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. Overall design: 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.

动物脑内的节律神经元（circadian neurons）调控着众多生理过程与行为，但这类神经元对睡眠调控的具体贡献尚不明晰。为弥补这一研究空白，我们借助单细胞RNA测序（single-cell RNA sequencing）技术，构建了全新且全面的果蝇时钟神经元（Drosophila clock neurons）转录组细胞类型全景图谱。本研究重点聚焦于一类功能未明的DN3神经元——这类神经元占果蝇脑内时钟神经元的绝大多数，此前几乎未被充分表征。与研究较为深入的时钟神经元相比，这类DN3神经元可分为12个转录组亚型，且具备独特的基因表达特征。我们进一步证实，此前未被表征的DN3亚型可通过G蛋白偶联受体（G protein-coupled receptor）TrissinR介导睡眠促进效应。本研究结果表明，时钟神经元对睡眠行为存在精密复杂的调控机制，并凸显了其在基因表达与功能特性上的显著多样性。
实验整体设计：我们构建了一种全新的split GAL4驱动株系（split GAL4 driver line），该株系可在几乎所有DN3神经元中特异性表达。为深入解析其基因表达谱特征，我们采用基于液滴的单细胞RNA测序（droplet-based single cell RNA sequencing）技术，在昼夜周期内的六个时间点获取了单细胞测序数据。