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Eye-antenna early L3 disc expression profiling in COX7a-LoF and Notch-GoF

ABSTRACT: Gene expression in larval, early third instar eye-antenna discs was assesed in genotypes with Notch Gain-of-Function (UAS-Delta or UAS-Notch[intra2]) over-expression or mitochondrial COX7a Loss-of-function (UAS-COX7a-RNAi) or a combination of both (UAS-Delta, UAS-COX7a-RNAi). The analysis revealed that, despite a strong genetic interaction between Notch pathway activation and knockdown of COX7a, no transcriptional cooperation or synergy was detectable in early L3 eye-antenna discs. Rather, COX7a knockdown induced a unique transcriptional signature, which further experiments revealed to be mediated by the transcription factor ATF4.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE148407 | GEO | 2020/04/10

REPOSITORIES: GEO

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Project description:Purpose: To compare transcriptomic changes in 3rd instar larval eye discs following sev-GAL4 driven down- or up- regulation of hsrω lncRNAs. Method: Eye disc total RNA profiles of wandering late third instar larvae of sev-GAL4>UAS-GFP, sev-GAL4>UAS-hsrωRNAi, sev-GAL4>EP3037 were generated by sequencing, in duplicate, using Illumina Hiseq2500 platform using 50bp pair-end reads, 6 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 RNAi for hsromega gene transcripts in eye discs resulted in differential expression of many genes, with 409 genes being down-regulated and 100 up-regulated, when compared with those in sev-GAL4>UAS-GFP eye discs. Compared to control eye discs in case of up regulation of hsrω RNA, 66 gene were up-regulated while 635 were down-regulated. Some of these (11 genes) were validated using qRT-PCR. Conclusion: Analysis of RNA-seq data revealed that a large proportion of transcripts were indeed similarly affected by down- or up-regulation of hsrω RNA in normal as well as activated Ras expression background. A comparison of transcriptomes of sev-GAL4>hsrωRNAi and sev-GAL4>EP3037 eye discs revealed that in each case the number of genes down regulated was more than those up-regulated. Interestingly, while 319 genes were commonly down regulated and 15 were commonly up-regulated in the two genotypes, only 2 genes showed opposing trends between sev-GAL4>UAS-hsrωRNAi and sev-GAL4>EP3037 eye discs.

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Eye-antenna early L3 disc expression profiling in COX7a-LoF and Notch-GoF

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