Extended Data Figure 4. Piezo regulate stem cell differentiation primarily through Ca²⁺ signaling, which is upstream of Notch, Ttk69, and the Achaete-Scute complex (AS-C).

a. Phenotypes associated with UAS-GFP (at 25°C or 32°C), UAS-Piezo^GFP together with Stim^RNAi, InsP3R^RNAi and N^ICD, and UAS-GFP together with Stim^RNAi, Stim^RNAi + Piezo^RNAi, InsP3R^RNAi, N^ICD, InsP3R over-expression (InsP3R^OE), and Orai over-expression (Orai^OE) (at 32°C). Overexpression of PiezoG^FP using esg-Gal4 did not show a significant phenotype at 25°C. By contrast, incubation at 32°C for 4 days showed an increased in the number of both esg⁺ cells and Pros⁺ EEs. Moderate over-expression of Piezo at 25°C had no significant effects. However, strong over-expression at 32°C caused an increase in both esg+ cells and EEs, which phenocopied the increase of cytosolic Ca2+ through SERCA reduction. All flies were incubated at the indicated temperature for 4–5 days before analysis. b. Statistics of the number of esg⁺ and Pros⁺ cells within 10,000 μm² area. Number of midgut areas quantified: n=30 (GFP 25°C), n=31 (GFP 32°C), n=25 (InsP3R^OE 32°C), n=27 (Orai^OE 32°C), n=31 (Stim-i 32°C), n=27 (Stim-i, Piezo-i 32°C), n=29 (InsP3R-i 32°C), n=29 (N-ICD 32°C). c. Average number of mitotic cells within the fly midgut from indicated genotypes were quantified. Number of midguts analyzed: n=20 (GFP 25°C), n=19 (GFP 32°C), n=20 (Piezo^GFP 25°C), n=19 (Piezo^GFP 32°C), n=18 (Serca-I, 32°C), n=18 (InsP3R^OE 32°C), n=24 (Orai^OE 32°C), n=19 (Stim-i 32°C), n=19 (Stim-i, Piezo-i 32°C), n=19 (Piezo^GFP, Stim-i 32°C), n=18 (InsP3R-i 32°C), n=18 (Piezo^GFP, InsP3R-i 32°C), n=17 (N-ICD 32°C), n=17 (Piezo^GFP, N-ICD 32°C). d,e. EE production induced by overexpression of Piezo^GFP is blocked by ase^RNAi (Acheate-Scute complex component asense). Number of midgut areas quantified: n=29 (Ctl), n=30 (ase-i). f,g. Expression of N^ICD in the presence of Serca^RNAi significantly reduced both stem cell proliferation and EE production. Meanwhile, knocking-down ase specifically blocks EE differentiation but not proliferation. Number of midgut areas quantified: n=27 (Ctl), n=24 (NICD), n=25 (ase-i). Even though ttk69 and AS-C knock down affect Piezo and Serca related phenotypes, we think that Ca²⁺ signaling probably does not directly affects Ttk69 or AS-C since previous studies have shown that Ttk69 and AS-C reduction can convert Notch-high EB cell into EEs²⁸, but neither Piezo over-expression nor Serca knockdown has any effect in EBs. h. MARCM clones of cells homozygous for FRT (Ctl), Piezo null allele (Piezo^KO), Stim-RNAi, Piezo^KO + Piezo^GFP, Piezo^KO + Pmca-RNAi, Piezo^KO + Serca-RNAi, Piezo^KO + O-fut-RNAi, and Piezo^KO + ttk69-RNAi. Rescue/reversion of the reduction of EEs in Piezo null clones by increasing cytosolic Ca²⁺ (by knocking down the Ca²⁺ export pump Pmca or endoplasmic reticulum Ca²⁺ ATPase Serca) or by reducing Notch activity (by knocking down its key processing enzyme O-fut, and knocking down EE cell fate repressor ttk69). All data are collected from at least two independent replicates and are expressed as mean + s.e.m.. P-values are calculated from two-tailed Student t-test with unequal variance. Scale bar, 50 μm.