University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2012 Regulation of Drosophila Rest: Activity Rhythms by a Microrna and Aging Wenyu Luo University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Family, Life Course, and Society Commons, Genetics Commons, and the Neuroscience and Neurobiology Commons Recommended Citation Luo, Wenyu, "Regulation of Drosophila Rest: Activity Rhythms by a Microrna and Aging" (2012). Publicly Accessible Penn Dissertations. 540. https://repository.upenn.edu/edissertations/540 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/540 For more information, please contact [email protected]. Regulation of Drosophila Rest: Activity Rhythms by a Microrna and Aging Abstract Although there has been much progress in deciphering the molecular basis of the circadian clock, major questions remain about clock mechanisms and about the control of behavior and physiology by the clock. In particular, mechanisms that transmit time-of-day signals from the clock and produce rhythmic behaviors are poorly understood. Also, it is not known why rest:activity rhythms break down with age. In this thesis, we used a Drosophila model to address some of these questions. We identified a pathway that is required downstream of the clock for rhythmic rest:activity and also explored the mechanisms that account for deterioration of behavioral rhythms with age. By investigating candidate circadian mutants identified in a previous genetic screen in the laboratory, we discovered a circadian function of a microRNA gene, miR-279. We found that miR-279 acts through the JAK/STAT pathway to drive rest:activity rhythms. These effects occur downstream of the clock and thus define a novel output circuit. In our work on aging, we found that although old flies have a longer period and an unstable phase, central clock function is robust. Thus, the deterioration also occurs downstream of the clock, in either the circadian output pathway or in the sleep homeostatic system. Indeed, a genetic manipulation that improves rhythms in old flies has been implicated independently in circadian rhythms and sleep. In addition, some environmental manipulations can strengthen sleep:wake cycles in old flies. In short, the studies described in this thesis contribute to our understanding of the molecular underpinnings of circadian output and suggest that such outputs are affected by aging. In the long term, these studies may contribute to finding treatments for clock dysregulation or age-related circadian disorders. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Amita Sehgal Second Advisor Thomas A. Jongens Keywords Aging, Circadian clock output, Circadian rhythms, JAK/STAT signaling, microRNA, PKA Subject Categories Family, Life Course, and Society | Genetics | Neuroscience and Neurobiology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/540 REGULATION OF DROSOPHILA REST:ACTIVITY RHYTHMS BY A MICRORNA AND AGING Wenyu Luo A DISSERTATION in CELL AND MOLECULAR BIOLOGY Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2012 Supervisor of Dissertation ____________________________________ Amita Sehgal, Ph.D. John Herr Musser Professor of Neuroscience Graduate Group Chairperson ____________________________________ Daniel S. Kessler, Ph.D. Associate Professor of Cell and Developmental Biology Dissertation Committee Nancy Bonini, Ph.D., Professor of Biology Thomas A. Jongens, Ph.D., Associate Professor of Genetics Zissimos Mourelatos, M.D., Associate Professor of Pathology and Laboratory Medicine David M. Raizen, M.D., Ph.D., Assistant Professor of Neurology ii DEDICATION This thesis is dedicated to my parents for their love and encouragement. iii ACKNOWLEDGEMENT I owe my deepest gratitude to my thesis advisor, Dr. Amita Sehgal, for providing advices, unreserved help and all possible support to my research and thesis writing, and for her patience, enthusiasm, kindness, and immense knowledge. I could not imagine having a better mentor for my PhD study. My special appreciation goes to Dr. Zhaohai Yang and Zhifeng Yue for generating the NE95-11-24 fly strain. Without them, the first part of this thesis would not have been possible. I would also like to thank my collaborators, Dr. Xiangzhong Zheng and Dr. Wen-Feng Chen, who made the second part of this thesis possible. I am very grateful to Dr. William Joiner, who introduced me into fly genetics and guided my rotation project. Also, I am indebted to many of my current and former colleagues in the Sehgal’s lab for their timely help with experiments and inspiring discussions. They include, but not limited to, Dr. Amanda Crocker, Dr. Shailesh Kumar, Dr. Yanshan Fang, Dr. Lei Bai, Dr. Mi Shi, Dr. Kanyan Xu, Dr. Dan Cavanaugh, Dr. Kyunghee Koh, Dr. Taichi Hara, Dr. Natalia Tulina, and Josephine Garban. It is my pleasure to thank my thesis committee, Dr. Thomas Jongens, Dr. Zissimos Mourelatos, Dr. Nancy Bonini, and Dr. David