Understanding how Connexin43 regulates joint formation in the regenerating zebrafish fin

摘要 滑膜关节对骨骼形态和功能至关重要，但容易患上诸如 骨性关节炎和损伤。虽然人们对起作用的关节的组成和力学有很多了解， 关于关节形成细胞(JFC)是如何在特定位置指定的，人们知之甚少 允许开发提供灵活性的关节。解决这一知识差距可以促进 通过靶向影响细胞命运的分子途径开发关节疾病的新疗法 决定。斑马鱼再生鳍是解决基本问题的重要模型系统 骨骼发育，包括JFC的规范和承诺。鳍的骨架是由骨头制成的 鳍条，每条鳍条由由关节隔开的骨段组成。因此，鳍是关节的丰富来源， 它们在典型的生长和再生过程中定期产生。成骨细胞和JFCs 构建鳍骨架来自位于侧鳍射线中的公共骨骼前体细胞(SPC) 间充质的我。Iovine实验室最近的研究提供了强有力的证据，表明连接蛋白43(Cx43)，通过GAP 连接细胞间通讯(GJIC)，通过抑制evx1的表达影响JFC规范。 Evx1是关节形成所必需的转录因子。这项提案的总体目标是揭示 Cx43-GJIC的性质，并揭示随后抑制evx1的机制。中环 这一假设是Cx43-GJIC通过转导细胞内的变化来影响关节的形成。 膜电位对SPC的作用；膜电位的变化进而触发基因的变化 影响evx1表达、JFC规范和关节形成的表达。这一假设是 根据先前的和初步的数据制定的。例如，Cx43-GJIC促进SIMPLET(SMP) 在SPC中的表达。SMP是将-连环蛋白带到细胞核所必需的。与Lef1或Tcf7合作 转录因子，-连环蛋白(直接或间接)抑制EVX1转录。这项提议的目的是 是用来检验中心假设的。第一个目标是测试膜静息电位的变化是否 足以影响SPC中的基因表达。这将通过驱动薄膜来实现 表达Cx43的细胞的超极化或去极化，以及SMP和evx1的表达 SPCS。目的2是确定影响evx1转录的顺式调控元件(Cres)。推定的Cres将 从evx1的BAC记者中删除，并将评估对记者表达的影响，以揭示可能 作用于evx1的增强剂或消音器。完成这些目标将极大地促进我们对 GJIC介导的影响细胞规格的信号，并将进一步提供对 JFC规范的时空调节。

Summary Synovial joints are critical for skeletal form and function, but are susceptible to debilitating diseases such as osteoarthritis and injury. While much is known about the composition and mechanics of functioning joints, relatively little is known about how joint-forming cells (JFCs) are specified in a particular location, thereby permitting the development of joints that provide flexibility. Addressing this gap in knowledge could facilitate the development of novel therapies for joint disease by targeting molecular pathways influencing cell fate decisions. The zebrafish regenerating fin is an important model system for addressing fundamental questions of skeletal development, including the specification and commitment of JFCs. The fin skeleton is made of bony fin rays, and each fin ray is made of bony segments separated by joints. Thus, the fin is a rich source of joints, which are produced regularly during typical outgrowth and during regeneration. The osteoblasts and JFCs that build the fin skeleton are derived from a common skeletal precursor cell (SPC) located in the lateral fin ray mesenchyme. Recent studies from the Iovine lab provide strong evidence that Connexin43 (Cx43), via gap junctional intercellular communication (GJIC), influences JFC specification by suppressing expression of evx1. Evx1 is a transcription factor required for joint formation. The overall objectives for this proposal are to reveal the nature of Cx43-GJIC, and to reveal the subsequent mechanism of evx1 suppression. The central hypothesis of this proposal is that Cx43-GJIC influences joint formation by transducing changes in membrane potential to the SPCs; changes in membrane potential in turn trigger changes in gene expression that influence evx1 expression, JFC specification, and joint formation. This hypothesis was formulated on the basis of prior and preliminary data. For example, Cx43-GJIC promotes simplet (smp) expression in SPCs. Smp is required to bring -catenin to the nucleus. In concert with Lef1 or TCF7 transcription factors, -catenin (directly or indirectly) suppresses evx1 transcription. The Aims of this proposal are designed to test the central hypothesis. Aim 1 is to test if changes in membrane resting potential are sufficient to influence gene expression in SPCs. This will be accomplished by driving membrane hyperpolarization or depolarization in Cx43-expressing cells, and evaluating smp and evx1 expression in SPCs. Aim 2 is to identify cis-regulatory elements (CREs) that influence evx1 transcription. Putative CREs will be deleted from a BAC reporter for evx1, and impacts on reporter expression will be assessed to reveal likely enhancers or silencers acting on evx1. Completion of these Aims will significantly advance our understanding of signals mediated by GJIC influencing cell specification, and will further provide novel insights into the spatiotemporal regulation of JFC specification.