Figure 1(A) Scheme for tracing intestinal progenitor cell number regulation in midgut with low intestinal renewal ('Low') using the lineage tracing ReDDM system. The strategy relies on minimizing exposure of flies to contaminated food by transferring flies to fresh food vials every two days ('Low') as compared with normal culturing conditions in which flies are typically transferred to fresh food vial every week. This 'Low' demand strategy effectively minimized intestinal renewal. (B) Scheme of the ReDDM tracing method (Antonello et al., 2015). This system uses two fluorescent transgenes with short- (membrane CD8::GFP, green) and long-term (nuclear RFP::H2B, red) stability and the Gal80 repressor (tubα1-Gal80ts) for temporal control of UAS-driven transgenes. Using esg-Gal4, the reporter transgenes are seen in the ISCs and EBs (detected as double labelled cells). Newly renewed ECs or EEs are traced by the nuclear RFP owing to the protein stability of RFP::H2B. (C) Quantification of renewed ECs (RFP-only labelled cells), left graph, and number of ISC and EB cells (double RFP+ GFP+ cells), right graph, in posterior midguts after 7, 14, or 21 days of tracing in normal ('Variable': gut scored n= 9, 8, 4) and low demand ('Low': n= 6, 4, 7) culturing conditions. Asterisks denote significances from one-way ANOVA with Bonferroni's Multiple Comparison Test (**, P = 0.0016; ****, P < 0.0001). Data information In the boxplots, the box extends from the 25th to 75th percentiles and whiskers show all points, min to max. The line in the middle of the box is plotted at the median (D) Quantification of mitosis (phospho-histone H3 (PH3)+ cells) in low demand midguts at the indicated time points. **, P = 0.0015; n.s. not significant from one-way ANOVA test. Data information: In the boxplots, the box extends from the 25th to 75th percentiles and whiskers show all points, min to max. The line in the middle of the box is plotted at the median (E) Representative adult midgut from control esgReDDM at day 14 after temperature shift in variable demand. ECs renewed are marked positively by persistent RFP labelling (red-only cells). (F and G) Few ECs had been renewed in 'Low' midguts after 14 (F) and 21 (G) days of tracing. Data information: Scale bars, 50µm. (H-J) Age-synchronized posterior midguts of control (esgReDDM>, H) and p35 overexpression in stem and progenitor cells (esgReDDM>p35, p35 I and J) at 7 days after temperature shift. Arrows in (H, I) point to newly differentiated ECs. In (J), tumour mass is found in the anterior midgut. Data information: Scale bars, 50µm. (K), Quantification of mitosis PH3+ cells in posterior midgut (pmg) (control: gut scored n=13 and esg>p35, p35: n=22]). Student t-test (**** = P < 0.0001). Data information: In the boxplots, the box extends from the 25th to 75th percentiles and whiskers show all points, min to max. (L), Diap1 is monitored by the GFP.4.3. (green) reporter (Djiane et al., 2013; Zhang et al., 2008). Diap1-GFP is detected in a subset of adult midgut esg+ cells that are negative for the mitotic marker PH3 (red, inset) and for EE marker Prospero (Pros, grey in inset). Diap1-GFP is not detected in mature EC (large nuclei cells, DAPI in blue). Data information: Scale bars, 50µm. (M-O), Diap1-GFP (green) co-localized almost 100% with the EB marker GBE-Su(H)-lacZ (red, M and O). Arrow points to a Diap1-GFP-negative GBE-Su(H)-lacZ-positive cell. Data information: Scale bars, 50µm.