DEVELOPMENTAL EXPRESSION OF HEME DEGRADATION GENES IN ZEBRAFISH (DANIO RERIO) SUGGEST NOVEL ROLES IN HEMATOPOIESIS AND EYE DEVELOPMENT by ANDREW HOLOWIECKI MATTHEW J. JENNY, COMMITTEE CHAIR JOHN YODER CAROL DUFFY STEVE MARCUS PATRICK FRANTOM A DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biological Sciences in the Graduate School of The University of Alabama TUSCALOOSA, ALABAMA 2015 Copyright Andrew Holowiecki 2015 ALL RIGHTS RESERVED ABSTRACT Heme is an important cofactor for numerous proteins, including hemoglobin used for oxygen transport. However, in its unbound form, heme is highly toxic and can perpetuate the formation of harmful reactive oxygen species (ROS). Thus, the heme degradation pathway, an evolutionarily conserved enzymatic pathway, facilitates the metabolism of heme into excretable by-products that may also have significant biological functions. Heme oxygenase (HO-1, HO-2) degrades heme into biliverdin (BV). Subsequently, BV is converted into the potent antioxidant bilirubin (BR) by biliverdin reductase (BVRa or BVRb). The ability of BR to efficiently quench ROS through a recycling reaction mediated by BVR is of great interest with strong medicinal implications. In support of this argument, individuals with slightly elevated levels of BR, a condition known as Gilbert’s syndrome, have lower incidences of diseases related to oxidative stress, such as cardiovascular disease. The goal of this research is to characterize the transcriptional regulation of genes involved in the heme degradation pathway. Chapter 2 provides a thorough characterization of expression of both sets of the HO-1 and HO-2 paralogs, as well as BVRa and BVRb, during normal development and in response to oxidative stress. It also provides new data on the sex-specific differences in expression of these genes under control and stressful conditions. The observed quantitative changes in divergent gene expression provide strong initial support for subfunction partitioning of the HO paralogs. Chapter 3 presents qualitative data on the expression patterns of HO-1a, BVRa and BVRb during development, conclusively demonstrating spatiotemporal patterns that are consistent with hematopoiesis. ii Additionally, in vivo promoter analysis demonstrates specific expression of HO-1a in eye lens epithelial cells during development. Finally, Chapter 4 presents functional data on the roles of GATA-1, the master regulator of erythroid development, and NRF2a, the master regulator of oxidative stress, on the spatiotemporal regulation of HO-1a, BVRa and BVRb during normal development and in response to oxidative stress. Furthermore, all three genes are expressed in developmental tissues that are highly sensitive to oxidative stress. In Chapter 5, we describe alternative experimental approaches, suggest future studies, and discuss these results in context with the current knowledge base. iii DEDICATION I dedicate this study to: My wife Kristy who has supported me through this, completion of this goal would not have been possible without your patience, love, and understanding. My daughter Lilly, who has been with me throughout this entire process, from birth she has looked over my data sets, critiqued my figures, and provided spot on advice on how to handle stress. My daughter Georgia, who entered the family half way through this project, your enthusiasm for life is contagious and inspirational. Your comedic timing is spot on. I love how you always listen to me prepare for seminars and repeat back “big science words”. My new son, Henry, I hope you will be proud of me. My mother, whom has set an example of what it means to work hard: first shift, second shift, or third shift for nearly 40 years at a steel plant. You continued to do this to help me during my time in graduate school and to help support me and my family. Clearly I could not have done this without you. My father, who passed away during my first year here, you instilled a strong sense of responsibility in me. I understand that it is a privilege to get to pursue an education. Thank you. My mother-in-law and father-in-law who have provided support and guidance: You have put in many miles to visit us down here. Thank you. iv LIST OF ABBREVIATIONS AND SYMBOLS AHR aryl hydrocarbon receptor ALM anterior lateral mesoderm AP-1 activator protein-1 ARNT aryl hydrocarbon receptor nuclear translocator proteins bHLH PAS basic-helix-loop-helix Per-ARNT-SIM protein family BVR biliverdin reductase BVRa biliverdin reductase alpha BVRb biliverdin reductase beta BV biliverdin BV-IXα alpha isomer of biliverdin BV-IX-β beta isomer of biliverdin BV-IXδ delta isomer of biliverdin BV-IXγ gamma isomer of biliverdin BR bilirubin BR-IXα alpha isomer of bilirubin BR-IX-β beta isomer of bilirubin bZip