Wnt/β-catenin Signaling Regulates Regeneration in Diverse Tissues of the Zebrafish Nicholas Stockton Strand A dissertation Submitted in partial fulfillment of the Requirements for the degree of Doctor of Philosophy University of Washington 2016 Reading Committee: Randall Moon, Chair Neil Nathanson Ronald Kwon Program Authorized to Offer Degree: Pharmacology ©Copyright 2016 Nicholas Stockton Strand University of Washington Abstract Wnt/β-catenin Signaling Regulates Regeneration in Diverse Tissues of the Zebrafish Nicholas Stockton Strand Chair of the Supervisory Committee: Professor Randall T Moon Department of Pharmacology The ability to regenerate tissue after injury is limited by species, tissue type, and age of the organism. Understanding the mechanisms of endogenous regeneration provides greater insight into this remarkable biological process while also offering up potential therapeutic targets for promoting regeneration in humans. The Wnt/β-catenin signaling pathway has been implicated in zebrafish regeneration, including the fin and nervous system. The body of work presented here expands upon the role of Wnt/β-catenin signaling in regeneration, characterizing roles for Wnt/β-catenin signaling in multiple tissues. We show that cholinergic signaling is required for blastema formation and Wnt/β-catenin signaling initiation in the caudal fin, and that overexpression of Wnt/β-catenin ligand is sufficient to rescue blastema formation in fins lacking cholinergic activity. Next, we characterized the glial response to Wnt/β-catenin signaling after spinal cord injury, demonstrating that Wnt/β-catenin signaling is necessary for recovery of motor function and the formation of bipolar glia after spinal cord injury. Lastly, we defined a role for Wnt/β-catenin signaling in heart regeneration, showing that cardiomyocyte proliferation is regulated by Wnt/β-catenin signaling. These data demonstrate a conserved role for Wnt/β-catenin signaling in promoting regeneration across multiple tissue types in the zebrafish, and further show conservation of some of these functions in human tissue. Table of Contents List of Figures List of Tables Glossary Acknowledgments Chapter One: Introduction 1.1 Regeneration 1.1.1 Historical Study 1.1.2 Phylogenetic Observations 1.2 Mechanisms of Regeneration 1.2.1 Whole Organism 1.2.2 Appendage 1.2.3 Spinal Cord 1.2.4 Heart 1.3 Wnt/β-catenin Signaling 1.3.1 Canonical Signaling Pathway 1.3.2 Functions in Regeneration 1.4 References Chapter Two: Cholinergic Activity is Required for Blastema Formation and Wnt/β-catenin Signaling in the Regenerating Zebrafish Caudal Fin 2.1 Introduction 2.2 Results 2.3 Discussion 2.4 Materials and Methods 2.5 References 2.6 Supplemental Material Chapter Three: Wnt/β-catenin signaling Promotes Glial Bridge Formation and Regeneration After Zebrafish Spinal Cord Injury 3.1 Introduction 3.2 Results 3.3 Discussion 3.4 Materials and Methods 3.5 References 3.6 Supplemental Material Chapter Four: Wnt/B-catenin Signaling Regulates Adult Zebrafish Cardiac Repair and Scar Remodeling 4.1 Introduction 4.2 Results 4.3 Discussion 4.4 Materials and Methods 4.5 References 4.6 Supplemental Material Chapter Five: Discussion and Future Directions 5.1 Summary 5.2 Future Work 5.3 Conclusions 5.4 References Compiled Bibliography List of Figures Chapter One: Introduction 1.1 Canonical Wnt signaling schematic Chapter Two: 2.1 Spinal cord transection impairs caudal fin regeneration. 2.2 BtxB administration inhibits fin ray regeneration localized to the injection site. 2.3 Spinal cord transection inhibits blastema formation and proliferation in the amputated fin. 2.4 Spinal cord transection inhibits Wnt/β-catenin signaling in the amputated fin. 2.5 Canonical Wnt8a overexpression rescues loss of regenerating rays after spinal cord transection. 2.S1 Spinal cord transection inhibits bone regeneration. 2.S2 Regaining muscle control in the tail fin rescues regeneration defects after spinal cord transection. 2.S3 Wound epithelium thickening is inhibited by spinal cord transection. 2.S4 mcherry transcripts are reduced at 1 day post amputation in spinal cord transected fish. 2.S5 Wnt8a activates Wnt/β-catenin signaling and increases bone length in the caudal fin after spinal cord transection. Chapter Three: 3.1 Wnt/β-catenin signaling is increased after spinal cord injury. 3.2 Wnt/β-catenin signaling is necessary for locomotor recovery after spinal cord injury. 3.3 Wnt/β-catenin signaling is necessary for bipolar glial cell formation. 3.4 Dkk overexpression does not regulate proliferation of glial progenitors after spinal cord injury. Chapter Four: 4.1 Cells undergoing Wnt/β-catenin signaling are present in injury site throughout regeneration 4.2 Dkk overexpression modulates extent of collagen scar formation after cardiac amputation 4.3 Dkk overexpression directly affects tissue expression profile of cardiac injury, healing, and regeneration genes. 