Effect of mifepristone on gene expression in Drosophila
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE64474 | GEO | 2015/01/18
SECONDARY ACCESSION(S): PRJNA271084
REPOSITORIES: GEO
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Project description:Illumina sequencing was used to assay the effect of mifepristone treatment on gene expression in adult Drosophila, including males, virgin females and mated females. Males of strain w[1118]; p53B[6] were crossed to virgins of w[1118]; rtTA(3)E2 and progeny males and virgins were collected over 48 hours. One half of the virgins were mated to w[1118] males at ratio of 1:1 virgins to males for 4 days. Mated females were then separated from the w[1118] males. The mated females, males and virgins females were then maintained at approximately 20 flies per vial, on food with and without supplementation with 160ug/ml mifepristone for 12 days. Total fly RNA was isolated from 20 animals per sample. Three replicate samples were generated for each type of flies: males, mated females and virgin females.
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Project description:Sex Peptide, a seminal fluid protein of Drosophila melanogaster males, elicits an array of post-mating responses in females, including decreased receptivity to re-mating, and increased egg laying, activity and food intake, with a preference for protein-rich food. To determine how one protein can have such widespread effects, we set out to dissect the genetic architecture of the female’s response to Sex Peptide, to determine whether Sex Peptide alters the expression of several regulators targeted to specific post-mating responses or acts on a pleiotropic regulator that controls multiple responses. We performed bulk RNA-seq of female heads at 10 time points within the first 24 hours after mating, sampling virgin females, females mated to control males and females mated to Sex Peptide-null males. Using this high-resolution time series, we identified mating- and Sex Peptide-dependent differentially expressed genes and discovered the presence of differentially used exons. We constructed gene regulatory networks using clustering and motif enrichment analyses, and identified cell types in which these changes might take place using deconvolution of our bulk RNA-seq dataset. One key network included metabolic genes which might change in expression in the female’s fat body. A second network included genes with neuronal functions, whose changes might be located in neurons or sensory organs in the female’s head. Within these networks we identified known molecular regulators of the circadian clock. Further, we found that many differentially expressed genes, and some differentially used exons, followed a circadian rhythm in virgin females, and that this rhythm was altered after mating with a Sex Peptide- male.
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Project description:In this analysis, we used microarrays to contrast genome-wide transcript levels in virgin versus mated females before and after infection. We repeated the entire experiment using female mutants that do not form a germline. We found that multiple genes involved in egg production show reduced expression in response to infection, and that this reduction is stronger in virgins than it is in mated females. In germline-less females, expression of egg-production genes was predictably low and not differentially affected by infection. We also identified several immune responsive genes that are differentially induced after infection in virgins versus mated females.
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Project description:Seminal fluid contains some of the fastest evolving proteins currently known. These seminal fluid proteins (Sfps) play crucial roles in reproduction, such as supporting sperm function, and – particularly in insects – modifying female physiology and behaviour. Identification of Sfps in small animals is challenging, and often relies on samples taken from the female reproductive tract after mating. A key pitfall of this method is that it might miss Sfps that are of low abundance due to dilution in the female-derived sample or rapid processing in females. Here we present a new and complimentary method, which provides added sensitivity to Sfp identification. We applied label-free quantitative proteomics to Drosophila melanogaster male reproductive tissue – where Sfps are unprocessed, and highly abundant – and quantified Sfps before and immediately after mating, to infer those transferred during copulation. We also analysed female reproductive tracts immediately before and after copulation to confirm the presence and abundance of known and candidate Sfps, where possible. Results were cross-referenced with transcriptomic and sequence databases to improve confidence in Sfp detection. Our data was consistent with 124 previously reported Sfps. We found 8 high-confidence novel candidate Sfps, which were both depleted in mated versus unmated males and identified within the reproductive tract of mated but not virgin females. We also identified 31 more candidates that are likely Sfps based on their abundance, known expression and predicted characteristics, and revealed that four proteins previously identified as Sfps are at best minor contributors to the ejaculate. The estimated copy numbers for our candidate Sfps were lower than for previously identified Sfps, supporting the idea that our technique provides a deeper analysis of the Sfp proteome than previous studies. Our results demonstrate a novel, high-sensitivity approach to the analysis of seminal fluid proteomes, whose application will further our understanding of reproductive biology.
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