PIONEER AXON GUIDANCE IN THE EMBRYONIC FOREBRAIN

描述（来自申请人的摘要）：本发明的长期目标是： 应用是为了增加我们的知识如何增长和指导 轴突在胚胎发育过程中受到调节。正常运作的神经系统 需要神经元之间的精确互连，而这个应用程序的目的是 解剖一组轴突的引导机制， 在小鼠前脑的纵向束， 连合（tpoc）。这篇文章是基础性的，因为它奠定了 随后是大量的上行和下行轴突。因此 tpoc对于理解基底节神经回路的形成至关重要 前脑 最近，我们发现，tpoc轴突作出严重的指导错误， 转录因子Pax6的胚胎突变体。这种突变体提供了一种 有机会发现早期模式基因，如Pax6，可能提供 引导线索形式的位置信息。主要方法将是 通过在发育中的神经元中创造Pax6的改变模式来挑战轴突， 个脑袋对轴突反应的分析将揭示Pax6在何处以及如何起作用， 影响轴突生长。为了改变Pax6的表达模式， 战略将被使用。一种新的电穿孔方法将用于靶向 完整胚胎中的基因表达，包括Pax6恢复到局部 Pax6突变体胚胎的区域，野生型中Pax6表达的扩增 胚胎，并通过靶向表达 Pax6的显性负等位基因。Pax6表达改变的影响将 也可以在具有野生型和Pax6的混合物的嵌合胚胎中进行检查 突变细胞，以及仅在一个细胞中选择性缺乏Pax6的突变小鼠 类型.在对Pax6下游靶点的初步分析中， Pax6依赖性细胞粘附分子R-cadherin的作用也将被 通过体外方法和胚胎电穿孔实验进行检查。 这项工作将有助于我们了解正常和突变 神经系统发育。发育缺陷导致了许多毁灭性的 神经系统疾病，并了解轴突寻路机制 和神经元模式可能最终导致预防策略， 出生缺陷的治疗。此外，轴突生长的机制， 发展可能会让我们深入了解神经系统的再生可能是如何 受创伤或疾病刺激。

DESCRIPTION (From the Applicant's Abstract): The long-term objective of this application is to increase our knowledge of how the growth and guidance of axons is regulated during embryonic development. The functioning nervous system requires precise interconnections between neurons, and this application aims to dissect the guidance mechanisms of a set of axons that pioneers the first longitudinal tract in the mouse forebrain, the tract of the postoptic commissure (tpoc). This tract is foundational, as it lays down the pathway followed by a multitude of later ascending and descending axons. Therefore the tpoc is crucial to understanding the formation of neural circuitry in the basal forebrain. Recently, we discovered that the tpoc axons make severe guidance errors in embryos mutant for the transcription factor Pax6. This mutant provides an opportunity to discover how early patterning genes, such as Pax6, might provide positional information in the form of guidance cues. The main approach will be to challenge the axons by creating altered patterns of Pax6 in the developing brain. Analysis of axon responses will reveal where and how Pax6 acts to influence axon outgrowth. To alter Pax6 expression patterns, several different strategies will be used. A new electroporation method will be used to target gene expression in intact embryos, including restoration of Pax6 to local regions of Pax6 mutant embryos, expansion of Pax6 expression in wild type embryos, and interference with endogenous Pax6 by targeted expression of dominant negative alleles of Pax6. The effect of altered Pax6 expression will also be examined in chimeric embryos with a mixture of wild type and Pax6 mutant cells, and in mutant mice that selectively lack Pax6 in only one cell type. In an initial analysis of downstream targets of Pax6, the axon guidance role of the Pax6-dependent cell adhesion molecule R-cadherin will also be examined by in vitro approaches and embryo electroporation experiments. This work will contribute to our knowledge of both the normal and mutant developing nervous system. Developmental defects underlie many devastating neurological disorders, and understanding the mechanisms of axon pathfinding and neuronal patterning may eventually lead to strategies for prevention or treatment of birth defects. In addition, the mechanisms of axon growth during development may give insight into how regeneration of the nervous system may be stimulated following trauma or disease.