Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of Drosophila eye antennal imaginal disc expressing either activated Ras or secreted Spitz

ABSTRACT: We have used microarrays to identify genes expressed and required for the second mitotic wave (SMW) during eye development. Eye discs expressing Spitz under the control of GMR Gal4 have no SMW as Spitz promotes G1 arrest, ectopic differentiation also occures. To control for the ectopic differentiation, Spi expressing eye antennal discs were compared to eye antennal discs expressing activated RasV12. In discs expresseding RasV12 under the control of GMRGal4 the SMW takes place normally prior to any ectopic differentiation. We used microarrays to detail the global programme of gene expression underlying cellularisation and identified distinct classes of up-regulated genes during this process. Experiment Overall Design: Drosophila eye antennal imaginal discs expressing either UAS RasV12 or UAS Spi under the control of GMRGal4 were dissected from 3rd instar larvae for RNA extraction and hybridization on Affymetrix microarrays.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Lucy Catherine Firth

PROVIDER: E-GEOD-6300 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Spitz from the retina regulates genes transcribed in the second mitotic wave, peripodial epithelium, glia and plasmatocytes of the Drosophila eye imaginal disc.

Firth Lucy C LC Baker Nicholas E NE

Developmental biology 20070503 2

Proliferation, differentiation, and other processes must be coordinated during the development of multi-cellular animals. A discrete and regulated cell division, the Second Mitotic Wave (SMW), occurs concomitantly with early cell fate decisions in the Drosophila developing retina. Signals from the Epidermal Growth Factor Receptor (EGFR) are required to promote cell cycle arrest of specified cells and antagonize S-phase entry in the SMW. Cells that do not receive any EGFR activity enter S-phase i ...[more]

PMID: 17553483

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Project description:Purpose: While studying possible interaction between ras and hsrω genes we found that down- or up- regulation hsrω RNA levels results in further enhancement in activity hyperactive Ras protein in eye discs of Drosophila melanogaster. In order to find out reason underlying this phenomena we checked difference in gene expression between eye discs expressing activated Ras along with alterations in hsrω RNA levels and those expressing activated Ras alone. Method: Eye disc RNA profiles of wandering late third instar larvae of sev-GAL4>UAS-GFP, sev-GAL4>UAS-hsrωRNAi, sev-GAL4>EP3037, sev-GAL4>UAS-RasV12, sev-GAL4>UAS-hsrωRNAi UAS-RasV12 and sev-GAL4>EP3037 UAS-RasV12 were generated by sequencing, in triplicate, using Illumina Hiseq2500 platform using 50bp pair-end reads, 12 samples per lane and each sample run across 2 lanes. This resulted in a sequencing depth of ~20 million reads. The resulting sequencing FastQ files were mapped to the Drosophila genome (dm6) using Tophat with Bowtie. The aligned SAM/BAM file for each was processed for guided transcript assembly using Cufflink 2.1.1 and gene counts were determined. Mapped reads were assembled using Cufflinks. Transcripts from all samples were subjected to Cuffmerge to get final transcriptome assembly across samples. In order to ascertain differential expression of gene transcripts between different samples, Cuffdiff 2.1.1 was used (Trapnell et al., 2012). Result: Using an optimized data analysis workflow, we mapped about 20 million sequence reads per sample to the Drosophila genome (dm6) and identified 15,905 transcripts with TopHat workflow in each genotypes studied. The sev-GAL4 driven expression of activated Ras in eye discs resulted in differential expression of many genes, with 374 genes being down-regulated and 138 up-regulated, when compared with those in sev-GAL4>UAS-GFP eye discs. Compared to only Ras expressing eye discs in case of down regulation of hsrω RNA in activated Ras background 148 gene were up-regulated while 434 were down-regulated. In eye discs expressing activated Ras alongwith over expression of hsrω RNA, 192 genes were up-regulated and 470 down-regulated compared to only activated Ras expressing eye discs. Some of these (11 genes) were validated using qRT-PCR. Conclusion: In activated Ras expressing eye discs, besides the expected increase in levels of transcripts of genes involved in cell growth, proliferation and differentiation, transcripts of many genes involved in RNA biosynthesis, metabolism and processing were up-regulated when compared with sev-GAL4>UAS-GFP eye discs. Levels of transcripts of none of the known members of Ras/Raf/MAPK signaling pathway were found to be significantly affected by changes in hsrω transcript levels in activated Ras expression background. Interestingly, several sn/snoRNAs along with a scaRNAs and some members of the RNA processing machinery were differentially expressed in activated Ras background and were further affected when the hsrω RNA levels were down-or up-regulated. In addition, while down-regulation of hsrω activity resulted in up-regulation of a few positive modulators of Ras signaling pathway, up-regulation of these transcripts caused down-regulation of the negative regulators of Ras/Raf/MAPK pathway, and thus resulting in increase in Ras activity in either cases.

Project description:Genomic enhancers regulate spatio-temporal gene expression by recruiting specific combinations of transcription factors (TFs). When TFs are bound to active regulatory regions, they displace canonical nucleosomes, making these regions biochemically detectable as nucleosome-depleted regions or accessible/open chromatin. Here we ask whether open chromatin profiling can be used to identify the entire repertoire of active promoters and enhancers underlying tissue-specific gene expression during normal development and oncogenesis in vivo. To this end, we first compare two different approaches to detect open chromatin in vivo using the Drosophila eye primordium as a model system: FAIRE-seq, based on physical separation of open versus closed chromatin; and ATAC-seq, based on preferential integration of a transposon into open chromatin. We find that both methods reproducibly capture the tissue-specific chromatin activity of regulatory regions, including promoters, enhancers, and insulators. Using both techniques, we screened for regulatory regions that become ectopically active during Ras-dependent oncogenesis, and identified 3778 regions that become (over-)activated during tumor development. Next, we applied motif discovery to search for candidate transcription factors that could bind these regions and identified AP-1 and Stat92E as key regulators. We validated the importance of Stat92E in the development of the tumors by introducing a loss of function Stat92E mutant, which was sufficient to rescue the tumor phenotype. Additionally we tested if the predicted Stat92E responsive regulatory regions are genuine, using ectopic induction of JAK/STAT signaling in developing eye discs, and observed that similar chromatin changes indeed occurred. Finally, we determine that these are functionally significant regulatory changes, as nearby target genes are up- or down-regulated. In conclusion, we show that FAIRE-seq and ATAC-seq based open chromatin profiling, combined with motif discovery, is a straightforward approach to identify functional genomic regulatory regions, master regulators, and gene regulatory networks controlling complex in vivo processes. FAIRE-Seq in Drosophila wild type eye-antennal imaginal discs (2 wt strains); ATAC-Seq in Drosophila wild type eye-antennal imaginal discs (3 wt strains) ; FAIRE-Seq in Drosophila Ras/Scrib induced eye disc tumors (1 early and 1 late); ATAC-Seq in Drosophila Ras/Scrib induced eye disc tumors (1 early and 1 late); ATAC-Seq in Drosophila eye discs with Unpaired over-expression (2 biological replicates); CTCF ChIP-seq in Drosophila eye discs; ChIP-seq input in Drosophila eye discs

Dataset Information

Transcription profiling of Drosophila eye antennal imaginal disc expressing either activated Ras or secreted Spitz

Publications

Spitz from the retina regulates genes transcribed in the second mitotic wave, peripodial epithelium, glia and plasmatocytes of the Drosophila eye imaginal disc.

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