Examining the role of chromatin modifications and gene expression in neurons is critical for understanding how the potential for behaviors are established and maintained. We investigate this question by examiningDrosophila melanogaster fru P1neurons that underlie reproductive behaviors in both sexes. We developed a method to purify cell-type-specific chromatin (Chromatag), using a tagged histone H2B variant that is expressed using the versatile Gal4/UAS gene expression system. Here, we use Chromatag to evaluate five chromatin modifications, at three life stages in both sexes. We find substantial changes in chromatin modification profiles across development and fewer differences between males and females. Additionally, we find chromatin modifications that persist in different sets of genes from pupal to adult stages, which may point to genes important for cell fate determination infru P1neurons. We generated cell-type-specific RNA-seq data sets, using translating ribosome affinity purification (TRAP). We identify actively translated genes infru P1neurons, revealing novel stage- and sex-differences in gene expression. We also find chromatin modification enrichment patterns that are associated with gene expression. Next, we use the chromatin modification data to identify cell-type-specific super-enhancer-containing genes. We show that genes with super-enhancers infru P1neurons differ across development and between the sexes. We validated that a set of genes are expressed infru P1neurons, which were chosen based on having a super-enhancer and TRAP-enriched expression infru P1neurons.

Drosophila reproductive behaviors are directed by fruitless neurons. A reanalysis of genomic studies shows that genes encoding dpr and DIP Immunoglobulin superfamily (IgSF) members are expressed in fru P1 neurons. We find that each fru P1 and dpr/DIP (fru P1 ∩ dpr/DIP) overlapping expression pattern is similar in both sexes, but there are dimorphisms in neuronal morphology and cell number. Behavioral studies of fru P1 ∩ dpr/DIP perturbation genotypes indicates that the mushroom body functions together with the lateral protocerebral complex to direct courtship behavior. A single-cell RNA-seq analysis of fru P1 neurons shows that many DIPs have high expression in a small set of neurons, whereas the dprs are often expressed in a larger set of neurons at intermediate levels, with a myriad of dpr/DIP expression combinations. Functionally, we find that perturbations of sex hierarchy genes and of DIP-ε change the sex-specific morphologies of fru P1 ∩ DIP-α neurons.

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.

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Examining the role of chromatin modifications and gene expression in neurons is critical for understanding how the potential for behaviors are established and maintained. We investigate this question by examining Drosophila melanogaster fru P1 neurons that underlie reproductive behaviors in both sexes. We developed a method to purify cell-type-specific chromatin (Chromatag), using a tagged histone H2B variant that is expressed using the versatile Gal4/UAS gene expression system. Here, we use Chromatag to evaluate five chromatin modifications, at three life stages in both sexes. We find substantial changes in chromatin modification profiles across development and fewer differences between males and females. We generated cell-type-specific RNA-seq data sets, using translating ribosome affinity purification (TRAP), and identify actively translated genes in fru P1 neurons, revealing novel stage- and sex-differences in gene expression. We compare chromatin modifications to the gene expression data and find patterns of chromatin modifications associated with gene expression. An examination of the genic features where chromatin modifications resides shows certain chromatin modifications are maintained in the same genes across development, whereas others are more dynamic, which may point to modifications important for cell fate determination in neurons. Using a computational analysis to identify super-enhancer-containing genes we discovered differences across development, and between the sexes that are cell-type-specific. A set of super-enhancer-containing genes that overlapped with those determined to be expressed with the TRAP approach were validated as expressed in fru P1 neurons.Author SummaryDifferences in male and female reproductive behaviors are pervasive in nature and important for species propagation. Studies of sex differences in the fruit fly, Drosophila melanogaster, have been ongoing since the early 1900s, with many of the critical molecular and neural circuit determinates that create sexually dimorphic behavior identified. This system is a powerful model to understand fundamental principles about the underpinnings of complex behavior at high resolution. In this study, we examine the gene expression and chromatin modification differences specifically in a set of neurons that direct male and female reproductive behaviors in Drosophila. We describe differences across development and between the sexes with the goal of understanding how the potential for behavior is created and maintained.