YAP regulates periosteal expansion in early fracture repair.
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ABSTRACT: Periosteal expansion is a key process in the early stages of bone fracture repair. The periosteum is typically quiescent, but upon fracture it expands, periosteal cells proliferate and contribute to the formation of a cartilaginous callus . The early expansion of the periosteum is tightly regulated at the transcriptional level. However, the molecular mechanisms behind periosteal expansion are unknown. Here, we show that Yes-Associated Protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) mediate periosteal expansion and periosteal cell proliferation. Bone fracture results in increased numbers of YAP-expressing periosteal cells. Deletion of YAP from Osterix (Osx) expressing periosteal cells impairs early periosteal expansion. Mechanistically, YAP regulates both cell intrinsic and extrinsic transcriptional programs that allow for periosteal expansion. Specifically, we identified Bmp4 as a cell extrinsic factor regulated by YAP, that rescues the impairment of periosteal expansion upon YAP deletion. Together, these data establish YAP mediated molecular mechanisms that allow for periosteal expansion in the early stages of fracture repair.
Project description:Fracture healing is a process that involves many cell populations. In this study we characterized gene expression in a subset of cells involved in fracture healing. αSMACreERT2 mice crossed with Ai9 reporter mice that express tdTomato fluorescent protein after Cre-mediated activation were used as an experimental model. αSMA-expressing cells were labeled by tamoxifen administration, then periosteal cells from the tibia were isolated two days later (controls), or tibial fractures were performed and periosteum/soft callus tissue was collected after 2 and 6 days. The tdTomato positive cell population was isolated by flow cytometry, and subjected to microarray analysis. Histology and cell surface marker analysis indicates that αSMACreERT2 labels a mainly mesenchymal population in the periosteum that expands after fracture, and contributes to both osteogenic and chondrogenic elements of the fracture callus. We were therefore able to examine gene expression in a defined population during the early stages of fracture healing. Total RNA was obtained from the tomato positive cells within the periosteal compartment of fractures from αSMACreERT2/Ai9 mice. Control animals were given 2 doses of tamoxifen, and periosteum was collected and labeled cells sorted (8-9 sex-matched mice per group). Fractures were performed after the second dose of tamoxifen, and tomato positive cells from periosteum/callus tissue were isolated 2 and 6 days after fracture (4-8 animals per sample pooled). 3 replicates for each sample are included.
Project description:Periosteum is a major source of skeletal stem/progenitor cells (SSPCs) during bone repair. However, the cellular composition of the periosteum is poorly characterized. Here, we provide single-cell RNAseq data of periosteal cells isolated by explant culture at steady state and at day 3 post-tibial fracture. We found that periosteal cell populations are heterogeneous containing several sub-populations of SSPCs. Upon fracture, SSPCs are activated by leaving their mesenchymal state, and engaging into fibrogenesis prior to chondrogenesis.
Project description:The outer coverings of the skeleton, or periosteum, are arranged in concentric layers and act as a reservoir for tissue specific bone progenitors. Cellular heterogeneity within this tissue depot is increasingly recognized. Here, Pdgfra reporter animals were used to highlight a subset of periosteal progenitor cells with high osteogenic potential. Pdgfra+ periosteal progenitor cells enhanced osteogenic differentiation in vitro and improved fracture healing and bone regeneration in vivo. Depletion of Pdgfra-expressing progenitor cells interferes with cortical appositional bone, periosteal bone formation in response to mechanical load, and during fracture repair. Pdgfra+ periosteal progenitors give rise to Nestin+ periosteal cells overtime, after fracture and upon transplantation.
Project description:Bone regeneration is a highly efficient process allowing scarless healing after injury. The periosteum, the outer layer of bones, is a critical source of skeletal stem/progenitor cells (SSPCs), as well as immune, endothelial and neural cells during bone repair. In our study, we generated a single-nuclei atlas of the murine periosteal response to bone fracture. We generated single nuclei datasets from uninjured periosteum and from injured periosteum and hematoma/fracture callus at days 5 and 7 post-injury from wild-type mice.
Project description:Fracture healing is a process that involves many cell populations. In this study we characterized gene expression in a subset of cells involved in fracture healing. αSMACreERT2 mice crossed with Ai9 reporter mice that express tdTomato fluorescent protein after Cre-mediated activation were used as an experimental model. αSMA-expressing cells were labeled by tamoxifen administration, then periosteal cells from the tibia were isolated two days later (controls), or tibial fractures were performed and periosteum/soft callus tissue was collected after 2 and 6 days. The tdTomato positive cell population was isolated by flow cytometry, and subjected to microarray analysis. Histology and cell surface marker analysis indicates that αSMACreERT2 labels a mainly mesenchymal population in the periosteum that expands after fracture, and contributes to both osteogenic and chondrogenic elements of the fracture callus. We were therefore able to examine gene expression in a defined population during the early stages of fracture healing.
