Regulation of limb synovial joint organization and function

滑膜关节对于骨骼运动和功能至关重要，其结构、组织、独特之处 组织和对疾病（包括骨关节炎）的易感性是众所周知的。然而，基本方面 它们的发育生物学仍不清楚。如果有的话，这些信息和见解可用于创建 新的关节组织修复策略。在胎儿的早期四肢中，骨骼原基最初是连续的并且 不受关节干扰。关节发育始于每个指定解剖部位的出现 间充质细胞，称为区间细胞，致密而致密，表达生长和- 分化因子 5 (Gdf5)。转基因小鼠的细胞谱系追踪和追踪表明，Gdf5 表达细胞和后代代表独特的干细胞，并随着时间的推移产生大部分（如果不是全部）关节组织， 包括关节软骨、韧带和滑膜内层。尽管这些内容是新的且具有广泛的相关性，但 研究尚不清楚。值得注意的是，关节形成过程中最不为人所知的过程之一是滑膜如何 空腔的形式，它是如何在压实的层间产生的，以及什么机制吸引和积累它 考虑到空腔的关键性质和重要作用，知识上存在着流动的、令人惊讶的差距。之前的研究 研究表明关节空化与局部透明质酸产生、蛋白酶表达和肌肉相关 运动，所有有助于区间细胞-细胞接触松弛的过程。但空腔本身是如何形成的 并放大？在初步研究中，我们发现空腔扩大需要主动和能量- 需要能够吸引流体并使相对的关节表面彼此远离的机构， 引出滑液腔空间。使用药理学方法，我们发现这些机制和 事实上，它们的活动是空腔形成和生长所必需的。这些和其他初步数据导致 中心假设是空化是一个由收敛和协调带来的逐步过程 不同的监管机制。我们提出了两个相互关联的目标，其中我们将开展单细胞 分析以描述所涉及的基因和上游调控机制（目标 1），并将测试 这些机制涉及产后关节维护、耐力以及结构和功能能力（目标 2）。 我们将利用包括分子遗传学在内的多种分析方法；组织形态计量学；显微CT； 单细胞RNA测序；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项目有望提供全新的 以及以前未曾预料到的关于关节空化和功能的数据和见解。四肢关节受到影响 目前的治疗方法仅部分有效且不持久的各种疾病。以其小说 概念和数据，当前的项目有望为创造更有效和持久的 关节疾病治疗策略。

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.