Figure 5A. Representative ReDDM labeling of a Drosophila Dronc heterozygous (+/-) intestine overexpressing an RNAi against Notch for 3 days; note the lack of fully differentiated Histone-RFP cells as EC (red) without esg expression (green, GFP). DAPI staining labels cell nuclei in panels All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity. Genotype: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / + B. Representative ReDDM labelling of a Drosophila Dronc mutant homozygous (-/-) intestine overexpressing an RNAi against Notch for 3 post temperature shift at 29 oC; note the lack of differentiated Histone-RFP cells as EC (red) without esg expression (green, GFP), as well as the increase of GFP-positive cells (compare A to B). Genotype: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry C. Quantification of the number of Histone-RFP cells normalised to DAPI (proxy of progenitor cell proliferation obtained from the experiments shown in A and B panels); note the statically significant increase in Histone-RFP positive cells in Dronc homozygous mutant intestines compared to controls (**; P =0.0080) (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, +/- n = 8, -/- n = 15). Error bars represent Standard Deviation of the mean. Genotypes: +/-: w1118 UAS-Notch-RNAi; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / + -/-: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry D. Drosophila Dronc heterozygous intestine overexpressing the Notch intracellular domain for 7 days post temperature shift at 29 oC; intestinal esg-positive progenitor cells (green (GFP) and red (Histone-RFP). Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra (Joaquin Navascués); TubG80ts UAS-Histone-RFP DroncKO / +G. Representative image of a Drosophila Dronc heterozygous intestines 7 days after transfer to 29 oC. esg expression in green (GFP) labels all intestinal progenitors cells, whilst Su(H)-lacZ (red) distinguishes a subpopulation of EBs within the esg-expressing cells. Genotype: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / + H. Representative image of a Drosophila Dronc KO intestine 7 days after transfer to 29 oC. Note, the transcription of Su(H) in large cells GFP (-), suggestive of an aberrant upregulation of Notch-signalling within fully differentiated ECs. Genotype: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry I. Representative image of a 7d Drosophila Dronc heterozygous intestines following a 16hr treatment with paraquat. Note the absence of large Su(H) positive, GFP (-) cells compared to (H). The white arrows indicate the enlarged area depicted in the insets. Genotypes: +/-: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / + -/-: w1118, Su(H)GBE-LacZ ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry