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<Abstract Centered> an ABSTRACT of the THESIS OF AN ABSTRACT OF THE DISSERTATION OF Michael Austin Garland for the degree of Doctor of Philosophy in Toxicology presented on June 14, 2019. Title: Transcriptomic Approaches for Discovering Regenerative and Developmental Regulatory Networks in Zebrafish Abstract approved: _____________________________________________________________________ Robert L. Tanguay Zebrafish are capable of fully regenerating organs and tissue such as their caudal fin, which is similar to a human regrowing an arm or a leg. In contrast, most mammals including humans have a greatly reduced capacity for wound healing. The ability of zebrafish to undergo this regenerative process, called epimorphic regeneration, hinges on the capacity to form a blastema at the wound site. The blastema quickly recapitulates the developmental processes involved in complex tissue formation to restore lost or damaged tissue. One key mechanism for inducing blastema formation is global repression of genes involved in tissue differentiation and maintenance. Induction of repressive factors, such as microRNAs (miRNAs), are involved in reprogramming cells during epimorphic regeneration. The upstream mechanism by which zebrafish undergo epimorphic regeneration remains elusive. Furthermore, while focus is shifting toward regulatory RNAs such as miRNAs, the full complement of their repressive activities is unknown. We took a transcriptomics approach to investigating epimorphic regeneration and fin development. Parallel sequencing of total RNA and small RNA samples was performed on regenerating fin tissue at 1 day post-amputation (dpa). Most miRNAs had increased expression, consistent with global repression of genes involved in cell specialization during de-differentiation. We identified predicted interactions between miRNAs and genes involved in transcriptional regulation, chromatin modification, and developmental signaling. miR-146a and miR-146b are anti- inflammatory miRNAs that were predicted to target eya4, which is involved in chromatin remodeling and innate immunity. miR-132-3p and miR-21 were predicted to cooperate in repression Bone morphogenic protein (BMP) signaling antagonists. Depletion of miR- 489 and miR-92b-3p were predicted to be involved in expression of cell cycle regulators mycn and foxm1, as well as other transcription factors associated with regeneration. We also identified Hedgehog and Scatter factor signaling as possibly targets of miRNAs regulation during regeneration. Next, we performed microarray analysis on tissue exposed to the glucocorticoid beclomethasone diproprionate (BDP). We identified cripto-1 as a highly upregulated gene flowing BDP exposure. Morpholino oligo knockdown of cripto-1 permitted regeneration during BDP exposure. We were able to phenocopy the effects of BDP exposure by exposing zebrafish to an Activin signaling inhibitor, SB431542. Promoter analysis identified binding sites for genes that were differentially expressed in our data. BDP exposure of murine embryonic stem cells confirmed that glucocorticoid-mediated induction of cripto-1 is conserved in mammals. Finally, we investigated a unique caudal fin duplication, called x-fin, resulting from embryonic exposure an exogenous ligand of the aryl hydrocarbon receptor (AHR), benzo[k]fluoranthene (BkF). We determined that the phenotype is Ahr2-dependent. We also determined that it is independent of hepatic metabolism using a hepatocyte ablation model. RNA seq analysis of trunk tissue at four time points of revealed that BMP and Fibroblast growth factor (FGF) ligands were robustly induced. Enrichment analysis revealed similarities to fin regeneration processes as well as inflammation. Altogether, we leveraged transcriptomics as a useful tool for identifying mechanisms associated with tissue regeneration and development. ©Copyright by Michael Austin Garland June 14, 2019 All Rights Reserved TRANSCRIPTOMIC APPROACHES FOR DISCOVERING REGENERATIVE AND DEVELOPMENTAL REGULATORY NETWORKS IN ZEBRAFISH by Michael Austin Garland A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented June 14, 2019 Commencement June 2020 Doctor of Philosophy dissertation of Michael Austin Garland presented on June 14, 2019. 