The Drosophila adult central brain contains 240 circadian neurons, of which there are more than 25 different neuron subtypes based on connectomic data. Recent single-cell RNA-seq characterization of these neurons "around the clock" also indicates a similar number of molecular subtypes of circadian neurons, but other conclusions from these transcriptomic studies warranted verifying and extending with other approaches. To address these limitations, we used three complementary approaches: 1) We used a genetic multiplexing strategy to profile the transcriptomes of circadian neurons from multiple time points in a single experiment, reducing confounding technical variation between timepoints; 2) Large numbers of single nuclei were sequenced (snRNA-seq), which was enabled because the method El-INTACT purifies nuclei from frozen heads; 3) We assayed 12 time points under both light-dark and constant darkness conditions. These approaches showed dramatic transcriptional differences between time points in many circadian neuron types and enhanced time-of-day gene expression analysis. The data indicate that most of this regulation is transcriptional and circadian. There were however a small number of light-dependent transcripts, including some that correspond to mammalian immediate-early genes. They probably play a role in the light-regulation of gene expression and behavior in specific neurons, perhaps circadian entrainment or phase-shifting. The results taken together provide a more comprehensive picture of gene expression heterogeneity within adult Drosophila circadian neurons including how intrinsic clock mechanisms and light cues are integrated across circadian neuron subtypes.
