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: We isolated Drosophila midgut cells : Delta+ intestinal stem cells (ISCs), Su(H)+enteroblasts (EBs), Esg+ cells (ISC+EB), Myo1A+Enterocytes (ECs), Pros+Enteroendocrine cells (EEs) and How+Visceral muscle cells (VM) from whole midguts to identify stem cell specific genes and study cell type specificities of midgut cells. We also isolated all the cell types from the 5 major regions (R1-R5) of the Drosophila midgut to study differences in cells in different regions. Methods: 3-7 day old female flies were dissected. Flies with GFP/YFP marking different cell types (using the GAL4-UAS system) were used to separate cells of the midgut.The midguts were dissociated with Elastase and FACS sorted using FACS AriaIII. RNA was extracted, amplified and sequenced. Whole midgut samples were sequenced on Illumina GAIIX and regional cell populations were sequenced on HiSeq2000. Methods:Raw fastqc reads were mapped to the Drosophila genome (Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa) using Tophat 2.0.9 at default (using boost_1_54_0, bowtie2-2.1.0, samtools-0.1.19). Methods: For differential expression analysis, DESeq (p-value adjustment 0.05 by method Benjamini-Hochberg) was used. The reads were normalized also to Reads per kilobase of transcript per million mapped reads (RPKM). Results: More than 50% of the genome is expressed in the adult midgut (FlyAtlas- Chintapalli et al., 2007), of these genes about 50% (2457) were differentially expressed (DE) between all 4 cell types (ISCs, EBs, ECs and EEs) atleast 2 folds with 95% confidence Results: 159 genes that were specifically enriched in ISCs, 509 genes were specifically repressed in ISCs Conclusions: Our study represents the first detailed analysis of Drosophila intestinal cell transcriptomes, with biologic replicates, generated by RNA-seq technology.Our data facilitates comparative investigations of expression profiles of cells and reveals novel stem cell genes. Further region specific profiling adds precision to the analysis of variances in the midgut regions. We identify transcriptional regulators and regional transcription factors which modulate the midgut physiology. The dataset will be a great resource for hypothesis generation, tool building and fine tuned studies on the Drosophila midgut. mRNA profiles of Drosophila intestinal cells from whole midguts and midgut regions were generated by Deep Sequencing. Whole midgut profiles were generated in triplicates (Illumina GAIIx, 72 bp read length) and regional cell type profiles were genrated in duplicates (HiSeq 2000, 50bp read length).

Project description:Purpose: We isolated Drosophila midgut cells : Delta+ intestinal stem cells (ISCs), Su(H)+enteroblasts (EBs), Esg+ cells (ISC+EB), Myo1A+Enterocytes (ECs), Pros+Enteroendocrine cells (EEs) and How+Visceral muscle cells (VM) from whole midguts to identify stem cell specific genes and study cell type specificities of midgut cells. We also isolated all the cell types from the 5 major regions (R1-R5) of the Drosophila midgut to study differences in cells in different regions. Methods: 3-7 day old female flies were dissected. Flies with GFP/YFP marking different cell types (using the GAL4-UAS system) were used to separate cells of the midgut.The midguts were dissociated with Elastase and FACS sorted using FACS AriaIII. RNA was extracted, amplified and sequenced. Whole midgut samples were sequenced on Illumina GAIIX and regional cell populations were sequenced on HiSeq2000. Methods:Raw fastqc reads were mapped to the Drosophila genome (Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa) using Tophat 2.0.9 at default (using boost_1_54_0, bowtie2-2.1.0, samtools-0.1.19). Methods: For differential expression analysis, DESeq (p-value adjustment 0.05 by method Benjamini-Hochberg) was used. The reads were normalized also to Reads per kilobase of transcript per million mapped reads (RPKM). Results: More than 50% of the genome is expressed in the adult midgut (FlyAtlas- Chintapalli et al., 2007), of these genes about 50% (2457) were differentially expressed (DE) between all 4 cell types (ISCs, EBs, ECs and EEs) atleast 2 folds with 95% confidence Results: 159 genes that were specifically enriched in ISCs, 509 genes were specifically repressed in ISCs Conclusions: Our study represents the first detailed analysis of Drosophila intestinal cell transcriptomes, with biologic replicates, generated by RNA-seq technology.Our data facilitates comparative investigations of expression profiles of cells and reveals novel stem cell genes. Further region specific profiling adds precision to the analysis of variances in the midgut regions. We identify transcriptional regulators and regional transcription factors which modulate the midgut physiology. The dataset will be a great resource for hypothesis generation, tool building and fine tuned studies on the Drosophila midgut.

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.

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Identification of endogenous changes in the transcriptome of young versus old Drosophila intestinal stem cells using Next Generation RNA Sequencing

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