The vertebrate skeleton forms predominantly by endochondral ossification (EO), where the cartilaginous model of the axial and appendicular skeleton, as well as of certain cranial bones, is replaced by bony trabeculae and marrow. The distinctive feature of this process is comprised of hypertrophic cartilage where EO initiates and collagen X is predominant.1 Emergence of hypertrophic cartilage defines each skeletal element where marrow forms. Since the marrow provides niches for blood cell differentiation, alterations in the cartilage-to-bone and marrow transition of EO may affect stromal and hematopoietic constituents. We demonstrate here that mice transgenic (Tg) for collagen X develop both skeletal and hematopoietic abnormalities, and that the latter likely arise as a consequence of disrupted collagen X function. These data reveal an unforeseen link between endochondral skeletogenesis and establishment of the marrow microenvironment prerequisite for hematopoiesis.During embryogenesis, EO initiates in cartilage with hypertrophy, and progresses by transforming a preexisting non-calcified avascular cartilage to a calcifiable one permissive to vascularization.

The link between endochondral skeletal development and hematopoiesis in the marrow was established in the collagen X transgenic (Tg) and null (KO) mice. Disrupted function of collagen X, a major hypertrophic cartilage matrix protein, resulted in skeletal and hematopoietic defects in endochondrally derived tissues. Manifestation of the disease phenotype was variable, ranging from perinatal lethality in a subset of mice, to altered lymphopoiesis and impaired immunity in the surviving mice. To exclude contribution of strain specific modifiers to this variable manifestation of the skeleto-hematopoietic phenotype, C57Bl/6 and DBA/2J collagen X congenic lines were established. Comparable disease manifestations confirmed that the skeleto-hematopoietic alterations are an inherent outcome of disrupted collagen X function. Further, colony forming cell assays, complete blood count analysis, serum antibody ELISA, and organ outgrowth studies established altered lymphopoiesis in all collagen X Tg and KO mice and implicated opportunistic infection as a contributor to the severe disease phenotype. These data support a model where endochondral ossification-specific collagen X contributes to the establishment of a hematopoietic niche at the chondro-osseous junction.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease in which extraskeletal (heterotopic) bone forms within tissues such as skeletal muscles, often in response to injury. Mutations in the BMP type I receptor ACVR1/ALK2 cause FOP by increasing BMP pathway signaling. In contrast to the growing understanding of the inappropriate formation of bone tissue within the muscle in FOP, much is still unknown about the regenerative capacity of adult diseased muscles. Utilizing an inducible ACVR1R206H knock-in mouse, we found that injured Acvr1R206H/+ skeletal muscle tissue regenerates poorly. We demonstrated that while two resident stem cell populations, muscle stem cells (MuSCs) and fibro/adipogenic progenitors (FAPs), have similar proliferation rates after injury, the differentiation potential of mutant MuSCs is compromised. Although MuSC-specific deletion of the ACVR1R206H mutation does not alter the regenerative potential of skeletal muscles in vivo, Acvr1R206H/+ MuSCs form underdeveloped fibers that fail to fuse in vitro. We further determined that FAPs from Acvr1R206H/+ mice repress the MuSC-mediated formation of Acvr1R206H/+ myotubes in vitro. These results identify a previously unrecognized role for ACVR1R206H in myogenesis in FOP, via improper interaction of tissue-resident stem cells during skeletal muscle regeneration.

The collagen X transgenic and null (ColX-Tg/KO) mice have revealed a link between endochondral ossification (EO) and hematopoiesis, and thus serve as model systems to study hematopoietic niches. The altered collagen X function in ColX-Tg/KO mice resulted not only in skeletal defects, which included changes in growth plate ultrastructure, altered localization of heparan sulfate proteoglycans (HSPG), and reduced trabecular bone, but also in hematopoietic defects, which included reduced B lymphocyte numbers throughout life without associated increases in B cell apoptosis. Consequently, the ColX-Tg/KO mice exhibited diminished in vitro and in vivo immune responses. Moreover, reduced expression of several hematopoietic and B lymphopoietic cytokines were measured from ColX-KO-derived hypertrophic chondrocyte and trabecular osteoblast cultures. Together, these data expand the current hematopoietic niche model by including the EO-derived extracellular matrix, for example, the collagen X/HSPG network, as well as the EO-derived hypertrophic chondrocytes and trabecular osteoblasts as hematopoietic signal mediating cells.

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