Abstract
Many sexually dimorphic behaviors exhibit a time-of-day preference, but our understanding of how sex shapes the molecular and circuit properties of central brain neurons remains incomplete. Here, we report a transcriptomic and circuit underpinning of sexual dimorphism within the Drosophila circadian network. By performing single-cell RNA sequencing of male and female clock neurons, we identify specific subsets of dorsal lateral neurons (LNds), dorsal neurons 1p (DN1ps) and dorsal neurons 3 (DN3s) with dramatic dimorphic gene expression profiles. These sex differences are driven in large part by cell-type-specific expression of genes involved in neural connectivity, particularly cell adhesion molecules (CAMs). We focused on the dimorphic Cry-negative E3 LNds and show that they form functionally active, synaptic connections with downstream doublesex-expressing pC1 and pCd neurons, central regulators of dimorphic behaviors. Moreover, we demonstrate that these connections are mediated at least in part by sex-enriched CAMs, dpr9 in males and dpr3 in females. Thus, our work reveals sexual differentiation mechanisms at both the molecular and circuit levels, identifying specific molecules that sculpt sex-specific pathways and link the circadian clock to dimorphic outputs.