Genetic Dissecting Serotonergic Phenotypes in C elegans

描述(申请人提供)：在终末分化过程中，每个神经元都必须选择一个递质。许多不同的神经元选择相同的递质，因此相同的神经递质可能具有许多不同的功能。尽管调控神经发生早期阶段的因素已经开始被阐明，但决定神经递质表型的遗传网络在很大程度上是未知的。本建议使用遗传学方法来确定决定线虫5-羟色胺能表型的基因，并研究5-羟色胺能神经元在代谢控制中的作用。本实验利用了5-羟色胺缺乏的蠕虫完全存活的优势，5-羟色胺能系统包括5类共9个神经元，为遗传和生理分析提供了简单性和多样性。首先，我们将识别调控5-羟色胺能神经元发育的基因。我们已经分离出两类影响特定5-羟色胺能神经元发育的突变。我们将评估这些突变是否阻止了神经元的生成，或者它们是否影响了最终神经元身份的特定方面。我们将克隆2-3个可能作用于末端分化的基因，并确定它们的表达模式。其次，我们将确定是否存在细胞特异性调节因子来指导5-羟色胺的合成。通过对5-羟色胺合成酶基因TPH-1启动子的分子解剖，我们将确定离散序列基序是否直接在特定神经元中表达TPH-1。我们将测试在部分5-羟色胺能神经元中表达TPH-1所需的POU转录因子UNC-86是否直接调节TPH-1。第三，我们将评估5-羟色胺能神经元在代谢控制中的作用。我们先前的研究表明，5-羟色胺调节转化生长因子-b和胰岛素神经内分泌途径，从而调节线虫的新陈代谢。我们将测试影响特定5-羟色胺能神经元的突变是否会影响特定的代谢调节途径，以及特定神经元中5-羟色胺生产的恢复是否可以挽救5-羟色胺缺陷突变体的代谢缺陷。第四，我们将使用遗传上位性分析来评估5-羟色胺和其他也调节新陈代谢的神经信号的相互作用。我们的目标是探索5-羟色胺信号如何整合来调节新陈代谢。5-羟色胺信号的调节异常与饮食失调、II型糖尿病和肥胖有关。鉴定决定线虫5-羟色胺能表型的基因，了解5-羟色胺能神经元在线虫代谢调节中的作用，将为研究人类5-羟色胺能神经元的发育和功能提供试剂。

DESCRIPTION (provided by applicant): During the terminal differentiation, every neuron must choose a transmitter. Many different neurons choose the same transmitter, thus the same neurotransmitter may subserve many different functions. Although factors regulating early stages of neurogenesis are beginning to be elucidated, the genetic network determining neurotransmitter phenotypes is largely unknown.This proposal uses genetic approaches to identify genes determining the serotonergic phenotype and to study the role of serotonergic neurons in metabolic control in C. elegans. The proposed experiments take advantages that serotonin-deficient worms are fully viable, and the serotonergic system, including 5 classes, a total of 9 neurons, offers both the simplicity and diversity for genetic and physiological analysis.First, we will identify genes regulating serotonergic neuron development. We have isolated 2 classes of mutations affecting the development of particular serotonergic neurons. We will assess if the mutations prevent the neuron generation, or if they affect particular aspects of the final neuronal identity. We will clone 2-3 genes likely acting in the terminal differentiation and determine their expression pattern. Second, we will determine if there are cell-specific regulators directing serotonin synthesis. Through molecular dissection of the promoter of the serotonin synthetic enzyme gene tph-1, we will determine if discrete sequence motifs direct tph-1 expression in particular neurons. We will test if the POU-transcription factor UNC-86 that is required for tph-1 expression in a subset of the serotonergic neurons directly regulates tph-1. Third, we will assess the role of serotonergic neurons in metabolic control. Our previous study indicated that serotonin regulates the TGF-b and insulin neuroendocrine pathways to modulate C. elegans metabolism. We will test if mutations affecting particular serotonergic neurons affect a specific metabolic regulatory pathway, and if restoration of serotonin production in particular neurons rescues the metabolic defects of serotonin deficient mutants. Fourth, We will use genetic epistasis analysis to assess the interaction of serotonin and other neural signaling that also regulate metabolism. The goal is to explore how serotonin signals are integrated to modulate metabolism.Dysregulations of serotonin signaling have been associated with eating disorders, type II diabetes, and obesity. Identification of genes that determine the serotonergic phenotype and understanding the role of serotonergic neurons in the regulation of metabolism in C. elegans will provide reagents to study the development and function of serotonergic neurons in human.