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Profiling of Esg genomic binding in the Drosophila midgut with DamID

ABSTRACT: Tissue stem cells divide asymmetrically to self-renew and generate differentiated progeny to maintain tissue homeostasis. Escargot (Esg) is a transcriptional repressor that is expressed in multiple stem cell populations in Drosophila melanogaster. Reduced esg function in intestinal stem cells (ISCs) causes a loss of ISCs and a biased differentiation of the daughter enteroblasts (EBs) toward the enteroendocrine (EE) cell fate. Loss of esg leads to an upregulation of differentiation factors and reduced Notch activity within EBs, indicating that Esg is a pivotal regulator of homeostasis in the intestine, supporting self-renewal to maintain a healthy stem cell pool as well as differentiation of progenitor cells. To identify potential transcriptional targets that mediate these phenotypes, in vivo DamID was used to generate the data deposited herein.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE55226 | GEO | 2014/11/18

SECONDARY ACCESSION(S): PRJNA238949

REPOSITORIES: GEO

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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).

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Profiling of Esg genomic binding in the Drosophila midgut with DamID

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