Fig. 3: Progenitor cell-produced Egr signals to Grnd in differentiated ECs.

Genetic manipulations targeting components of the JNK pathway in different cell types of adult fly midguts were conducted with the following drivers: Dl^ts for ISCs (i, m), esg^ts for progenitors (ISCs and EBs) (a–c, g, k, l, and o), Su(H)GBE^ts for EBs (j, n), Myo1A^ts (d) or mex^ts for ECs (h), and prosV1^ts for EEs (e). Flies were raised at 18 °C and 2–3-day-old adult females were shifted from 18–29 °C for various durations (as indicated in panels) before treatments (P.e. or 250 μM bleomycin) and subsequent dissections. Midguts were stained with anti-pH3 antibodies, and ISC mitoses were quantified by counting pH3⁺ cells. Quantifications of data shown in (a–e and g–n) represent the mean ± SD (two-tailed unpaired t-test, ^nsP > 0.05, *P = 0.0356 (g), **P = 0.0032 (g), ****P < 0.0001). N values in individual panels indicate the number of midguts examined. a UAS-egr^[w] represents a standard UAS line with the egr cDNA is cloned into the pUAST vector⁵, while UAS-egr^regg1 is a GS transposon insertion line (GS9830) that contains UAS enhancers inserted into the promoter region of egr⁵. f The expression levels of grnd and wgn were examined using available RNA-seq datasets from our laboratory. DESeq2 was used for count normalization (plotted) and differential expression analysis (n = 3 replicates, error bars indicate mean ± SD, **** adjusted p value < 0.0001) of grnd and wgn in the adult fly midgut with/without P.e. infection. o Overexpression of grnd in progenitors was driven by esg^ts at 29 °C for 2 days. Midguts were stained with anti-GFP/-lacZ antibodies, with nuclei labeled in blue. Puc-lacZ^E69 was used to indicate the activation of JNK signaling. Yellow arrowheads indicate ISCs/EBs and white arrowheads indicate ECs. Images in (o) are representative of two independent experiments. Scale bars: 15 μm. Source data are provided as a Source Data file.