Project description:Distinctions between craniofacial and axial muscles exist from the onset of development and throughout adulthood. The masticatory muscles are a specialized group of craniofacial muscles that retain embryonic fiber properties throughout adulthood, suggesting that the developmental origin of these muscles may govern a pattern of expression that differs from limb muscles. To determine the extent of these differences, expression profiling of total RNA isolated from the masseter and tibialis anterior (TA) muscles of adult female mice was performed, which identified transcriptional changes in unanticipated functional classes of genes in addition to those associated with fiber type. In particular, the masseters displayed a reduction of transcripts associated with load-sensing and anabolic processes, and heightened expression of genes associated with stress. Consistent with these observations were a significantly smaller fiber cross-sectional area in masseters, significantly elevated load-sensing signaling (phosphorylated Focal Adhesion Kinase (FAK)), and increased apoptotic index in masseters compared to TA muscles. Based on these results, we hypothesize that masticatory muscles may sense and respond to load differently than limb muscles, where the drive for anabolic processes is reduced, and cell stress mediated processes are enhanced. These results establish a novel classification for the masseter muscle in the spectrum of skeletal muscle allotypes, and may provide insight into the molecular basis for specific muscle-related pathologies associated with masticatory muscles. Keywords: skeletal muscle, developmental origin, craniofacial muscles
Project description:Fibroblasts are the most common cell type found in connective tissues, known to play major roles in development, homeostasis, regeneration and disease. Although specific fibroblast subpopulations were associated with different biological processes, the mechanisms and unique activities underlying their diversity has not been thoroughly examined. Turning to skeletal muscle development, we set to dissect the variation of muscle-resident fibroblasts (mrFibroblasts). Our results demonstrate mrFibroblasts diversify following the transition from embryonic to fetal myogenesis prior to birth. We find mrFibroblast segregate into two major subpopulations occupying distinct niches, with interstitial fibroblasts residing between the muscle fibers, and delineating fibroblasts sheathing the muscle mass. We further show these subpopulations entail distinct cellular dynamics and transcriptomes. Notably, we find mrFibroblast subpopulations exert distinct regulatory roles on myoblast proliferation and differentiation. Finally, we demonstrate this diversification depends on muscle contraction. Altogether, these findings establish mrFibroblast diversify in a spatio-temporal embryonic process into distinct cell types, entailing different characteristics and roles.
Project description:Distinctions between craniofacial and axial muscles exist from the onset of development and throughout adulthood. The masticatory muscles are a specialized group of craniofacial muscles that retain embryonic fiber properties throughout adulthood, suggesting that the developmental origin of these muscles may govern a pattern of expression that differs from limb muscles. To determine the extent of these differences, expression profiling of total RNA isolated from the masseter and tibialis anterior (TA) muscles of adult female mice was performed, which identified transcriptional changes in unanticipated functional classes of genes in addition to those associated with fiber type. In particular, the masseters displayed a reduction of transcripts associated with load-sensing and anabolic processes, and heightened expression of genes associated with stress. Consistent with these observations were a significantly smaller fiber cross-sectional area in masseters, significantly elevated load-sensing signaling (phosphorylated Focal Adhesion Kinase (FAK)), and increased apoptotic index in masseters compared to TA muscles. Based on these results, we hypothesize that masticatory muscles may sense and respond to load differently than limb muscles, where the drive for anabolic processes is reduced, and cell stress mediated processes are enhanced. These results establish a novel classification for the masseter muscle in the spectrum of skeletal muscle allotypes, and may provide insight into the molecular basis for specific muscle-related pathologies associated with masticatory muscles. Experiment Overall Design: Tissues were isolated from normal adult female mice (C57Bl/6), age 6 months. Paired comparisons between masseter and tibialis anterior muscles were performed on all present genes using "Significance Analysis of Microarrays" (SAM) to identify differentially expressed genes between masticatory and axial muscles.
Project description:Mapping the transcriptional landscape of human embryonic skeletogenesis at single-cell resolution during limb bud and primary ossification center (POC) formation. We found significant heterogeneity of stromal cells within the limb bud mesenchyme that specified proximal-distal and anterior-posterior patterning. Embryonic skeletal stem and progenitor cells first appeared during POC formation, which were highly enriched by CADM1 expression and could differentiate into osteoblasts, chondrocytes and periosteal mesenchymal stromal cells.
