Antarctic Fish and MicroRNA Control of Development and Physiology

冰鱼生活在寒冷的南极海域，具有独特的特征，如没有红细胞，心脏增大，血管直径大，骨密度低，脂肪滴扰乱肌肉。这些特征对其他动物是有害的。在生活在温暖水域的哺乳动物和鱼类中，与这些特征有关的器官的发育受到编码特定蛋白质的基因的调节，但蛋白质的产生速度往往受到称为microRNAs(MiRNAs)的短RNA分子的调节。编码蛋白质的基因必须首先制作一个RNA拷贝，而实际的蛋白质是由这个RNA拷贝中间产物组成的。MiRNAs通过与RNA中间体结合并中断其蛋白质的生产来调节蛋白质的量。MiRNAs与RNA的结合强烈依赖于温度。在生活在淡水冰点以下的海洋中的南极鱼类中，还没有研究过miRNAs对基因的调控。该项目将比较南极鱼和暖水鱼中的miRNA调节1)，以了解miRNAs如何在持续寒冷中调节基因表达；以及2)在没有红细胞、心脏增大和骨密度降低的南极冰鱼中，与含有红细胞、正常心脏和致密骨骼的密切相关的南极鱼相比。该项目将对国内和全球的科学和社会产生广泛的影响。首先，这将是对南极鱼类基因调控重要因素(MiRNAs)的首次研究，这是整个南大洋生态的重要组成部分，并将揭示这些鱼类可能如何应对南极水域变暖。其次，它将通过在南极野外季节期间举行视频会议，并在研究实验室主办学生对南极鱼类的调查，将南极科学带给当地另类市中心高中的未被充分代表的高中生。MicroRNAs(MiRNAs)是温带动物发育和生理调节基因表达的关键转录后调节因子。虽然miRNAs通过与信使RNAs(MRNAs)结合来发挥作用，这是一个对温度高度敏感的过程，但迄今尚未在南极动物中研究miRNAs，包括在南大洋占主导地位的无尾类鱼类。本项目将比较1)南极和温带鱼类的miRNA调节，以了解miRNA调节在适应持续寒冷中的作用；以及2)底栖、密骨、红血的Nototheniods和高浮力、嗜骨、白血的银鱼，以了解在血红蛋白基因和红细胞的丧失、心脏和血管增大的起源以及由于骨骼矿化减少和脂肪沉积增加而引起的浮力增加的进化之后，专门器官中的miRNA的调节。目的1是验证南极鱼类为应对持续寒冷而进化出与miRNA相关的基因组专门化的假说。该项目将把四种南极有线虫与两种温带有线虫和两种温带实验室物种进行比较，以检验以下假设：(A)南极鱼类通过失去祖先基因和/或获得新基因来进化miRNA基因组图谱，(B)表达与耐寒有关的miRNAs，以及(C)通过改变miRNA基因表达来响应温度变化。目的2是为了验证一种假设，即从红血海底栖息的祖先进化而来的冰鱼伴随着一种改变的miRNA基因组谱系、序列和/或表达。该项目将检验以下假设：(A)银鱼中的miRNAs在序列上和/或在银鱼专门化中的表达上进化，包括头肾(红细胞的起源)；心脏(血管系统的变化)；头盖骨和胸带(骨密度降低)；以及骨骼肌(脂肪沉积)，以及(B)根据对斑马鱼miRNAs同源基因的功能测试确定，在银鱼专业化中进化的miRNAs具有与其在银鱼中衍生的角色有关的祖先功能。该程序将利用高通量转录和新型软件分离、测序和确定miRNAs和mRNAs的表达。结果将显示在被推向生理极端的脊椎动物中，microRNA系统是如何进化的，并为全球变暖最快地区的关键物种的前景提供洞察力。

Icefish live in frigid Antarctic seas, and have unique traits such as the absence of red blood cells, enlarged hearts, large diameter blood vessels, low bone mineral densities, and fat droplets that disrupt their muscles. These features would be harmful in other animals. In mammals and fish inhabiting warm waters, development of organs involved in these traits is modulated by genes that encode specific proteins, but the rate of protein production is often regulated by short RNA molecules called microRNAs (miRNAs). Genes that code for proteins must first make an RNA copy, and the actual protein is made from this RNA copy intermediate. MiRNAs regulate the amount of protein that is made by binding to the RNA intermediate and interrupting its production of protein. Binding of miRNAs to RNA depends strongly on temperature. Regulation of genes by miRNAs has not been studied in Antarctic fish, which live in seas with temperatures below the freezing point of fresh water. This project will compare miRNA regulation 1) in Antarctic fish vs. warm-water fish to learn how miRNAs regulate gene expression in constant cold; and 2) in Antarctic icefish with no red blood cells, enlarged hearts, and reduced bone density vs. closely related Antarctic fish containing red blood cells, normal hearts, and dense bones. The project will have broad impacts to science and society nationally and globally. First, this will be the first study of important factors in gene regulation (miRNAs) in Antarctic fish, which are an essential component of the entire ecology of the Southern Ocean, and will shed light on how these fish might respond to the warming of Antarctic waters. Second, it will bring Antarctic science to under-represented high school students at a local alternative downtown high school by conducting video conferences during the Antarctic field seasons and hosting student investigations of Antarctic fish in the research laboratory. microRNAs (miRNAs) are key post-transcriptional regulators of gene expression that modulate development and physiology in temperate animals. Although miRNAs act by binding to messenger RNAs (mRNAs), a process that is strongly sensitive to temperature, miRNAs have yet not been studied in Antarctic animals, including Notothenioid fish, which dominate the Southern Ocean. This project will compare miRNA regulation in 1) Antarctic vs. temperate fish to learn the roles of miRNA regulation in adaptation to constant cold; and in 2) bottom-dwelling, dense-boned, red-blooded Nototheniods vs. high buoyancy, osteopenic, white-blooded icefish to understand miRNA regulation in specialized organs after the evolution of the loss of hemoglobin genes and red blood cells, the origin of enlarged heart and vasculature, and the evolution of increased buoyancy, which arose by decreased bone mineralization and increased lipid deposition. Aim 1 is to test the hypothesis that Antarctic fish evolved miRNA-related genome specializations in response to constant cold. The project will compare four Antarctic Notothenioid species to two temperate Notothenioids and two temperate laboratory species to test the hypotheses that (a) Antarctic fish evolved miRNA genome repertoires by loss of ancestral genes and/or gain of new genes, (b) express miRNAs that are involved in cold tolerance, and (c) respond to temperature change by changing miRNA gene expression. Aim 2 is to test the hypothesis that the evolution of icefish from red-blooded bottom-dwelling ancestors was accompanied by an altered miRNA genomic repertoire, sequence, and/or expression. The project will test the hypotheses that (a) miRNAs in icefish evolved in sequence and/or in expression in icefish specializations, including head kidney (origin of red blood cells); heart (changes in vascular system), cranium and pectoral girdle (reduced bone mineral density); and skeletal muscle (lipid deposition), and (b) miRNAs that evolved in icefish specializations had ancestral functions related to their derived roles in icefish, as determined by functional tests of zebrafish orthologs of icefish miRNAs in developing zebrafish. The program will isolate, sequence, and determine the expression of miRNAs and mRNAs using high-throughput transcriptomics and novel software. Results will show how the microRNA system evolves in vertebrate animals pushed to physiological extremes and provide insights into the prospects of key species in the most rapidly warming part of the globe.