Regulation of limb synovial joint organization and function

滑膜关节对于骨骼运动和功能是至关重要的，并且它们的结构、组织、独特的结构和功能是关节的重要组成部分。 组织和对疾病（包括骨关节炎）的易感性是很好理解的。然而， 它们的发育生物学仍不清楚。如果可用，这些信息和见解可用于创建 新的关节组织修复策略。在早期胚胎肢体中，骨骼原基最初是连续的， 不受关节的干扰。关节发育始于每个规定的解剖部位的出现， 间充质细胞，称为间带，是密集和压实，并表达生长和 分化因子5（Gdf 5）。在转基因小鼠中的细胞谱系追踪和追踪表明，Gdf 5 表达细胞和后代代表独特的干细胞并随时间产生大多数（如果不是全部）关节组织， 包括关节软骨、韧带和滑膜衬里。尽管这些建议和其他建议是新的和广泛相关的， 研究留下了很多不清楚的东西。值得注意的是，关节形成中最不了解的过程之一是滑膜如何形成关节。 空腔的形式，它如何可以在压实的夹层内创建，以及什么机制吸引和积累其 鉴于空洞的关键性质和重要作用，我们在知识上存在着流动性和令人惊讶的差距。以前的研究 显示关节空洞与局部透明质酸产生、蛋白酶表达和肌肉萎缩有关。 运动，所有过程都有助于细胞间接触松弛。但空洞本身是如何形成的呢 扩大？在初步研究中，我们发现，腔扩大需要活性和能量- 需要能够吸引流体并使相对的铰接表面彼此隔开的机构， 引起滑膜腔间隙。使用药理学方法，我们发现这些机制和 它们的活性实际上是空腔形成和生长所必需的。这些和其他初步数据导致 中心假设，空化是一个逐步的过程所带来的收敛和协调 不同的监管机制。我们提出了两个相互关联的目标，其中我们将进行单细胞 分析以描述相关基因和上游调控机制（目标1），并将测试 这些机制在产后关节维持、耐力以及结构和功能能力方面发挥作用（目标2）。 我们将利用不同的分析方法，包括分子遗传学，组织形态学，microCT， 单细胞RNAseq;原位杂交;组织分离;细胞培养和细胞分级分离;和成像 量化和重建。这个高风险高回报的R21项目预计将提供全新的 以及以前未被怀疑的数据和对关节空化和功能的见解。四肢关节受到以下因素的影响 目前的治疗方法仅部分有效且不持久的各种疾病。以其新颖 概念和数据，目前的项目承诺铺平道路，创造更有效和持久的 关节疾病的治疗策略。

The synovial joints are critical for skeletal motion and function, and their structure, organization, distinct tissues and susceptibility to diseases, including osteoarthritis, are well understood. However, basic aspects of their developmental biology remain unclear. If available, such information and insights could be used to create new joint tissue repair strategies. In early fetal limbs, the skeletal primordia are initially continuous and uninterrupted by joints. Joint development starts with emergence at each prescribed anatomical site of mesenchymal cells, called the interzone, that are dense and compacted and express growth-and- differentiation-factor 5 (Gdf5). Cell lineage tracing and tracking in transgenic mice showed that the Gdf5 expressing cells and progenies represent unique stem cells and produce most, if not all, joint tissues over time, including articular cartilage, ligaments and synovial lining. Though new and broadly relevant, these and other studies left much unclear. Notably, one of the least understood processes in joint formation is how the synovial cavity forms, how it can be created within the compacted interzone and what mechanisms attract and accrue its fluid, surprising gaps in knowledge given the cavity’s critical nature and essential roles. Previous studies showed that joint cavitation is associated with local hyaluronate production, protease expression and muscle motion, all processes contributing to interzone cell-cell contact relaxation. But how does the cavity itself form and enlarge? In preliminary studies, we have found that cavity enlargement requires active and energy- requiring mechanisms able to attract fluid and distance the opposing articulating surfaces from each other, eliciting a synovial cavity space. Using pharmacological approaches, we have found that these mechanisms and their activities are in fact required for cavity formation and growth. These and other preliminary data lead to the central hypothesis that cavitation is a stepwise process brought about by convergence and coordination of distinct regulatory mechanisms. We propose two interrelated Aims in which we will carry out single cell analyses to delineate genes involved and upstream regulatory mechanisms (Aim 1) and will test the roles of these mechanisms in postnatal joint maintenance, endurance and structural and functional capacities (Aim 2). We will make use of diverse analytical approaches including molecular genetics; histomorphometry; microCT; single cell RNAseq; in situ hybridization; tissue isolation; cell cultures and cell fractionation; and imaging quantification and reconstruction. This high risk-high return R21 project is expected to provide wholly novel and previously unsuspected data and insights into joint cavitation and function. Limb joints are affected by various diseases for which current treatments are only partially effective and not long-lasting. With its novel concepts and data, the present project promises to pave the way for the creation of more effective and enduring strategies for joint disease therapy.