined with adhesive material application, clodronate treatment markedly delayed wound healing and localized hair regrowth in males, whereas these responses were comparatively preserved in females. These findings show that physiological hair cycling and adhesive material-induced hair regeneration exhibit distinct sex differences and suggest that the localized regenerative response is more macrophage-dependent in males than in females.

Osteoarthritis is a leading cause of chronic pain and disability, which lacks disease-modifying treatment. Given the complex multi-tissue and multifactorial drivers behind disease progression, effective treatments will require simultaneously targeting several mechanisms underlying joint degeneration and pain. Here, we developed and evaluated a combinatorial gene therapy, consisting of a high-capacity adenoviral vector carrying two therapeutic genes to target distinct pathological mechanisms: inflammation (IL-1Ra) and chondrocyte health (PRG4). Intra-articular delivery of this treatment improved functional, structural, and pain outcomes in murine and equine osteoarthritis models. In addition, treatment normalized inflammatory environments in joint tissues, as well as in the dorsal root ganglia (DRG) known to harbor joint-innervating sensory neurons. Moreover, gene therapy reversed OA-induced molecular signatures of neural hyperexcitability, suggesting amelioration of peripheral sensitization. Collectively, these findings support combinatorial gene therapy as a promising treatment for osteoarthritis, while identifying neuroinflammatory signatures for correction of disease progression and pain. One Sentence SummaryA single intra-articular injection of a combinatorial gene therapy slows OA progression and reduces pain in small and large animal models.

The extracellular matrix (ECM) stiffness influences many physiological and pathological processes. Cells can sense mechanical changes within the ECM through mechanosensors, most notably through the focal adhesion kinase (FAK). Using human-derived data and 2D/3D engineered models, we identified a ubiquitous and uncharacterized FAK isoform lacking exon 4 (FAK{Delta}e4). We demonstrated that FAK{Delta}e4 splicing is regulated by substrate stiffness in a biphasic manner, in which an optimal stiffness is required for a maximal FAK{Delta}e4 expression. We further showed that FAK{Delta}e4 dictates at which stiffness optimal migration speed and invasion occur, impairs focal adhesion dynamics and maturation, and shifts its autophosphorylation and downstream YAP nuclear translocation toward lower stiffness compared to the canonical FAK isoform. Moreover, the FAK{Delta}e4 expression levels to canonical FAK determine at which stiffness cells will converge during durotaxis. Our results reveal how FAK{Delta}e4 acts as a fine-tuning mechanosensing switch and reframe our understanding of mechanotransduction.

Aging is a primary risk factor for osteoarthritis (OA), yet the mechanisms that preserve extracellular matrix integrity in long-lived connective tissues remain poorly defined. The function of articular cartilage depends on maintaining extracellular matrix integrity over a lifetime of mechanical use. Here we identify failure of matrix stabilization as an initiating mechanism of age-associated OA. Cartilage-specific deletion of decorin in mature mice disrupted the superficial collagen II fibrillar network, reduced aggrecan retention, and impaired poroelastic fluid pressurization by 9 months of age, preceding substantial transcriptional changes in resident chondrocytes. With advancing age, these matrix defects culminated in cartilage erosion, fibrotic remodeling, and spontaneous OA by 18 months. Mechanistically, decorin attenuated force-induced collagen II fibril realignment and reinforced the superficial fibrillar network, preserving matrix architecture under sustained physiological loading. These findings establish decorin-mediated extracellular matrix stabilization as a critical determinant of cartilage longevity and support a matrix-first model of age-associated degeneration.

Adult zebrafish efficiently regenerate skeletal muscle following different types of injury; however, the molecular programs involved in repair after extensive cryoinjury remain to be comprehensively characterized. Here, we explored the transcriptomic response of adult zebrafish skeletal muscle at 7 days post cryoinjury (dpci), a stage marked by ongoing tissue clearance, progenitor expansion, myogenic differentiation, and nascent myofiber formation, and compared it with phase-matched stab wound injury. Cryoinjury induced a broad transcriptional response, with 5,330 differentially expressed genes. Integrated enrichment and protein-protein interaction analyses revealed that, at 7 dpci, zebrafish skeletal muscle functions as an integrated regenerative system in which immune remodeling, progenitor expansion, myogenic differentiation, extracellular matrix reconstruction, mechanotransduction, biosynthetic adaptation, proteostasis, and intracellular trafficking operate simultaneously. In parallel, mature sarcomeric and oxidative metabolic programs were suppressed, consistent with ongoing tissue reconstruction and structural immaturity. Comparison with stab-wounded skeletal muscle revealed substantial transcriptional conservation, as 612 of 717 stab-wound-responsive genes (85%) were also differentially expressed after cryoinjury. Shared upregulated genes formed coherent functional modules related to proliferation, extracellular matrix organization and signaling, immune regulation, muscle differentiation, and protein processing. Thus, distinct injury modalities converge on a common regenerative program, while cryoinjury elicits a quantitatively broader transcriptional response. These findings support a conserved regenerative architecture of adult zebrafish skeletal muscle repair, in which interconnected biological modules act coordinately, with the breadth of transcriptional engagement reflecting regenerative demand.

