Study of Neuropeptide F Function in Drosophila

描述（申请人摘要）：最近的分子和遗传研究 表明神经肽Y（NPY）介导的信号通路， 在包括蠕虫和人类在内的动物中是保守的， 摄食行为然而，其调节机制尚不清楚。 这项计划的长期目标是了解果蝇的作用 神经肽F（NPF），一种人类神经肽Y同源物，在调节摄食行为中， 潜在机制及其与脊椎动物的功能关系 神经肽Y家族分子。果蝇是唯一的无脊椎动物模型 已经鉴定了NPY及其受体的同源物的生物体。使用 整体原位RNA杂交和免疫细胞化学，我们发现， dNPF在中枢神经系统和肠道中都有表达。 脑支配食管下、胸和腹神经节，因此可能 作为神经递质，调节大脑对进食行为的控制。我们 还发现糖的味觉刺激诱导了两个细胞中dNPF的表达， 幼虫食管下神经节的额外神经元。这个表达仍然是 糖戒断后长时间强，暗示dNPF介导碳水化合物 调节果蝇中枢神经系统。dNPF中肠表达通过食物摄取而增强， 因饥饿而减少。其中一种假定的dNPF受体表达于 幼虫的肠、脑和唾液腺。因此，dNPF也可以用作 一种肠道激素，协调各种动物的摄食相关活动， 组织中在本申请中，我们建议直接测试 dNPF在转基因果蝇中的应用，具体目标如下：1） 通过控制性转染构建功能获得性转基因果蝇幼虫 异位表达dNPF并分析其摄食行为; 2）克隆和鉴定dNPF基因， 使用UAStauGFP报告基因表征dNPF的顺式调节元件 系统; 3）通过靶向构建功能丧失型转基因幼虫， dNPF的功能敲除和确定脑的特定作用， 食管下神经节和中肠dNPF的摄食行为。这些研究 应该可以提供关于进食信号机制的一般性见解 在无脊椎动物以及包括人类在内的高等动物中的调节。

DESCRIPTION (applicant's abstract): Recent molecular and genetic studies indicate that a neuropeptide Y (NPY) mediated signaling pathway, which is conserved among animals including worms and humans, is involved in regulating feeding behavior. However, the mechanism for its regulation is not understood. The long term goal of this proposal is to understand the role of Drosophila neuropeptide F(NPF), a human NPY homolog, in modulating feeding behavior, the underlying mechanism(s), and its functional relationship to vertebrate neuropeptide Y family molecules. Drosophila is the only invertebrate model organism where homologs of NPY and its receptors have been identified. Using whole mount in situ RNA hybridization and immunocytochemistry, we showed that dNPF is expressed in both CNS and gut In larval CNS, dNPFergic neurons of the brain innervate subesophageal, thoracic, and abdominal ganglia, and may thereby act as neurotransmitters for mediating brain control of feeding behavior. We also found that gustatory stimulation by sugars induces dNPF expression in two additional neurons of larval subesophageal ganglion. The expression remains strong long after sugar withdrawl, implicating dNPF in mediating carbohydrate conditioning of fly CNS. The dNPF midgut expression is enhanced by food uptake, and reduced by starvation. One of the putative dNPF receptors is expressed in larval gut, brain and possibly salivary glands. Thus, dNPF may also function as a gut hormone that coordinates feeding associated activities in diverse tissues. In this application, we propose to directly test the specific roles of dNPF in transgenic flies by addressing the following specific aims: 1) Constructing gain-of-function transgenic Drosophila larvae through controlled ectopic dNPF expression and analyzing their feeding behaviors; 2) Cloning and characterization of cis regulatory elements of dNPF using UAStauGFP reporter system; 3) constructing loss-of-function transgenic larvae through targeted functional knockout of dNPF and determining the specific roles of brain, subesophageal ganglion and midgut dNPF in feeding behavior. These studies should provide general insights into the signaling mechanism for feeding regulation in invertebrates as well as higher animals including humans.