Transcriptional profiling of Drosophila male-specific P1 (pC1) neurons

Abstract

In Drosophila melanogaster, the P1 (pC1) cluster of male-specific neurons both integrates sensory cues and drives or modulates behavioral programs such as courtship, in addition to contributing to a social arousal state. The behavioral function of these neurons is linked to the genes they express, which underpin their capacity for synaptic signaling, neuromodulation, and physiology. Yet, P1 (pC1) neurons have not been fully characterized at the transcriptome level. Moreover, it is unknown how the molecular landscape of P1 (pC1) neurons acutely changes after flies engage in social behaviors, where baseline P1 (pC1) neural activity is expected to increase. To address these two gaps, we use single cell-type RNA sequencing to profile and compare the transcriptomes of P1 (pC1) neurons harvested from socially paired versus solitary male flies. Compared to control transcriptome datasets, we find that P1 (pC1) neurons are enriched in 2,665 genes, including those encoding receptors, neuropeptides, and cell-adhesion molecules (dprs/DIPs). Furthermore, courtship is characterized by changes in ~300 genes, including those previously implicated in regulating behavior (e.g. DopEcR, Octβ3R, Fife, kairos, rad). Finally, we identify a suite of genes that link conspecific courtship with the innate immune system. Together, these data serve as a molecular map for future studies of an important set of higher-order and sexually-dimorphic neurons.

Keywords: Courtship Behavior; Drosophila melanogaster; Transcriptomics.

Figure 1.. Single cell-type RNA sequencing of fru+ P1 (pC1) neurons.

(A) P1 (pC1) neurons comprise roughly 2% of fruitless+ (fru+) neurons and 0.02% of all neurons in the central brain. (B) Schematic of courtship assay and protocol for harvesting P1 (pC1) neurons and RNA amplification. Male flies were isolated (unpaired) or placed with a conspecific female for 30 minutes (paired) prior to harvesting P1 (pC1) neurons (10–12 neurons per male). (C) Volcano plot of genes that are differentially expressed in P1 (pC1) neurons (orange). (D) Heatmap of gene expression. Each row denotes a gene with an expected expression pattern in P1 (pC1) neurons, organized by function: house-keeping genes, neuron-specific genes, astrocyte-specific genes, and sex-specific genes. Each column denotes an independent dataset. Unpaired and paired whole-head control datasets were previously published (35). The two female whole-brain control datasets were generated alongside P1 (pC1) neurons and previously published (36). cpm, counts per million.

Figure 2.. Receptor gene expression profile of P1 (pC1) neurons.

Each row denotes a single receptor-encoding gene, organized by receptor type: neuromodulatory, sensory, neuropeptide, and hormone receptors, with 4 genes (Lrp4, Eph, LpR1, LpR2) categorized as “other”. Each column denotes an independent dataset (as in heatmap in Figure 1). Genes enriched in P1 (pC1) are annotated in orange.

Figure 3.. Neuropeptide gene expression profile of P1 (pC1) neurons.

Each row denotes a single neuropeptide-encoding gene. Each column denotes an independent dataset (as in heatmap in Figure 1). Genes enriched in P1 (pC1) are annotated in orange.

Figure 4.. Gene expression differences between P1 (pC1) and fruitless+ neurons.

(A) Venn diagram of 1,326 genes that are upregulated in both P1 (pC1) and fruitless+ neuron transcriptome datasets (60). (B) Number of the 1,326 shared genes that are upregulated in brain, hindgut, and testis-derived tissue according to FlyAtlas AffyCall. (C) Number of the 1,326 shared genes that are downregulated in brain, hindgut, and testis-derived tissue. (D) Expression pattern of dpr/DIP genes in P1 (pC1) and control datasets. Each row denotes a single dpr/DIP gene. Each column denotes an independent dataset (as in heatmap in Figure 1). Genes enriched in P1 (pC1) are annotated in orange.

Figure 5.. Expression of genes that are upregulated in paired male flies.

The top 100 P1 (pC1) genes with higher expression in paired male flies compared to unpaired male flies. Each row denotes a single gene and each column denotes an independent dataset (as in heatmap in Figure 1). Gene expression was z-scored for each gene and genes were hierarchically clustered based on their expression pattern across animals.

Figure 6.. Expression of genes that are downregulated in paired male flies.

The top 89 P1 (pC1) genes with lower expression in paired male flies compared to unpaired male flies. Each row denotes a single gene and each column denotes an independent dataset (as in heatmap in Figure 1). Gene expression was z-scored for each gene and genes were hierarchically clustered based on their expression pattern across animals.

Figure 7.. Differential gene expression associated with courtship

Venn diagram of genes identified in this study with differential expression associated with courtship pairing, compared with two studies that identified genes with differential expression in the head following courtship pairing (35, 80). Genes with a putative role in the immune-response are highlighted.