Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2011 Transcriptional Regulation of Photoreceptor Development in the Zebrafish Retina Karen Alvarez-Delfin Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES TRANSCRIPTIONAL REGULATION OF PHOTORECEPTOR DEVELOPMENT IN THE ZEBRAFISH RETINA By KAREN ALVAREZ-DELFIN A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy Degree Awarded: Summer Semester, 2011 The members of the committee approve the Dissertation of Karen Alvarez-Delfin defended on June 23th, 2011. _______________________________________ James M. Fadool Professor Directing Dissertation _______________________________________ Michael Blaber University Representative _______________________________________ Lloyd M. Epstein Committee Member _______________________________________ Wu-min Deng Committee Member _______________________________________ Laura R. Keller Committee Member Approved: _____________________________________ P. Bryant Chase, Chair, Department of Biological Science The Graduate School has verified and approved the above-named committee members. ii To my parents and Amalia María iii ACKNOWLEDGEMENTS I would like to sincerely acknowledge the following: The funding sources through all graduate school: NIH Grant IY017753, pre-doctoral Kirschstein NRSA from the National Eye Institute, the Department of Biological Science for financial support and Publication and M. Menzel awards, the National Eye Institute for full support to attend the Vision Course in the MBL, the Association for Research in Vision and Ophthalmology (ARVO) for travel grant, FSU for the Dissertation Research Grant and the Leslie N. Wilson-Delores Auzenne Assistanship, and the Bryan W. Robinson Endowment for the Neurosciences of the Tallahassee Memorial Foundation. The zebrafish and retina scientific communities for sharing reagents and fish lines. The FSU Biological Science Department; Kimberly A. Riddle and Thomas J. Fellers from the Biological Science Imaging Facility for great assistance; Cheryl Pye, Brian Washburn, Rani Dhanarajan and Steve Miller from the Core Facility for assisting with cloning, sequencing and good advising; Ms. Judy Bowers for invaluable help and guidance; the Clerical Office, especially to Ms. Linda Sims, Ms. Bobbie Weston and Ms. Virginia Hellman; Ms Anne B. Thistle for manuscript editing; Charles Badland for friendship and wonderful assistance with art work and photography; Dr. Debra Fadool, Dr. Hank Bass and Dr. Lisa Lyons and their lab members for being great neighbors and supporters; Dr. Fanxiu Zhu and his lab members for assistance with the reporter experiments, especially the PhD student Qiming Liang; my officemates, all the CMB, MOB, Biomedical and Neuroscience graduate student for the friendship. My major professor and advisor Dr. James M. Fadool for his excellent guidance and support, also for the understanding and patience. Thanks for always encourage me to take on new challenges, but also letting me be independent. The members of my doctoral committee: Dr. Laura Keller, Dr. Lloyd Epstein, Dr. Wu-min Deng and Dr. Michael Blaber for their valuable comments, suggestions and advising. All members of the J.M. Fadool’s laboratory for friendship and support; the former members: Dr. Ann C. Morris, Gina Johnson, Katie Loughlin, Jamal Barlett, Haley Robinson and Alex Csizinszky; the current members, Madison Grant, Orly Guerra, Liz Ball, Tyler Scott, especially to my friends Mailin Sotolongo and Carole Saade for their help, support and for sharing good and bad times. My family and all my friends, some of them have been far geographically, but always very encouraging and enthusiastic about my success. I greatly thank my parents Rosa María Delfín and Rafael Alvarez for always give me inspiration to accomplish my goals, for the unconditional love and support through my life and especially during these years in graduate school. iv TABLE OF CONTENTS List of Tables ................................................................................................................................ vii List of Figures.............................................................................................................................. viii Abbreviations.................................................................................................................................. x Abstract.......................................................................................................................................... xi 1. CHAPTER ONE-INTRODUCTION......................................................................................1 1.1 Zebrafish as Animal Model ...........................................................................................1 1.2 Eye Development...........................................................................................................3 1.2.1 Retinal Development .........................................................................................3 1.2.2 Retinal Development in Zebrafish.....................................................................4 1.2.3 Photoreceptor Cell Type Specification and Regulation of Gene Expression ....6 1.3 T-box Family of Genes in Development .....................................................................10 1.3.1 Tbx2 in Retinal Development..........................................................................12 1.4 Six Genes in Development...........................................................................................13 1.4.1 Six Genes in Eye and Retinal Development....................................................14 1.4.2 The Six3/6 Subfamily in Zebrafish ..................................................................16 2. CHAPTER TWO-TBX2B IS REQUIRED FOR ULTRAVIOLET PHOTORECEPTOR CELL SPECIFICATION DURING ZEBRAFISH RETINAL DEVELOPMENT.......................19 2.1 Introduction..................................................................................................................19 2.2 Material and Methods ..................................................................................................20 Zebrafish Maintenance.....................................................................................20 Polymerase Chain Reaction (PCR), cloning and sequencing ..........................20 Immunochemistry............................................................................................21 Whole-mount in situ Hybridization .................................................................22 Cell Transplantation.........................................................................................22 Optokinetic Response Assay (OKR) ...............................................................22 Mapping...........................................................................................................22 Cell Quantification...........................................................................................22 Cell Death ........................................................................................................23 2.3 Results..........................................................................................................................23 2.4 Discussion....................................................................................................................35 3. CHAPTER THREE-TRANSCRIPTIONAL REGULATION OF PHOTORECPETOR GENES...........................................................................................................................................39 3.1 Introduction..................................................................................................................39 3.2 Material and Methods ..................................................................................................41 Plasmids...........................................................................................................41 Cell Culture......................................................................................................42 v Luciferase Reporter Assay...............................................................................42 3.3 Results..........................................................................................................................42 3.4 Discussion....................................................................................................................47 4. CHAPTER FOUR-THE TRANSCRIPTION FACTOR SIX7 REGULATES ROD NUMBER DURING ZEBRAFISH RETINAL DEVELOPMENT ..............................................50 4.1 Introduction..................................................................................................................50 4.2 Material and Methods ..................................................................................................53 Zebrafish Maintenance.....................................................................................53 Cloning and Sequencing..................................................................................53 Embryo Injections............................................................................................53 RT-PCR............................................................................................................53 Whole-mount in