Using fish, and FISH, to interrogate the vertebrate opsin repertoire

使用鱼和 FISH 来探究脊椎动物视蛋白库

• 批准号: RGPIN-2019-05609
• 负责人: Taylor, John
• 金额: $ 2.04万
• 依托单位: University of Victoria
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=715619
• 关键词: Using; fish; FISH; interrogate; vertebrate

The longterm aim of my research program is to advance our understanding of opsin gene function and of vertebrate light sensitivity. I am especially interested in non-visual opsins and extra-ocular light sensitivity. We have known for sometime that opsin expression is not limited to eyes and that other tissues are light sensitive, but studies connecting these two observations are rare. In addition, no studies to date adequately explain opsin repertoire size or expression domain diversity. The ancestor of all vertebrates had at least 23 opsins, five visual opsins and 18 non-visual opsins. These repertoires are larger than would seem necessary for colour vision and circadian rhythm. In addition, opsin expression patterns are puzzling; opsins are expressed in human skin, brain, lung, liver, heart, and kidney and zebrafish express opsins in gills, heart, brain and testes. Visual and non-visual opsins are also expressed in zebrafish embryos well before eyes develop. The research proposed here includes the study of repertoire and expression variation in three fish species adapted to peculiar light exposures. Genome sequences, in-situ hybridization and digital PCR data will be collected and analyzed. I will work on three distinct projects. 1) Genome sequencing of Phreatobius cisternarum, a troglomorphic, subterranean catfish from the Amazonian region of Brazil. The goal of this project is to characterize the opsin repertoire and thereby identify opsins retained in a species adapted to life in perpetual darkness. Genome sequences will also be used to study the molecular basis of troglomorphism in general, and will be used to quantify population structure providing insight into the connectedness of subterranean aquatic habits in this region of Brazil, 2) We will quantify the levels of opsin expression during Anoplopoma fimbria (sablefish) development. This species has at least 27 opsins, but lives below the photic zone (i.e., at a depth of 200 m to 2,000 m). We predict that this large opsin repertoire is utilized primarily during development. We will use digital PCR to quantify expression in i) adults caught at depth and ii) a diversity of developmental stages (starting with fertilized eggs) in individuals from Golden Eagle Sable Fish, the only sable fish aquaculture facility in Canada. 3) We will use in-situ hybridization to characterize opsin expression in the brain of Platichthys stellatus, the starry flounder. This is a local and abundant species and, unlike most other flatfish, occurs in left- and right-sided morphs. Opsin expression in zebrafish brain is perfectly symmetrical (Davies et al., 2015). If these opsins are light sensors, we predict that expression domains will be asymmetric in flatfish, whose brains are essentially lying on their side. We also predict that left- and right-sided starry flounder will differ from one another. The research described engages a post-doc, 2 PhDs and 10 undergraduates.

我的研究计划的长期目的是提高我们对Opsin基因功能和脊椎动物光敏感性的理解。 我对非视觉运动和眼外光灵敏度特别感兴趣。我们已经有时知道Opsin表达不限于眼睛，并且其他组织对这两种观察结果的研究很少，但是很少见。此外，迄今为止，没有任何研究能够充分解释Opsin曲目的大小或表达域的多样性。所有脊椎动物的祖先至少有23个Opsins，5种视觉蛋白和18种非视觉蛋白酶。 这些曲目比色觉和昼夜节律所需的要大。另外，Opsin表达模式令人困惑。 Opsins在人的皮肤，脑，肺，肝脏，心脏，肾脏，肾脏和斑马鱼Express Opsins中表达。在眼睛发育之前，视觉和非视觉蛋白在斑马鱼胚胎中也表达。 此处提出的研究包括对适合特殊光暴露的三种鱼类的曲目和表达变化的研究。将收集和分析基因组序列，原位杂交和数字PCR数据。 我将研究三个不同的项目。 1）Phreatobius Cisternarum的基因组测序，这是来自巴西亚马逊地区的Troglomormormormormorphic，Subterranean cat鱼。 该项目的目的是表征Opsin曲目，从而确定在永久黑暗中适应生命的物种中保留的Opsins。基因组序列也将用于研究一般的trog型形态学的分子基础，并将用于量化种群结构，以洞悉巴西这个地区地下水生习惯的连接性，2）我们将量化烟丝蛋白酶表达的水平（Sobabria fimbria（Sableflia sablefis Fish））的发育。该物种至少有27个OPSIN，但生活在光学区域以下（即，深度为200 m至2,000 m）。我们预测，这种大型的Opsin曲目主要在开发过程中使用。我们将使用数字PCR来量化i）在深度捕获的成年人和ii）来自Golden Eagle Sable Fish的个体中的多种发育阶段（从受精卵开始），Golden Eagle Sable Fish，这是加拿大唯一的貂鱼水产养殖设施。 3）我们将使用原位杂交来表征星际平原（Starry The Stellatus）的大脑中的Opsin表达。这是一个局部和丰富的物种，与大多数其他扁平鱼不同，发生在左侧和右侧的形态中。 斑马鱼大脑中的Opsin表达完全对称（Davies等，2015）。 如果这些动蛋白是光传感器，我们预测表达域将在Flatfish中不对称，其大脑基本上躺在它们的侧面。我们还预测，左侧和右侧的星空比目鱼将彼此不同。该研究描述的是在大约2个博士学位和10名本科生中参与。