Transcriptomic DN3 clock neuron subtypes regulate Drosophila sleep

Circadian neurons within animal brains orchestrate myriad physiological processes and behaviors, but the contribution of these neurons to the regulation of sleep is not well understood. To address this deficiency, we leveraged single-cell RNA sequencing to generate a new and now comprehensive census of transcriptomic cell types of Drosophila clock neurons. We focused principally on the enigmatic DN3s, which constitute most fly brain clock neurons and were previously almost completely uncharacterized. These DN3s are organized into 12 clusters with unusual gene expression features compared to the more well-studied clock neurons. We further show that previously uncharacterized DN3 subtypes promote sleep through a G protein–coupled receptor, TrissinR. Our findings indicate an intricate regulation of sleep behavior by clock neurons and highlight their remarkable diversity in gene expression and functional properties. We developed a novel split GAL4 driver line that is expressed in almost all of the DN3 neurons. To learn more about their gene expression profiles, we utilized a droplet-based single cell RNA sequencing method and generated six time points of single cell data around the clock.