A simple animal model for characterizing gene regulatory control of an immune response by Eric Chun Hei Ho A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Medical Biophysics University of Toronto © Copyright by Eric Chun Hei Ho 2015 A simple animal model for characterizing gene regulatory control of an immune response Eric Chun Hei Ho Doctorate of Philosophy Department of Medical Biophysics University of Toronto 2015 Abstract A robust but tightly regulated system of innate gut immunity is required for maintaining gut homeostasis. Given the complex nature of the mammalian gut, a simple yet phylogenetically relevant model is useful to decipher the underlying molecular controls that sustain this balance. Here I use the purple sea urchin larva as a morphologically simple model for these studies. I approach this problem with three interrelated strategies: (1) Characterization of larval immunocytes based on morphology, cell behaviour and gene expression, (2) analysis of gene activity in a model of gut-associated immune response to bacteria and (3) regulatory analysis of selected response genes. I characterize five larval cell types that are active in gut immunity and develop a gut associated infection model using the bacterium Vibrio diazotrophicus. RNA-seq analysis of this model is used to identify immune genes that are differentially expressed over the course of infection. Member of the sea urchin IL-17 multigene family are identified as immune response genes. One subfamily, SpIL-17-I, is expressed early upon exposure to V. diazotrophicus ii in the mid- and hindgut and is then attenuated. BAC-based GFP transgenes and smaller reporter constructs recapitulate endogenous SpIL-17-I expression. Regulatory regions are defined upstream and downstream of the IL-17 coding sequence. Deletions of critical elements results in a decrease or absence of IL-17 expression but never ectopic expression or expression in the absence of immune stimulus. Downstream function of IL-17 was investigated by perturbing its receptors using Morpholino antisense oligonucleotides. In addition to physiological changes in the larva, expression of several immune effector genes are downregulated in IL-17R perturbed embryos. Collectively this work suggests that a core network of regulatory circuitry is shared with the vertebrates in the gut associated immune response even in this simple larval form. This can then form the basis for more elaborate network analysis of gut immunity in future studies using this model. iii Acknowledgments First of all, I would like to express my deepest gratitude to my supervisor, Dr. Jonathan Rast, for providing me with this wonderful opportunity. I am especially thankful for the mentorship and guidance that he has provided over this long journey. He has instilled many invaluable scientific knowledge as well as life lessons that will undoubtedly have a profound effect for the rest of my life. I would also like to thank my committee members, Dr. Phillip Poussier and Dr. Richard Wells, for providing invaluable guidance throughout the years. Their ideas and critiques have helped improve the scope and direction of my project. Members of the Rast lab, both past and present, have been instrumental in the completion of my thesis as well as making my experience more enjoyable. Firstly, I would like to express my deepest appreciation to Dr. Kate Buckley for all her help; without her expertise and advice, I would not have been able to finish my project. Other members of the lab that I would specifically like to thank are Dr. Cynthia Solek for her tutorage during the start of my PhD, Catherine Schrankel for her friendship and keeping the lab lively, Nick Schuh for bringing a different prospective, and Dr. Casey Wang for being the motherly figure that every labs need (as well as being the in-situ guru). I have met many friends along the way. I would like to thank Linda for all her help and friendship; Elena and Mei for all their silly moments, Samantha for all her advice, and Shane for our lively conversations. My family has been a tremendous source of support throughout my PhD studies. I would like to thank my parents for being so helpful and understanding. They were always there, good iv or bad, throughout my whole life. I will forever be grateful for all their help. Without them, I would not have been able to achieve the things that I have done in my life. Also, I would like to thank my sister, Cindy. She is always there to help me get out of “interesting” situations that I somehow get myself into. Lastly, I would also like to thank Aunt Catherine for all the caring that she has shown towards me and my wife. Finally, I would like to thank my wife Loksum. I will always treasure our lively scientific discussion which helped keep the project interesting. I would not have been able to complete this thesis without her love and encouragement. v Table of Contents Acknowledgments .................................................................................................................... iv Table of Contents ..................................................................................................................... vi List of Tables .......................................................................................................................... xii List of Figures ........................................................................................................................ xiii List of Appendices ...................................................................................................................xv List of Abbreviations.............................................................................................................. xvi Contributions from others and publications ............................................................................ xix Chapter 1 Introduction: Host-pathogen interactions and immune diversity across phyla ............ 1 An evolutionary perspective on the immune response............................................................ 1 Cellular, innate, and adaptive immune responses ............................................................... 1 Pathogens, symbionts and the origins of immune complexity ............................................ 3 Diversity of immune recognition mechanisms across animal phyla .................................... 4 Immune cells across animal phylogeny .............................................................................. 8 A systems-level approach to studying immunity ................................................................ 9 The gut-associated immune response ....................................................................................13 Gut immunity in vertebrates .............................................................................................13 Crosstalk between the microbiota and the host immune system ........................................16 The role of sequencing data in comparative immunology .....................................................18 vi The sea urchin immune system as a model for immune regulation ........................................19 Sea urchin adult immunity ................................................................................................19 Adult sea urchin immunocytes ..........................................................................................19 Immune genes encoded in the sea urchin genome .............................................................24 Summary ..............................................................................................................................28 Chapter 2 Cellular and molecular immune responses in the sea urchin larva .............................31 Introduction ..........................................................................................................................31 Sea urchins have a biphasic lifecycle that includes a feeding larval stage ..........................31 Larvae are characterized by simple morphology ...............................................................32 Larval immune cells are derived from two precursor populations .....................................34 The sea urchin larva as a simple, systems-level model for immunity .................................34 Materials and Methods .........................................................................................................37 Animals and larval culture ................................................................................................37 Isolation and identification of larval-associated bacterial species ......................................37 Larval bacterial exposure ..................................................................................................38 Neutralizing bacteria for larval exposure ..........................................................................39 Intrablastocoelar injection.................................................................................................39 Microscopy and time-lapse analysis..................................................................................40 Transcriptome analysis .....................................................................................................40 Whole mount in situ hybridization (WMISH) ...................................................................41 vii Results .................................................................................................................................41