Transcript profiling of the cone-only Nrl-knockout retina using custom cDNA- microarrays: identification of novel targets of Nrl and of signaling pathways involved in photoreceptor function by Jindan Yu A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biomedical Engineering) in The University of Michigan 2004 Doctoral Committee: Professor Anand Swaroop, Co-Chair Assistant Professor Alan J. Hunt, Co-Chair Associate Professor Thomas M. Glaser Professor Alfred O. Hero Professor Peter F. Hitchcock To my mother and my husband ii ACKNOWLEDGMENTS Many people helped me throughout my degree. My dissertation committee, including Drs. Anand Swaroop, Alan J. Hunt, Tom M. Glaser, Alfred O. Hero and Peter F. Hitchcock, has continuously supported and advised me over the last couple years. I am especially indebted to my thesis advisor, Dr. Anand Swarop, for his guidance in both my professional development and personal growth. I am grateful to past and present members of Swaroop lab for providing me a wonderful environment. Particularly, Rafal Farjo was involved in sequencing and annotating of 10,000 clones and printing microarray slides. James S. Friedman and Shirley He performed the chromatin immunoprecipitation assay. James also commented on the writing of this dissertation. Sean P. MacNee picked up thousands of clones and conducted PCR amplification for printing microarray slides. Mohammad I. Othman managed the microarray facility and helped in optimizing microarray hybridization protocols. Sepideh Zareparsi was involved in testing the effects of starting amounts of total RNA on microarray hybridization and provided helpful discussion on this dissertation. My undergraduate student, Rusha Patel, carried out some of the real-time PCR experiments. I also thank Shigeo Yoshida for teaching me molecular biology and Alan J. Mears for valuable discussions. In addition, thanks are due to many other friends and colleagues, including Brian J. Akerley, Deborah M Eadie, Debashis Ghosh, David Hicks, Dider Job, David H. Kohn, Mirna Mustapha, Donald G. Puro, Terry Speed, Maria E. Steele and Sandra A. Staneff. iii Lastly, I am grateful to my husband, Chang-Sheng, for being incredibly supportive throughout. I thank my late parents, my sister and brother for their love. I have also been very fortunate to have my mother-in-law’s help in taking care of my baby daughter. I want to express my extreme gratitude to her for the love she has extended to me. I also thank Emily for being such a sweet daughter. iv TABLE OF CONTENTS DEDICATION................................................................................................................... ii ACKNOWLEDGMENTS...............................................................................................iii LIST OF FIGURES.........................................................................................................vii LIST OF TABLES...........................................................................................................ix CHAPTER 1. INTRODUCTION TO THE STUDY.............................................................1 1.1 Problem Statement.....................................................................1 1.2 Purpose of Study........................................................................2 1.3 Specific Aims.............................................................................2 1.4 Dissertation Outline ...................................................................3 1.5 References................................................................................10 2. REVIEW OF THE LITERATURE ON PHOTORECEPTOR DIFFERENTIATION AND FUNCTION.................................................14 2.1 Eye Anatomy and Structure.....................................................14 2.2 Photoreceptor Function............................................................17 2.3 Retinal Development and Photoreceptor Differentiation ........25 2.4 Nrl and the Nrl Knockout ........................................................26 2.5 Photoreceptor Degeneration and Cone Survival......................30 2.6 Microarray Technology ...........................................................30 2.7 Summary..................................................................................33 2.8 References................................................................................34 3. ANNOTATION AND ANALYSIS OF 10,000 ESTS FROM DEVELOPING MOUSE EYE AND ADULT RETINA .........................39 3.1 Abstract....................................................................................39 3.2 Introduction..............................................................................40 3.3 Materials and Methods.............................................................42 3.4 Results......................................................................................45 3.5 Discussion................................................................................64 3.6 References................................................................................70 4. GENERATION OF EYE-GENE MICROARRAYS..................................73 4.1 Abstract....................................................................................73 4.2 Introduction..............................................................................73 4.3 Methods....................................................................................75 v 4.4 Results......................................................................................79 4.5 Discussion................................................................................86 4.6 References................................................................................88 5. EVALUATION AND OPTIMIZATION OF PROCEDURES FOR TARGET LABELING AND HYBRIDIZATION OF CDNA MICROARRAYS........................................................................................91 5.1 Abstract....................................................................................91 5.2 Introduction..............................................................................92 5.3 Methods....................................................................................93 5.4 Results and Discussion ............................................................99 5.5 References..............................................................................107 6. ALTERED EXPRESSION OF GENES OF THE BMP/SMAD AND WNT/CALCIUM SIGNALING PATHWAYS IN THE CONE-ONLY NRL-KNOCKOUT MOUSE RETINA, REVEALED BY GENE PROFILING USING CUSTOM CDNA MICROARRAYS......................................................................................110 6.1 Summary................................................................................110 6.2 Introduction............................................................................111 6.3 Materials and Methods...........................................................114 6.4 Results....................................................................................118 6.5 Discussion..............................................................................133 6.6 References..............................................................................138 7. GENE EXPRESSION PROFILES OF RETINA DURING POSTNATAL DEVELOPMENT AND COMPARISON TO THE NRL-/- RETINA................................................................................144 7.1 Summary................................................................................144 7.2 Introduction............................................................................145 7.3 Materials and Methods...........................................................147 7.4 Results....................................................................................150 7.5 Discussion..............................................................................160 7.6 Reference ...............................................................................165 8. CONCLUSIONS ..........................................................................................170 8.1 Concluding Remarks..............................................................170 8.2 Implications............................................................................171 8.3 Future Directions ...................................................................172 8.4 References..............................................................................173 vi LIST OF FIGURES Figures Figure 2.1. The anatomy and structure of the adult human eye............................15 Figure 2.2. The structure of the retina...................................................................16 Figure 2.3. The structure of photoreceptors..........................................................18 Figure 2.4. The phototransduction cascade...........................................................20 Figure 2.5. Retinoid cycles. ..................................................................................22 Figure 2.6. The retinal cellular pathways..............................................................24 Figure 2.7. A model for photoreceptor differentiation. ........................................27 Figure 2.8. Opsin immunohistochemistry of the wild-type (+/+), Nrl heterozygous (+/−), and Nrl knockout (−/−) retina. ...........................29 Figure 2.9. Schematic overview of cDNA microarray technology. .....................32 Figure 3.1. Schematic representation