Analysis of microRNA expression reveals convergent evolution of the molecular control of diapause in annual killifishes

Emanuel Barth¹Mario Baumgart²Luca Dolfi³Rongfeng Cui⁴Marco Groth²Roberto Ripa⁵Aurora Savino⁶Dario Riccardo Valenzano²Matthias Platzer²Manja Marz^1*Alessandro Cellerino^2*

• ¹Friedrich Schiller University Jena, Jena, Thuringia, Germany
• ²Leibniz Institute on Aging, Fritz Lipmann Institute (FLI), Jena, Germany
• ³Laboratory of Biology, Scuola Normale Superiore, Pisa, Italy
• ⁴Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong, China
• ⁵Max Planck Institute for Biology of Ageing, Cologne, North Rhine-Westphalia, Germany
• ⁶Human Technopole, Milano, Lombardy, Italy

Background: Diapause is a condition of developmental arrest in anticipation of adverse environmental conditions present in a large number of diverse taxa. Diapause is a key adaptation that enabled the colonization of ephemeral habitats subject by annual killifishes. Diapause can occur at three different developmental stages, but Diapause II (DII), which occurs during somitogenesis, is the primary point of developmental arrest. Physiologically, Diapause II is associated with the arrest of the cell cycle in G1 and deeply reduced oxygen consumption and protein synthesis. Diapause is not obligatory, and some embryos can go through an alternative developmental pathway into direct development. Diapause has evolved independently several times in killifish clades from Africa and South America, enabling identifying possible molecular determinants of diapause by comparative expression analysis. Here, we compare microRNA expression profiles of annual killifishes during DII with non-annual killifish in a comparable stage of morphological development. Results: We used smallRNA-Seq to quantify microRNA expression annual- and non-annual killifish species from three independent clades and from direct-developing embryos of the annual killifish Nothobranchius furzeri. We analyzed the expression of broadly conserved microRNAs and microRNAs that appear to have evolved in the killifish lineage.We found several microRNAs that showed convergent regulation in the three different clades, and for some microRNAs also a phenomenon of switch in the prevalent form between 3p and 5p or vice versa was noted. In addition, we detected a significant overlap between the microRNA regulation during diapause and aging.Particularly interesting is the regulation of the miR-430 family. These microRNAs represent the second most expressed microRNA family in the killifish embryos, and diapause is associated with dramatic down-regulation of the prevalent 3p form and up-regulation of the minor 5p form. Analysis of recently sequenced 45 low-coverage killifish genomes revealed that the miR-430 locus contains a lower number of copies in annual- as opposed to non-annual killifish. Conclusions: The Evolution of diapause is reflected in the convergent evolution of microRNA regulation in killifishes. A prominent feature is a dramatic down-regulation of miR-430 expression that could be partially explained with a reduction of its copy numbers in the genome.