Abstract
To achieve reproductive success, an organism must integrate environmental information and coordinate interactions between functionally distinct neuronal and non-neuronal cell types. In Drosophila, courtship and mating involve sexually distinct neurons that serve as hubs of stimulus integration, leading to sexually dimorphic behaviors essential for reproduction. These behavioral outputs include courtship rituals, mating, aggression, mate receptivity, oogenesis, oviposition, and locomotor activity. Many of these dimorphic physiological and behavioral outputs follow daily rhythms or time-of-day preferences. These behavioral outputs are thought to be regulated by endogenous timekeepers: the circadian neurons. Clock neurons, a heterogeneous group of ~240 cells in the Drosophila brain, are the master regulators of circadian rhythms. They contribute to circadian and non-circadian processes, with their unique gene expression shaping behavioral outputs. However, the role of sexually dimorphic gene expression in these neurons in regulating dimorphic behaviors remains poorly understood.
The work presented here focuses on identifying sex differences in the transcriptome of circadian neurons and how gene expression might influence sexually dimorphic behaviors. We find that sexually dimorphic transcriptomic features are not ubiquitous across the circadian circuit, but at least four circadian cell types exhibit sex-specific gene expression. These include subsets of dorsal lateral neurons (LNds) and dorsal neurons (DNs), whose function in circadian regulation remains largely enigmatic. The sexually differentiated gene expression profiles of these neurons indicate enrichment of neural connectivity molecules, including neuropeptides, G-protein coupled receptors (GPCRs), and cell adhesion molecules (CAMs). Additionally, we identified synaptic connections from the dimorphic LNds to two central regulators of sexually dimorphic behavioral outputs: pC1 and pCd neurons in both sexes. This connection appears to be, at least in part, mediated by sex-specific CAMs in the LNds.
Further, we profiled the transcriptome of male and female circadian neurons under two sexually dimorphic paradigms: female post-mating response and male rival-induced mating duration. Our analysis suggests that clock neuron function and gene expression play a role in regulating sex-specific behaviors. In the female post-mating response dataset, we found several DNs, particularly the DN1a cell type, upregulate GPCRs and stress-response genes following mating. In the male rival-induced mating duration dataset, we observed male-male social interactions upregulate signaling genes involved in tyramine and octopamine synthesis and calcium ion binding activity. In contrast, social isolation upregulates genes involved in axon guidance and heterochromatin formation. Additionally, we identified candidate genes in the dimorphic male LNds that may modulate rival-induced mating duration.
By generating these datasets, we uncovered sexually dimorphic gene expression profiles of previously uncharacterized circadian subsets and identified a circadian transcriptional response under two sexually dimorphic behavioral paradigms. These datasets could help find new interactions and mechanisms that enhance our understanding of how functionally distinct neurons collaborate in regulating sexual reproduction.