AN ABSTRACT OF THE DISSERTATION OF Prarthana Shankar for the degree of Doctor of Philosophy in Toxicology presented on February 19, 2021. Title: Elucidating the Signaling Events Downstream of Aryl Hydrocarbon Receptor Activation in Zebrafish Abstract approved: _____________________________________________________ Robyn L. Tanguay All animals have developed the critical ability to detect, respond to, and detoxify a large array of environmental chemicals and stressors that can cause adverse health effects. Important examples of landmark contaminants around the world are polycyclic aromatic hydrocarbons (PAHs) and dioxins, both of which can act via the aryl hydrocarbon receptor (AHR), a protein receptor and ligand activated transcription factor. The AHR is conserved across multiple phyla, is required for proper development, and mediates the adverse developmental effects of several PAHs across vertebrates, including humans. While a plethora of research has been conducted investigating PAH toxicity, significant challenges still persist around our lack of understanding of the diversity in toxicity mechanisms, especially due to our narrow focus on select PAHs and few biomarkers. Our knowledge of AHR-regulated mechanisms of PAH toxicity is still in its infancy. The zebrafish (Danio rerio) is an established model organism in toxicological and biomedical sciences that is well-suited for investigating AHR-regulated biological processes, especially due to the presence of a functional ortholog (AHR2) of the mammalian AHR. To this end, the overall objective of my dissertation is to leverage the zebrafish model to characterize and classify PAHs, and further our understanding of the downstream signaling events upon AHR activation. To achieve this, I first compiled a comprehensive review (Chapter 2) spanning twenty years of AHR research in zebrafish. The review demonstrates the magnitude of research that has been conducted navigating the complexity of AHR signaling as it relates to zebrafish exposure to PAHs and other xenobiotic AHR ligands, such as 2,3,7,8-Tetrachlorodibenzodioxin (TCDD). The chapter identifies significant knowledge gaps such as our lack of understanding of how different PAHs differentially alter the AHR signaling cascade and AHR’s crosstalk with other signaling pathways. To answer these questions, I conducted two studies (Chapters 3 and 4) that leveraged large compendiums of RNA sequencing data collected from developing zebrafish exposed to a diverse array of chemicals. Chapter 3 compares 16 individual PAHs and couples transcriptomic and developmental toxicity data to both characterize and classify PAHs. Using the Context Likelihood of Relatedness algorithm, I identified two major groups of PAHs, one being more developmentally toxic, predominantly activating AHR2, and leading to transcriptional profiles that had both similarities and differences. One important finding was that the expression of cyp1a (an AHR-dependent phase I metabolic enzyme commonly used as a biomarker for AHR activation) was more indicative of transcriptional profiles and not developmental toxicity phenotypes, suggesting the need for additional biomarkers that can better predict toxicity outcomes. Chapter 3 leverages a novel gene co-expression approach in zebrafish to compare and contrast PAHs and TCDD (AHR2 Activators), along with a large array of diverse flame retardant chemicals (FRCs). I found that the AHR2 Activators and FRCs localized to distinct regions of the network, with the FRCs associated with broad neurobehavioral and vascular developmental processes. On the contrary, the AHR2 Activators localized to one region of the network that was primarily associated with chemical metabolism processes. Guided by the network, I identified several new members of the AHR2 signaling pathway that should be investigated in future research. Both Chapters 3 and 4 leveraged co-expression analyses to narrow down on several potentially important candidates for biomarkers of PAH exposure. In Chapter 5, I investigate one such AHR-regulated gene, wfikkn1 (WAP, Follistatin/Kazal, Immunoglobulin, Kunitz And Netrin Domain Containing 1). I found that the expression of wfikkn1 was AHR2-dependent in developing zebrafish exposed to TCDD. Using a combination of CRISPR-Cas9 to generate a mutant zebrafish line, and transcriptomic, proteomic, and high-throughput neurobehavioral assays, I discovered that this AHR2- regulated gene has potentially important roles in muscle developmental processes. Upon exposure to TCDD, wfikkn1 mutant zebrafish had a significantly altered transcriptome