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.
More...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.
Overall design: Total mRNA were extracted from the ISCs of C, M, FC and FM group of flies. The mRNA profiles were generated in duplicates using Hiseq3000.
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