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

我的研究计划的长期目标是促进我们对视蛋白基因功能和脊椎动物光敏感性的理解。 我对非视觉视蛋白和眼外光敏感性特别感兴趣。我们已经知道视蛋白的表达不仅限于眼睛，其他组织也对光敏感，但将这两种观察结果联系起来的研究很少。此外，迄今为止还没有研究充分解释视蛋白库大小或表达结构域多样性。所有脊椎动物的祖先至少有23个视蛋白，5个视觉视蛋白和18个非视觉视蛋白。 这些剧目是大于似乎必要的色觉和昼夜节律。此外，视蛋白表达模式令人困惑;视蛋白在人皮肤、脑、肺、肝、心脏和肾中表达，而斑马鱼在鳃、心脏、脑和睾丸中表达视蛋白。视觉和非视觉视蛋白也在眼睛发育之前的斑马鱼胚胎中表达。这里提出的研究包括三种鱼类适应特殊光暴露的剧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。 我将致力于三个不同的项目。1)来自巴西亚马逊河流域的一种穴居地下鲶鱼--Phreatobius cisternarum的基因组测序。 该项目的目标是表征视蛋白库，从而识别适应永久黑暗生活的物种中保留的视蛋白。基因组序列也将被用来研究一般troglomorphism的分子基础，并将被用来量化人口结构，提供洞察巴西的这个地区的地下水生习性的连通性。2）我们将量化Anoplopoma fimbria（sablefish）发展过程中的视蛋白表达水平。该物种具有至少27种视蛋白，但生活在透光区以下（即，深度为200米至2,000米）。我们预测，这个大的视蛋白库主要是在开发过程中使用。我们将使用数字PCR来量化表达i）在深度捕获的成年人和ii）来自加拿大唯一的黑貂鱼水产养殖设施金鹰黑貂鱼个体的发育阶段（从受精卵开始）的多样性。3)我们将使用原位杂交来表征Platichthys stellatus，星状比目鱼大脑中的视蛋白表达。这是一个本地和丰富的物种，不像大多数其他比目鱼，发生在左侧和右侧的变种。 斑马鱼脑中的视蛋白表达是完全对称的（Davies等人，2015年）。 如果这些视蛋白是光传感器，我们预测比目鱼的表达域将是不对称的，因为比目鱼的大脑基本上是侧躺的。我们还预测，左侧和右侧的星空比目鱼将彼此不同。本研究涉及一名博士后、2名博士和10名本科生。

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.