Determining the role of sema3d during fin regeneration

描述(申请人提供)：骨骼形态发生需要整合多种信号以协调生长和构图。例如，细胞增殖必须与成骨细胞和关节形成细胞的分化相协调。然而，负责协调多个细胞事件的机制在很大程度上是未知的。我们使用斑马鱼再生鳍作为模型系统的分析已经确定了两个鳍长度突变，这两个突变为深入了解鳍生长和骨骼图案的协调提供了洞察。短小的 FIN(Sof B123)突变体由于短鳍射线段的发育(即关节过早形成)而具有短鳍。相反，另一个长鳍(Alf Dty86)突变体由于随机关节破坏而显示出鳍过度生长和过长的节段。有趣的是，sof b123和alf dty86在缝隙连接基因连接蛋白43(Cx43)的表达上也显示出重要的和相反的差异。Sof b123表型是由Cx43亚型突变引起的，而Alf dty86的关节失效表型是Cx43过度表达的结果。事实上，Cx43在Alf dty86再生鳍上的击倒拯救了关节的形成。总之，这些和其他数据表明，Cx43功能既促进了鳍的生长，又抑制了关节的形成。此外，人类Cx43的突变会导致眼齿-指骨发育不良，这是一种以颅面和远端肢体骨骼的形态缺陷为特征的综合征。因此，Cx43在骨骼形态发生中表现出保守但未知的功能。为了从分子上了解Cx43如何介导其对鳍骨骼生长和模式的影响，我们鉴定了Cx43下游表达的基因。有趣的是，分泌信号素的基因Sema3d在再生鳍中以依赖于Cx43的方式表达。引人注目的是，Sema3d的敲除导致Alf dty86的鳍长度缩短，细胞增殖减少，片段长度减少，关节形成挽救，类似于Cx43-敲除表型。综上所述，我们的发现表明，Sema3d与Cx43在调节鳍生长和关节形成的共同途径中发挥作用。信号素是进化上保守的一大类信号分子的成员，最好的理解是为轴突和血管提供指导线索。最近的研究表明，信号素在发育过程中广泛表达，并启动了细胞结果的广泛多样性。信号素功能的丧失会导致人类疾病，包括癌症进展、神经退行性疾病、多发性硬化症和类风湿性关节炎。因此，信号素在正常和病理发育中起着广泛的作用。这项建议的目的是确定操纵Sema3d信号对依赖Cx43的细胞结果的影响，并确定Sema3d下游信号通路的组成部分。这一研究结果将揭示Cx43-Sema3d在鳍再生过程中的作用机制，并将为更全面地确定Cx43和Sema3d之间的功能关系以及脊椎动物骨骼生长和模式协调所需的分子途径(S)提供初步数据。

DESCRIPTION (provided by applicant): Skeletal morphogenesis requires the integration of multiple signals for coordinated growth and patterning. For example, cell proliferation must be coordinated with the differentiation of osteoblasts and joint-forming cells. However, the mechanisms responsible for coordinating multiple cellular events are largely unknown. Our analyses using the zebrafish regenerating fin as a model system have identified two fin length mutants that provide insight into the coordination of fin growth and skeletal patterning. The short fin (sof b123) mutant has short fins due to the development of short fin ray segments (i.e. or premature joint formation). In contrast, the another long fin (alf dty86) mutant exhibits fin overgrowth and overlong segments due to stochastic joint failure. Interestingly, sof b123and alf dty86 also exhibit important and opposing differences in the expression of the gap junction gene connexin43 (cx43). The sof b123phenotypes are caused by hypomorphic mutations in cx43, while the joint failure phenotype in alf dty86 is the result of cx43 over-expression. Indeed, cx43 knockdown in alf dty86regenerating fins rescues joint formation. Together, these and other data suggest that Cx43 function both promotes fin growth and suppresses joint formation. Moreover, mutations in human CX43 cause oculodentodigital dysplasia, a syndrome characterized by morphological defects of the craniofacial and distal limb skeletons. Thus, Cx43 exhibits conserved, but unknown, functions in skeletal morphogenesis. In order to understand molecularly how Cx43 mediates its effects on growth and patterning of the fin skeleton, we identified genes expressed downstream of cx43. Interestingly, the gene for a secreted semaphorin, sema3d, is expressed in the regenerating fin in a cx43-dependent manner. Strikingly, knockdown of sema3d leads to reduced fin length, reduced cell proliferation, reduced segment length, and rescue of joint formation in alf dty86, similar to the cx43-knockdown phenotypes. Together, our findings suggest that sema3d functions in a common pathway with cx43 to regulate fin growth and joint formation. Semaphorins are members of a large evolutionarily conserved class of signaling molecules, best understood as providing guidance cues for axons and blood vessels. More recent studies revealed that semaphorins are expressed broadly during development and initiate a wide diversity of cellular outcomes. Loss of semaphorin function leads to human diseases including cancer progression, neurodegenerative diseases, multiple sclerosis, and rheumatoid arthritis. Thus, semaphorins contribute broadly to normal and pathological development. The aims of this proposal are to identify the cx43-dependent cellular outcomes affected by manipulating Sema3d signaling and to identify the components of the signaling pathway downstream of Sema3d. Results obtained from this proposal will reveal the underlying mechanism of Cx43-Sema3d action during fin regeneration, and will serve as preliminary data for a more comprehensive proposal on defining the functional relationship between Cx43 and Sema3d, and the molecular pathway(s) required for the coordination of growth and patterning of the vertebrate skeleton.