Cellular and molecular mechanisms regulating synovial joint development

项目概要和摘要 滑膜关节对于身体运动和生活质量至关重要。它们的滑液腔和富含润滑剂的液体 允许不受阻碍的关节运动和功能，并提供组织保护和营养。虽然这些 滑膜关节生物学的各个方面都已广为人知，但对于腔及其液体实际上是如何运作的却知之甚少 在胚胎发生过程中发育。在胎儿早期阶段，肢体骨骼原基由连续的 没有关节的软骨结构。关节发育始于“间带”的出现，即组织 由表达生长和分化因子 5 基因 (Gdf5) 的间充质细胞组成。我们之前 表明 Gdf5 细胞后代随着时间的推移产生大多数关节组织，并且滑液腔在 中间地带。由于带间细胞最初是相互附着的，空化过程 必须涉及它们沿着预期关节线的物理分离，以促进创建充满液体的 空腔。先前的研究表明，区间细胞在空化时间前后产生透明质酸（HA），并且 这伴随着局部组织中富含 HA 的基质的积累。 HA的主要组成部分 细胞外基质和滑液，在组织稳态中发挥重要作用。在我的初步 研究中，我发现在空化发生之前，小鼠胚胎四肢中的区间细胞表达透明质酸 合酶 2（HAS2，“HA 合成器”）和跨膜蛋白 2 (TMEM2)，一种细胞表面透明质酸酶 特异性地将高分子量 HA 裂解成中间片段和生物活性片段。我也 发现，从形态上看，空化是随着沿着预期方向形成微腔而开始的。 关节线，当袋合并形成单个滑液时很快就完成了 空腔。这个过程在发育中的膝盖中非常快，但在手指中则较慢。这些和其他小说 数据引出了我的中心假设，即联合空化是由不同但不同的因素的汇聚引起的 协调的生物过程。因此，目标 1 是确定 HAS2 和 TMEM2 在联合治疗中的作用 使用转基因小鼠模型进行空化。我将有条件地删除域间的 Has2 和/或 Tmem2 细胞（使用 Gdf5Cre 小鼠）并对所得突变胚胎进行详细分析。目标 2 是确定 空化的细胞和分子机制。我将研究下游信号通路作为回应 HA 大小的变化以及由此产生的与细胞表面 CD44 受体的相互作用，调节 HA 代谢 滑膜关节的发育和长期维护。该项目将为机制提供新的见解 潜在的联合发展和空穴。根据K01机制，该项目将使我能够 获得骨骼发育和分子生物学方面的新专业知识，并将其与我以前的知识相结合 生物工程培训。这种在两个不同但相互关联的领域的专业知识的独特组合将允许 我要建立一个与我的导师不同的独立职业，并创造新颖的治疗工具来修复 并再生软骨以治疗骨关节炎（OA）等关节疾病。

Project Summary and Abstract Synovial joints are essential for body motion and quality of life. Their synovial cavity and lubricant-rich fluid permit unhindered joint motion and function and provide tissue protection and nourishment. While these aspects of synovial joint biology are well understood, little is known about how the cavity and its fluid actually develop during embryogenesis. At early fetal stages, the limb skeletal primordia are composed of continuous cartilaginous structures without joints. Joint development starts with appearance of an “interzone”, a tissue made of mesenchymal cells expressing the growth and differentiation factor 5 gene (Gdf5). We previously showed that Gdf5+ cell progenies produce most joint tissues over time and the synovial cavity forms in the middle of the interzone. Because the interzone cells are initially attached to each other, the cavitation process must involve their physical separation along the prospective articular line to facilitate the creation of a fluid-filled cavity. Previous studies indicated that interzone cells produce hyaluronan (HA) around the cavitation time, and this is accompanied by accumulation of a HA-rich matrix in local tissues. HA is a major component of extracellular matrix and synovial fluid and plays important roles in tissue homeostasis. In my preliminary studies, I found that just before cavitation onset, interzone cells in mouse embryo limbs express hyaluronan synthase 2 (HAS2, ‘the HA synthesizer’) and transmembrane protein 2 (TMEM2), a cell surface hyaluronidase that specifically cleaves high molecular weight HA into intermediate and biologically-active fragments. I also discovered that, morphologically, cavitation initiates with formation of microlumens along the prospective articular line and is completed soon afterwards when the pockets coalesce to generate a single one synovial cavity. This process is extremely rapid in the developing knee but is slower in digits. These and other novel data lead to my central hypothesis that joint cavitation is brought about by convergence of diverse but coordinated biological processes. Accordingly, Aim 1 is to determine the role of HAS2 and TMEM2 in joint cavitation using genetically modified mouse models. I will conditionally delete Has2 and/or Tmem2 in interzone cells (using Gdf5Cre mice) and subject resulting mutant embryos to detailed analysis. Aim 2 is to determine cellular and molecular mechanisms of cavitation. I will investigate downstream signaling pathways in response to changes in HA sizes and resulting interactions with cell surface CD44 receptor, regulating HA metabolism in synovial joint development and long-term maintenance. The project will provide novel insights into mechanisms underlying joint development and cavitation. In line with the K01 mechanism, the project will allow me to acquire new expertise in skeletal developmental and molecular biology and to integrate it with my previous training in bioengineering. This unique combination of expertise in two distinct but interrelated fields will allow me to establish an independent career distinct from my mentors and create novel therapeutic tools to repair and regenerate cartilage for the treatment of joint conditions such as osteoarthritis (OA).