Determining the role of sema3d during fin regeneration

确定 sema3d 在鳍再生过程中的作用

• 批准号: 8446293
• 负责人: Mary Kathryn Iovine
• 金额: $ 6.96万
• 依托单位: LEHIGH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2014-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8446293
• 关键词: Affect; Axon; Biological Assay; Biological Models; Blood Vessels; Bone Growth; Cell Differentiation process; Cell Proliferation; Cell Proliferation Regulation; Cells; Connexin 43; Cues; Data; Defect; Development; Distal; Event; Exhibits; Failure; Gap Junctions; Genes; Goals; Growth; Human; Joints; Lead; Length; Limb structure; Location; Mediating; Molecular; Morphogenesis; Morphology; Multiple Sclerosis; Mutation; Natural regeneration; Neurodegenerative Disorders; Outcome; Pathway interactions; Pattern; Phenotype; Rheumatoid Arthritis; Role; Semaphorins; Shapes; Signal Pathway; Signal Transduction; Signaling Molecule; Skeleton; Syndrome; Tissues; Zebrafish; cell type; craniofacial; gain of function; human disease; insight; loss of function; member; mutant; oculodentodigital dysplasia; osteoblast differentiation; premature; receptor; skeletal; tumor progression

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.

描述（由申请人提供）：骨骼形态发生需要整合多种信号以协调生长和形成模式。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。我们的分析使用斑马鱼再生鳍作为模型系统已经确定了两个鳍长突变体，提供深入了解鳍生长和骨骼图案的协调。短 鳍（sof b123）突变体由于短鳍条节的发育（即过早的关节形成）而具有短鳍。相反，另一个长鳍（alf dty 86）突变体表现出鳍过度生长和过长的节由于随机关节故障。有趣的是，sof b123和alf dty 86在差距连接基因连接蛋白43（cx43）的表达上也表现出重要的相反的差异。sof的b123表型是由cx43的亚型突变引起的，而alf dty 86的关节衰竭表型是cx43过度表达的结果。事实上，alf dty 86再生鳍中cx43的敲除挽救了关节的形成。总之，这些和其他数据表明，Cx43功能既促进鳍生长，又抑制关节形成。此外，人类CX43的突变导致眼齿指发育不良，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在骨骼形态发生中具有保守但未知的功能。为了从分子水平上了解Cx43如何介导其对鳍骨架生长和图案化的影响，我们鉴定了Cx43下游表达的基因。有趣的是，分泌的信号蛋白基因sema 3d在再生鳍中以cx43依赖的方式表达。引人注目的是，在alf dty 86中，sema 3d的敲低导致鳍长度减少，细胞增殖减少，节段长度减少，关节形成的拯救，类似于cx43敲低的表型。总之，我们的研究结果表明，sema 3d功能与cx43在一个共同的途径，以调节鳍的生长和关节的形成。脑信号蛋白是一个进化上保守的信号分子大类的成员，最好的理解为提供指导线索的轴突和血管。最近的研究表明，semaphorins在发育过程中广泛表达，并启动多种多样的细胞结果。脑信号蛋白功能的丧失导致人类疾病，包括癌症进展、神经退行性疾病、多发性硬化和类风湿性关节炎。因此，semaphorins有助于广泛的正常和病理发展。该提案的目的是确定受操纵Sema 3d信号传导影响的cx43依赖性细胞结果，并确定Sema 3d下游信号传导途径的组成部分。本研究的结果将揭示Cx43-Sema 3d在鳍再生过程中的作用机制，并为更全面地确定Cx43和Sema 3d之间的功能关系以及脊椎动物骨骼生长和图案化协调所需的分子途径提供初步数据。