Project description:Our aim is to analyze how does the lack of each of the Ca2+ channels, involved in excitation-contraction coupling in skeletal muscle - RYR1 and Cav1.1, affect gene expression in embryonic mouse limb skeletal muscle during secondary myogenesis. We extracted total RNA from the limb skeletal muscle of WT, heterozygous RYR1-/+ and Cav1.1+/-, and homozygous RYR1-/- and Cav1.1-/- mutants from 3 litters (n = 3 for each group) at days E14.5 and E18.5 and subjected it to microarray analyses.
Project description:Despite recent advances in pluripotent stem cell-based approaches to induce skeletal cells, recapitulating human limb skeletal development in terms of structure and longitudinally oriented growth remains an unresolved challenge. Here, we report a method to differentiate human pluripotent stem cells into region-specific skeletal organoids harboring GDF5+PRG4+ interzone/articular chondrocyte progenitors (IZ/ACPs) and SP7+ growth plate chondrocytes (GPCs) via PRRX1⁺ limb-bud mesenchymal cells. Comparative analysis demonstrated marked similarities of IZ/ACP and GPC organoids to the human embryonic limb, and graft fate and regenerative capacity in vivo were further characterized. We also mimicked the limb skeletal developmental process in a spatially structured manner by vertically positioning two IZ/ACP organoids at both ends of a GPC organoid to generate a human skeletal assembloid. Notably, this human skeletal assembloid recapitulated endochondral ossification with longitudinal skeletal growth upon transplantation. In summary, our study provides a novel research platform for human limb skeletal development and disease.
Project description:Despite recent advances in pluripotent stem cell-based approaches to induce skeletal cells, recapitulating human limb skeletal development in terms of structure and longitudinally oriented growth remains an unresolved challenge. Here, we report a method to differentiate human pluripotent stem cells into region-specific skeletal organoids harboring GDF5+PRG4+ interzone/articular chondrocyte progenitors (IZ/ACPs) and SP7+ growth plate chondrocytes (GPCs) via PRRX1⁺ limb-bud mesenchymal cells. Comparative analysis demonstrated marked similarities of IZ/ACP and GPC organoids to the human embryonic limb, and graft fate and regenerative capacity in vivo were further characterized. We also mimicked the limb skeletal developmental process in a spatially structured manner by vertically positioning two IZ/ACP organoids at both ends of a GPC organoid to generate a human skeletal assembloid. Notably, this human skeletal assembloid recapitulated endochondral ossification with longitudinal skeletal growth upon transplantation. In summary, our study provides a novel research platform for human limb skeletal development and disease.
Project description:Despite recent advances in pluripotent stem cell-based approaches to induce skeletal cells, recapitulating human limb skeletal development in terms of structure and longitudinally oriented growth remains an unresolved challenge. Here, we report a method to differentiate human pluripotent stem cells into region-specific skeletal organoids harboring GDF5+PRG4+ interzone/articular chondrocyte progenitors (IZ/ACPs) and SP7+ growth plate chondrocytes (GPCs) via PRRX1⁺ limb-bud mesenchymal cells. Comparative analysis demonstrated marked similarities of IZ/ACP and GPC organoids to the human embryonic limb, and graft fate and regenerative capacity in vivo were further characterized. We also mimicked the limb skeletal developmental process in a spatially structured manner by vertically positioning two IZ/ACP organoids at both ends of a GPC organoid to generate a human skeletal assembloid. Notably, this human skeletal assembloid recapitulated endochondral ossification with longitudinal skeletal growth upon transplantation. In summary, our study provides a novel research platform for human limb skeletal development and disease.
Project description:Despite recent advances in pluripotent stem cell-based approaches to induce skeletal cells, recapitulating human limb skeletal development in terms of structure and longitudinally oriented growth remains an unresolved challenge. Here, we report a method to differentiate human pluripotent stem cells into region-specific skeletal organoids harboring GDF5+PRG4+ interzone/articular chondrocyte progenitors (IZ/ACPs) and SP7+ growth plate chondrocytes (GPCs) via PRRX1⁺ limb-bud mesenchymal cells. Comparative analysis demonstrated marked similarities of IZ/ACP and GPC organoids to the human embryonic limb, and graft fate and regenerative capacity in vivo were further characterized. We also mimicked the limb skeletal developmental process in a spatially structured manner by vertically positioning two IZ/ACP organoids at both ends of a GPC organoid to generate a human skeletal assembloid. Notably, this human skeletal assembloid recapitulated endochondral ossification with longitudinal skeletal growth upon transplantation. In summary, our study provides a novel research platform for human limb skeletal development and disease.