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

描述（由申请人提供）：目前没有有效的治疗方法来改善人类的轴突再生。因此，需要对线虫、苍蝇和小鼠等模式生物进行基础研究，以更好地了解调节再生的生物学机制。最近的研究表明，保守的DLK-1信号通路是再生的关键调节因子。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。该提案旨在鉴定由DLK-1信号传导调节的基因（目的1），并确定这些基因中哪些对于确定受损神经元的再生潜力是重要的（目的2）。这些发现将扩大对DLK-1通路的理解，并有可能确定轴突再生的新机制。该提案在模式生物C中使用了创新方法。elegans来鉴定在再生中起作用的DLK-1信号传导的转录靶点。基因转录的研究通常会识别大量的靶点，但通常很难分析几个以上的候选基因的功能。本提案采用四种策略来解决这一难题。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。第二，一种新的方法是用来纯化细胞的转录谱，使分析被定向到一个单一的神经元类型，GABA运动神经元。第三，通过使用一种新的RNAi技术，基因只在GABA神经元中被敲低，用于功能分析，避免了混淆效应，甚至能够研究必需基因。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通路对基因表达的调节如何影响轴突再生。此外，这项研究将作为未来调查的蓝图， 连接神经损伤、细胞信号、基因转录和轴突再生的机制。

DESCRIPTION (provided by applicant): 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 understanding 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 s 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.