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 _____________________________________________________________________ Michael Austin Garland, Author ACKNOWLEDGEMENTS After five years, I have a lot to be thankful for. First, I would like to thank my mentor, Dr. Robert Tanguay, who has been there every step of the way and provided me with all the opportunities I needed to succeed. I would also like to thank my committee: Dr. Siva Kolluri, who supported me during my laboratory rotation and offered encouragement through the years; Dr. Susan Tilton, who counseled me on bioinformatics matters; Dr. Stacey Harper, who helped me better understand the chemical side of my projects; and Dr. Claudia Maier, who jumped in at the last minute as Graduate Council Representative and offered new perspectives. Importantly, I would like to thank my co-authors for making these works possible. In particular, due credit must go to Dr. Jill Franzosa, Dr. Mitra Geier, and Dr. Sumitra Sengupta for their roles in moving these projects forward. During my rotation in the Kolluri Lab, I had the privilege of working with Dr. Hyo Sang Jang, whose thoughtfulness and guidance has been unmatched. At SARL, I would like to thank Dr. Lisa Truong for her advice, consultations, and guidance. I greatly appreciate the fish room support from Carrie Barton and the microinjection/lab support from Jane La Du. I was also lucky enough to receive mentorship from Dr. Derik Haggard and Dr. Gloria Garcia, whose positive outlook and assistance in the lab was indispensible. I also thank Prarthana Shankar, Lindsay Denluck, Jamie Minick, Holly Dixon, Cassie Nix, Jeremy Larson, Joesen Shen, and Claudia Santillan for their camaraderie. To the fish room and HTS crew, formerly including Greg Gonnerman: Without you, this would have been a whole lot harder. Thank you for all your efforts and indulging my last-minute requests. I would also like to thank Dr. Sean Bugel and Dr. Josie Bonventre, whose friendship and adventures completely changed my experience in Oregon. At ALS, I was lucky enough to get to know Yvonne Chang, Dan Elson, Martin Pearce, Carolyn Poutasse, and everyone else in the department whose friendship and support I will never forget. I received immeasurable support from my friends and family. I always looked forward to catching up with Willie Levy (rest in peace, 1989-2018) and Thomas Winberry whenever I could make it home to Redding, which was always a breath a fresh air. I would not have been able to make my first steps into academics without the endless support and encouragement from my mom, dad, brother, sister in law, and my nieces. Last but not least, I would also like to thank Chomper and Ember, whose manuscript contributions were edited out before submission. CONTRIBUTION OF AUTHORS In all chapters, Dr. Robert Tanguay contributed to intellectual formulation, writing, and study design. Chapter 2: Michael Garland analyzed and interpreted the data, and prepared the manuscript. Jill Franzosa conceived the experimental design. Cheryl Dunham, Chris Sullivan, Jason Wendler, and Joseph Brown contributed to the RNA read processing and differential expression. Susan Tilton provided bioinformatics support. Chapter 3: Michael Garland performed bioinformatic analysis, interpreted results, and prepared the manuscript. Sumitra Sengupta designed/performed laboratory experiments and bioinformatics analysis, and prepared the manuscript. Lijoy Mathew performed laboratory experiments and provided editorial comments. Lisa Truong provided editorial comments. Jane La Du assisted with microinjections. Esther de Jong and Aldert Piersma performed the embryonic stem cell experiments. Chapter 4: Michael Garland and Mitra Geier designed/performed laboratory experiments and prepared the manuscript. Sean Bugel contributed to experimental design and provided editorial comments. Cheryl Dunham, Joseph Brown, and Susan Tilton provided bioinformatics support. TABLE OF CONTENTS Page Chapter 1 – Introduction .....................................................................................................2 Vertebrate limb development ............................................................................... 2 Zebrafish and Epimorphic regeneration ............................................................... 4 Repression of gene expression by miRNAs ......................................................... 7 Transcriptomics approaches for investigating regeneration ................................ 8 Dissertation objective ......................................................................................... 10 Chapter 2 – Parallel sequencing predicts novel regulatory miRNA-mRNA interactions during blastema formation in zebrafish .................................................................11
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