Friday, November 22, 2024
11/22/2024
Project Summary In mammals, cartilage is predominantly an embryonic tissue: the vast majority of cartilage is replaced by bone during the processes of hypertrophy and ossification, with cartilage persisting in relatively few places within the adult skeleton (e.g. in joints, as articular cartilage). Articular cartilage is an aneural, avascular tissue with very limited capacity for spontaneous repair, hence the prevalence in humans of cartilage pathologies like osteoarthritis. In contrast, cartilaginous fishes (sharks, skates, and rays) have undergone an evolutionary loss of bone, instead possessing a skeleton that is composed entirely of pre-hypertrophic cartilage, and that remains cartilaginous throughout life. Understanding how cartilaginous fishes arrest skeletal development prior to chondrocyte hypertrophy will shed new light on transcriptional features that could be employed for in vitro engineering of stable chondrocytes for the treatment of human cartilage injuries. Stem cell-based approaches for the treatment of articular cartilage injuries are currently hindered by the relative instability of mammalian cartilage cells (chondrocytes) in vitro and upon implantation into an injury site. In this project, the molecular development of cartilage in the little skate (Leucoraja erinacea) will be studied to discover gene expression correlates of their permanent cartilaginous skeleton. This project will begin with the use of single-cell RNA-sequencing to identify genes that are differentially expressed between developing and differentiated skate and mammalian chondrocytes (Aim 1), and that may underlie arrest prior to hypertrophy in the former. ATAC-seq and comparative genomic approaches will then be used to test for variation in non-coding regions (i.e. putative enhancers) that might account for divergent gene expression during skate and mammalian skeletogeneis (Aim 2). Finally, skate-inspired molecular manipulations (CRISPR/Cas9 genome editing and/or transgenesis) will be incorporated in an in vitro model of mammalian skeletogenesis, in order to achieve a permanent and stable chondrocyte cell state from mammalian mesenchymal progenitors (Aim 3). This project will capitalize on the unique properties of the skate skeleton, and on the biological resources, facilities, and expertise that are available at the Marine Biological Laboratory in Woods Hole. By taking an evolution-inspired tissue engineering approach, this project will contribute to the development of novel in vitro techniques for cartilage engineering and for the treatment of mammalian skeletal pathologies.
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