PIONEER AXON GUIDANCE IN THE EMBRYONIC FOREBRAIN

描述(摘自申请者摘要)：该项目的长期目标 应用是为了增加我们的知识是如何成长和引导的 轴突在胚胎发育过程中受到调节。功能正常的神经系统 需要神经元之间精确的互连，这项应用旨在 解剖一组先于第一个轴突的轴突的引导机制 小鼠前脑内的纵束，视后束 连带(TPOC)。这块区域是基础的，因为它铺设了道路。 然后是大量后来上升和下降的轴突。因此， TPOC对于理解基底神经回路的形成至关重要。 前脑。 最近，我们发现TPOC轴突在 转录因子Pax6突变的胚胎。这个突变体提供了一种 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。主要的方法将是 在发育过程中通过改变Pax6的模式来挑战轴突 大脑。分析轴突反应将揭示Pax6在哪里以及如何作用于 影响轴突生长。要改变Pax6的表达模式，有几种不同的 将使用策略。将使用一种新的电穿孔方法来瞄准 完整胚胎中的基因表达，包括将Pax6恢复到局部 野生型Pax6突变胚胎区及Pax6表达的扩增 胚胎，并通过靶向表达对内源性Pax6的干扰 Pax6的显性负性等位基因。Pax6表达改变的影响将 也可以在野生型和Pax6混合的嵌合体胚胎中进行检测 突变细胞，以及仅在一个细胞中选择性缺失Pax6的突变小鼠 打字。在对Pax6下游靶点的初步分析中，轴突引导 依赖Pax6的细胞黏附分子R-钙粘附素的作用也将是 通过体外方法和胚胎电穿孔实验进行检测。 这项工作将有助于我们了解正常的和突变的 发育中的神经系统。发育缺陷是许多毁灭性因素的基础 神经功能障碍和轴突寻路机制的理解 神经元模式最终可能导致预防或 治疗出生缺陷。此外，轴突生长的机制在 发展可能会让我们洞察神经系统的再生是如何 在创伤或疾病后受到刺激。

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.