Mechanisms of Circadian Clock Output

描述(申请人提供)：我们对昼夜节律起搏神经元用来组织日常运动行为的信号机制感兴趣。近年来，在确定细胞自主时钟机制的分子基础方面取得了巨大的进展，这种机制是昼夜节律的基础。这一定义使人们能够识别大脑中的初级生物钟神经元。反过来，这提供了重新审视与行为的神经基础有关的基本问题的机会。这个研究项目以细胞、分子和遗传技术为特色，以了解信号是如何从初级起搏器传递到反应神经元的。此前，我们发现神经肽PDF是果蝇体内主要的昼夜节律递质。遗传证据表明，它是初级时钟细胞(LN-v神经元)使用的最重要的信号。我们将通过进行实验来扩展这些信息，这些实验旨在询问PDF在大脑中何时何地发出昼夜节律信号。进一步，我们将鉴定和鉴定PDF的受体(S)。对PDF受体的定义将允许描述哪些神经元件和哪些电路对昼夜节律递质做出反应。我们也可以开始问，由于PDF受体激活而调节的长期后果(信号转导通路)是什么。由于昼夜节律信号发生在如此长的时间基础上(许多小时)，我们假设PDF信号可能包括转录调节。我们已经确定了几个基因，它们的表达水平依赖于pdf，并且每天循环。我们现在建议展示它们作为PDF靶标在调节昼夜节律信号中的作用。最后，我们对果蝇头部的昼夜节律基因表达进行了广泛的全基因组微阵列筛选。我们已经确定了22个昼夜节律基因，其中15个是在昼夜节律生物学背景下的新基因。我们假设这些昼夜节律基因中的大多数参与了昼夜节律输出机制，并提出了解决这种可能性的实验方案。

DESCRIPTION (provided by applicant): We are interested in signaling mechanisms used by circadian pacemaker neurons to organize daily locomotor behavior. There has been tremendous progress in recent years in defining the molecular basis of the cell autonomous clockwork mechanism that underlies circadian timekeeping. That definition has permitted the identification of the primary circadian clock neurons within the brain. In turn, that has presented the opportunity to re-examine fundamental issues concerning the neural basis of behavior. This research program features cellular, molecular and genetic techniques to understand how signals are passed from the primary pacemakers to responsive neurons. Previously we showed that the neuropeptide PDF is the principal circadian transmitter in Drosophila. Genetic evidence indicates it is the most important signal used by the primary clock cells (the LN-v neurons). We will extend that information by performing experiments that are designed to ask where and when PDF signals circadian time within the brain. Further, we will identify and characterize the receptor(s) for PDF. Definition of the PDF receptor will permit a description of which neural elements and which circuits respond to the circadian transmitter. We can also begin to ask what are the long-term consequences (signal transduction pathways) that are modulated as a result of PDF receptor activation. Because circadian signaling occurs on such a long time base (many hours), we hypothesize that PDF signaling may include transcriptional regulation. We have identified several genes whose expression levels are pdf-dependent and which cycle on a daily basis. We propose now to demonstrate their role as PDF targets for the mediation of circadian signaling. Finally, we have performed an extensive genome-wide microarray screen for circadian gene expression in the Drosophila head. We have identified 22 circadian genes, of which 15 are novel in the context of circadian biology. We hypothesize that most of these circadian genes participate in circadian output mechanisms, and propose experiments to address this possibility