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一个基本特征，需要细胞在复杂和动态的分子环境中精确导航。这一过程中的错误可能会导致人类发育过程中严重的解剖学和认知障碍。虽然在这些缺陷中受影响的一些基因是已知的，但指导迁移的分子机制仍然知之甚少，部分原因是脊椎动物准备的复杂性。这个问题可以在蛾Manduca的肠道神经系统(ENS)中得到解决，在该系统中，一组已识别的迁移神经元(EP细胞)及其通路(内脏肌带)在整个发育过程中仍然是唯一可访问的。尽管存在明显的形态差异，但该系统中的神经元发育采用了与哺乳动物系统相似的信号传递过程，从而允许对活胚胎中的迁移进行机制分析。具体地说，Fasciclin II(MFAs II)已被确定为EP细胞的重要指导信号。和脊椎动物的NCAM一样，MFAs Il是免疫球蛋白相关的黏附受体家族的成员。MFAs II是正常迁移所必需的，它的两种亚型(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。细胞内注射异构体特异的RNA和质粒将被用来诱导非通路肌肉中异位MFAs II的表达，而反义寡核苷酸和RNAi探针将被注射来阻断内源性MFAs II的表达，以测试这种受体是否既是必要的，也是支持迁移的充分条件。神经元的迁移行为也将在MFAs Il转基因细胞的原代培养中进行测试。平行实验将被用来测试每种异构体如何影响神经元的运动。其中一个亚型已被证明与酪氨酸激酶有关，其身份和功能将通过免疫共沉淀、显微测序和培养操作来确定。柱联GST融合蛋白和双杂交方法将用于MFAs Il相互作用蛋白的功能独立筛选。这些实验应该有助于深入了解这类重要的指导信号在体内调节神经元迁移的基本机制。

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.