4.4 Dkk overexpression reduces activity of renewed cardiomyocytes in wound area. 4.5 Wnt/ β-catenin signaling promotes cell proliferation in human embryonic stem cell- derived cardiomyocytes. 4.S1: mCherry+ cells are reduced in fish overexpressing Dkk after heart amputation. Chapter Five: 5.1 Tissue specific, inducible CRISPR/Cas9 Experimental Design. 5.2 Strategies for tissue specific, inducible inhibition of Wnt/β-catenin signaling. List of Tables Chapter Two: 2.S1 qPCR Primers Chapter Three: 3.S1 qPCR Primers Chapter Four: 4.S1 qPCR Primers Glossary BtxB – Botulinum Toxin B DPA – Days Post Amputation DPT – Days Post Transection GRP - Glial-Restricted Precursor hESC - human Embryonic Stem Cell NPC - Neural Progenitor Cell PCR - Polymerase Chain Reaction Acknowledgments I would like to thank the many people who helped make this work possible. First and foremost, my advisor Randall Moon has provided me with the opportunity to pursue my interests, always offering his support and guidance. I would also like to thank my committee members, who generously gave their time, consideration, and ideas to help me grow as a scientist. I would like to thank all the members, past and present, of the Moon lab. In particular, I would like to thank Jason Berndt and Jeremy Rabinowitz for their mentorship in experimental design, technical expertise, and helpful comments throughout my career in the Moon lab. I would also like to thank Timothy Petrie, Cristi Stoick-Cooper, and Peter Hofsteen, who are responsible for some of the data in chapter 4. To my parents, thank you for the endless encouragement and support. You have provided me with me so much, none of this would have been possible without you. Your enthusiasm for my work was so important to me, I am forever grateful for what you have given me. Finally, I must thank my wife, Robin. You had the responsibility of sitting through so many talks and data analysis sessions that you could describe my projects without any problems. You have been my source of daily support and encouragement, and the love of my life. Thank you for all you have done, and all you continue to do. 1 – Introduction 1.1 – Regeneration 1.1.1 Historical Study The loss of tissue, due to injury or disease, is a debilitating and potentially lethal problem for animals. However, some animals are capable of regenerating new cells to replace lost tissue, preserving function and life. Some of the earliest writings on the phenomena of tissue regeneration can be traced to Aristotle, who referred to lizard tail regeneration in his writings (Aristotle et al. 1965). The formalized study of regeneration is also considered to be the birth of experimental biology, advancing upon the observational cataloguing of previous generations. Abraham Trembley published the first report on experimental regenerative biology, documenting the regenerative ability of hydra, a basal freshwater cnidarian capable of regenerating its entire body after amputation (Lenhoff, Lenhoff, and Trembley 1986). This work was followed up by Charles Bonnet, who observed similar traits in annelid worms (Bonnet 1745). Lazzaro Spallanzani published the first work on tadpole and salamander regeneration, studying limb, digit, and tail regeneration (Spallanzani 1768). These foundational works helped develop the study of biology generally and regeneration specifically, and led to questions still grappled with today, including why are some animals and tissues capable of regeneration and not others, and what are the mechanisms that govern regeneration. 1.1.2 Phylogenetic Observations The ability to regenerate tissues is observed throughout phyla, though this capacity is diminished in less basal animals. Numerous invertebrate species are capable of extensive regeneration after injury. The hydra, for example, is capable of regenerating its entire body after amputation, leading to 2 viable hydra after injury. Planaria, invertebrate flatworms, are also capable of regenerating their entire body from a small portion of their body. This ability to regenerate an entire organism is not observed in vertebrates. Vertebrate regeneration can be observed in several classes of animals, including fish (zebrafish), amphibians (salamanders), and mammals (mice). Adult zebrafish possess the ability to regenerate several tissues after injury, including caudal fin, heart, spinal cord, retina, and brain (Stoick-Cooper, Moon, and Weidinger 2007). This ability to regenerate numerous tissue types, coupled with its genetic tractability, make the zebrafish a popular model for studying the molecular signals necessary for regeneration. Similarly,