Unknown,Transcriptomics,Genomics,Proteomics

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The Wright Stuff: Reimagining Path Analysis Reveals Novel Components of the Sex Determination Hierarchy in Drosophila Melanogaster

ABSTRACT: 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.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Michelle Arbeitman

PROVIDER: E-GEOD-67400 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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:Background: RNA-seq is revolutionizing the way we study transcriptomes. mRNA can be surveyed without prior knowledge of gene transcripts. Alternative splicing of transcript isoforms and the identification of previously unknown exons are being reported. Initial reports of differences in exon usage, and splicing between samples as well as quantitative differences among samples are beginning to surface. Biological variation has been reported to be larger than technical variation. In addition, technical variation has been reported to be in line with expectations due to random sampling. However, strategies for dealing with technical variation will differ depending on the magnitude. The size of technical variance, and the role of sampling are examined in this manuscript. Results: Independent Solexa/Illumina experiments containing technical replicates are analyzed. When coverage is low, large disagreements between technical replicates are apparent. Exon detection between technical replicates is highly variable when the coverage is less than 5 reads per nucleotide and estimates of gene expression are more likely to disagree when coverage is low. Although large disagreements in the estimates of expression are observed at all levels of coverage. Conclusions: Technical variability is too high to ignore. Technical variability results in inconsistent detection of exons at low levels of coverage. Further, the estimate of the relative abundance of a transcript can substantially disagree, even when coverage levels are high. This may be due to the low sampling fraction and if so, it will persist as an issue needing to be addressed in experimental design even as the next wave of technology produces larger numbers of reads. We provide practical recommendations for dealing with the technical variability, without dramatic cost increases. Three independent samples of D. melanogaster female heads were collected with each sample representing a unique pool of biological material. Each sample was prepared according to manufacturer's instructions and then the same library was run on two lanes of a Solexa/Illumina flow cell, resulting in two technical replicates for each biological replicate, runs were 36 base-pair paired end.

Project description:Background: RNA-seq is revolutionizing the way we study transcriptomes. mRNA can be surveyed without prior knowledge of gene transcripts. Alternative splicing of transcript isoforms and the identification of previously unknown exons are being reported. Initial reports of differences in exon usage, and splicing between samples as well as quantitative differences among samples are beginning to surface. Biological variation has been reported to be larger than technical variation. In addition, technical variation has been reported to be in line with expectations due to random sampling. However, strategies for dealing with technical variation will differ depending on the magnitude. The size of technical variance, and the role of sampling are examined in this manuscript. Results: Independent Solexa/Illumina experiments containing technical replicates are analyzed. When coverage is low, large disagreements between technical replicates are apparent. Exon detection between technical replicates is highly variable when the coverage is less than 5 reads per nucleotide and estimates of gene expression are more likely to disagree when coverage is low. Although large disagreements in the estimates of expression are observed at all levels of coverage. Conclusions: Technical variability is too high to ignore. Technical variability results in inconsistent detection of exons at low levels of coverage. Further, the estimate of the relative abundance of a transcript can substantially disagree, even when coverage levels are high. This may be due to the low sampling fraction and if so, it will persist as an issue needing to be addressed in experimental design even as the next wave of technology produces larger numbers of reads. We provide practical recommendations for dealing with the technical variability, without dramatic cost increases. Three independent samples of D. simulans male heads were collected with each sample representing a unique pool of biological material. Each sample was prepared according to manufacturer's instructions and then the same library was run on two lanes of a Solexa/Illumina flow cell, resulting in two technical replicates for each biological replicate, runs were 36 base-pair paired end.

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The Wright Stuff: Reimagining Path Analysis Reveals Novel Components of the Sex Determination Hierarchy in Drosophila Melanogaster

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