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.
