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

描述(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形成的机制，最终将这一知识应用于了解神经发育障碍。活动依赖的过程被认为是早期神经发育的重要机制，电突触被认为在神经元网络的建立和完善中发挥着不可或缺的作用。电突触是通过神经细胞之间的缝隙连接通讯形成的，这种通讯允许电流的直接转移。负责缝隙连接形成的蛋白质，连接蛋白，与许多神经发育过程有关。然而，越来越多的证据表明，连接蛋白不仅起到造孔的作用，还可能参与其他细胞内和细胞外的相互作用，这些作用可以对发育产生深远的影响。本研究的目的是通过探索这些蛋白复合体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所起的作用。本研究利用斑马鱼模型来确定神经元特异性连接蛋白35(Cx35)是否在脊髓发育中起基础作用。这两个特定的目标将检验以下假设：(1)斑马鱼脊髓的正常发育需要Cx35的发育调控表达，以及(2)Cx35介导神经发育的机制可能是通过孔功能和/或细胞内相互作用来执行的。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、简单的遗传操作、活体成像和药理学方法。目的1应用原位杂交(ISH)和免疫组织化学(IHC)方法检测Cx35基因的表达谱和吗啡敲除，以确定Cx35是否是脊髓细胞正常发育所必需的，从而阐明需要Cx35才能正常发育的细胞网络。目的利用药理学方法分离缝隙连接和表达Cx35突变形式的转基因株系，以确定Cx35的发育控制是否由孔依赖和/或孔非依赖的分子过程所介导。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的神经元网络和决定细胞发育中的重要性的理解。

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.