Project description:Bone regeneration relies on the activation of skeletal stem cells (SSCs) that still remain poorly characterized. Here, we show that periosteum contains SSCs with high bone regenerative potential compared to bone marrow stromal cells/skeletal stem cells (BMSCs) in mice. Although periosteal cells (PCs) and BMSCs are derived from a common embryonic mesenchymal lineage, post-natally PCs exhibit greater clonogenicity, growth and differentiation capacity than BMSCs. During bone repair, PCs can efficiently contribute to cartilage and bone, and integrate long-term after transplantation. Molecular profiling uncovers genes encoding Periostin and other extracellular matrix molecules associated with the enhanced response to injury of PCs. Periostin gene deletion impairs PC functions and fracture consolidation. Periostin-deficient periosteum cannot reconstitute a pool of PCs after injury demonstrating the presence of SSCs within periosteum and the requirement of Periostin in maintaining this pool. Overall our results highlight the importance of analyzing periosteum and PCs to understand bone phenotypes.
Project description:We present a transcriptomic analysis that provides a better understanding of regulatory mechanisms within the healthy and injured periosteum leading to novel translational approaches for bone healing. The focus of this work is on the early regulatory control of bone healing by completing a transcriptomic analysis of forming periosteal callus cells on day 3 post fracture. Based on our previous work indicating that induced Notch1 signaling in osteoprogenitors leads to better healing, we contrasted samples in which Notch 1 intracellular domain (NICD1) is overexpressed by periosteal stem/progenitor cells with control unperturbed periosteum. We determined molecular mechanisms and changes in skeletal stem/progenitor cells (SSPC) and other cell populations within callus including hematopoietic lineages. Notch ligands were differentially expressed in endothelial and mesenchymal populations, with Dll4 restricted to endothelial cells while Jag1 expressed by various mesenchymal populations. When targeting deletion of Dll4 in SSPCs using a-smooth muscle actin (aSMACreER) in mesenchymal cells there was no phenotype, while deletion in EC using Cdh5CreER exhibited negative effects on the early fracture healing. Translation of these observations into clinically relevant model of bone healing revealed the positive effects of combination of Notch ligands delivery with currently used osteogenic inducer BMP2.
Project description:The periosteum contains cells which function as a reservoir of stem cells and progenitors and contribute to cortical expansion during growth, cortical bone homeostasis and repair. However, the local or paracrine factors that govern stem cell renewal and differentiation within the periosteal niche remains elusive. Cathepsin K (Ctsk) together with additional cell surface markers marks a subset of stem cells in the periosteum (PSC) which possess self-renewal ability and inducible multipotency. These PSCs produce osteoblasts mediating periosteal bone formation and fracture repair. Sfrp4 is expressed in periosteal Ctsk-lineage cells and using CtskCre mice that are either wild type or Sfrp4-/-, we report here that Sfrp4 deletion decreases the pool of PSCs, impairs their self-renewal, their ability to give rise to their derivatives and their clonal multipotency for differentiation into osteoblasts and chondrocytes in vitro and formation of bone organoids in vivo. Bulk RNA sequencing analysis in Ctsk-lineage PSCs demonstrated that Sfrp4 deletion leads to downregulation of signaling pathways associated with skeletal development, positive regulation of bone mineralization and wound healing. Sfrp4 deletion hampers the Ctsk-lineage PSC response and recruitment after bone injury and leads to an impaired periosteal response. Periosteal Ctsk-lineage cells respond to PTH(1-34) treatment with an increase in the % of PSCs, a response not seen in the absence of Sfrp4. Importantly, bone histomorphometry analysis showed that in the absence of Sfrp4, PTH(1-34)-dependent increase in cortical thickness, periosteal bone formation is markedly impaired.
Project description:Congenital pseudarthrosis of the tibia (CPT) is a severe pediatric disorder affecting children. CPT is characterized by tibial bowing at birth leading to spontaneous fracture and fibrous non-union. Our study showed the presence of biallelic inactivation of the NF1 gene in the periosteum and in periosteal skeletal/stem progenitor cells (pSSPCs) at the pseudarthrosis site.