We analysed five predefined programmes across three human tendon transcriptomic contexts: acute exercise-associated fibroblast pseudobulks (E-MTAB-15400; 4 control and 4 exercise samples), mechanically stretched donor-derived cells (GSE150482; 3 normal and 3 tendinopathy donors), and paired lesional versus grossly normal-appearing tendon (GSE26051; 23 donor pairs). Inference was conducted at the sample, donor or donor-pair level. Non-stimulated GSE150482 single-cell RNA sequencing comprised only N1 and D1, whereas stretched single-cell RNA sequencing comprised N1-N3 and D1-D3; the conditions also used Drop-seq and 10X Chromium, respectively. A donor-replicated disease-by-stimulation interaction is therefore not estimable. Within-context Benjamini-Hochberg adjustment supported acute mechanical-response, extracellular-matrix (ECM) and protein-folding differences and paired-lesion collagen, integrin and ECM differences, but no stretched donor-background difference. Exact label permutations, a conservative global 15-test adjustment, leave-one-unit analyses, a Fib1-Fib4 pooled sensitivity and a paired exact Wilcoxon sensitivity qualified these findings. Integrin was fully nested in ECM, while collagen overlapped 52/79 genes with ECM. Collagen fibril organization showed the most directionally consistent matrix-associated pattern across the three non-equivalent contrasts, without establishing a disease-specific loading response or causal mechanism. The analysis demonstrates why public tendon datasets must be integrated by estimand and biological inference unit rather than pooled as interchangeable replications.

Desmin is the major intermediate filament protein of muscle, yet its potential nuclear functions remain poorly understood. Here, we investigated the nuclear localization and organization of endogenous desmin in human skeletal muscle cells using quantitative confocal microscopy, biochemical fractionation, targeted MRM/SRM proteomics, and computational structural modeling. Desmin localized to the nuclear compartment and exhibited significantly stronger colocalization with emerin and lamin A/C than with lamin B1. Nuclear fractionation and targeted proteomics independently confirmed endogenous nuclear desmin, while protein-protein docking predicted structurally plausible interactions with all three nuclear envelope proteins, supporting a context-dependent nuclear interaction network. Together, these findings identify the nuclear envelope as a functional platform for desmin and suggest that nuclear desmin contributes to nucleo-cytoskeletal communication and the organization of mechanically responsive nuclear architecture during myogenesis. This work expands the current understanding of desmin biology and provides a framework for investigating its role in desmin-related muscle disease.

Skin, the bodys largest mechanosensitive organ, relies on a tension gradient across epidermal layers to maintain structure and function, but how mechanical force contributes to epidermal development and disease pathogenesis is poorly understood. By anchoring intermediate filaments (IF) to the plasma membrane, desmosomes, the most abundant intercellular junctions in the epidermis, help create a supracellular scaffolding that provides mechanical resilience to the tissue. However, the contribution of the desmosome-IF network to the epidermal response to mechanical strain remains unknown. Here we show that the desmosome-IF connection is not only required to induce a proper cellular mechano-response but is actively strengthened in response to stretch through the PP2A-mediated phospho-regulation of the cytoskeletal linker protein desmoplakin (DP). Additionally, we show in human skin dephosphorylated DP localizes to high tension layers, suggesting this mechano-response mechanism is coordinated with the epidermal tension gradient. Furthermore, in models of Carvajal syndrome, a cardio-cutaneous disorder caused by truncating DP mutations, cells lose mechano-responsive behavior and exhibit abnormal morphology in high-tension epidermal layers. Together, these findings identify the DP-IF network as a key component of the response to mechanical strain and show that its disruption compromises epidermal homeostasis and contributes to disease pathogenesis.

