Drosophila Sex Hierarchy Regulated Gene Expression in Adult Head and Central Nervous System Tissues
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ABSTRACT: The Drosophila sex determination hierarchy controls all aspects of somatic sexual differentiation, including sex-specific differences in adult morphology and behavior. To gain insight into the molecular-genetic specification of reproductive behaviors and physiology, we identified genes expressed in the adult head and central nervous system that are regulated downstream of sex-specific transcription factors encoded by doublesex (dsx) and fruitless (fru). We used a microarray approach and identified 54 genes regulated downstream of dsx. Furthermore, based on these expression studies we identified new modes of DSX-regulated gene expression. We also identified 90 and 26 genes regulated in the adult head and central nervous system tissues, respectively, downstream of the sex-specific transcription factors encoded by fru. In addition, we present molecular-genetic analyses of two genes identified in our studies, calphotin (cpn) and defective probocisis response (dpr), and begin to describe their functional roles in male behaviors. We show that dpr and dpr-expressing cells are required for the proper timing of male courtship behaviors. Keywords: genetic modification
Project description: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.
Project description:Adult reproductive behaviors in Drosophila melanogaster males and females are vastly different. Yet, neurons that express sex-specifically spliced fruitless transcripts (fru P1) underlie these behaviors in both sexes. How the same set of neurons can drive such different behaviors is an important and unresolved question in developmental genetics. A particular challenge is that fru P1-expressing neurons are only 2-5% of the adult nervous system, making studies of adult head tissue, or even the whole brain, unlikely to yield informative inferences. Translating Ribosome Affinity Purification (TRAP) identifies the actively translated pool of mRNAs from fru P1-expressing neurons and allowed us to conduct a sensitive, cell-specific assay of gene expression. The male and female fru P1-expressing neurons have a shared set of 1,642 genes with enriched TRAP transcripts that form a distinct repertoire, relative to TRAP analyses of all neurons in the adult head. Further, there are a striking number of genes (3,147) that have sex-biased TRAP enrichment in fru P1-expressing neurons. Yet, most of these genes (3,107) have only male-biased TRAP enrichment. This suggests an underlying mechanism to generate dimorphism in behavior, with the transcript repertoire that specifies female behaviors present in both sexes and a large additional set of genes with expression in the male dramatically altering the pattern. Thus, these additional genes invoke the male-specific behaviors by establishing cell fate in the same context of gene expression observed in females. These results suggest a possible global mechanism for how distinct behaviors can arise in different environments, from a shared set of neurons. Libraries were prepared from five independent biological replicates, from TRAP adult heads samples from males and females, and the mRNA input from adult heads from males and females. For each experimental condition, approximately 1,000 flies that were 8 to 24 hours post-eclosion were used.
Project description:Chromatin modifications have essential roles in directing nervous system development and behavior. Drosophila courtship is an ideal model for understanding the relationships between chromatin modifications, gene expression, sexual dimorphisms in neuroanatomy, and reproductive behaviors. These behaviors are largely innate and are under the control of sex determination hierarchy genes doublesex (dsx) and fruitless (fru) that encode sex-specific transcription factors. Transcripts from the fru P1 promoter are sex-specifically spliced, resulting in male-specific proteins, FruM, which are required for male courtship behaviors. FruM has been shown to form a complex with Bonus/TIF1, a transcriptional cofactor, and interact with chromatin modifying proteins. This indicates that sex-differential modification of the chromatin landscape contributes to creating and maintaining the potential for sexually dimorphic behavior. To examine chromatin modifications genome-wide in fru P1-expressing neurons, we have developed a method to purify chromatin in a cell-type-specific manner using a tagged histone H2B under UAS control, “Chromatag”, paired with sequential ChIP-seq. Here, we evaluate five chromatin modifications: H3K27ac, H3K4me3, H3K36me3, H3K9me3, and H3K27me3 to investigate fru P1-expressing neurons at three developmental stages in both sexes. These modifications have been well characterized for their roles in transcriptional activation or repression. We observe greater changes in chromatin modification profiles across developmental stages than between sexes. In addition, we have generated cell-type specific RNA-seq data sets, using Translating Ribosome Affinity Purification (TRAP), to examine gene expression in fru P1-expressing neurons of both sexes. Informed by these data sets, we have identified several genes with roles in neuronal function and examined their expression in fru P1-expressing neurons, revealing sexually dimorphic expression patterns. Altogether, this work offers insights into stage and sex-differences in chromatin modifications and gene expression in fru P1-expressing neurons.
