Purdue University Purdue e-Pubs Open Access Dissertations Theses and Dissertations January 2016 THE ROLE OF NADPH OXIDASE IN NEURITE OUTGROWTH AND ZEBRAFISH NEURODEVELOPMENT Cory J. Weaver Purdue University Follow this and additional works at: https://docs.lib.purdue.edu/open_access_dissertations Recommended Citation Weaver, Cory J., "THE ROLE OF NADPH OXIDASE IN NEURITE OUTGROWTH AND ZEBRAFISH NEURODEVELOPMENT" (2016). Open Access Dissertations. 1476. https://docs.lib.purdue.edu/open_access_dissertations/1476 This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. Graduate School Form 30 Updated 12/26/2015 PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By Cory J. Weaver Entitled THE ROLE OF NADPH OXIDASES IN NEURITE OUTGROWTH AND ZEBRAFISH NEURODEVELOPMENT For the degree of Doctor of Philosophy Is approved by the final examining committee: Daniel M. Suter Chair Yuk Fai Leung Christopher J. Staiger Joseph P. Ogas To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy of Integrity in Research” and the use of copyright material. Approved by Major Professor(s): Daniel M. Suter Daoguo Zhou 5/14/2016 Approved by: Head of the Departmental Graduate Program Date i THE ROLE OF NADPH OXIDASES IN NEURITE OUTGROWTH AND ZEBRAFISH NEURODEVELOPMENT A Dissertation Submitted to the Faculty of Purdue University by Cory J. Weaver In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy i August 2016 Purdue University West Lafayette, Indiana ii “For me, it is far better to grasp the Universe as it really is than to persist in delusion, however satisfying and reassuring.” - Carl Sagan, The Demon-Haunted World: Science as a Candle in the Dark ii iii ACKNOWLEDGEMENTS My project would not have been possible without tremendous support from many people. I first would like to acknowledge my graduate mentor, Dr. Daniel M. Suter. His support and guidance are irreplaceable. He pushed me to be a better scientist, mentor and communicator, and gave me the confidence to pursue a unique research path. I am very grateful and those lessons will remain with me throughout my life. I have also benefited from an excellent advisory committee. Dr. Yuk Fai Leung, my committee chair, graciously provided space in the animal facility to conduct the zebrafish work. Dr. Chris Staiger, Dr. Joseph Ogas and Dr. Paul Collodi provided critical discussion and poignant criticism to advance the quality of my research. I appreciate that each committee member was dedicated to my professional advancement and this was evident in their continued push for excellence in experimental quality and scientific communication. I would also like to thank all of the labs involved in the care and maintenance of the fish facility, but especially Deborah Biesemeier for her tireless effort to keep the iii facility in excellent condition. Without the dedication of all involved, my project and so many others would be impossible. I have had the privilege of being a teaching assistant for several courses during my time at Purdue. My teaching experience deepened my scientific knowledge, improved iv my communication and gave me the confidence to pursue a career in science. I would specifically like to thank Dr. Peter Hollenbeck, Dr. John Anderson, Dr. Susan Karcher, Dr. Laurie Iten, Dr. Clark Gedney and Deborah Anderson for giving me the opportunity to teach in their courses. I would like to extend my gratitude to former lab members Dr. Vidhya Munnamalai and Dr. Yingpei He for their mentoring and support. I would also like to thank our current post-doc and graduate students Dr. Ahmad Athammneh, Yuan Ren, Haley Roeder and Aslihan Terzi. It has been a privilege to be a part of such an insightful and talented group. Finally, none of this work would have been possible without the overwhelming support of my family. I especially want to extend my deepest gratitude to my wife. Her unwavering encouragement and confidence inspired me regardless of the circumstance. I admire you more than you know. It is a profound failure of language that all I can say is thank you. To my daughter, your intelligence and curiosity leave nothing out of your reach. I am proud of the person you are and excited to see the person you will become. iv v TABLE OF CONTENTS Page LIST OF TABLES .............................................................................................................. x LIST OF FIGURES ........................................................................................................... xi LIST OF ABBREVIATIONS .......................................................................................... xiii ABSTRACT .................................................................................................................... xvii CHAPTER 1. INTRODUCTION ..................................................................................... 1 1.1 Reactive oxygen species as cellular signals .......................................................... 1 1.2 Sources of ROS and downstream effectors .......................................................... 3 1.2.1 Redox regulation of actin ............................................................................. 5 1.2.2 Redox regulation of microtubules ................................................................ 7 1.3 Linking extracellular signaling to redox control of the cytoskeleton ................... 8 1.4 Introduction of the NOX isoforms ...................................................................... 10 1.4.1 NOX2 ......................................................................................................... 10 1.4.2 NOX1 ......................................................................................................... 18 1.4.3 NOX3 ......................................................................................................... 21 1.4.4 NOX4 ......................................................................................................... 22 1.4.5 NOX5 ......................................................................................................... 25 1.4.6 DUOX1/2 ................................................................................................... 25 v 1.5 Additional roles for NOX in the nervous system................................................ 27 1.6 Overview of experimental work ......................................................................... 28 CHAPTER 2. METHODS .............................................................................................. 32 2.1 General methods ................................................................................................. 32 2.1.1 Zebrafish housing and breeding ................................................................. 32 2.1.2 Mounting embryos in 1% low melting point agarose ...................................... 33 vi Page 2.1.3 Preparation of 7.5% N-Phenylthiourea stock ............................................. 34 2.1.4 Determination of zebrafish Nox domains ................................................. 34 2.2 Methods for Chapter 3 ........................................................................................ 35 2.2.1 Aplysia bag cell neuronal culture .............................................................. 35 2.2.2 Live cell imaging and neurite outgrowth analysis ..................................... 35 2.2.3 Immunolabeling and fluorescent imaging ................................................. 37 2.2.4 ROS imaging .............................................................................................. 38 2.2.5 Image Processing, Fluorescence Intensity and Colocalization Analysis ... 39 2.2.6 Statistical Analysis ..................................................................................... 40 2.3 Methods for Chapter 4 ........................................................................................ 40 2.3.1 Quantitative PCR ....................................................................................... 40 2.3.2 in situ hybridization ................................................................................... 42 2.4 Methods for Chapter 5 ........................................................................................ 45 2.4.1 Inhibitor treatment of live embryos ........................................................... 45 2.4.2 Immunolabeling ......................................................................................... 45 2.4.3 Live-imaging of transgenic lines ............................................................... 47 2.4.4 Hydrogen peroxide rescue ......................................................................... 47 2.4.5 gRNA design and synthesis ....................................................................... 48 2.4.6 gRNA injection .......................................................................................... 51 vi 2.4.7 Genotyping ................................................................................................. 52 2.4.8 Generation of stable mutant lines .............................................................. 54 2.4.9 Measurements of retinal and tectal