ABSTRACT: 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.