Project description:Chromatin modifications have essential roles in directing nervous system development and behavior. Drosophila courtship is an ideal model for understanding the relationships between chromatin modifications, gene expression, sexual dimorphisms in neuroanatomy, and reproductive behaviors. These behaviors are largely innate and are under the control of sex determination hierarchy genes doublesex (dsx) and fruitless (fru) that encode sex-specific transcription factors. Transcripts from the fru P1 promoter are sex-specifically spliced, resulting in male-specific proteins, FruM, which are required for male courtship behaviors. FruM has been shown to form a complex with Bonus/TIF1, a transcriptional cofactor, and interact with chromatin modifying proteins. This indicates that sex-differential modification of the chromatin landscape contributes to creating and maintaining the potential for sexually dimorphic behavior. To examine chromatin modifications genome-wide in fru P1-expressing neurons, we have developed a method to purify chromatin in a cell-type-specific manner using a tagged histone H2B under UAS control, “Chromatag”, paired with sequential ChIP-seq. Here, we evaluate five chromatin modifications: H3K27ac, H3K4me3, H3K36me3, H3K9me3, and H3K27me3 to investigate fru P1-expressing neurons at three developmental stages in both sexes. These modifications have been well characterized for their roles in transcriptional activation or repression. We observe greater changes in chromatin modification profiles across developmental stages than between sexes. In addition, we have generated cell-type specific RNA-seq data sets, using Translating Ribosome Affinity Purification (TRAP), to examine gene expression in fru P1-expressing neurons of both sexes. Informed by these data sets, we have identified several genes with roles in neuronal function and examined their expression in fru P1-expressing neurons, revealing sexually dimorphic expression patterns. Altogether, this work offers insights into stage and sex-differences in chromatin modifications and gene expression in fru P1-expressing neurons.
Project description:Chromatin modifications have essential roles in directing nervous system development and behavior. Drosophila courtship is an ideal model for understanding the relationships between chromatin modifications, gene expression, sexual dimorphisms in neuroanatomy, and reproductive behaviors. These behaviors are largely innate and are under the control of sex determination hierarchy genes doublesex (dsx) and fruitless (fru) that encode sex-specific transcription factors. Transcripts from the fru P1 promoter are sex-specifically spliced, resulting in male-specific proteins, FruM, which are required for male courtship behaviors. FruM has been shown to form a complex with Bonus/TIF1, a transcriptional cofactor, and interact with chromatin modifying proteins. This indicates that sex-differential modification of the chromatin landscape contributes to creating and maintaining the potential for sexually dimorphic behavior. To examine chromatin modifications genome-wide in fru P1-expressing neurons, we have developed a method to purify chromatin in a cell-type-specific manner using a tagged histone H2B under UAS control, “Chromatag”, paired with sequential ChIP-seq. Here, we evaluate five chromatin modifications: H3K27ac, H3K4me3, H3K36me3, H3K9me3, and H3K27me3 to investigate fru P1-expressing neurons at three developmental stages in both sexes. These modifications have been well characterized for their roles in transcriptional activation or repression. We observe greater changes in chromatin modification profiles across developmental stages than between sexes. In addition, we have generated cell-type specific RNA-seq data sets, using Translating Ribosome Affinity Purification (TRAP), to examine gene expression in fru P1-expressing neurons of both sexes. Informed by these data sets, we have identified several genes with roles in neuronal function and examined their expression in fru P1-expressing neurons, revealing sexually dimorphic expression patterns. Altogether, this work offers insights into stage and sex-differences in chromatin modifications and gene expression in fru P1-expressing neurons.
Project description:Male and female reproductive behaviors in Drosophila melanogaster are vastly different, but the neurons that express sex-specifically spliced fruitless transcripts (fru P1) underlie these behaviors in both sexes. How this set of neurons can generate such different behaviors between the two sexes is an unresolved question. A particular challenge is that fru P1-expressing neurons comprise only 2-5% of the adult nervous system, and so studies of adult head tissue or whole brain may not reveal crucial differences. Translating Ribosome Affinity Purification (TRAP) identifies the actively translated pool of mRNAs from fru P1-expressing neurons allowing a sensitive, cell-type-specific assay. Genes with TRAP mRNAs that are detected in both sexes are four times as likely to be male biased (1,210) as female biased (331) in fru P1-expressing neurons. This suggests a potential mechanism to generate dimorphism in behavior. The male-biased genes may direct male behaviors by establishing cell fate in a similar context of gene expression observed in females. These results suggest a possible global mechanism for how distinct behaviors can arise from a shared set of neurons.
