UNIVERSITY OF CALIFORNIA, SAN DIEGO Phenotypic Characterization of Estrogen-related Receptor Gamma Mutant Mice A Dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biomedical Sciences by William Arthur Alaynick Committee in charge: Professor Ronald M. Evans, Chair Professor Lawrence S.B. Goldstein, Co-Chair Professor Bruce A. Hamilton Professor Samuel L. Pfaff Professor Nicholas Spitzer Professor Anthony Wynshaw-Boris 2006 © William Arthur Alaynick, 2006 All rights reserved ii The dissertation of William Arthur Alaynick is approved, and it is acceptable in quality and form for publication on microfilm: University of California, San Diego 2006 iii “No one can be a great thinker who does not recognize, that as a thinker it is his first duty to follow his intellect to whatever conclusions it may lead. Truth gains more even by the errors of one who, with due study and preparation, thinks for himself, than by the true opinions of those who only hold them because they do not suffer themselves to think.” John Stuart Mill This dissertation is dedicated to my parents, Susan and Michael, who have lent me their finest qualities. iv TABLE OF CONTENTS Signature Page ..............................................................................................................................................iii Dedication .....................................................................................................................................................iv Table of Contents ...........................................................................................................................................v List of Figures ...............................................................................................................................................vi Acknowledgements ....................................................................................................................................viii Vita ...............................................................................................................................................................xi Abstract of the Dissertation .........................................................................................................................xii Chapter 1. Introduction .......................................................................................................................1 Chapter 2. Generation and Initial Characterization of ERRγ-null Mice ..........................................80 Chapter 3. Motor and Central Pattern Generator Defects ................................................................89 Chapter 4. Cardiac Defects .............................................................................................................113 Chapter 5. Genomic Analyses ........................................................................................................132 Chapter 6. Conclusion ....................................................................................................................146 Appendix I. Cellular and Functional Classification of Target Genes ...............................................157 Appendix II. QPCR Results ...............................................................................................................166 v LIST OF FIGURES Chapter 1 Figure 1.1 The Nuclear Hormone Receptor Superfamily ..................................................................3 Figure 1.2 Structural Differences in ERRs .........................................................................................5 Figure 1.3 Nuclear Receptor Response Elements ..............................................................................7 Figure 1.4 Northern Blot Analysis of ERR Expression in Mouse ...................................................10 Figure 1.5 RT-PCR Analysis of ERRγ Expression ..........................................................................54 Figure 1.6 Detection of ERRγ in the CNS by X-gal Staining ..........................................................56 Chapter 2 Figure 2.1 Targeting Strategy and PCR Genotyping of ERRγ Mutant Mice ...................................81 Figure 2.2 ERRγ is Essential During Early Postnatal Life ...............................................................81 Figure 2.3 ERRγ-null Mice Die Neonatally from Inanition .............................................................82 Figure 2.4 Whole Mount X-gal Staining of E10.5 Embryos ............................................................83 Figure 2.5 Whole Mount X-gal Staining of E12.5 and E13.5 ERRγLacZ/LacZ Embryos ....................83 Figure 2.6 ERRγ is Expressed in the Parvalbumin-positive Cells of the DRG ................................84 Figure 2.7 ERRγ is Expressed in Muscle Spindles and Golgi Tendon Organs ................................85 Figure 2.8 ERRγ is Expressed in Slow Twitch Muscles ..................................................................86 Figure 2.9 Bone Development is Grossly Normal in ERRγ-null Mice ............................................87 Chapter 3 Figure 3.1 Canonical Markers of Ventral Spinal Cord Neuronal Development ..............................90 Figure 3.2 Electrophysiologic Recording of CPG Activity .............................................................91 Figure 3.3 Loss of ERRγ Disturbs Motor Function .........................................................................93 Figure 3.4 Increased Spontaneous Bursting in ERRγ-null Cord ......................................................96 Figure 3.5 Superimposed Pattern of Alternating Suppression: SPAS ..............................................98 Figure 3.6 Sarcosine Attenuates SPAS ............................................................................................99 Figure 3.7 Split-cord Attenuates SPAS ..........................................................................................101 Figure 3.8 Co-localization of Developmental Markers with ERRγ ...............................................103 vi Figure 3.9 ERRγ Defines a Novel Class of Ventral Interneurons ..................................................104 Figure 3.10 Characterization of ERRγ in Spinal Cord .....................................................................105 Figure 3.11 ERRγ-null Cords Have Reduced Contralateral Projections ..........................................106 Chapter 4 Figure 4.1 Electrocardiographic Analysis of ERRγ Mice ..............................................................115 Figure 4.2 ECG Reveals Prolonged QRS and QT with Loss of ERRγ ..........................................117 Figure 4.3 Altered ECG Morphologies in ERRγ-null Mice ...........................................................118 Figure 4.4 ERRγ-null Cardiomyocytes Have Reduced Sodium Current .......................................119 Figure 4.5 QPCR Analysis of Ion Channels in Heart .....................................................................120 Figure 4.6 Altered Gap Junction Expression in ERRγ-null Heart .................................................121 Figure 4.7 Sodium Channel SCN5A is Not Alternatively Spliced in ERRγ-null Heart ...............122 Figure 4.8 ERRγ-null Cardiomyocytes Have Normal Fatty-Acid Oxidation ..................................123 Figure 4.9 Reduced Ventricular Myocardial Mass in ERRγ-null Mice ...........................................124 Figure 4.10 ERRγLacZ/LacZ Mice Develop Fasting Lactic Acidosis ...................................................125 Figure 4.11 mtDNA:nDNA Alterations with Loss of ERRγ ............................................................126 Figure 4.12 Mitochondrial Substrate Utilization is Not Altered by Loss of ERRγ ..........................127 Figure 4.13 Key Electron Transport Proteins Are Not Altered in ERRγLacZ/LacZ Mice ....................128 Figure 4.14 Biochemical Analysis of ERRγLacZ/LacZ Mitochondria ..................................................128 Table 4.1. Electrocardiographic Values .........................................................................................117 Chapter 5 Figure 5.1 Complimentary Genomic Analysis ...............................................................................139 Chapter 6 Figure 6.1 Transcription Factors Controlling Mitochondrial Biogenesis ......................................155 vii ACKNOWLEDGEMENTS My first question of physiology was asking my mother, “If one were in water up to their nose, would their stuffy nose clear to allow breathing?” I don’t recall her answer, but I would like to think she enjoyed the question. I was aware of my own physiology prior to this. My father’s asking why his three- year-old son did not want to join him in the pool was answered by, “Because I can’t swim and I can’t breathe under water.” Why all this concern over breathing around water? Over the years I have asked my (nearly) tireless mother countless questions about physiology and medicine. I poured over my parents’ medical texts and have been keenly aware of Guinea Worm, Grave’s Syndrome, and the down-sides of a penetrating wound—all brought to life by the Technicolor hand of Frank Netter. When I finally gained the courage to attempt academics in 1995, I spent a formative six weeks with my father at his Emergency Medicine practice. This culminated with my suggesting that the monocularly blind patient before us might have had a penetrating wound to the optic nerve. This fortunately (?) turned