GENETIC SIGNATURES OF THE RETINA IN HEALTH AND DISEASE by DEBARSHI MUSTAFI Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Krzysztof Palczewski Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY AUGUST 2013 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of DEBARSHI MUSTAFI candidate for the Ph.D degree *. (signed) Dr. Johannes Von Lintig (chair of the committee) Dr. Krzysztof Palczewski Dr. Robert A. Bonomo Dr. George Dubyak Dr. Andreas Engel Dr. Vera Moiseenkova-Bell Dr. Jonathan E. Sears (date) ___ May 31, 2013_______________ *We also certify that written approval has been obtained for any proprietary material contained therein ii TABLE OF CONTENTS LIST OF TABLES……………………………………………………………………...viii LIST OF FIGURES…………………………………………………………………….....x ACKNOWLEDGMENTS………………………………………………………...….…xiv LIST OF ABBREVIATIONS…………………………………………………………..xvi ABSTRACT……………………………………………………………………………….1 CHAPTER 1: THE GENOTYPE TO PHENOTYPE QUESTION UNDERLYING VISION………………………………………………………………...2 1.1 The eye 1.1.1 Features of the eye across species……………………………………………....3 1.1.2 The retinal layer and the role of rod and cone photoreceptors………………….5 1.1.3 The importance of cone cells in the context of retinal disease………………….6 1.2 Cone phototransduction signaling 1.2.1 The first steps in visual response by cones………………………………….7 1.2.2 Structural dynamics of cone opsins during phototransduction……………...9 1.3 Evolutionary dynamics of the eye 1.3.1 Evolution of retinal circuitry……………………………………………….12 1.3.2 Evolution of cone photoreceptors……………………………………….…13 1.4 Structural features of cone photoreceptors 1.4.1 Cone disc renewal………………………………………………………….16 1.4.2 Cone disc morphogenesis………………………………………………….19 1.5 Mammalian models to better elucidate cone cell structure and function iii 1.5.1 Mouse models to study cone cells……………………………………………..22 1.5.2 Other mammalian rodent species for study of cone cells……………………...23 1.6 The genotypic to phenotype connection in the retina in the context of disease 1.6.1 High resolution phenotypic methods to reveal retinal architecture………..24 1.6.2 High throughput sequencing to reveal global gene expression patterns in the retina………………………………………………………………………25 1.7 Project approach……………………………………………………………………26 Figures…………………………………………………………………………...……...28 CHAPTER 2: PROGRESSIVE DEGENERATION IN MONOGENIC ENHANCED S-CONE SYNDROME IS DRIVEN BY ABBERANT RETINAL HOMEOSTASIS…………………………………………………………...38 2.1 The monogenic disease enhanced S-cone syndrome 2.1.1 The human condition and the corresponding mouse model of disease……39 2.2 Rationale and methodology to elucidate the degenerative component of ESCS 2.2.1 Rationale for research approach……………………………………………41 2.2.2 Materials and methods……………………………………………………..42 2.3 Results 2.3.1 Phenotypic features of human ESCS patients and the relationship of these features to the Nrl-/- mouse model………………………………………56 2.3.2 Transcriptome analysis by Illumina based RNA-Seq of retinas from Wt and Nrl-/- mice………………………………………………………………..59 2.3.3 Verification of sequencing data by RT-PCR………………………………60 iv 2.3.4 Characterization of differentially expressed transcripts…………………...61 2.3.5 Disrupted ESCS retinal architecture and patchy loss of photoreceptors…..63 2.3.6 ESCS photoreceptors exhibit abnormal accumulations of material……….64 2.3.7 Aberrant distribution of disc membranes influences abnormal packing architecture of ESCS photoreceptors…………………………………………….65 2.3.8 Evidence for aberrant phagocytosis in ESCS disease……………………...67 2.3.9 ESCS phenotype attributed to photoreceptor abnormalities rather than a RPE defect…………………………….…………………………………..69 2.4 Discussion and conclusions………………………………………………………...70 Tables…………………………………………………………………………………....77 Figures…………………………………………………………………………………..82 CHAPTER 3: DIFFERENTIAL BACKGROUND GENETIC NETWORKS DRIVE MULTIGENIC AGE-RELATED RETINAL DEGENERATION.………102 3.1 The multi-genic etiology of age-related retinal degeneration (ARD) 3.1.1 Age-related pathology in the eye and the genetic contributions Influencing disease…………………………………………………………….103 3.2 Rationale and methodology to multi-genic contributions driving ARD 3.2.1 Rationale for research approach………………………………………….104 3.2.2 Materials and methods……………………………………………………105 3.3 Results 3.3.1 A/J genetic background mice undergo pronounced age−related retinal degeneration……………………………………………………….…….109 v 3.3.2 ARD in A/J mice is accompanied by inflammatory cell infiltration and RPE cell pathology…………………………………………………………110 3.3.3 RNA−Seq reveals differential genetic background contributions to the transcriptome……………………………………………………………..112 3.3.4 Pathway analysis highlights inflammatory priming coupled with impaired retinal homeostatic cellular pathways in 1−month−old A/J mice before retinal pathology is evident……………………………………………...114 3.3.5 Inflammatory priming in A/J retina is exacerbated with age………..