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

描述(申请人提供)：目前还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、苍蝇和老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的基因表达来调节再生。这项建议试图确定受DLK-1信号调节的基因(目标1)，并确定这些基因中哪些对确定受损神经元的再生潜力(目标2)重要。这些发现将扩大对DLK-1途径的理解，并有可能确定轴突再生的新机制。这项建议在模式生物秀丽线虫中使用了一种创新的方法来确定DLK-1信号的转录靶标，该信号在再生中发挥作用。基因转录的研究通常会识别大量的靶点，但S往往很难分析选定的几个候选基因的功能。这项提案使用了四种策略来解决这一困难。首先，通过使用新的遗传背景，重点分析了单一信号途径-DLK-1途径的转录效应。其次，使用了一种新的方法来纯化细胞以进行转录图谱分析，使分析能够针对单一的神经元类型，即GABA运动神经元。第三，通过使用一种新的RNAi技术，只需敲除GABA神经元中的基因进行功能分析，就可以避免混淆效应，甚至能够研究必要的基因。第四，用单神经元激光切断法对GABA神经元进行功能分析。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.