Genetic Basis of Metabolic Evolution in the Cave Fish Astyanax Mexicanus The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:40050145 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Gene�c Basis of Metabolic Evolu�on in the Cave fish Astyanax mexicanus A disserta�on presented by Ariel Cacayuran Aspiras to The Division of Medical Sciences in par�al fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biological and Biomedical Sciences Harvard University Cambridge, Massachuse�s May 2018 ©2018 by Ariel Cacayuran Aspiras All rights reserved. iii Dissertation Advisor: Dr. Clifford Tabin Ariel Cacayuran Aspiras Genetic Basis of Metabolic Evolution in the Cave fish Astyanax mexicanus Abstract Organisms evolve to thrive in new environments. In spite of the role metabolism plays in adaptation, the genetic basis for metabolic variation remains poorly understood. Here we use independently derived populations of Astyanax mexicanus, a Mexican tetra, to interrogate the genetic basis of extreme metabolic variation between surface and cave adapted populations. In the cave environment, food is much more scarce than in nutrient-rich rivers. Cave populations of Astyanax mexicanus rely on sporadic input of food from outside the cave. As a result, cave fish populations have evolved a suite of metabolic traits such as: starvation resistance, hyperphagia, hyperglycemia, and insulin resistance. We examined starvation resistance and hyperphagia in cave fish, finding that all cave populations had elevated triglycerides and reduced weight loss while fasting. Intriguingly, only a subset of the cave populations exhibited hyperphagia. Using a candidate gene approach, we identified a conserved coding mutation in melanocortin 4 receptor (mc4r), specific to hyperphagic populations. A comparison of signaling efficiency demonstrate that the cave allele had reduced maximal response and reduced basal activity relative to the surface allele in vitro. We further show that the cave allele contributes to hyperphagia and starvation resistance in vivo. We also investigated variation in blood glucose regulation among cave and surface populations. We observed a dysregulation in blood glucose homeostasis and insulin resistance in a subset of cave populations. Those populations were carrying a mutation in insulin receptor that leads to reduced ligand binding in vitro and contributes to elevated blood glucose. Surface/cave hybrids carrying the allele are heavier than non-carriers and zebrafish genetically iv engineered with the cave allele are heavier and insulin resistant. Humans with an identical mutation have a severe form of insulin resistance and reduced life span. However, cave fish populations with this mutation have a similar life span to surface counterparts and do not accumulate advanced glycation end products typically associated with the progression of diabetes-associated pathologies. Together, these studies provide new insight on how complex metabolic systems such blood glucose and weight regulation evolve and vary among populations. v Table of Contents Abstract ........................................................................................................................................................ iii Acknowledgements ...................................................................................................................................... vi Dedication .................................................................................................................................................. viii Chapter 1 Introduction ................................................................................................................................. 1 Summary: .................................................................................................................................................. 2 Metabolic evolution: ................................................................................................................................. 2 The Cave fish System ................................................................................................................................ 7 Overview of the dissertation: ................................................................................................................. 10 Works Cited ............................................................................................................................................. 12 Chapter 2 melanocortin 4 receptor ............................................................................................................. 21 Abstract ................................................................................................................................................... 23 Introduction ............................................................................................................................................ 24 Results ..................................................................................................................................................... 25 Discussion................................................................................................................................................ 34 Works Cited ............................................................................................................................................. 38 Materials and Methods: .......................................................................................................................... 41 Chapter 3 insulin receptor ........................................................................................................................... 44 Methods .................................................................................................................................................. 59 Works Cited ............................................................................................................................................. 67 Chapter 4 Concluding Discussion ................................................................................................................ 70 Appendix 1: QTL on blood glucose.............................................................................................................. 81 Appendix 2: Chapter 2 supplemental data ................................................................................................. 96 Appendix 3: Chapter 3 extra data ............................................................................................................. 111 vi Acknowledgements I would not be here today without my mom, Rose Aspiras and my guardians, Michael and Eileen Harte. My mom emigrated from the Philippines when I was 2 to work as nurse. When I was 5, she brought me to United States, so that I could be raised in the land of opportunity. My mom worked at least two fulltime jobs to provide for me and for her family back in the Philippines. When I was 9, she passed away. I would have been sent back, but Michael and Eileen took me in and raised me. They were friends with my mom and had babysat for me in the past. But in truth, none of us were prepared for the challenges that awaited us. They had just sent their son James to college and were ready to have the nest be empty. Yet, they took me in anyways and raised me as one of their own. I’m eternally grateful to all three of my parents. I owe my love for scientific research to Dr. David Angelini. Dr. A is a smart, generous and patient mentor. He gave me confidence in my work and allowed me to explore any questions I found interesting. I need to also thank Dr. David Carlini and Dr. Daniel Fong from American University. They taught me how to be critical of my work, how to present my data, and the value of collaboration. I owe my love for cave biology to Dr. Fong. He took me caving, which initiated a collaborative side project with Dr. A. He also invited me to the weekly cave club margarita meetings with the premier D.C. area cave biologists Dr. David Culver and Dr. Bill Jeffery. Dr. A, Dr. Carlini and Dr. Fong all took an interest in me and went above and beyond what a mentor had to do. I wouldn’t have made it to Harvard without them. I knew I wanted to join Dr. Clifford Tabin’s lab well before my first rotation. While I was applying for PhD programs, I saw him give a talk at a Society of Developmental Biology meeting, when he was receiving a mentorship award. I just remember feeling amazed by his talk and feeling like I just wanted vii to be a part of his team. I’m so grateful that he allowed me to join his lab. Cliff has been such a generous and supportive mentor to me and I will miss being in his lab. I am also grateful to my DAC members, Dr. Matt Harris, Dr. Spiegelman, Dr. David Reich, and Dr. Naar. They have always provided me with great feedback and council. One of the key strengths of Cliff and Dr. Connie Cepko’s lab is its people. I am so thankful that I have spent my PhD surrounded by the most generous, intelligent lab mates. I want to especially thank Yash, Brian M, Brian R, Tyler K, Tyler H, Cem, John D, Megan, and Gene for being there to talk science