Identification of transcriptional determinants of dendritic patterning

描述（由申请人提供）：疼痛感触发对有害伤害的自我保护反应。疼痛是由专门的感觉神经元感知的，这些神经元被称为伤害感受器，它们利用复杂的树突网络来检测有害的刺激。伤害感受神经元的这些特征在动物中普遍观察到，因此可能依赖于在整个发育过程中控制伤害感受器形态发生的保守遗传程序。控制这些复杂结构的产生的机制知之甚少，但最近在果蝇中的研究已经确定，感觉神经元的形态是由令人惊讶的多样化的转录因子阵列定义的。然而，下游基因的身份及其通过这些转录网络的编排在很大程度上是未知的。在这里，我们建议解决这个问题，利用一个强大的基因表达谱的方法来确定目标的转录因子，控制模型感觉神经元的形态发生。在线虫中，C.在线虫中，PVDL和PVDR是单对双侧对称的神经元，它们用规则排列的高度分支的树突包裹身体，这些树突在活体动物中可以用荧光（GFP）标记物容易地可视化。PVDs对强机械力（“粗糙触摸”）作出反应以触发撤回反射，这是一种指示伤害性功能的行为。我们已经使用了细胞特异性微阵列分析策略，以确定在PVD神经元中高度表达的转录本。具体而言，我们将：（1）使用可用的突变体和RNAi克隆来测试该数据集中的转录因子基因在PVD树突状形态发生中的作用;（2）使用mRNA标记方法来揭示保守的LIM同源结构域蛋白MEC-3下游的基因，MEC-3是PVD树突状分支的确定决定子;和（3）将该mRNA标记策略扩展到目标1中鉴定的至少五个另外的关键转录因子基因。这些转录因子靶基因的数据集将为将来的研究提供基础，将转录控制与树突状分支的细胞生物学联系起来。我们相信，我们的方法代表了一个有用的替代直接研究哺乳动物组织，而且，它提供了一个独特的强大的战略，确定分子的基本，保守的作用，伤害感受器形态发生。公共卫生相关性：调节疼痛感觉的专门神经细胞利用直接位于皮肤下的一系列复杂的意大利面条样感觉过程来检测有害刺激。为了便于识别控制这些感觉网络的基因，我们提出了一种在线虫C。elegans，一种简单的模式生物，具有一对PVD感觉神经元，其显示出刻板且容易可见的神经过程阵列。这项工作的结果应该揭示类似的人类基因在创造感觉神经元阵列中起着关键作用，因此可能导致治疗破坏人类感觉神经元发育的损伤或疾病。

DESCRIPTION (provided by applicant): The sensation of pain triggers self-protective responses to noxious insults. Pain is perceived by specialized sensory neurons called nociceptors that utilize elaborate webs of dendritic processes to detect harmful stimuli. These features of nociceptive neurons are universally observed in animals and are thus likely to depend on conserved genetic programs that control nociceptor morphogenesis throughout phylogeny. The mechanisms that govern the creation of these complex structures are poorly understood but recent studies in Drosophila have established that sensory neuron morphology is defined by surprisingly diverse arrays of transcription factors. The identities of downstream genes and their orchestration by these transcriptional networks are largely unknown, however. Here, we propose to solve this problem by exploiting a powerful gene expression profiling method to identify targets of transcription factors that control morphogenesis of a model sensory neuron. In the nematode, C. elegans, a single pair of bilaterally symmetric neurons, PVDL and PVDR envelop the body with a regular array of highly branched dendrites that can be readily visualized with a fluorescent (GFP) marker in living animals. The PVDs respond to strong mechanical force ("harsh touch") to trigger a withdrawal reflex, a behavior indicative of nociceptive function. We have used a cell specific microarray profiling strategy to identify transcripts that are highly expressed in PVD neurons. Specifically, we will: (1) Use available mutants and RNAi clones to test transcription factor genes in this data set for roles in PVD dendritic morphogenesis; (2) Use the mRNA tagging method to reveal genes downstream of the conserved LIM- homeodomain protein, MEC-3, an established determinant of PVD dendritic branching and; (3) Extend this mRNA tagging strategy to at least five additional key transcription factor genes identified in Aim 1. These data sets of transcription factor target genes will provide a foundation for future studies that link transcriptional control to the cell biology of dendritic arborization. We believe that our approach represents a useful alternative to direct studies of mammalian tissues and moreover, that it offers a uniquely powerful strategy for identifying molecules with fundamental, conserved roles in nociceptor morphogenesis. PUBLIC HEALTH RELEVANCE: Specialized nerve cells that mediate the sensation of pain utilize an elaborate array of spaghetti-like sensory processes directly beneath the skin to detect noxious stimuli. To facilitate the identification of genes that govern the creation of these sensory networks, we propose a genetic approach in the nematode C. elegans, a simple model organism with a single pair of PVD sensory neurons that display a stereotypical and readily visible array of nerve processes. The results of this work should reveal similar human genes with crucial roles in the creation of sensory neuron arrays and therefore potentially lead to treatments for injuries or diseases that disrupt human sensory neuron development.