Guidance of neuronal migration in Embryonic Development

描述（由申请人提供）：定向神经元迁移是发育中的神经系统的基本特征，需要通过复杂和动态的分子环境精确导航细胞。这一过程中的错误可能导致人类发育中严重的解剖和认知障碍。虽然这些缺陷中受影响的一些基因是已知的，但引导迁移的分子机制仍然知之甚少，部分原因是脊椎动物制备的复杂性。这个问题可以在蛾Manduca的肠神经系统（ENS）中解决，其中一组确定的迁移神经元（EP细胞）及其通路（内脏肌带）在整个发育过程中保持独特的可访问性。尽管有明显的形态差异，在这个系统中的神经元发育采用类似的信号转导过程中发现在哺乳动物系统中，允许在活胚胎迁移的机制分析。具体地，成束蛋白II（MFas II）已被鉴定为EP细胞的重要引导因子。与其脊椎动物对应物NCAM一样，MFas II是免疫球蛋白相关粘附受体家族的成员。MFas II是正常迁移所必需的，其两种亚型（跨膜和GPI连接）在迁移神经元及其通路中表现出不同的表达模式。将使用同种型特异性RNA和质粒的细胞内注射来诱导非途径肌肉中的异位MFas II表达，而将注射反义寡核苷酸和RNAi探针来阻断内源性MFas II表达，以测试该受体是否是支持迁移所必需的且足够的。还将在MFas II转染的细胞上的原代培养物中测试神经元的迁移行为。平行实验将用于测试每种亚型如何影响神经元运动。其中一种亚型已被证明与酪氨酸激酶相关，其身份和功能将通过免疫共沉淀，微测序和培养中的操作来确定。柱连接的GST融合蛋白和双杂交方法将用于MFas II相互作用蛋白的功能独立筛选。这些实验应该提供洞察的基本机制，这类重要的指导线索调节体内神经元迁移。

DESCRIPTION (provided by applicant): Directed neuronal migration is an essential feature of the developing nervous system, requiring the precise navigation of cells through a complex and dynamic molecular environment. Errors in this process can give rise to severe anatomical and cognitive disorders in human development. Although some of the genes affected in these defects are known, the molecular mechanisms that guide migration remain poorly understood, in part due to the complexity of vertebrate preparations. This issue can be addressed in the enteric nervous system (ENS) of the moth Manduca, in which an identified set of migratory neurons (the EP cells) and their pathways (visceral muscle bands) remain uniquely accessible throughout development. Despite obvious morphological differences, neuronal development in this system employs similar signaling processes as found in mammalian systems, permitting a mechanistic analysis of migration in living embryos. Specifically, fasciclin II (MFas II) has been identified as an essential guidance cue for the EP cells. Like its vertebrate counterpart NCAM, MFas Il is a member of the immunoglobulin related adhesion receptor family. MFas II is required for normal migration, and its two isoforms (transmembrane and GPI-linked) exhibit distinct patterns of expression in the migratory neurons and their pathways. Intracellular injections of isoform-specific RNA and plasmids will be used to induce ectopic MFas II expression in non-pathway muscles, while antisense oligonucleotides and RNAi probes will be injected to block endogenous MFas II expression to test whether this receptor is both necessary and sufficient to support migration. The migratory behavior of the neurons will also be tested in primary culture on MFas Il-transfected cells. Parallel experiments will be used to test how each isoform affects neuronal motility. One of the isoforms has been shown to associate with a tyrosine kinase, whose identity and function will be determined by co-immunoprecipitation, microsequencing, and manipulations in culture. Column-linked GST fusion proteins and 2-hybrid approaches will be used in function-independent screens of MFas Il-interacting proteins. These experiments should lend insight into the fundamental mechanisms by which this important class of guidance cues regulate neuronal migration in vivo.