…...115 3.3.6 Marginally−expressed retinal homeostasis proteins exhibit abnormal RPE localization in A/J mice…………………………………………………...116 3.4 Discussion and conclusions…………………………………………………….....117 Tables…………………………………………………………………………………..124 Figures………………………………………………………………………………….129 CHAPTER 4: THE ROLE OF NON-CODING RNAs IN VISUAL FUNCTION……………………………………………………………..156 4.1 Delineating roles of long intergenic non-coding RNAs in the adult retina 4.1.1 Long intergenic non-coding RNAs and their possible physiological roles……………………………………………………………...157 4.2 Rationale and methodology to reveal evolutionary conservation of lincRNAs across species as a determinant of functional preservation in the eye 4.2.1 Rationale for research approach………………………………………….158 4.2.2 Materials and methods……………………………………………………159 vi 4.3 Results 4.3.1 RNA−Seq identifies eye lincRNAs that exhibit sequence conservation in mammals and those that exhibit conservation in the human retina and macular region………………………………………………………………….164 4.3.2 Tissue and eye compartment expression of conserved lincRNAs………..166 4.3.3 Expression of some conserved lincRNAs is localized to specific retinal layers…………………………………………………………...167 4.3.4 Genetic loci and in silico analyses of promoter motifs highlight possible roles of lincRNAs in adult retinal homeostasis……………………………..….168 4.4 Discussion and conclusions……………………………………………..………...169 Tables…………………………………………………………………………………..174 Figures……………………………………………………….………………………...181 CHAPTER 5: CONCLUSIONS AND FUTURE DIRECTIONS…...………….......188 REFERENCES…………………………………………………...…………………....199 vii LIST OF TABLES Table1: GO term breakdown of transcript reads across different RNA-Seq experiments with Wt and Nrl-/- tissues…………………………………………………..77 Table 2: Fold changes of selected transcripts in Nrl-/- relative to Wt tissue across different experiments…………………………………………………………………….78 Table 3: Transcript levels of visual cycle proteins in Nrl-/- relative to Wt tissue across different RNA-Seq runs………………………………………………..…………79 Table 4: Transcript levels (in FPKM) of putative phagocytosis proteins in Nrl-/- relative to Wt tissue across different RNA-Seq runs……………………...…………….81 Table 5: GoTerm breakdown of transcript reads across different RNA−Seq experiments with from 1-month-old A/J, BALB/c and B6 mice whole eye tissue….....124 Table 6: Differential expression profile of genes in the A/J and B6 mouse eye and SNP analysis of differentially expressed genes……………………………..……..125 Table 7: Transcript reads (FPKM) of selected genes from A/J, BALB/c, and B6 mice as well as from Long-Evans rat and Nile rat eyes………………………..127 Table 8: Profile of 18 conserved lincRNAs in the eye across species…………………174 Table 9: Profile of 18 conserved lincRNAs in the retina across species………………175 Table 10: Transcript abundance (FPKM) in the eye and retina of B6 mice…………..176 Table 11: Profile of 18 conserved lincRNAs in 4 biological replicates of macaque macula tissue…………………………………………………………………177 Table 12: Semi-quantitative RT-PCR of conserved lincRNAs in organs and eye compartments of 1 month old C57BL/6 mice………………………………………….178 viii Table 13: Semi-quantitative RT-PCR of conserved lincRNAs in mice with different retinal environments……………………………………………………………………179 Table 14: In silico promoter analysis of conserved lincRNAs reveals binding sites for transcription factors that drive retinal processes……………………………...………..180 ix LIST OF FIGURES Figure 1: Differences in photoreceptors and their arrangement in the retina……….....28 Figure 2: Distribution of photoreceptors in the eye………………………………...….29 Figure 3: The steps in cone phototransduction……………………………………..….30 Figure 4: Structure and renewal of rod and cone discs …………………….…………31 Figure 5: Homology model of S-cone (blue) opsin …………………..……………….32 Figure 6: Evolution of photoreceptors …………………………………..…………….33 Figure 7: Schematics of retinal sample preparation and FIB-SEM based experimentation…………………………………………………………………………34 Figure 8: Experimental set-up for photoreceptor observation with FIB-SEM technology……………………………………………………………………………....35 Figure 9: Schematic of SBF-SEM experimentation……………………………...……36 Figure 10: High-throughput sequencing reveals global gene expression patterns……..37 Figure 11: Key features of human ESCS disease and the Nrl-/- mouse model……..…..82 Figure 12: ESCS photoreceptors of Nrl-/- mice display aberrant packing……...………84 Figure 13: Reproducibility of murine eye RNA-Seq experiments and globally differentially expressed genes detected between Wt and Nrl-/- whole eyes…………….85 Figure 14: RNA-Seq of Wt and Nrl-/- retinas reveals