Embryonic Expression and Roles of Neuronal Connexin 35 in Zebrafish Spinal Cord

斑马鱼脊髓中神经元连接蛋白 35 的胚胎表达和作用

• 批准号: 8198999
• 负责人: Tara Carlisle
• 金额: $ 2.88万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8198999
• 关键词: Adult; Antibodies; Biochemical; Biology; Cell Communication; Cells; Collection; Communication; Complex; Connexins; Coupled; Coupling; Cytoskeleton; Data; Development; Disease; Electrical Synapse; Embryo; Embryonic Development; Extracellular Matrix; Gap Junctions; Genes; Goals; Green Fluorescent Proteins; Image; Immunohistochemistry; In Situ Hybridization; Investigation; Knowledge; Leukoencephalopathy; Life; Link; Mediating; Messenger RNA; Modeling; Molecular; Molecular Probes; Molecular Profiling; Motor Neurons; Mus; Mutation; Nervous system structure; Neuraxis; Neurodevelopmental Disorder; Neuroglia; Neurologic; Neurons; Pattern; Phenocopy; Phenotype; Play; Population; Process; Production; Protein Family; Proteins; Reporter; Role; Series; Signal Transduction; Spinal; Spinal Cord; Staging; Symptoms; Techniques; Testing; Transgenic Organisms; Uncoupling Agents; Zebrafish; autism spectrum disorder; cellular development; connexin 36; connexin pore; extracellular; genetic manipulation; human disease; insight; knock-down; mRNA Expression; member; mutant; nervous system disorder; neurodevelopment; oculodentodigital dysplasia; postnatal; promoter; protein complex; relating to nervous system; research study; tool; zebrafish development

DESCRIPTION (provided by applicant): A goal of neurodevelopmental biology is to identify mechanisms by which normal paterning of the nervous system occurs to ultimately apply this knowledge to understanding neurodevelopmental disorders. Activity-dependent processes have been recognized as important mechanisms for early neurodevelopment and electrical synapses have been suggested to play an integral role in the establishment and refinement of neuronal networks. Electrical synapses are formed with gap junction communications between neuronal cells that allow for the direct transfer of current. The proteins responsible for most gap junction formations, connexins, have been implicated in a number of neurodevelopmental processes. However, increasing evidence indicates connexins play more than a pore-forming role, but may also participate in other intracellular and extracellular interactions that can have profound effects on development. The goal of the current study is to determine the role neuronal specific connexins play in neurodevelopment by exploring the many possible mechanisms by which these protein complexes may mediate developmental control. This study takes advantage of the Danio rerio (zebrafish) model to determine whether the neuronal specific connexin 35 (Cx35) plays a fundamental role in the development of the spinal cord. The two specific aims will test the hypotheses that (1) proper development of the zebrafish spinal cord requires developmentally regulated expression of Cx35 and (2) the mechanism by which Cx35 mediates neurodevelopment may be executed by pore function and/or intracellular interactions. The experimental approach utilizes a number of tools available in zebrafish, such as the availability of many transgenic lines, easy genetic manipulation, live- imaging, and pharmacological approaches. Aim 1 will use in situ hybridization (ISH) and immunohistochemistry (IHC) to determine the cx35 mRNA expression profile and morpholino knockdown to determine if Cx35 is required for proper development of spinal cord cells in order to elucidate the cellular networks that require Cx35 for proper development. Aim 2 wil use a pharmacological approach to uncouple gap junctions and the production of transgenic lines that express mutant forms of Cx35 to determine whether Cx35 developmental control is mediated by pore-dependent and/or pore-independent molecular processes. The role of connexin proteins in early development has not been fuly explored. Much of the current knowledge regarding connexins in the nervous system is on their role in fine-tuning already established networks. Investigating the role of connexins in early development may help deepen our understanding of the importance of these proteins in establishing complex neuronal networks and determining cellular development. PUBLIC HEALTH RELEVANCE: Some human diseases with neurological symptoms have been linked to mutations in connexin genes, such as oculodentodigital dysplasia (ODDD) and a recessive hypomyelinating leukoencephalopathy called Pelizaeus- Merzbacher-like disease (PMLD). Additionally, electrical synapses are responsible for generating synchrony among coupled cells and atypical neural synchrony has been suggested to play a role in some developmental neurological diseases, such as Autism Spectrum Disorders. However, the mechanisms by which connexins mediate neurodevelopmental control is largely unknown, but investigations into the role connexins play in patterning the nervous system may provide insight into some neurological disease processes.

描述（由申请人提供）： 神经发育生物学的一个目标是确定神经系统发生正常模式化的机制，以最终将这些知识应用于理解神经发育障碍。活动依赖性过程已被认为是早期神经发育的重要机制，并且电突触已被认为在神经元网络的建立和完善中发挥不可或缺的作用。电突触由神经元细胞之间的间隙连接通信形成，允许电流的直接传递。负责大多数间隙连接形成的蛋白质，连接蛋白，已经涉及许多神经发育过程。然而，越来越多的证据表明，连接蛋白发挥更多的孔形成的作用，但也可能参与其他细胞内和细胞外的相互作用，可以有深远的影响发育。本研究的目的是通过探索这些蛋白复合物可能介导发育控制的许多可能机制来确定神经元特异性连接蛋白在神经发育中的作用。 本研究利用斑马鱼模型来确定神经元特异性连接蛋白35（Cx 35）是否在脊髓发育中起基础作用。这两个特定的目标将测试的假设，（1）斑马鱼脊髓的正常发育需要发育调节表达的Cx 35和（2）的机制，Cx 35介导的神经发育可能是由孔功能和/或细胞内的相互作用。实验方法利用了斑马鱼中可用的许多工具，例如许多转基因品系的可用性、容易的遗传操作、活体成像和药理学方法。目的1将使用原位杂交（ISH）和免疫组织化学（IHC），以确定Cx 35 mRNA的表达谱和吗啉敲低，以确定是否Cx 35是需要适当的发展，以阐明脊髓细胞的细胞网络，需要Cx 35的适当发展。目的2：利用药理学方法分离缝隙连接，并制备表达Cx 35突变体的转基因株系，以确定Cx 35的发育控制是否由孔依赖性和/或非孔依赖性分子过程介导。 连接蛋白在早期发育中的作用尚未得到充分的研究。目前关于神经系统中连接蛋白的大部分知识都是关于它们在微调已经建立的网络中的作用。研究连接蛋白在早期发育中的作用可能有助于加深我们对这些蛋白在建立复杂神经网络和决定细胞发育中的重要性的理解。 公共卫生相关性： 一些具有神经症状的人类疾病与连接蛋白基因的突变有关，例如眼齿指发育不良（ODDD）和称为Pelizaeus-Merzbacher样疾病（PMLD）的隐性髓鞘生成不足白质脑病。此外，电突触负责在耦合细胞之间产生同步，并且非典型神经同步已经被认为在一些发育神经系统疾病中发挥作用，例如自闭症谱系障碍。然而，连接蛋白介导神经发育控制的机制在很大程度上是未知的，但对连接蛋白在神经系统模式化中所起作用的研究可能会提供对一些神经疾病过程的深入了解。