Bichromatic exon-reporters reveal voltage-gated Ca2+-channel spliceisoform diversity across Drosophila neurons in vivo

Every neuron contains the same genomic information but its complement of proteins is the product of countless neuron-specific steps including pre-mRNA splicing. Despite advances in RNA sequencing techniques, pre-mRNA splicing biases that favor one isoform over another are largely inscrutable in live neurons in situ . Here, in Drosophila , we developed bichromatic fluorescent reporters to investigate alternative splicing of cacophony (cac) – a gene that codes the pore-forming α 1 -subunit of the primary neuronal voltage-gated Ca 2+ channel (VGCC). These reporters revealed a neuron-specific pattern of exon biases, highly consistent from one animal to the next, suggesting that each neuron splices a unique and consistent portfolio of VGCC isoforms. Stereotypical patterns were observed within motor neurons and multidendritic sensory neurons of female larvae, and also within mushroom body Kenyon cells of female adults. In a validation step we demonstrated that exon splice bias reporting was not dependent on the choice of fluorophores. Additionally, functional properties of the female larval motor neuron terminals could be generally reconciled with the functional properties predicted for the reported exon bias. The application of this technology to a large gene such as cac provides a precedence for effective exon-reporter design for other Drosophila genes. Significance Statement Ca 2+ ions are ubiquitous messengers in the nervous system and the channels that gate their passage across membranes play a prominent role in nervous system function. In flies, as in humans, genes that code for Ca 2+ channels give rise to different Ca 2+ channel variants through the process of alternative gene splicing. However, at the level of individual cells in living tissue, the splicing process is largely inscrutable, making it difficult to elucidate the consequences of alternative splicing in either health or disease. Here, in the fruit fly brain, we demonstrate a technique that expresses fluorescent proteins of different colors according to the bias of the splicing process that yields different Ca 2+ channel variants in different cells in the live animal.