and larval neurobehavioral processes compared to wildtype zebrafish, suggestive of its additional role in AHR-regulated neurodevelopment. These data also highlight potential crosstalk between AHR and other signaling pathways such as Estrogen Receptor signaling via wfikkn1, which should be investigated in future studies. Overall, within this dissertation, I leveraged the advantages of the zebrafish model organism to characterize and classify PAHs by their transcriptomic and developmental toxicity responses. I also investigated the functional role of a novel AHR-dependent gene that might contribute to AHR-regulated toxicity responses. Increasing our knowledge of biological processes and mechanisms associated with AHR activation can help us better understand how PAHs cause toxicity, which will lead to more guided risk assessment measures and opportunities for susceptibility research. Copyright by Prarthana Shankar February 19, 2021 All Rights Reserved Elucidating the Signaling Events Downstream of Aryl Hydrocarbon Receptor Activation in Zebrafish by Prarthana Shankar A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented February 19, 2021 Commencement June 2021 Doctor of Philosophy dissertation of Prarthana Shankar presented on February 19, 2021 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. Prarthana Shankar, Author ACKNOWLEDGEMENTS Quoting Aristotle’s words, “The more you know, the more you realize you don’t know.” This has been the essence of my incredible graduate school learning experience at Oregon State University. As my time here comes to an end, I remind myself that this journey would not have been possible without the support of many, many people. First and foremost, I would like to extend my sincere gratitude to my graduate mentor, Dr. Robyn Tanguay, for her patient guidance throughout my time here. In addition to the immense scientific knowledge I have gained from her, I have also learned to work systematically yet efficiently, while never being the roadblock for anyone. I would like to thank all previous and current members of my committee: Dr. Kim Anderson, Dr. Thomas Sharpton, Dr. Susan Tilton, and Dr. Jaga Giebultowicz, and Dr. Kate Shilke for their critical feedback and guidance. I also extend my gratitude to the entire faculty and administrative staff in the Department of Environmental and Molecular Toxicology, particularly Mary Mucia, Cheyenne Pozar, and Joey Carson for their continued help and support. All the members of the Sinnhuber Aquatic Research Laboratory have been a part of my academic family for the last 4.5 years and I would not have succeeded without their help. I extend my sincere thanks to Dr. Lisa Truong for being my life guru, for always being available, for providing me with much-needed reality-checks, and for teaching me to make (almost) perfect figures. I gratefully acknowledge Jane LaDu for being my favorite bench- mate and for patiently putting up with all my questions and requests over the last few years. I will definitely miss our IHC conversations by the Keyence. Special thanks to Dr. Gloria Garcia for taking me on as a mentee when I first started as a graduate student, and for showing me the wonders of molecular biology bench work. I would also like to acknowledge Carrie Barton for all her help with fish husbandry, Kimberly Hayward for patiently dealing with my million complicated ZAAP requests, Dr. Michael Simonich for his incredible support editing my manuscripts, and Connor Leong and Dante Perone for assistance with the juvenile and adult behavior studies. I thank Dr. Subham Dasgupta for all his help editing my dissertation and providing the much-needed jellybeans for all the long nights. A huge shout out to Brian Head for putting up with all my loooong experimental rants, for troubleshooting with me, and for being my forever dissertation buddy. Special thanks to other Tanguay Lab graduate and post-doctoral trainees, Joeshen Shen, Chenglian Bai, Lindsay Wilson, Yvonne Rericha, Dr. Mitra Geier, Dr. Mike Garland, Dr. Courtney Roper, and Dr. Delia Shelton, for all the help, guidance, lab get- togethers, and fun hallway conversations. Over the years, I have had the fortunate chance to collaborate with and be mentored by several incredibly knowledgeable and talented scientists. I would like to extend my gratitude to Dr. Ryan McClure for not only being the mastermind behind all of our network studies, but for also being the most easiest and approachable of collaborators to work with. I also thank Dr. Katrina Waters for always asking me the hard but right questions at my posters,