basic leucine zipper DNA binding domain Cd cadmium v cDNA complementary deoxyribonucleic acid CHT caudal hematopoietic tissue CMP’s common myelo-erythroid progenitors CRISPRs clustered regularly interspaced short palindromic repeats CUL3 cullin3 CYP1A cytochrome-p4501A DA dorsal aorta DDC duplication, degeneration, and complementation model DIG digoxigenin EDCs endocrine-disrupting chemicals eGFP enhanced green fluorescent protein EHT endothelial-to-hematopoietic transition EMPs erythomyeloid progenitors ETS E26 transformation-specific family FIH-1 factor inhibiting hypoxia inducible factor 1α GRPX glutathione peroxidase GRX glutathione reductase GSH reduced glutathione GSSG oxidized glutathione Hif1α hypoxia inducible factor-1α HO heme oxygenase HO-1 heme oxygenase 1 HO-2 heme oxygenase 2 vi HOOH hydrogen peroxide HRE hypoxia response element HSCs hematopoietic stem cells HSF heat shock factor HSP heat shock protein ICM intermediate cell mass IDHc cytosolic isocitrate dehydrogenase IDHm mitochondrial isocitrate dehydrogenase INRF2 inhibitor of nuclear factor erythroid 2-related factor 2 ISVs intersegmental veins Keap1 kelch-like ECH-associated protein 1 KOH potassium hydroxide Le lens LTZ lens transition zone mCherry red fluorescent protein (“m” monomer conformation) MAPK mitogen-activated protein kinase MEPS Mass Embryo Production System MO morpholino MTF-1 metal-regulatory transcription factor 1 NAD nicotinamide adenine dinucleotide NADH nicotinamide adenine dinucleotide (reduced form) NADP nicotinamide adenine dinucleotide phosphate NADPH nicotinamide adenine dinucleotide phosphate (reduced form) vii NES nuclear export signal NLS nuclear localization signal NFκB nuclear factor–κB NNT nicatinamide nucleotide transhydrogenase NRF2 nuclear factor erythroid 2-related factor 2 OD o-dianisidine OP olfactory placode PBI posterior blood island PBS phosphate buffered saline PBST phosphate buffered saline with 0.1% tween PCBs polychlorinated biphenols PFA paraformaldehyde PL posterior lens PLM posterior lateral mesoderm PMBC primordial midbrain channel PPP pentose phosphate pathway PQQ pyrroloquinoline quinone PRDX peroxiredoxin qPCR quantitative polymerase chain reaction RBC red blood cell ROS reactive oxygen species RPE retinal pigment epithelium SOD superoxide dismutase viii S/T/K serine, threonine, tyrosine kinase TALENs transcription activator-like effector nucleases tBHQ tert-butylhydroquinone TCDD dioxin VEGF vascular endothelial growth factor UROD uroporphyrinogen decarboxylase WBC’s white blood cell XRE xenobiotic response element ix ACKNOWLEDGEMENTS First I would like to thank Dr. Matthew Jenny for taking a chance on me, providing guidance, and continually challenging me. Additionally, I would like to thank my committee members: Your dedication to teaching in the classroom played a big part in my success. Many of my ideas originated from classroom discussions. Finally, I would like to acknowledge members of the Jenny lab. x CONTENTS ABSTRACT .................................................................................................................................... ii DEDICATION ............................................................................................................................... iv LIST OF ABREVIATIONS AND SYMBOLS ...............................................................................v ACKNOWLEDGEMENTS .............................................................................................................x LIST OF TABLES ....................................................................................................................... xiv LIST OF FIGURES .......................................................................................................................xv 1 UTILIZING ZEBRAFISH (DANIO RERIO) AS A DEVELOPMENTAL AND TOXICOLOGICAL MODEL ........................................................1 1.1 Introduction ...............................................................................................................................1 1.2 Zebrafish as a Developmental, Toxicological, and Disease model ..........................................2 1.3 The Zebrafish Genome and Subfunction Partitioning ..............................................................4 1.4 Oxidative Stress and Redox Signaling ......................................................................................5 1.5 Antioxidant Defense Mechanisms ............................................................................................6 1.6 Transcriptional Regulators of Stress Response and their Roles in Development ....................8 1.7 NRF2 is the Master Regulator of Oxidative Stress ...................................................................8