CHARACTERIZING THE ROLE OF GENE REGULATORY FACTORS IN AGING DROSOPHILA PHOTORECEPTORS
Aging is associated with a decline in visual function and increased prevalence of ocular disease, correlating with changes in the transcriptome and epigenome of cells in the eye. The extended photoreceptor cell lifespan, in addition to its high metabolic needs due to phototransduction, makes it critical for these neurons to continually respond to the stresses associated with aging by mounting an appropriate gene expression response. My work, in collaboration with fellow lab members and colleagues, has focused on better understanding the regulatory mechanisms that result in age-dependent transcriptional changes in photoreceptors, and if these changes not only correlate with but cause the decrease in function with age. In order to better characterize photoreceptor specific changes my initial work first focused on expanding the gene expression toolkit for eye specific expression. In chapter 1 we describe a previously unnoticed sevenless mutation present in the majority of the TRiP RNAi collection. In chapter 2 we characterized the currently available eye- and photoreceptor-specific binary expression system drivers in Drosophila. Using a luciferase and fluorescent reporter, we characterized the relative expression and cell type-specificity of each driver in the 10-day old adult eye. Also, wecharacterized the expression pattern of these drivers in various developmental stages. We then compared several Gal4 drivers from the Bloomington Drosophila Stock Center (BDSC) including GMR-Gal4, longGMR-Gal4 and Rh1-Gal4 with newly developed Gal4 and QF2 drivers that are specific to different cell types in the adult eye. In addition, we generated drug-inducible Rh1-GSGal4 lines and compared their induced expression with an available GMR-GSGal4 line. Although both lines had significant induction of gene expression measured by luciferase activity, Rh1-GSGal4 was expressed at levels below the detection of the fluorescent reporter by confocal microscopy, while GMR-GSGal4 showed substantial reporter expression in the absence of drug by microscopy. This study systematically characterized and compared a large toolkit of eye- and photoreceptor-specific drivers, while also uncovering some of the limitations of currently available expression systems in the adult eye.
In chapter 3, we sought to untangle the more general neuronal age-dependent transcriptional signature of photoreceptors with that induced by light stress. To do this, we aged flies or exposed them to various durations of blue light, followed by photoreceptor nuclei-specific transcriptome profiling. Using this approach, we identified genes that are both common and uniquely regulated by aging and light induced stress. Whereas both age and blue light induce expression of DNA repair genes and a neuronal-specific signature of death, both conditions result in downregulation of neurotransmitters important for synaptic transmission. Interestingly, blue light uniquely induced genes that directly counteract the overactivation of the phototransduction signaling cascade. Lastly, unique gene expression changes in aging photoreceptors included the downregulation of genes involved in membrane potential homeostasis and mitochondrial function, as well as the upregulation of immune response genes. We proposed that light stress contributes to the aging transcriptome of photoreceptors, but that there are also other environmental or intrinsic factors involved in age-associated photoreceptor gene expression signatures.
In chapter 4, we sought to test if age-associated changes in gene expression patterns in the eye directly contribute to the increased risk of retinal degeneration. To do this, we performed a targeted photoreceptor specific RNAi screen in Drosophila to identify gene regulatory factors that result in premature, age-dependent retinal degeneration. From an initial set of 155 RNAi lines each targeting a unique gene and spanning a diverse set of gene regulatory factors, we identified 18 high confidence target genes whose decreased expression in adult photoreceptors leads to premature and progressive retinal degeneration. The 18 target genes were enriched for factors involved in the regulation of transcription initiation, pausing, and elongation, suggesting that these processes are essential for maintaining the health of aging photoreceptors. To identify the genes regulated by these factors, we profiled the photoreceptor transcriptome in a subset of lines. Strikingly, two of the 18 target genes, Spt5 and domino, show substantially similar changes in gene expression to those observed with advanced age.
Together, our data suggests that dysregulation of the mechanisms involved in transcription initiation and elongation plays a key role in shaping the transcriptome of aging photoreceptors. Further, our findings indicate that the age-dependent changes in gene expression not only correlate, but might also contribute to increased risk of retinal degeneration.