Single-cell transcriptome profiles of Drosophila fruitless-expressing neurons from both sexes

Abstract

Drosophila melanogaster reproductive behaviors are orchestrated by fruitless neurons. We performed single-cell RNA-sequencing on pupal neurons that produce sex-specifically spliced fru transcripts, the fru P1-expressing neurons. Uniform Manifold Approximation and Projection (UMAP) with clustering generates an atlas containing 113 clusters. While the male and female neurons overlap in UMAP space, more than half the clusters have sex differences in neuron number, and nearly all clusters display sex-differential expression. Based on an examination of enriched marker genes, we annotate clusters as circadian clock neurons, mushroom body Kenyon cell neurons, neurotransmitter- and/or neuropeptide-producing, and those that express doublesex. Marker gene analyses also show that genes that encode members of the immunoglobulin superfamily of cell adhesion molecules, transcription factors, neuropeptides, neuropeptide receptors, and Wnts have unique patterns of enriched expression across the clusters. In vivo spatial gene expression links to the clusters are examined. A functional analysis of fru P1 circadian neurons shows they have dimorphic roles in activity and period length. Given that most clusters are comprised of male and female neurons indicates that the sexes have fru P1 neurons with common gene expression programs. Sex-specific expression is overlaid on this program, to build the potential for vastly different sex-specific behaviors.

Transcriptional profiling ofDrosophilamale-specific P1 (pC1) neurons

“…The diuretic hormone Dh44 contributes to nutrient sensing (54), aggression (55), and stress responses (56). We find that P1 (pC1) neurons differentially express Dh44 , in keeping with a recent analysis of fruitless+ neurons showing that Dh44 is expressed in a population of fruitless+ cholinergic neurons (48). Moreover, the morphology of some of these neurons resembles P1 (pC1).…”

“…Several of these genes are expressed in fruitless+ neurons or contribute to courtship behaviors. For example, Dh31 and Ilp7 are highly expressed in fruitless+ neurons of both sexes (48). And, Neuropeptide F ( NPF ) regulates courtship drive in male flies (45).…”

Let's talk about sex: Mechanisms of neural sexual differentiation in Bilateria

“…More recently, molecular characterizations of sexually differentiated neurons have identified hundreds of genes with sex‐specific, cell‐type‐specific expression (Brovkina et al, 2021; P. B. Chen et al, 2019; Gegenhuber et al, 2022; D.‐W. Kim et al, 2019; Moffitt et al, 2018; Palmateer et al, 2023; Welch et al, 2019). Finally, direct neuronal targets of ERα were recently described for the first time, in mice, by Gegenhuber et al (2022); many targets are involved in neuronal development processes like synapse organization and regulation of ion transport.…”

“…Fru M regulates hundreds of enhancers and has different effectors in different populations of neurons. Many of these enhancers are located near genes for cell surface molecules or other transcription factors (Brovkina et al, 2021; Palmateer et al, 2023). One known target of Fru M is the robo1 gene in males; the differential repression of this gene leads to different neuronal morphology between sexes (Ito et al, 2016).…”

“…Fru, TRA‐1, ERα, and AR are all expressed in diverse neuronal populations and mediate heterogeneous changes to neuronal characteristics across different cell types and brain regions. The transcriptional targets of these factors are beginning to be identified and are cell‐type‐specific, which begins to describe how individual transcription factors can have such pleiotropic effects on neuronal and circuit features across the life span (Brovkina et al, 2021; Gegenhuber et al, 2022; Krause et al, 2021; Palmateer et al, 2023). Nevertheless, a tremendous amount of work remains to be done to determine how cell‐type‐specific changes in gene expression induced by sex‐specific transcription factors produce sex differences in neurons, circuits, and behaviors.…”