Fig. 5
RNA-Seq indicates Klu represses Notch targets and EE differentiation genes. a Overview of the experiment: esgts GFP+ cells either expressing kluRNAi or UAS-kluFL were sorted in triplicate and their transcriptome was compared to control esgts GFP cells (see Methods for more details). b qRT-PCR analysis of sorted cells for Klu and the critical EE fate regulators Scute (sc) and Prospero (pros). c qRT-PCR analysis of klu mRNA expression with a primer pair that targets the endogenous 3′ UTR coding sequence (klu UTR) and a primer pair that targets the coding region only (klu CDS). For b and c, cDNA was derived from three replicates/genotype, each replicate containing mRNA isolated from esgts GFP+ cells from 100 midguts/genotype. Data are plotted as mean ± SEM. d Overlap of upregulated genes in kluRNAi and downregulated genes in UAS-kluFL. e Volcano plots comparing expression of a selection of genes from the overlap of 81 genes shown in d. Most genes upregulated in the kluRNAi vs control set (left) are downregulated in the UAS-klu vs control set (right). f–j Klu represses Da-dependent miraP-GFP expression in ISC and EB. f, g Control miraP-GFP expression is high in ISCs (arrowheads) and EBs (arrows). h miraP-GFP was slightly increased in kluRNAi midguts. i UAS-kluFL expression resulted in reduced levels of miraP-GFP. Representative areas of posterior midgut are shown. n = 3 animals examined/genotype in f–i. j GFP intensity of miraP-GFP-positive cells for the genotypes in f–i by FACS in a separate experiment. n = 50 midguts per genotype. Scale bar = 50 µm
