Novel clock neuron subtypes regulate temporal aspects of 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 about half of the 75 pairs of clock neurons in the fly brain 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 different DN3 subtypes with distinct projection patterns promote sleep at specific times of the day through a common 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, projection patterns and functional properties.