Transcriptional Profiling of Specific Cell Types in the Drosophila Visual System During Development and in Response to Light Stress
Multicellular organisms contain a highly heterogeneous mixture of cell types within each tissue that complicate gene expression analyses. To analyze cell-specific gene expression profiles, it is necessary to isolate specific cell types with high purity and at sufficient yields for downstream transcriptional profiling. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in a tissue, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). In this dissertation, I describe an alternative approach to characterizing cellspecific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells. Nuclei in the specific cell type of interest in the visual system of Drosophila melanogaster are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. This method has been applied to study cell type-specific gene regulation in glia during visual development and in adult photoreceptors under light stress. Glia are required for proper targeting of photoreceptor axons during development of the Drosophila visual system. However, the factors that regulate their migration and control proper axon targeting are not fully characterized. The Spt-Ada-Gcn5 acetyltransferase complex (SAGA) plays an important role in visual development in flies. SAGA deubiquitinase activity is required for proper photoreceptor axon targeting since mutations in nonstop and sgf11 that disrupt this activity, causing targeting defects. The SAGA deubiquitinase module is composed of four conserved subunits: Nonstop, Sgf11, E(y)2 and Ataxin 7. Notably, disrupting SAGA deubiquitinase activity results in the human progressive visual disorder spinocerebellar ataxia type 7 (SCA7). Glial cells play a crucial role in both the neuronal connectivity defect in nonstop and sgf11 flies, and in the retinal degeneration in SCA7 patients. Thus, we sought to identify the gene targets of SAGA deubiquitinase activity in glia. We used our nuclei affinity-purification technique to profile the transcriptomes of glia from wild-type, nonstop and sgf11 larval optic lobes and showed that SAGA deubiquitinase activity is required for proper expression of 16% of actively transcribed genes in glia, especially genes involved in proteasome function, protein folding and axon guidance. We further show that SAGA deubiquitinase-activated gene Multiplexin (Mp), is required in glia for proper photoreceptor axon targeting. Mutations in the human ortholog of Mp, COL18A1, have been identified in a family with a SCA7-like progressive visual disorder, suggesting that defects in the expression of this gene in SCA7 patients could play a role in the retinal degeneration that is unique to this ataxia. Following development, photoreceptor neurons must function throughout the lifespan of the organism, ranging from up to 100 days for Drosophila melanogaster to 100 years for humans. Aging is the primary risk factor for neurodegenerative disorders and ocular diseases such as Alzheimer’s disease and macular degeneration. There is a consistent pattern of induction of stress response pathways in the aging retina of Drosophila, mice and humans. While the stress response might play a neuroprotective role in the aging brain, the direct contribution of stress to the transcriptional changes observed in aging photoreceptors is not known. Light itself, although essential for vision, poses a stress to the visual system through photogeneration of reactive oxygen species (ROS). Increasing age correlates with increased sensitivity to blue light in Drosophila photoreceptors. Thus, blue light provides a model system in which to determine how aging affects the transcriptional response to stress in photoreceptor neurons. We have characterized the transcriptional response to blue light in photoreceptors from day 1 and 6 old flies to identify genes that are differentially regulated in response to blue light in aging photoreceptors. Strikingly, blue light exposure induces similar transcriptional changes to those observed in 40 day old photoreceptor neurons, suggesting that light stress accounts for a subset of the transcriptional changes observed in aging photoreceptors. These studies suggest that blue light provides a paradigm to characterize the interplay between stress and aging in Drosophila photoreceptors.
Weake, Purdue University.
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