Extended Data Figure 8: Over-feeding triggers stem-cell proliferation and an increase in EEs.

a, Schematic illustration of fly midguts from control (5% sucrose) or methylcellulose (5% sucrose plus 10% methylcellulose) fed flies. b, ‘Smurf’ assay of flies fed on both control and methylcellulose food shows no damage to gut integrity. Two independent replicates showed similar results. c, d, Image of a midgut of a fly fed on methylcellulose food. The cell proliferation phenotype is associated with an increase in midgut diameter but not food content. Data are from 23 midgut areas from 2 independent experiments for each condition. e, f, Midguts from flies fed methylcellulose with no increase in gut diameter show no change in phenotype compared with control. Data are from 31 regions (control) and 28 regions (MC) from three independent experiments. g, h, Feeding-induced cell proliferation produces more Piezo⁺ cells, which differentiate into EEs. White arrowheads denote newborn EEs. Data are from 27 areas from 2 independent experiments for each condition. i, j, Feeding-induced midgut enlargement triggers a significant increase in the enteroendocrine precursor/Piezo⁺ cell number. Data are from n = 30 (control) and n = 32 (MC) midgut areas from 2 independent replicates. k, l, Feeding-trigged stem-cell proliferation and EE increases are blocked in the Piezo-null (Piezo^KO) mutant. Data are from n = 27 (control) and n = 32 (Piezo^KO) midgut areas from 2 independent replicates. m, Linage-tracing experiment (using Piezo-GAL4) under overfed conditions shows a significant increase in cell number (2–3) in the same cluster compared to tracing result under control conditions, suggesting that either more Piezo cells were created from ISCs or more Piezo⁺ cells divide to create more progeny. Arrowheads denote cells positive for both GFP and Pros. n, Images of live midguts from the following conditions/genotypes: control, methylcellulose fed without midgut diameter increase (normal size), methylcellulose fed with enlarged midgut diameter, methylcellulose fed with Piezo-i and enlarged midgut diameter, and methylcellulose fed with InsP3R-i + Stim-i and enlarged midgut diameter. o, Representative traces of Ca²⁺ oscillations in Dl⁺ stem cells of flies from indicated treatment/genotypes. Data are from 3 independent experiments for each genotype/condition. p, q, Ca²⁺ oscillation frequency and GCaMP/RFP intensity ratio of 30 cells from three individual guts for each genotype. Mean ± s.e.m. is displayed in red. Enlarged midgut of fly fed on methylcellulose shows reduced Ca²⁺ oscillation frequency but increased average cytosolic Ca²⁺ level. Methylcellulose alone does not trigger any significant change in Ca²⁺ activity. Knockdown of Piezo or of both Stim and InsP3R blocks this feeding-induced increase in cytosolic Ca²⁺. Knockdown of InsP3R or Stim alone has no significant effect on cytosolic Ca²⁺ (data not shown), which is probably due to the reduced expression levels of Dl-GAL4 compared with esg-GAL4. The change in Ca²⁺ activity in enlarged midguts of methylcellulose-fed flies is similar to some cells in the bleomycin-damaged midguts (Extended Data Fig. 6f, g). However, most cells from enlarged midguts of methylcellulose-fed flies still oscillate, which is different from stem cells in bleomycin-treated midguts, in which a large portion of cells maintain a constant high level of Ca²⁺ (Extended Data Fig. 6f, g). Data are mean + s.e.m. P values are from a two-tailed Student’s t-test with unequal variance. Scale bars, 50 μm (e, i, k, n), 25 μm (g) and 10 μm (m).

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