Bichromatic Exon-Reporters Reveal Voltage-Gated Ca2+-Channel Splice-Isoform 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 α₁ subunit of the primary neuronal voltage-gated Ca²⁺ 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.