The organismal landscape of clock cells and circadian gene expression in Drosophila
Circadian rhythms time physiological and behavioral processes to 24-hour cycles. It is generally assumed that most cells contain self-sustained circadian clocks that drive circadian rhythms in gene expression that ultimately generating circadian rhythms in physiology. While those clocks supposedly act cell autonomously, current work suggests that in Drosophila some of them can be adjusted by the brain circadian pacemaker through neuropeptides, like the Pigment Dispersing Factor (PDF). Despite these findings and the ample knowledge of the molecular clockwork, it is still unknown how circadian gene expression in Drosophila is achieved across the body . Here, we used single-cell and bulk RNAseq data to identify cells within the fly that express core-clock components.  Surprisingly, we  found that less than a third of the cell types in the fly are enriched for core-clock genes. Moreover, we identified Lamina wild field (Lawf) and Ponx-neuro positive (Poxn) neurons as putative new circadian neurons. In addition, we found several cell types that don't express core clock components but are highly enriched for cyclically expressed mRNAs. Strikingly, these cell types  express the PDF receptor (Pdfr),  suggesting that PDF drives rhythmic gene expression in many cell types in flies. Other cell types express both core circadian clock components and Pdfr, suggesting that in these cells, PDF regulates the phase of rhythmic gene expression. Together, our data suggest three different mechanisms generate daily gene expression in cells and tissues: an endogenous canonical molecular clock, PDF signaling-driven expression, or a combination of both. Overall design: w1118 flies were entrained for at least 3 days 25 °C and dissected at six different timepoints (ZT3, ZT7, ZT11, ZT15, ZT19, and ZT23). RNA from the fly brains was extracted using TRIzol reagent (Sigma, T9424) and treated with DnaseI (NEB, M0303L). We used 150 ng of RNA as input for preparing 3' RNA sequencing libraries following CelSeq2 protocol (97,98), changing the UMI to 6 bases. Sequencing was performed on Illumina NextSeq 500 system