AN ABSTRACT OF THE DISSERTATION OF Sumitra Sengupta for the degree of Doctor in Philosophy in Toxicology presented on June 4, 2010. Title: Chemical and Biological Approaches to Identify Vertebrate Tissue Regeneration Pathways Abstract approved: ______________________________________________________________________ Robert L. Tanguay Abstract Numerous human conditions would be improved if therapies to encourage tissue regeneration were available. The goal of regenerative medicine is to encourage the body`s intrinsic ability to repair and restore tissues lost by disease, injury or aging. While certain vertebrates have the inherent capacity to regenerate, mammals do not. To study tissue regeneration we developed an early life stage zebrafish model. Through comparative global mRNA expression analysis in regenerating tissues isolated from adult caudal fins, hearts and larval fins, we discovered that raldh2 is induced across all regenerating platforms and its expression is critical during early stages of regeneration. Our studies determined the role of Wnt and Fgf in larval regenerating tissue, establishing the early life stage model as a powerful platform to study regeneration. Utilizing this model we developed a rapid in vivo larval regeneration assay to identify small molecule modulators of regeneration. Our initial screening of a 2000 member FDA approved drug library identified eight glucocorticoids (GCs) that inhibited regeneration. We chose beclomethasone dipropionate (BDP) as a representative glucocorticoid receptor (GR) ligand and performed mRNA expression analysis in BDP exposed fin regenerates to identify downstream effectors of GR that are required to block tissue regeneration. Bioinformatic analysis revealed that Cripto-1 mRNA expression increased significantly following BDP exposure. We hypothesized that misexpression of Cripto-1, an Activin inhibitor, was necessary for GR ligands to block tissue regeneration. Suppression of Cripto-1 by morpholino or retinoic acid exposure restored regeneration in the presence of BDP supporting our hypothesis. Our chemical biological screen also identified 21 glucocorticoids that activated GR but did not impact regeneration. We hypothesized that differences in ligand structure induced alternate GR conformational changes and these structural differences resulted in distinct regenerative activity. Docking studies identified that ligands with large substitutions at position17 induce an energetically stable active GR confirmation that correlates with the blocking of tissue regeneration. Our research identified novel GR ligands with cortisol backbones and bulky C17 substitutions that confirmed our hypothesis. Collectively, our results demonstrate the power of the larval zebrafish regeneration model to understand the pathways that permit tissue regeneration. ©Copyrighted by Sumitra Sengupta June 4, 2010 All Rights Reserved Chemical and Biological Approaches to Identify Vertebrate Tissue Regeneration Pathways by Sumitra Sengupta A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented June 4, 2010 Commencement June 2011 Doctor of Philosophy dissertation of Sumitra Sengupta presented on June 4, 2010. APPROVED: Major Professor, representing Toxicology Head of the Department of Environmental and Molecular Toxicology Dean of the Graduate School I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. Sumitra Sengupta, Author ACKNOWLEDGEMENTS I would like to thank Environmental and Molecular Toxicology department for giving me this opportunity to obtain an excellent training. My deepest gratitude to Dr. Robert Tanguay for being such an excellent mentor. I would like to thank him for being patient, guiding me and supporting me as and when required. I will always look up to him as a great source of inspiration. I also want to thank Dr. Lijoy K. Mathew who has guided me throughout my graduate school career by being my second mentor. I will always be thankful to Jane La Du for all her guidance intellectually and spiritually and for being my home away from home. Lisa Truong and Hao Truong has to be thanked for being such an excellent technical support. I would like to thank the entire Tanguay lab and my friends here at OSU who have always supported me and encouraged me throughout my training. I want to thank all my committee members, namely, Dr. Mark Leid, Dr. Siva Kolluri, Dr. Andrew Buermeyer and Dr. Emily Ho for serving in my graduate committee, helping me with the fellowship grants and guiding me throughout my graduate studies. I also want to thank Partiban Jesudason and Jessica Perry for helping me with various experiments. Most importantly, I want to thank my parents, my family members, friends, and Miss Debi Mukherjee who has been a great support for me all through my studies. Finally, I want to dedicate my thesis to Dr. Sukta Das, who has been an excellent teacher and source of inspiration and courage. CONTRIBUTION OF AUTHORS This dissertation is a collective effort of many collaborators from different universities and all Tanguay lab members. In Chapter 1, Dr. Lijoy K Mathew primarily prepared the manuscript. Jill A Franzosa helped with the retinoic acid exposure experiments. In Chapter 2, Dr. Lijoy K Mathew assisted me with the microarray analysis and quantitative- PCR experiments. Jane LaDu provided technical support for this project including assistance with morpholinos microinjections and animal care. Lisa Truong provided assistance with processing of the microarray data. Hao Truong provided technical assistance in the preparation of the manuscript. In Chapter3, William Bisson conducted the docking studies. Dr. Randall Peterson provided us with the 2000-member small molecule library for rapid throughput regeneration assay. TABLE OF CONTENTS Page CHAPTER 1 - Introduction................................................................................. 1 Tissue Regeneration .................................................................. 1 Molecular Signaling During Zebrafish Caudal Fin Regeneration ........................................................................ 5 Early Life Stage Fin Regneration Model ..................................... 8 Chemical Genetics and Regeneration ........................................ 9 Rapid in vivo Larval Regeneration Assay ................................. 10 Glucocorticoids ......................................................................... 11 Structure Activity Relationship and Regenerative Medicine ............................................................. 13 References ............................................................................... 15 CHAPTER 2 – Comparative Expression Profiling Reveals an Essential Role for Raldh2 in Epimorphic Regeneration ........................... 21 Abstract .................................................................................... 22 Introduction .............................................................................. 23 Materials and Methods ............................................................. 25 Results ..................................................................................... 31 Discussion ................................................................................ 40 References ............................................................................... 45 Acknowledgement .................................................................... 48 TABLE OF CONTENTS (Continued) Page CHAPTER 3 – Glucocorticoid-dependent Cripto-1 Induction Inhibits Epimorphic Tissue Regeneration ............................................. 77 Abstract .................................................................................... 78 Introduction .............................................................................. 79 Materials and Methods ............................................................. 82 Results ..................................................................................... 86 Discussion ................................................................................ 90 References ............................................................................... 93 CHAPTER 4 – The Regenerative Activity of Glucocorticoids is Dictated by Alternate Ligand Structures .............................. 134 Abstract .................................................................................. 135 Introduction ............................................................................ 136 Materials and Methods ........................................................... 138 Results ................................................................................... 143 Discussion .............................................................................. 148 References ............................................................................. 156 CHAPTER 5 – Conclusions ............................................................................ 165 LIST OF FIGURES Figure Page 2- 1. Comparative genomic analysis during zebrafish generation ................. 49 2- 2. In situ localization of raldh2 in the larval and adult regenerating fin tissue. ............................................................................................... 50 2- 3. Inhibition of RA signaling impairs wound epithelium and blastema