Understanding how Cx43 regulates joint formation in the zebrafish fin

 描述（由申请人提供）：滑膜关节对骨骼的形态和功能至关重要，但易患骨关节炎等衰弱性疾病和损伤。虽然关于功能性关节的组成和力学已经知道很多，但定义关节形成细胞如何在特定位置指定的分子机制仍然在很大程度上未知。我们使用斑马鱼再生鳍来解决有关骨骼发育的基本问题，包括鳍条关节的规格和承诺。事实上，在关节形成的初始阶段，鳍条关节和滑膜关节之间存在重要的相似性，这表明鳍条关节中的发现将适用于滑膜关节。例如，滑膜关节是通过形成间带而在不间断的软骨成分中开始的。中间区是软骨细胞的离散带，其在未来关节的部位凝聚并指导关节形成的后期阶段。鳍条节理同样是由一组凝聚在未来节理位置的细胞在一个不间断基质区域中发起的。这些细胞代表鳍条关节间区，并同样出现指导关节形态发生。在任何假定的关节中，中间层的细胞如何决定启动关节形成，仍然是我们知识中的一个空白。对两种鳍长突变体，短鳍（sof b123）和另一种长鳍（alf dty 86）的评估表明，差距连接蛋白连接蛋白43（Cx 43）决定了鳍条连接的位置。例如，sof b123突变体表现出由过早的关节形成引起的短鳍条节。相比之下，alf dty86由于随机接合失效而平均表现出较长的片段。值得注意的是，cx43敲低挽救了alf dty86中的关节形成。这些发现表明cx43抑制关节形成。该提议的基本假设是，需要Cx43功能的瞬时废除以允许关节形成细胞的定型，从而允许关节形成。本提案的两个目的是提供支持这一假设的切实证据。在目标1中，我们提出证明阻止Cx43的废除会导致关节失败。这将通过产生以热诱导方式过表达Cx43的转基因系来实现，并使用药物治疗来防止Cx43周转。在目标2中，我们提出揭示联合形成细胞的承诺的分子要求。这将通过操纵关节形成细胞（即evx 1）或成骨细胞形成细胞（即osx）中所需基因的表达来实现，以揭示关节形成细胞定型的功能要求。类似于目标1，将确定是否需要消除Cx43以用于联合形成细胞的定型。在这两个目标中，我们将进一步测试的假设，间隙连接细胞间通讯所需的Cx43功能在联合承诺和联合形成。总之，完成这些目标将揭示关键的机械细节至关重要，我们的理解的初始事件的关节形成和模式的脊椎动物骨骼。

 DESCRIPTION (provided by applicant): Synovial joints are critical for skeletal form and function, but are susceptible to debilitating diseases such as osteoarthritis and to injury. While much is known regarding the composition and mechanics of functioning joints, the molecular mechanisms defining how joint-forming cells are specified in a particular location remain largely unknown. We use the zebrafish regenerating fin to address fundamental questions regarding skeletal development, including the specification and commitment of fin ray joints. Indeed, there are important similarities between fin ray joints and synovial joints during the initial stages of joint formation, suggesting that findings in the fin ray joints will be applicable to synovial joins. For example, the synovial joint is initiated in uninterrupted cartilaginous elements by the formation of the interzone. The interzone is a discrete band of chondrocytes that condense at the site of the future joint and guides later stages of joint formation. The fin ray joint is similrly initiated in a region of uninterrupted matrix by a group of cells that condense at the location of the future joint. These cells represent the fin ray joint interzone, and similarly appear to guide joint morphogenesis. How the cells of the interzone decide to initiate joint formation, in any presumptive joint, remains a gap in our knowledge. Evaluation of two fin length mutants, short fin (sof b123) and another long fin (alf dty86), suggest that the gap junction protein Connexin43 (Cx43) determines where a fin ray joint will be produced. For example, the sof b123 mutant exhibits short fin ray segments caused by premature joint formation. In contrast, alf dty86 exhibits long segments on average due to stochastic joint failure. Notably, cx43- knockdown rescues joint formation in alf dty86. These findings suggest that cx43 suppresses joint formation. The underlying hypothesis of this proposal is that transient abrogation of Cx43 function is required to permit the commitment of joint-forming cells, and thereby permit joint formation. The two aims of this proposal are to provide tangible evidence in support of this hypothesis. In Aim 1 we propose to demonstrate that preventing the abrogation of Cx43 causes joint failure. This will be accomplished by generating a transgenic line that overexpresses Cx43 in a heat-inducible manner, and to prevent Cx43 turnover using pharmacological treatments. In Aim 2 we propose to reveal the molecular requirements for the commitment of joint-forming cells. This will be accomplished by manipulating the expression of genes required in either joint- forming cells (i.e. evx1) or osteoblast-forming cells (i.e. osx) to reveal the functional requirements for joint- forming cell commitment. Similarly to Aim 1, it will be determined if abrogation of Cx43 is required for the commitment of joint-forming cells. In both Aims, we will further test the hypothesis that gap junctional intercellular communication is required for Cx43 function during both joint commitment and joint formation. Together, completion of these aims will reveal key mechanistic details critical for our understanding of the initial events of joint formation and patterning in the vertebrate skeleton.