Raizen, for their helpful suggestions and comments during my study. Finally, my gratitude also goes to Dechun Chen, Kiet Luu, and Mallory Sowcik for their technical support, and Sue Kelchner for arranging lab events. iv ABSTRACT REGULATION OF DROSOPHILA REST:ACTIVITY RHYTHMS BY A MICRORNA AND AGING Wenyu Luo Thesis Advisor: Amita Sehgal, Ph.D. Although there has been much progress in deciphering the molecular basis of the circadian clock, major questions remain about clock mechanisms and about the control of behavior and physiology by the clock. In particular, mechanisms that transmit time-of- day signals from the clock and produce rhythmic behaviors are poorly understood. Also, it is not known why rest:activity rhythms break down with age. In this thesis, we used a Drosophila model to address some of these questions. We identified a pathway that is required downstream of the clock for rhythmic rest:activity and also explored the mechanisms that account for deterioration of behavioral rhythms with age. By investigating candidate circadian mutants identified in a previous genetic screen in the laboratory, we discovered a circadian function of a microRNA gene, miR-279. We found that miR-279 acts through the JAK/STAT pathway to drive rest:activity rhythms. These effects occur downstream of the clock and thus define a novel output circuit. In our work on aging, we found that although old flies have a longer period and an unstable phase, central clock function is robust. Thus, the deterioration also occurs downstream of the clock, in either the circadian output pathway or in the sleep homeostatic system. Indeed, a genetic manipulation that improves rhythms in old flies has been implicated v independently in circadian rhythms and sleep. In addition, some environmental manipulations can strengthen sleep:wake cycles in old flies. In short, the studies described in this thesis contribute to our understanding of the molecular underpinnings of circadian output and suggest that such outputs are affected by aging. In the long term, these studies may contribute to finding treatments for clock dysregulation or age-related circadian disorders. vi TABLE OF CONTENTS Title Page i Dedication ii Acknowledgement iii Abstract v Table of Contents vi List of Tables viii List of Figures ix Chapter I: Introduction 1 Figures 32 Chapter II: Regulation of Circadian Behavioral Output via a MicroRNA-JAK/ STAT Circuit 34 Abstract 35 Introduction 35 Methods 37 Results 42 vii Discussion 55 Acknowledgement 59 Tables and Figures 61 Chapter III: Old flies Have a Robust Central Oscillator but Weaker Behavioral Rhythms That Can Be Improved by Genetic and Environmental Manipulations 92 Abstract 93 Introduction 93 Methods 96 Results 100 Discussion 108 Acknowledgement 114 Tables and Figures 116 Chapter IV: Conclusions and Perspectives 145 Bibliography 155 viii LIST OF TABLES Chapter II Table 2-1. Alterations in Levels of miR-279 Disrupt Locomotor Rhythms in Flies 61 Table 2-2. miR-279 Acts through the JAK/STAT Pathway to Regulate Locomotor Activity Rhythms 63 Supplemental Table S2-1. Putative miR-279 Targets Predicted by Three Computational Algorithms 90 Chapter III Table 3-1. Characterization of Circadian Behavior of Aging Flies 116 Table 3-2. Reduced Pka-C1 Expression Partially Rescues Circadian Behavioral Rhythms 117 ix LIST OF FIGURES Chapter I Figure 1-1. A Schematic Diagram Showing Drosophila Cerebral Cells Expressing PERIOD 32 Figure 1-2. A Scheme Illustrating Current Knowledge about Molecular Machinery of the Drosophila Circadian Clock 33 Chapter II Figure 2-1. Overexpression of miR-279 Disrupts Locomotor Activity Rhythms 65 Figure 2-2. Deletion of miR-279 Reduces Behavioral Rhythmicity without Changing PER Cycling in Small LNvs 67 Figure 2-3. Unpaired (Upd) Is a Direct Target of miR-279 69 Figure 2-4. The JAK/STAT Signaling Pathway Is Required for Locomotor Activity Rhythms 72 Figure 2-5. JAK/STAT Signaling Is downstream of the Central Clock and the PDF Receptor (PDFR) 74 Figure 2-6. JAK/STAT Signaling and miR-279 Are in a Circadian Output Circuit 77 Supplemental Figure S2-1. Effects of Expressing miR-279 in Adults, Using tim and miR-279 GAL4 Drivers 79 Supplemental Figure S2-2. miR-279 Does Not Affect Clock Function, nor Is It Regulated by the Clock 81 Supplemental Figure S2-3. Identification of Upd as a Target of miR-279 83 Supplemental Figure S2-4. Upd-Expressing Neurons Are Necessary for Circadian Activity Rhythms 85 Supplemental Figure S2-5. Daily Expression of JAK/STAT Components in Fly Brains 87 Supplemental Figure S2-6. Small LNvs Send PDF-Containing Dorsal Projections to the Dendrites of Upd-Expressing Neurons 89 Chapter III Figure 3-1. Characteristics of Circadian Behavior in Young and Old Flies 118 Figure 3-2. The Circadian Morning Peak of Activity Is Weakened in Old Flies 120 Figure 3-3. The Molecular Circadian Clock Is Dampened in Peripheral Tissues of Old Flies 122 Figure 3-4.