The role of circadian clocks in photoreceptor cell development, maintenance and function

项目摘要/摘要 在视网膜中，生理和功能的许多方面由昼夜节律(24小时)时钟和时钟控制 故障会影响信息处理和细胞存活。然而，在以下方面存在着根本的差距 了解时钟如何控制健康和患病视网膜组织的功能通路。续 这一差距的存在代表着一个重要的问题，因为在它被填补之前，对 生物钟故障和视网膜疾病之间的联系机制在很大程度上仍不清楚。 我们的长期目标是更好地了解生物钟是如何控制 视网膜中的视觉处理。这个项目的目标是确定生物钟如何在 光感受器(即视杆、视锥和表达Opn4/黑素蛋白的视网膜神经节细胞或ipRGC)控制着 这些细胞的开发、维护和/或功能。我们的中心假设是生物钟是 存在于大多数视网膜细胞类型中，每种细胞类型的时钟控制着视网膜发育的特定方面和 通过一条受限的时钟通路发挥作用。我们的中心假设是基于我们的 拥有该领域的初步数据和最新出版物。拟议研究的理由是通过 通过基因沉默时钟机制，特别是在一种光感受器细胞类型中，我们将能够将这一点联系起来 细胞类型的时钟与视网膜发育和功能相关的不同时钟通路。我们有 开发了新的转基因小鼠品系，并产生了强大的初步数据。我们将追求两个目标 具体目标：1)表征视网膜特异性和光感受器细胞类型特异性时钟缺陷小鼠 模型；以及2)在光感受器时钟的控制下识别候选基因和信号通路。 在第一个目标下，对视网膜组织和各种电生理和行为进行形态分析 方法将用于已经创建的条件性时钟不足的鼠标线。在第二个目标下， 我们将结合锥体、视杆或ipRGC的荧光辅助单细胞分选(FACS)、RNA测序 和分析、荧光RNA原位杂交(FISH)和免疫细胞化学。此外，我们 已经制定了一项计划，将RNAseq数据分析优先于几个感兴趣的特定生物过程。 这项拟议的研究具有重要意义，因为它有望垂直推进和扩大对 生物钟如何控制视网膜的发育和功能，并将提供关键的缺失信息 关于所涉及的时钟路径。最终，这样的知识有可能增加我们的 了解控制光感受器维护的一般规则以及导致 他们在老年性黄斑变性等退行性疾病中的功能障碍。

PROJECT SUMMARY/ABSTRACT In the retina, many aspects of physiology and function are controlled by circadian (24-h) clocks, and clock malfunction impinges on information processing and cell viability. However, there is a fundamental gap in understanding how clocks control functional pathways in both healthy and diseased retinal tissue. Continued existence of this gap represents an important problem because, until it is filled, understanding of the mechanisms that link circadian clock malfunction and retinal disorders will remain largely incomprehensible. Our long-term goal is to better understand how circadian clocks control development and maintenance of visual processing in the retina. The objective of this project is to determine how circadian clocks within photoreceptors (i.e. rods, cones and opn4/melanopsin-expressing retinal ganglion cells or ipRGCs) control the development, maintenance and/or function of these cells. Our central hypothesis is that circadian clocks are present in most retinal cell types and each cell type's clock controls specific aspects of retinal development and function through a restricted clock pathway. Our central hypothesis has been formulated on the basis of our own preliminary data and recent publications in the field. The rationale for the proposed research is that by genetically silencing the clock mechanism specifically in a photoreceptor cell type, we will be able to link this cell type's clock to distinct clock pathways associated with retinal development and function. We have developed new genetically modified mouse lines and generated strong preliminary data. We will pursue two Specific Aims: 1) Characterize retina-specific and photoreceptor cell type-specific clock-deficient mouse models; and 2) Identify candidate genes and signaling pathways under the control of the photoreceptor clocks. Under the first aim, morphological analysis of retinal tissue and a variety of electrophysiological and behavioral approaches will be used in the conditional clock-deficient mouse lines already created. Under the second aim, we will combine Fluorescence-Assisted single-Cell Sorting (FACS) of cones, rods or ipRGCs, RNA sequencing and analysis, Fluorescence RNA In Situ Hybridization (FISH), and immuno-cytochemistry. In addition, we have established a plan to prioritize RNAseq data analysis to a few specific biological processes of interest. The proposed research is significant because it is expected to vertically advance and expand understanding of how circadian clocks control retinal development and function and will provide critical missing information about the clock pathways involved. Ultimately, such knowledge has the potential to increase our understanding of the general rules governing the maintenance of photoreceptors and of the events leading to their malfunction in degenerative diseases such as age-related macular degeneration.