Fig. 4: The age mosaic induced by EI prevents the abrupt onset of aging hyperplasia.

a Upper, schematic illustration of the method to track the turnover of puparium labeled H2B-RFP⁺ ECs in UT and EI (bleomycin-fed 24 h at 4 d) midguts. Lower, representative images of the tracing pattern of H2B-RFP⁺ ECs in UT and EI midguts at 6 d, 10 d and 14 d. Midgut regions were selected from R4C/R5A. Newly generated ECs (white arrowheads) are RFP^- and visualized by the large polyploid DAPI staining. b Cartoon showing the distribution of puparium-labeled RFP⁺ (red) ECs versus newly generated RFP^- (blue) ECs in the UT and EI midgut during aging. The red color, representing the amount of H2B-RFP in the EC nuclear, fades with age. Green cells are ISC-EB pairs. c Upper, schematic illustration of the second H2B-RFP labeling and tracing of ECs. Lower, representative images of the tracing pattern of RFP⁺ ECs in UT and EI midguts after 22 d. White arrowheads indicate the newly generated RFP^- ECs, yellow arrowheads indicate PH3⁺ cells. d Cartoon showing the abrupt collective turnover of RFP⁺ ECs from 37 d to 42 d, contrasting with the formation of the age mosaic of EI midgut. e Statistics of the ratio of RFP⁺ ECs to total ECs in (a) and (c). The red stripe indicates the duration of EC labeling. n, numbers of regions, UT: n = 24 (4 d), 26 (6 d), 25 (10 d), 25 (14 d), 26 (27 d), 30 (37 d), 26 (42 d); EI: n = 22 (4 d), 21 (6 d), 24 (10 d), 24 (14 d), 24 (27 d), 30 (37 d), 21 (42 d). f Statistics of the number of PH3⁺ mitotic cells in UT and EI midgut at 42 d. n, numbers of midguts. n = 16 (UT), 14 (EI). Statistics of the ratio of RFP⁺ ECs to total ECs (g) and representative images of the tracing pattern of H2B-RFP⁺ ECs in UT and Myo1A^ts>Reaper midguts (h). n, numbers of regions. UT: n = 10 (5 d), 17 (7 d), 11 (10 d), 12 (13 d), 13 (16 d); EI: n = 16 (5 d), 24 (7 d), 22 (10 d), 29 (13 d), 22 (16 d). White arrowheads indicate occasional newly formed ECs in the UT midgut. i A model illustrates how EI induced EC age mosaic prevents the abrupt onset of aging hyperplasia. Data are mean ± SEM. Significance was determined using two-tailed unpaired t test. Scale bars, 20 μm. Source data are provided as a Source Data file.