Molecular control of chondrocyte hypertrophy: an evolutionary approach

项目摘要 在哺乳动物中，软骨主要是胚胎组织：绝大多数软骨被骨取代 在肥大和骨化的过程中，软骨在骨内相对较少的地方持续存在， 成年人的骨骼（如关节中的关节软骨）。关节软骨是一种无神经、无血管的组织， 有限的自发修复能力，因此在人类中普遍存在软骨病变， 骨关节炎相反，软骨鱼类（鲨鱼、冰鞋和鳐）经历了一次进化损失， 骨，而不是拥有一个完全由前肥大软骨组成的骨骼， 终生保持软骨。了解软骨鱼类如何阻止骨骼发育 软骨细胞肥大将揭示新的转录特征，可用于体外 用于治疗人软骨损伤的稳定软骨细胞的工程化。 用于治疗关节软骨损伤的基于干细胞的方法目前受到以下因素的阻碍： 哺乳动物软骨细胞（软骨细胞）在体外和植入损伤部位后的相对不稳定性。 在本项目中，我们将研究小鳐软骨的分子发育， 发现与其永久性软骨骨骼相关的基因表达。这个项目将开始与 使用单细胞RNA测序来鉴定在细胞间差异表达的基因， 发育和分化的溜冰鞋和哺乳动物软骨细胞（目标1），这可能是基础逮捕 在前一个肥大之前。ATAC-seq和比较基因组方法将用于 检测非编码区（即推定的增强子）中可能导致不同基因的变异 在滑冰和哺乳动物骨骼发育过程中的表达（Aim 2）。最后，受滑板启发的分子 将在体外将CRISPR/Cas9基因组编辑和/或转基因操作（CRISPR/Cas9基因组编辑和/或转基因）并入到细胞中。 哺乳动物骨骼发生模型，以获得永久和稳定的软骨细胞状态 来自哺乳动物间充质祖细胞（Aim 3）。 这个项目将利用冰鞋骨架的独特特性，以及生物资源， 伍兹霍尔海洋生物实验室的设施和专业知识。通过采取 进化启发的组织工程方法，该项目将有助于开发新的体外 用于软骨工程和用于治疗哺乳动物骨骼病变的技术。

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.