Project description:To investigate the in vivo gene expression changes in Drosophila intestinal stem cells (ISCs) when OXPHOS is disrupted and the role of FOXO in regulating this process, we dissected midguts from 4 groups of adult flies: "esg-GAL4,tub-GAL80TS,UAS-nlsGFP/UAS-mitoXhoI;UAS-pAbp-FLAG/+ & homoplasmic mt:CoIsyn" (C group), "esg-GAL4,tub-GAL80TS,UAS-nlsGFP/UAS-mitoXhoI;UAS-pAbp-FLAG/+ & heteroplasmic mt:CoIT300I" (H group, also called M group), "esg-GAL4,tub-GAL80TS,UAS-nlsGFP/UAS-mitoXhoI;UAS-pAbp-FLAG/UAS-FOXO-IR & homoplasmic mt:CoIsyn" (FC group), "esg-GAL4,tub-GAL80TS,UAS-nlsGFP/UAS-mitoXhoI;UAS-pAbp-FLAG/UAS-FOXO-IR & heteroplasmic mt:CoIT300I" (FH group, also called FM group), ~200 midguts per group. The flies were initially obtained and maintained at 18 °C till 2 days after eclosion and were then heat-shocked at 37 ºC for 2 hours and maintained in 29 °C for 5 days. Thus, the ISCs of H and FH group flies were carrying the lethal homoplasmic mitoXhoI-resistant mt:CoIT300I mitochondrial DNAs (mtDNAs) with the wild-type mtDNAs eliminated by mitoXhoI and the OXPHOS disrupted. Meanwhile, the ISCs of C and FC group flies were still carrying wild-type-like but mitoXhoI-resistant mt:CoIsyn mtDNAs. Through the mRNA tagging method with the expressed FLAG-tagged poly(A)-binding protein (pAbp) and anti-FLAG affinity pull-down, We purified and compared mRNA transcriptome profiles (RNA-seq) of different ISCs groups in duplicates. We found about 10% of the mRNAs we detected were either up-regulated or down-regulated in the OXPHOS-disrupted (M group) ISCs compared with control (C group). The down-regulated pool included many genes involved in cell differentiation, proliferation, and growth regulation. The up-regulated pool was enriched in genes participating in various metabolic processes, including glycolysis and fatty acids synthesis. This result suggests a possible metabolic adaptation in response to the OXPHOS deficiency in M group ISCs. Importantly, knockdown of FOXO in OXPHOS-disrupted ISCs restored the expression level of more than 50% of the up- or down-regulated genes, indicating that transcriptional response to OXPHOS deficiency is partially mediated by FOXO. We also noticed that genes whose expressions were altered in OXPHOS-disrupted ISCs included many related to Notch signaling, indicating the mis-regulation of this signaling in these ISCs.

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.

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 29°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.

Project description:Purpose: identify global changes in gene expression in thorax tissues caused by the presence of gut tumors generated by yki expression in intestine stem cells. Note that the thorax of adult flies is composed mainly by muscle tissue but fat body is also present. Changes in gene expression do not exclusively reflect the muscle transcriptome Methods: To extract total RNAs for RNA-Seq experiment, we used 8-10 thoraces dissected out from both esg-Gal4, tub-Gal80ts, UAS-GFP/+ (Con) and esg-Gal4, tub-Gal80ts, UAS-GFP/UAS-yki-act (yki) flies, making sure the gut of these flies was completely removed. Crosses were kept at 18°C to avoid expression of yki during development. Adult males were collected every 24-48 hours and then shifted to 29°C to induce gut yki-tumors. To assess transcriptional changes during the recovery phase, flies were moved to 18°C after a period of 14 days at 29°C in order to shutdown yki expression in the gut and suppress tumor growth. 6-day time points were analyzed during the recovery phase and included T20R, T26R, T32R and T38R. After assessing RNA quality with Agilent Bioanalyzer, rRNA depletion was performed. Then, sequencing libraries constructed with Illumina TruSeq RNA prep kit were sequenced using. We multiplexed samples in each lane, which yields targeted number of single-end 75 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 r6.30) using STAR. With the uniquely mapped reads, we quantified gene expression levels using Cufflinks (FPKM values). Next, differentially expressed genes between experimental and control data were analyzed with DESeq2. Results: Gene list enrichment analysis of the downregulated muscle transcriptome by yki overexpression in intestine stem cells revealed a striking enrichment of multiple metabolic processes impinging on carbohydrate metabolism, amino acid metabolism, and energy metabolism. Interestingly, target genes of Foxo, a transcription factor inhibited by insulin/IGF signaling, are enriched in the upregulated thorax transcriptome of ISCs yki overexpression flies and downregulated during recovery (T20R, T26R, T32R and T38R). In particular, induction of InR and Thor, well-characterized targets of Foxo, are validated with qPCR. Also, the transcription factor REPTOR that is modulated by mTOR signaling and regulates metabolism, was found to be upregulated in thoraces of flies with yki-tumors and downregulated during recovery. Conclusions: Our study discovered that the generation of yki-tumors in the gut drives the upregulation of REPTOR in the thorax of adult flies.

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Piwi knockdown in Drosophila Intestinal Stem Cells

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