Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2017 Insights into retinal cell fate determination in vertebrates using transcriptomic profiling and genome editing Rebecca Chowdhury Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Developmental Biology Commons, Genetics Commons, and the Molecular Biology Commons Recommended Citation Chowdhury, Rebecca, "Insights into retinal cell fate determination in vertebrates using transcriptomic profiling and genome editing" (2017). Graduate Theses and Dissertations. 16512. https://lib.dr.iastate.edu/etd/16512 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Insights into retinal cell fate determination in vertebrates using transcriptomic profiling and genome editing by Rebecca Chowdhury A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Molecular, Cellular and Developmental Biology Program of Study Committee: Jeffrey Trimarchi, Major Professor Jeffrey Essner Clark Coffman Donald Sakaguchi Basil Nikolau Iowa State University Ames, Iowa 2017 Copyright ©Rebecca Chowdhury, 2017. All rights reserved. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS..………………………………………………………………………………………………..vi ABSTRACT..…………………………………………………………………………………………………………………..vii CHAPTER 1. GENERAL INTRODUCTION..…………………………………………………………………………1 1. Anatomy of the eye……………………………………………………………………………………………………...1 1.1 Anterior section of the eye…………………………………………………………………………………………1 1.1.1 Cornea and iris……………………………………………………………………………………………………….1 1.1.2 Lens……………………………………………………………………………………………………………………….3 1.2 Posterior section of the eye: retina………………………………………………………………………….....3 2. Development of the vertebrate eye………………………………………………………………………………5 2.1 Eye development in mammals……………………………………………………………………………….......5 2.2 Eye development in zebrafish…………………………………………………………………………………….7 2.3 Factors important for development of the vertebrate eye……………………………………………8 3. Retinal development in vertebrates……………………………………………………………………………10 3.1 Introduction to the retina………………………………………………………………………………………...10 3.2 Competence and cell fate determination…………………………………………………………………..12 3.3 Models of cell fate determination……………………………………………………………………………..13 iii 3.4 Factors involved in retinal cell fate regulation…………………………………………………………..15 3.4.1 Atoh7…………………………………………………………………………………………………………………...15 3.4.2 The NeuroD family……………………………………………………………………......................................17 3.4.3 Dlx1/2………………………………………………………………………………………………………………….17 3.4.4 Ikzf family……………………………………………………………………………….........................................18 3.4.5 Other factors………………………………………………………………………………………………………...18 3.4.6 MicroRNAs…………………………………………………………………………………………………………...19 4. Morphology and functions of the retinal neurons...............................................................................20 4.1 Photoreceptors…………………………………………………………………………..........................................20 4.2 Bipolar cells…………………………………………………………………………………………………………….22 4.3 Amacrine cells…………………………………………………………………………………………………………23 4.4 Horizontal cells……………………………………………………………………………………………………….24 4.5 Ganglion cells……………………………………………………………………………..........................................24 5. Guidance of retinal axons to their target within the brain……………………………………………26 6. Conclusions..........................................................................................................................................................31 7. References............................................................................................................................................................32 CHAPTER 2. SINGLE CELL PROFILING OF ATOH7-EXPRESSING RETINAL CELLS...................43 Abstract.......................................................................................................................................................................43 iv Introduction..............................................................................................................................................................44 Materials and Methods.........................................................................................................................................47 Results.........................................................................................................................................................................52 Discussion..................................................................................................................................................................60 References.................................................................................................................................................................64 Figures and Legends.............................................................................................................................................71 CHAPTER 3. TRIM GENES IN THE RETINA: EXPRESSION AND FUNCTIONAL CHARACTERIZATION …………………………………………………………………………………………………...82 Abstract.......................................................................................................................................................................82 Introduction..............................................................................................................................................................83 Materials and Methods.........................................................................................................................................86 Results.........................................................................................................................................................................94 Discussion...............................................................................................................................................................103 Acknowledgements…………………………………………………………………………………………………….106 References...............................................................................................................................................................106 Figures and Legends...........................................................................................................................................113 v CHAPTER 4. USING GENOME EDITING TO INVESTIGATE VERTEBRATE RETINAL DEVELOPMENT IN ZEBRAFISH…….............................................................................................................125 Abstract....................................................................................................................................................................125 Introduction...........................................................................................................................................................126 Materials and Methods......................................................................................................................................130 Results……………....................................................................................................................................................136 Discussion…………………………………………………………………………………………………………………..145 Acknowledgements…………………………………………………………………………………………………….149 References...............................................................................................................................................................150 Figures and Legends...........................................................................................................................................159 CHAPTER 5. GENERAL DISCUSSION...........................................................................................................170 Introduction……………………………………………………………………………………………………………….170 Retinal transcriptomics in Atoh7+ cells………………………………………………………………………..172 Functional studies using Trim9 mouse mutants…………………………………………………………...173 Functional studies using zebrafish mutants………………………………………………………………….174 Conclusion………………………………………………………………………………………………………………….177 References……………………………………………………………………………..…………………………………...179 vi ACKNOWLEDGEMENTS I would like to thank my committee members, Dr. Jeffrey Essner, Dr. Clark Coffman, Dr. Don Sakaguchi, and Dr. Basil Nikolau for their insights, guidance, and support throughout the course of this research. I am very grateful to my mentor, Dr. Jeffrey Trimarchi. His guidance every step of the way, patience and unwavering support has made this possible. He was someone I have always been able to rely on whenever I needed help. His immense knowledge, enthusiasm and motivation has made this a rewarding experience for me. I would also like to thank members of the Trimarchi lab, who played instrumental roles in my work. Jillian Goetz taught me a lot when I first joined lab. Lauren Laboissonniere, was an amazing colleague and friend, and someone who I have always been able to discuss anything and everything with. I would also like to thank present and past undergraduate students who were instrumental in my projects. Courtney Smith, Abbie Burney,