Autosomal dominant gain-of-function mutations in platelet-derived growth factor receptor beta (PDGFRb) cause overgrowth of the skeleton and other connective tissue in Kosaki overgrowth syndrome. However, the target cell type and signaling pathways underlying PDGFRb-driven overgrowth are unknown. Normal postnatal growth is controlled by pituitary-secreted growth hormone (GH), which activates the STAT5 transcriptional factor to upregulate insulin-like growth factor 1 (IGF1). To investigate the role of the GH-STAT5-IGF1 pathway in PDGFRb-related overgrowth, we generated mice with a PDGFRb gain-of-function mutation in skeletal and fibroblast lineages, which resulted in STAT5 activation and gigantism. Conditional deletion of Stat5ab in connective tissue lineages rescued skeletal overgrowth and keloid-like fibrosis in the skin. Conditional deletion of GH receptor (Ghr) did not rescue overgrowth, indicating the physiological activator of STAT5 is not required for overgrowth. However, deletion of Igf1, the STAT5 target gene, and its receptor, Igf1r, in connective tissue, rescued the overgrowth phenotype. These findings demonstrate a GHR-independent STAT5-IGF1 signaling pathway in mutant connective tissue cells, which mediates PDGFRb-driven overgrowth in mice and potentially in humans with similar PDGFRB mutations.

Background: Pressure ulcers often develop complex three-dimensional morphologies that extend beyond the visible wound surface. Subsurface extensions such as tunneling and undermining create hidden cavities that complicate clinical assessment and wound management. Despite their clinical relevance, the prevalence and spatial characteristics of these subsurface wound morphologies have not been well characterized at scale. Methods: We performed a registry-based analysis using data from the LIFT-OFF Pressure Ulcer Registry, which captures longitudinal clinical documentation of pressure ulcers treated in routine care. The registry included approximately 18,000 patients with 32,000 documented pressure ulcers. Spatial characteristics of tunneling and undermining were analyzed using measurements recorded during routine wound assessments, including tract length, direction, and circumferential extent. Directional and circumferential distributions of subsurface defects were examined to characterize wound geometry. Results: Tunneling was present in 764 of 14,700 full-thickness pressure ulcers (5.2%), whereas undermining occurred in 2,293 wounds (15.6%). Tunneling tracts were typically short and exhibited directional clustering relative to the wound bed. In contrast, undermining demonstrated broader circumferential distributions and frequently involved larger subsurface separations beneath the wound margin. Both morphologies demonstrated distinct spatial patterns across anatomical locations and wound stages. Conclusion: Tunneling and undermining are common subsurface features of pressure ulcers and exhibit distinct spatial geometries. Whereas tunneling manifests as directional tract-like extensions, undermining more frequently produces circumferential tissue separation beneath wound margins. Improved characterization of subsurface wound architecture may enhance assessment of wound complexity and provide information not captured by surface measurements alone. Future studies should evaluate whether these features contribute to wound severity assessment, prognosis, and risk stratification.

Individuals with fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder caused by mutations in the bone morphogenetic protein receptor ACVR1 (also known as ALK2), experience progressive and severely debilitating endochondral heterotopic ossification (HO). Previous studies have identified fibro-adipogenic progenitors (FAPs) as a major cellular source of HO in FOP mouse models. Here, we used mRNA sequencing of FAPs isolated from injured muscle of FOP mice to capture the earliest gene expression changes associated with the transformation of muscle tissues into bone. Whereas the transcriptomes of mutant and wild-type FAPs were indistinguishable prior to injury and at 1 day post injury, dramatic dysregulation of gene expression was observed at 3 and 5 days after injury. Surprisingly, gene expression changes suggested early specification of osteochondral lineages by 3 days post injury, approximately 3 to 4 days before the appearance of histologically identifiable cartilage. We identified dysregulation of genes encoding components of major signaling pathways, cell cycle regulators, extracellular matrix components, and hypoxia response mediators, and showed that these gene expression changes are temporally associated with abnormal population growth of mutant FAPs. We further demonstrated that treatment of FOP mice with either an anti-activin A monoclonal antibody or the ACVR1 kinase inhibitor BLU-782 (IPN60130; fidrisertib) normalized the expression of the majority of dysregulated FAP genes. However, the relative efficacy of the drugs differed, and distinct sets of overlapping genes were normalized by the treatments. The substantial transcriptional changes caused by these drugs in FAPs from normally regenerating muscle were also enumerated and described. These findings define early transcriptional events associated with pathogenic FAP reprogramming and provide a molecular framework for understanding the shared and distinct effects of activin A and ACVR1 inhibition.