Many different functions are regulated by circadian rhythms, including those orchestrated by discrete clock neurons within animal brains. To comprehensively characterize and assign cell identity to the 75 pairs of Drosophila circadian neurons, we optimized a single-cell RNA sequencing method and assayed clock neuron gene expression at different times of day. The data identify at least 17 clock neuron categories with striking spatial regulation of gene expression. Transcription factor regulation is prominent and likely contributes to the robust circadian oscillation of many transcripts, including those that encode cell-surface proteins previously shown to be important for cell recognition and synapse formation during development. The many other clock-regulated genes also constitute an important resource for future mechanistic and functional studies between clock neurons and/or for temporal signaling to circuits elsewhere in the fly brain.

A sensitivity of the circadian clock to light/dark cycles ensures that biological rhythms maintain optimal phase relationships with the external day. In animals, the circadian clock neuron network (CCNN) driving sleep/activity rhythms receives light input from multiple photoreceptors, but how these photoreceptors modulate CCNN components is not well understood. Here we show that the HofbauerBuchner eyelets differentially modulate two classes of ventral lateral neurons (LNvs) within the Drosophila CCNN. The eyelets antagonize Cryptochrome (CRY)-and compound-eye-based photoreception in the large LNvs while synergizing CRY-mediated photoreception in the small LNvs. Furthermore, we show that the large LNvs interact with subsets of "evening cells" to adjust the timing of the evening peak of activity in a day length-dependent manner. Our work identifies a peptidergic connection between the large LNvs and a group of evening cells that is critical for the seasonal adjustment of circadian rhythms.

The genus Drosophila contains over 2,000 species that, stemming from a common ancestor in the Old World Tropics, populate today very different environments [1, 2] (reviewed in [3]). We found significant differences in the activity pattern of Drosophila species belonging to the holarctic virilis group, i.e., D. ezoana and D. littoralis, collected in Northern Europe, compared to that of the cosmopolitan D. melanogaster, collected close to the equator. These behavioral differences might have been of adaptive significance for colonizing high-latitude habitats and hence adjust to long photoperiods. Most interestingly, the flies' locomotor activity correlates with the neurochemistry of their circadian clock network, which differs between low and high latitude for the expression pattern of the blue light photopigment cryptochrome (CRY) and the neuropeptide Pigment-dispersing factor (PDF) [4-6]. In D. melanogaster, CRY and PDF are known to modulate the timing of activity and to maintain robust rhythmicity under constant conditions [7-11]. We could partly simulate the rhythmic behavior of the high-latitude virilis group species by mimicking their CRY/PDF expression patterns in a laboratory strain of D. melanogaster. We therefore suggest that these alterations in the CRY/PDF clock neurochemistry might have allowed the virilis group species to colonize high-latitude environments.

Cryptochromes are flavoproteins, structurally and evolutionarily related to photolyases, that are involved in the development, magnetoreception, and temporal organization of a variety of organisms. Drosophila CRYPTOCHROME (dCRY) is involved in light synchronization of the master circadian clock, and its C terminus plays an important role in modulating light sensitivity and activity of the protein. The activation of dCRY by light requires a conformational change, but it has been suggested that activation could be mediated also by specific "regulators" that bind the C terminus of the protein. This C-terminal region harbors several protein-protein interaction motifs, likely relevant for signal transduction regulation. Here, we show that some functional linear motifs are evolutionarily conserved in the C terminus of cryptochromes and that class III PDZ-binding sites are selectively maintained in animals. A coimmunoprecipitation assay followed by mass spectrometry analysis revealed that dCRY interacts with Retinal Degeneration A (RDGA) and with Neither Inactivation Nor Afterpotential C (NINAC) proteins. Both proteins belong to a multiprotein complex (the Signalplex) that includes visualsignaling molecules. Using bioinformatic and molecular approaches, dCRY was found to interact with Neither Inactivation Nor Afterpotential C through Inactivation No Afterpotential D (INAD) in a light-dependent manner and that the CRY-Inactivation No Afterpotential D interaction is mediated by specific domains of the two proteins and involves the CRY C terminus. Moreover, an impairment of the visual behavior was observed in fly mutants for dCRY, indicative of a role, direct or indirect, for this photoreceptor in fly vision.