Unknown,Transcriptomics,Genomics,Proteomics

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Indirect Flight Muscle (IFM) expression profiling of actin and myosin nulls in Drosophila melanogaster

ABSTRACT: Expression profiling of IFMs from 1-2 day old adult male flies of 3 genotypes: Canton-S, IFM-specific actin null (Act88FKM88) and IFM-specific myosin null (Mhc7). Results provide insight into how muscles respond to the absence of major scaffold proteins, and whether these transcriptional responses are filament-specific or generic to the tissue. RNA was extracted from the IFMs of 1-2 day old adult male flies of 3 genotypes (Canton-S, Act88FKM88, Mhc7), labeled, and hybridized on Affymetrix GeneChip Drosophila Genome 2.0 microarrays. 9 microarray experiments from 3 independent biological replicates of each genotype. Data was analyzed using RMA method, and significance was tested using SAM version 3.02, with cut-off parameters of 10% FDR and minimum 2-fold change in expression levels.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Aditi Madan

PROVIDER: E-GEOD-70252 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: localized aberrant cell proliferation induced by activation of Yki in ISCs impairs muscle functions. To gain insight into the cross talk between intestine and muscle, we performed a transcriptomic analysis of thoracic muscles in ISC overproliferation flies. Methods: To extract total RNAs for RNA-Seq experiment, we used 10 thoraces dissected out from both esg-Gal4, tub-Gal80ts, UAS-GFP/+ (Con) and esg-Gal4, tub-Gal80ts, UAS-GFP/UAS-Yki-act (Yki) flies incubated for 8 days at 29Â°C. After assessing RNA quality with Agilent Bioanalyzer, mRNAs were enriched by poly-A pull-down. Then, sequencing libraries constructed with Illumina TruSeq RNA prep kit were sequenced using Illumina HiSeq2000 at the Columbia Genome Center (http://systemsbiology.columbia.edu/genome-center). We multiplexed samples in each lane, which yields targeted number of single-end 100 bp reads for each sample, as a fraction of 180 million reads for the whole lane. Sequence reads were mapped back to the Drosophila genome (flybase genome annotation version r5.51) using Tophat. With the uniquely mapped reads, we quantified gene expression levels using Cufflinks (FPKM values). Next, we performed data normalization on the read counts and applied a negative binomial statistical framework using the Bioconductor package DESeq to quantify differential expression between experimental and control data. Results: Gene list enrichment analysis of the downregulated muscle transcriptome by ISCs Yki overexpressioin revealed a striking enrichment of multiple metabolic processes impinging on carbohydrate metabolism, amino acid metabolism, metabolism of vitamins and cofactors, and metabolism of xenobiotics by cytochrome P450. Interestingly, target genes of Foxo, a transcription factor inhibited by insulin/IGF signaling, are enriched in the upregulated muscle transcriptome of ISCs Yki overepxression flies. In particular, induction of InR and Thor, well-characterized targets of Foxo, are validated with qPCR. Conclusions: Our study represents ISCs overproliferation induced by Yki overepxression remotedly regulates muscle function and gene expression probally via modulation of insulin signaling in muscle. Our results show that RNA-seq offers a comprehensive evaluation of signaling network and biological process in organ communication. Thoraces mRNA profiles of both esg-Gal4, tub-Gal80ts, UAS-GFP/+ (Con) and esg-Gal4, tub-Gal80ts, UAS-GFP/UAS-Yki-act (Yki) flies incubated for 8 days at 29Â°C were generated by deep sequencing, in replicate, using Illumina HiSeq2000.

Dataset Information

Indirect Flight Muscle (IFM) expression profiling of actin and myosin nulls in Drosophila melanogaster

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