(A–H) Representative images of the posterior midgut of male or female esgts > w1118 (A, E), esgts > N RNAiKK (B, F), esgts > N RNAiTrip (C, G) and esgts > UAS NDN animals, stained with pH3 antibody in red to mark mitotic cells. esg-GFP is shown in green, nuclei are stained with DAPI (blue). Animals were raised at 18°C to adulthood and then incubated at 29°C for 15 days. (I) Quantification of pH3-positive cells per adult midgut of the indicated genotypes after 15 days at 29°C. (J–O) Representative images of the posterior midgut of male or female esgts UAS-Cas9.P2 pCFD6-se2x (J, M), esgts UAS-Cas9.P2 pCFD3-N (K, N) and esgts UAS-Cas9.P2 pCFD6-N2x (L, O) flies stained with pH3 antibody in red and nuclei are stained with DAPI (blue). Animals were raised at 18°C, after eclosion mutagenesis was induced for 5 days at 29°C, followed by 18°C for 15 days. Unlike in the RNAi condition, no GFP is visible due to repression by Gal80 at 18°C. (P) Quantification of pH3-positive cells per adult midgut of the indicated genotypes at 18°C for 30 days after inducing CRISPR/Cas9 mediated mutagenesis. Significant differences in the number of pH3-positive cells between Notch RNAi or knock-out group (esgts > N RNAiKK, esgts > N RNAitrip, esgts;UAS-Cas9 >N gRNA PCFD3 or esgts;UAS-Cas9 >N-gRNA1184) and the control groups. (esgts > w1118 or esgts;UAS-Cas9 >sepia gRNA) are indicated with asterisks (*p<0.05; **p<0.01; ***p<0.001; two-tailed T-test) Scale bars: 30 µm (A–H, J–O). Note that the total number of mitotic cells in the RNAi and CRISPR conditions cannot be directly compared, as animals were raised at different temperatures.