Project description:Drosophila melanogaster undergoes a complete metamorphosis, during which time the larval male and female forms transition into sexually dimorphic, reproductive adult forms. To understand this complex morphogenetic process at a molecular-genetic level, we performed whole genome microarray analyses. In somatic tissues at 48 hour After Puparium Formation (APF), 173 sex-biased transcripts that likely function downstream of the doublesex (dsx) branch of the sex determination hierarchy were identified. The mode of regulation of the sex-specific isoforms of DSX (DSX-F and DSX-M) was examined. It was determined that for most downstream targets, DSX-F and DSX-M regulate gene expression in the same manner, but that one isoform acts as a more potent regulator. Keywords: wild type; genetic modification
Project description:The Drosophila sex determination hierarchy consists of a splicing cascade with sex-specific transcription directing somatic sexual dimorphism. Our understanding of this pathway, and many others, is incomplete. Here we pioneer an approach to expand our knowledge of gene regulatory networks (GRNs) by leveraging natural genetic variation. This approach is generalizable to any natural population, including humans. Two studies from Drosophila female head tissue were used – the DSPR collection (alleles from 15 natural variants) and F1-hybrid collection (alleles from heterozygotes of 75 isogenic lines crossed to w1118) – in a structural equation model (SEM) analysis. We expanded the sex hierarchy GRN by adding novel links among genes in the pathway and by adding novel genes to the pathway. A link from fruitless (fru) to Sex-lethal (Sxl) was found in both populations, which is supported by the presence of fru binding sites in the Sxl locus. The splicing factors male-specific lethal 2 and Rm62 were correctly identified as downstream targets of Sxl. There were 754 additional candidate genes for an expanded sex hierarchy GRN. These candidates were enriched for genes with sex-biased splicing and many components of the spliceosome were placed in the GRN. As with other population-genetic analyses, the number of alleles limits the number of observable interactions. Network expansion was only clear in the F1-hybrid population, which has an average of twice as many alleles as the DSPR population. Independent studies of doublesex and transformer mutants support many novel connections, including evidence for a link between the sex hierarchy and metabolism, with the inclusion of Insulin-like receptor in the sex hierarchy GRN. RNA sequencing was performed on mRNA derived from adult male or female heads, for a total of 9 samples. These samples included females that produce the male isoform of dsx [w/+;DsxD/dsxm+r15 (XX)], and two dsx mutants: females [w/+; dsxm+r15/dsxd+r3 (XX)] and males [w;dsxm+r15/dsxd+r3 (XY)]. Two wild type genotypes (Berlin and Canton-S) were sequenced at the same time, but have previously been published as part of GSE50515. There were at least 3 replicates from biological samples.
Project description:In Drosophila,male courtship behaviour serves as an excellent paradigm to study how innate behaviors are controlled by the nervous system. These behaviors are in large part regulated by the gene fruitless (fru). fru encodes a set of putative transcription factors that promote male sexual behaior by controlling the development of sexually-dimorphic neuronal circuitry. Little is known about how Fru proteins function at the level of transcriptional regulation. To characterize the roles of fru sex-specific isoforms in specifying male behavior, we generated novel isoform-specific mutants, and used a genomic approach to identify direct Fru isoform targets during development. We demonstrate that all Fru isoforms directly target genes involved in the development of the nervous system, with individual isoforms exhibiting unique binding specificities. We observe that fru behavioral phenotypes are specificed by either a single, or combination, of isoforms. Finally, we illustrate the utility of these data for the identification of novel sexually dimorphic genoic enhancers, and novel downstream regulators of male-sexual behavior.
Project description:In Drosophila,male courtship behaviour serves as an excellent paradigm to study how innate behaviors are controlled by the nervous system. These behaviors are in large part regulated by the gene fruitless (fru). fru encodes a set of putative transcription factors that promote male sexual behaior by controlling the development of sexually-dimorphic neuronal circuitry. Little is known about how Fru proteins function at the level of transcriptional regulation. To characterize the roles of fru sex-specific isoforms in specifying male behavior, we generated novel isoform-specific mutants, and used a genomic approach to identify direct Fru isoform targets during development. We demonstrate that all Fru isoforms directly target genes involved in the development of the nervous system, with individual isoforms exhibiting unique binding specificities. We observe that fru behavioral phenotypes are specificed by either a single, or combination, of isoforms. Finally, we illustrate the utility of these data for the identification of novel sexually dimorphic genoic enhancers, and novel downstream regulators of male-sexual behavior. 3 replicates per condition, one dye swap; per condition: one control, one experimental per replicate. No processed data for GSM1261882 and GSM1261883 are available.