Transcriptomic analysis of the effect of dark-rearing on Astyanax mexicanus A thesis submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements of the degree of Master of Science in the Department of Biological Sciences of the College of Arts & Sciences by Connor R. Sears B.S. Biology, University of Kentucky, 2015 Committee: Dr. Joshua Gross, chair Dr. Eric Tepe Dr. Dennis Grogan March 4th, 2019 THESIS ABSTRACT This thesis examines the role of light in global RNA architecture. To accomplish this, the blind Mexican cavefish, Astyanax mexicanus, has been reared in total darkness for ~5 years. This species, which encompasses both river-dwelling and cave-dwelling morphotypes, is notable for the dramatically different phenotypes apparent in each of these morphotypes. The cavefish, in contrast to surface fish, exhibits a dramatic reduction of eyes and pigmentation in addition to an expansion of non-visual sensory systems, amongst other traits. Additionally, lighting conditions experienced by the cave and surface populations of fish differ dramatically between the extreme darkness of the cave and the diurnal light cycle of surface waters. This project has examined the effect of light on gene expression by investigating differential expression seen when a cavefish is restored to “Natural Conditions”—a comparative paradigm of lighting condition encompassing total darkness for cavefish and a diurnal light/dark cycle for surface fish. By examining the role of light in gene expression in this system, it expands the paradigm of how surface fish were able to successfully colonize the cave environment. Global gene expression is least similar under Natural Conditions by correlative comparison, and the number of genes differentially expressed between the two morphotypes is greatly expanded. This expansion of differentially expressed genes represents a subset of expression that is likely inducible by dark lighting conditions. The functional repertoire of this gene set implicates the circulatory and olfactory systems as potential down- stream targets of light-affected gene expression. Additional comparisons of gene expression were made at locations of QTL markers which have been implicated in eye or pigmentation reduction in the cavefish compared to the surface fish. Comparisons between cave and surface fish reared on Light/Dark Conditions are not the same as comparisons conducted on Natural (photic) Conditions when examined both globally and functionally. This comparison provides a greater understanding ii of how these inherently regressive traits may have been lost in the dark environment, identifying novel candidate genes mediating eye or pigmentation loss. In sum, examining the effect of light on gene expression in this system grants a better understanding of the complex genetic architecture accompanying life in the extreme cave environment. This study illuminates the need to understand the effect that rearing under light and non-natural conditions has on animals whose natural environment is one of total darkness. iii iv THESIS ACKNOWLEDGEMENTS As my graduate studies come to a close, I want to take the time to acknowledge the people that have been instrumental to my success here at the University of Cincinnati. First and foremost, I would like to thank my advisor, Dr. Joshua Gross. Through his instruction and guidance, and with much patience, he has provided me with the skills to succeed not only in the laboratory setting, but also in life. You have helped in invaluable and incalculable ways, but most importantly you have helped me hone a critical eye to better my own work, and you have taught me how to be a kind and patient mentor. Thank you to Dr. Dennis Grogan, for the help and ideas you have provided along this journey. To Dr. Eric Tepe, thank you for your support along the last 2+ years, and for the opportunity to teach under your guidance. Thank you to the many other faculty members of UC, including (but not limited to) Drs. Josh Benoit, Dan Buchholz, and Stephanie Rollmann, who have encouraged me not only as a scientist but as a professional, and provided me with many opportunities to succeed. I would like to thank my family for pushing me to succeed and for always cheering for me in my studies and in my life choices. I would not have made it to the end of this degree without my mother, Debbie Sears, who has taught me to be a strong, independent woman and who has best demonstrated how to surround myself with people who support me unconditionally. Mom, I have watched you build a life that I admire, and you have been instrumental in my success in completing this thesis. I also wish to thank the entire Phillips family—my aunts, uncles, cousins, and grandparents—for being my allies, support system, and role models—few people are fortunate enough to have an extended family this amazing. A huge thank you goes to my father, Terry Sears and my grandmother Nina Sears, for their enthusiastic curiosity about my work. Dad, thank you v for your unwavering support of and confidence in my academic pursuits. You have always assumed my success and told me to shoot for the moon, without in any way believing that I will land amongst the stars. Both my oldest friends and the friends that I have made here have been essential to my success along this journey. To my lab-mates-turned-friends, Mandy Powers, Danny Berning, Tyler Boggs, Heidi Luc, and Amy Manning, thank you so much for your support, insightful comments, and honest desire to help me better my work. I cannot begin to tell you all how much I appreciate everything that you have given me and done for me during my time here. To Julianne Horn, my chosen sister, you have always believed in me and supported me, but seeing your drive first-hand as you ceaselessly pursued your dreams gave me more inspiration to pursue my own than you even know. To Cody Patterson, I truly do not know how I would have done any of this without you. Thank you for being my best friend and my partner through this wild journey and for indulging every crazy dream I have for the future. Lastly, and perhaps most importantly, thank you to my fur-babies Brody and Remy for their unconditional love—to you both, I promise more walkies now. Thank you to the many organizations who have generously supported this work through funding including the Wieman-Benedict Award, Department of Biological Sciences, University of Cincinnati to CRS and a grant from the National Science Foundation (DEB-1457630) to JBG. vi TABLE OF CONTENTS THESIS ABSTRACT ................................................................................................................... ii THESIS ACKNOWLEDGEMENTS .......................................................................................... v LIST OF TABLES AND FIGURES ............................................................................................ 1 INTRODUCTORY STATEMENTS ........................................................................................... 2 REFERENCES .............................................................................................................................. 6 Transcriptomic assessment of dark-reared Astyanax mexicanus cavefish reveals dramatic alterations to global RNA architecture ABSTRACT .............................................................................................................................. 11 INTRODUCTION .................................................................................................................... 12 METHODS ............................................................................................................................... 17 RESULTS ................................................................................................................................. 23 DISCUSSION ........................................................................................................................... 28 CONCLUSIONS ...................................................................................................................... 43 ACKNOWLEDGEMENTS ...................................................................................................... 44 REFERENCES ......................................................................................................................... 45 GENERAL CONCLUSIONS .................................................................................................... 59 REFERENCES ............................................................................................................................ 61 VISUAL MATERIALS .............................................................................................................. 62 TABLES ....................................................................................................................................... 75 vii LIST OF TABLES AND FIGURES Figure 1: Comparisons between morphotypes are most divergent under Natural Conditions Figure 2: More genes are under-expressed in cavefish, regardless of rearing conditions Figure 3: Rearing under Natural Conditions reveals novel changes to gene expression Figure 4: Enriched GO terms under Light/Dark Conditions reveal prototypic cave phenotypes Figure 5: Enriched GO terms under Natural Conditions reveal terms referring to circulatory and olfactory systems Figure 6: Vision related genes map closely to vision related QTL Figure 7: Pigmentation related genes map near pigmentation related QTL Figure 8: