Characterization of the Role of Differential Gene Expression and RNA Editing in Drosophila Tonic and Phasic Motoneuron Diversity

Abstract

How differential regulation of gene expression programs across neuronal subtypes leads to their unique morphology, membrane excitability and synaptic properties is poorly understood. Protein expression and function within neurons depends on numerous transcriptional and post-transcriptional regulatory steps, which can be controlled at a single cell level. Using modern single cell sequencing approaches, the transcriptional and post-transcriptional regulatory programs for many cell subtypes have recently been analyzed. In this thesis, I have compared and analyzed the distinct transcriptomes of the tonic Ib and phasic Is glutamatergic motoneuron subtypes in the Drosophila 3rd instar larvae. These neurons display distinct morphological and electrophysiological properties, in addition to being easily accessible for imaging, electrophysiology, and genetic approaches, making them an ideal model system to test how programs of gene regulation can lead to distinct morphological and functional synaptic properties. First, we sequenced RNA from 105 Ib and 101 Is single neurons to define the transcriptome for each neuronal subtype. Comparison of gene expression levels revealed ~800 differentially expressed genes (DEGs) across multiple gene classes, including intracellular Ca²⁺ buffers, signaling ligands and transcription factors, synaptic cleft proteins, and ion channels. Perturbation of these genes produced cell-type-specific morphological and electrophysiological defects, indicating that these genes are important in determining cell-type-specific characteristics of the two neuronal classes. Next, I analyzed how a specific type of posttranscriptional modification, RNA editing, was differentially regulated across the transcriptome in each neuronal subtype. Out of ~14,000 genes in the Drosophila genome, only a small number (324 sites in 215 genes) were canonically RNA edited at a robust level in these motoneurons. The majority of the edits caused significant missense mutations in protein coding domains, indicating RNA editing could play a direct role in modulating protein function. Editing levels varied considerably between single neurons, although some edits were found in almost every cell and some sites were edited to ~100% in each cell with editing. 42 edits occurred in evolutionarily conserved regions of the encoded proteins, suggesting they may play important roles in protein function. Finally, 26 of the 324 sites discovered were differentially edited between Ib and Is subtypes. Taken together, our data indicate that cell-type-specific programs of transcriptional regulation set initial cell-specific characteristics, but that post-transcriptional and post-translation regulation through RNA editing and other mechanisms act to fine-tune effective expression levels and function for a subset of the proteome. Our transcriptomic and RNA editing data provide a resource for future experiments to determine the role of specific DEGs and RNA editing events in determining cell-type-specific diversity and function of Drosophila larval motoneurons.