Determining the role of REV-ERBs in the SCN and liver circadian hierarchy

项目摘要/摘要 维持和同步整个身体的昼夜节律对于新陈代谢健康和 动态平衡。在哺乳动物中，下丘脑视交叉上核(SCN)起着昼夜节律的作用 调节器，但肝脏等器官拥有调节昼夜基因表达的细胞自主钟 以特定于组织的方式。REV-ERBα和β是核受体的重要组成部分 哺乳动物的分子时钟，使新陈代谢和昼夜节律同步。来自The Lazar的已发布数据 使用REV-ERBα/β肝细胞双基因敲除小鼠的实验室表明，有不同的集合 肝脏中具有不同程度细胞自主性的基因。事实上，数百个正常振荡的基因丢失了 在HepDKO条件下的节律性，但保留了几乎相同数量的昼夜振荡基因 肝脏的节律性。这意味着这些基因有非细胞自主的节律，由 来自SCN的信号。有几种建议的SCN派生的输出可以指示肝脏的节律性， 但通过迷走神经的肝总支(CHB)神经支配是一种研究较少的选择。迷走神经 神经是大脑和外周之间的一种既定的沟通方式，可以整合昼夜节律 进食/禁食状态的节律。然而，CHB在SCN和SCN之间传递昼夜节律信号的作用 肝脏还有待检查。因此，我假设来自SCN时钟的REV-ERB介导的信号是 对于调节保留在肝脏中的基因是必要的，而慢性乙肝作为一个整体 这些调控信号的传播者。具体目标1将描述肝脏生理学和 在SCN和肝脏中敲除REV-ERBS的REV-ERBS缺失小鼠模型的节律性 并进行昼夜节律和代谢表型实验。我也会收集 每隔3小时取自这些动物的组织，持续24小时，比较分子和基因调控表型 由使用基于下一代测序(NGS)的组学技术的REV-ERBS DKO产生。特定的 目标2将确定CHB在传递REV-ERB介导的昼夜节律信号中的作用 经迷走神经切断术(CHBx)诱导的小鼠慢性乙型病毒性肝炎。我将进行昼夜节律和代谢表型分析 每3小时收集一次组织，持续24小时进行实验，比较分子和基因的调控 基于NGS组学的CHBx的表型。我预测基因表达的节奏和 HepDKO小鼠的调节以及行为和代谢节律性将受到严重干扰 不含SCN Rev-ERBS，并对CHBx作出响应。这里概述的实验不仅提供了重要的 了解昼夜节律和代谢节律同步的进展，但提供了实质性的 利用Lazar实验室独特的REV-ERB DKO小鼠模型和他们的 在最先进的NGS工具方面的专业知识，以探索REV-ERB冥想的节奏性的作用。

PROJECT SUMMARY/ABSTRACT The maintenance and synchrony of circadian rhythms throughout the body is essential for metabolic health and homeostasis. In mammals, the hypothalamic suprachiasmatic nucleus (SCN) acts as the master circadian regulator, but organs such as the liver possess cell-autonomous clocks that regulate circadian gene expression in a tissue-specific manner. The REV-ERBα and β nuclear receptors are important components of the mammalian molecular clock that synchronize metabolic and circadian rhythms. Published data from the Lazar lab using a REV-ERBα/β hepatocyte double knock-out (HepDKO) mouse suggests that there are distinct sets of genes in the liver with varying degrees of cell-autonomy. Indeed, hundreds of normally oscillating genes lose rhythmicity under HepDKO conditions, but a nearly equal number of circadian oscillating genes retained rhythmicity in HepDKO livers. This implies that these genes have non-cell-autonomous rhythms dictated by signals from the SCN. There are several proposed SCN-derived outputs that can dictate hepatic rhythmicity, but innervation via the common hepatic branch (CHB) of the vagus nerve an understudied option. The vagus nerve is an established mode of communication between the brain and periphery that can integrate circadian rhythms of fed/fasting state. However, the role of the CHB in relaying circadian signals between the SCN and liver has yet to be examined. Therefore, I hypothesize that REV-ERB-mediated signals from the SCN clock are necessary for regulation of genes retained in HepDKO livers and that the CHB acts as an integral communicator of these regulatory signals. Specific Aim 1 will characterize hepatic physiology and rhythmicity in a mouse model lacking REV-ERBs in the SCN and liver by knocking out REV-ERBs in the SCN of HepDKO animals and performing circadian and metabolic phenotyping experiments. I will also collect tissues from these animals every 3 hours for 24 hours to compare molecular and gene regulatory phenotypes resulting from REV-ERBs DKO using next generation sequencing (NGS)-based -omics techniques. Specific Aim 2 will determine the role of the CHB in relaying REV-ERB-mediated circadian signals by severing the CHB through vagotomy (CHBx) in HepDKO mice. I will perform circadian and metabolic phenotyping experiments and collect tissues every 3 hours for 24 hours to compare molecular and gene regulatory phenotypes resulting from CHBx using NGS-based -omics. I predict that the rhythm of gene expression and regulation as well as behavioral and metabolic rhythmicity in HepDKO mice will be significantly disrupted without SCN REV-ERBs and in response to CHBx. The experiments outlined herein not only provide significant advances in the understanding of circadian and metabolic rhythm synchronization but provide substantial training opportunities for me by utilizing the Lazar lab's unique REV-ERB DKO mouse models and their expertise in state-of-the-art NGS tools to probe the role of REV-ERB-meditated rhythmicity.