Photoreceptor Transcriptome Analysis in Zebrafish A Dissertation Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy with a Major in Neuroscience in the College of Graduate Studies University of Idaho by Chi Sun Major Professor: Dr. Deborah Stenkamp Committee members: Dr. Peter Fuerst, Dr. Gordon Murdoch, Dr. Michael Varnum Department administrator: Dr. James Nagler December 2017 ii Authorization to Submit Dissertation This dissertation of Chi Sun, submitted for the degree of Doctor of Philosophy with a Major in Neuroscience and titled “Photoreceptor Transcriptome Analysis in Zebrafish,” has been reviewed in final form. Permission, as indicated by the signatures and dates below, is now granted to submit final copies to the College of Graduate Studies for approval. Major professor: _________________________________ Date:____________ Dr. Deborah Stenkamp Committee members: _________________________________ Date:____________ Dr. Peter Fuerst _________________________________ Date:____________ Dr. Gordon Murdoch _________________________________ Date:____________ Dr. Michael Varnum Department administrator: _________________________________ Date:____________ Dr. James Nagler iii Abstract The mammalian retina does not possess the capacity of intrinsic generation of functional replacement upon the loss of retinal neurons, and functional replacement of these cells in human patients is not medically feasible at present. Identification of regulatory targets controlling retinal neurogenesis and regeneration in zebrafish can potentially provide new directions to treat retinal pathologies as well as to induce potential retinal regeneration in humans. Fortunately, next-generation sequencing (NGS) provides powerful systems-based analysis of cellular pathways, and has been used to analyze these regulatory targets in this study. The goals of this dissertation are to develop a methodology of isolating photoreceptors of different subtypes, to analyze NGS-derived photoreceptor transcriptome profiling (RNA- seq), and to study functions of selected transcripts in zebrafish photoreceptor development and maintenance. iv Acknowledgements Appreciation is credited to my advisor Dr. Deborah L. Stenkamp for her constant guidance throughout the course of my doctoral degree. In addition, I would also like to thank my committee members for their invaluable advice given to my research. Various funding agencies have provided important financial resources to this research, including NIH R01 EY012346 (DLS), NIH R21 EY026814 (DLS), a Technology Access Grant (CS and DLS) available through Idaho’s NIH-INBRE program (supported by NIH P20 GM103408), and the Malcolm and Carol Renfrew Faculty Fellowship (DLS). I would like to acknowledge Dr. James Fadool for providing the XOPS:eGFP zebrafish, Dr. Ann Morris for the XOPS:mCFP zebrafish, Dr. Rachel Wong for providing trβ2:tdTomato transgenic line, Dr. Shoji Kawamura for the sws1:GFP, rh2-2:GFP, lws:Pac(H) zebrafish, and Dr. Pamela Raymond for sws2:mCherry zebrafish.. I am very grateful to Ms. Ruth Frey and Mr. Carlos Galicia, the scientific aides of Dr. Stenkamp’s laboratory, for the technical assistance, to Ms. Ann Norton of the UI IBEST Optical Imaging Core for assistance with FACS, to Ms. Melissa Oatley for assistance with FACS at WSU CRB FACS Core, to Mr. Dan New and Dr. Sam Hunter of the UI IBEST Genomics Core for RNA-Seq experiments and assistance with bioinformatics, to Dr. Diana Mitchell for retinal lesioning experiments, to University of Utah Mutation Generation and Detection Core for assistance with CRISPR-Cas9 techniques. A research collaboration with Dr. Anand Swaroop’s lab, NEI, NIH, was arranged for RNA-Seq experiments of regenerated photoreceptors and subsequent bioinformatics. v Table of Contents Authorization to Submit Dissertation ....................................................................................... ii Abstract ................................................................................................................................. iii Acknowledgements ............................................................................................................... iv Table of Contents ................................................................................................................... v List of Figures ....................................................................................................................... vii List of Tables ......................................................................................................................... ix List of Abbreviations ............................................................................................................... x Chapter 1. Introduction ........................................................................................................ 1 Specific Aims .............................................................................................................. 1 Significance ................................................................................................................ 2 Zebrafish Retina and Zebrafish as a Model ................................................................. 3 Retinal Regeneration in Zebrafish .............................................................................. 8 Factors involved in Vertebrate Retinal Neurogenesis ............................................... 10 RNA-Seq Experiments ............................................................................................. 12 References ............................................................................................................... 13 Chapter 2. Identification of transcripts within rod photoreceptors of the zebrafish retina ................................................................................................................................... 17 Abstract .................................................................................................................... 17 Background .............................................................................................................. 18 Methods .................................................................................................................... 20 Results...................................................................................................................... 24 Discussion ................................................................................................................ 31 Conclusion ................................................................................................................ 33 References ............................................................................................................... 34 vi Figures and Tables ................................................................................................... 38 Chapter 3. Isolation of photoreceptors from mature, developing, and regenerating zebrafish retinae and microglia from regenerating zebrafish retinae for gene expression analysis .......................................................................................................... 55 Abstract .................................................................................................................... 55 Introduction ............................................................................................................... 56 Materials and Supplies .............................................................................................. 59 Detailed Methods ...................................................................................................... 61 Results...................................................................................................................... 67 Potential Pitfalls and Troubleshooting ....................................................................... 74 Discussion ................................................................................................................ 78 References ............................................................................................................... 80 Figures and Tables ................................................................................................... 83 Chapter 4. Zebrafish rxrγa mutants: generation, validation, and preliminary characterization of opsin expression phenotype .......................................................... 110 Abstract .................................................................................................................. 110 Introduction ............................................................................................................. 111 Methods .................................................................................................................. 114 Results.................................................................................................................... 118 Discussion and Future Studies ............................................................................... 121 References ............................................................................................................. 123 Figures and Tables ................................................................................................. 127 vii List of Figures Figure 2.1 ............................................................................................................................