Identification of transcriptional targets of the DLK-1 axon regeneration pathway

目前还没有有效的治疗方法来改善人类的轴突再生。因此，基础研究 模型生物，如线虫，苍蝇，和小鼠，需要提供一个更好的理解， 调节再生的生物机制。最近的研究表明，保守的DLK-1 信号通路是再生的关键调节器。在线虫、苍蝇和小鼠中，DLK-1通路 通过调节受损神经元中的基因表达来调节再生。这项建议旨在确定 通过DLK-1信号传导（Aim 1）调节的基因，并确定这些基因中哪些对DLK-1信号传导（Aim 1）重要。 确定受损神经元的再生潜力（目的2）。这些发现将扩大 DLK-1通路的理解，并有可能确定轴突的新机制， 再生 该提案在模式生物C中使用了创新方法。识别转录靶点 DLK-1信号在再生中起作用。基因转录的研究通常会发现大量的 目标，但它往往是难以分析的功能，几个以上的选定的候选人。这项建议 用四种策略来解决这个难题。首先，通过使用新的遗传背景，分析集中在 一个单一的信号通路-DLK-1通路的转录效应。其次，一种新颖的方法是 用于纯化细胞进行转录谱分析，使分析能够针对单一神经元类型， GABA运动神经元第三，通过使用一种新的RNAi技术， 神经元的功能分析，避免混淆效应，使研究甚至必要的基因。 第四，使用单神经元激光轴突切断术在GABA神经元中进行功能分析。 这些实验将详细分析DLK-1通路如何调节基因表达 影响轴突再生此外，这项研究将作为未来调查的蓝图， 神经损伤、细胞信号传导、基因转录和轴突再生之间的联系机制。

There is currently no effective treatment to improve axon regeneration in humans. Thus, basic research in model organisms such as nematodes, flies, and mice is needed to provide a better undertanding of the biological mechanisms that regulate regeneration. Recent work has demonstrated that the conserved DLK-1 signaling pathway is a critical regulator of regeneration. In nematodes, flies, and mice, the DLK-1 pathway regulates regeneration by modulating gene expression in injured neurons. This proposal seeks to identify the genes that are regulated by DLK-1 signaling (Aim 1), and to determine which of these genes are important for determining the regenerative potential of the injured neuron (Aim 2). These findings will expand understanding of the DLK-1 pathway, and have the potential to identify novel mechanisms for axon regeneration. This proposal uses an innovative approach in the model organism C. elegans to identify transcriptional targets of DLK-1 signaling that function in regeneration. Studies of gene transcription typically identify large numbers of targets, but it is often difficult to analyze the function of more than a few selected candidates. This proposal uses four strategies to address this difficulty. First, by using novel genetic backgrounds, analysis is focused on the transcriptional effects of a single signaling pathway-the DLK-1 pathway. Second, a novel approach is used to purify cells for transcriptional profiling, enabling analysis to be directed to a single neuronal type, the GABA motor neurons. Third, by using a novel RNAi technique, genes are knocked down only in GABA neurons for functional analysis, avoiding confounding effects and enabling the study of even essential genes. Fourth, functional analysis is performed using single-neuron laser axotomy in GABA neurons. These experiments will provide a detailed analysis of how modulation of gene expression by the DLK-1 pathway affects axon regeneration. In addition, this study will serve as a blueprint for future investigations into the mechanisms that link nerve injury, cellular signaling, gene transcription, and axon regeneration.