Mechanical regulation of stem-cell differentiation by the stretch-activated Piezo channel

Abstract

Somatic stem cells constantly adjust their self-renewal and lineage commitment by integrating various environmental cues to maintain tissue homeostasis. Although numerous chemical and biological signals have been identified that regulate stem-cell behaviour, whether stem cells can directly sense mechanical signals in vivo remains unclear. Here we show that mechanical stress regulates stem-cell differentiation in the adult Drosophila midgut through the stretch-activated ion channel Piezo. We find that Piezo is specifically expressed in previously unidentified enteroendocrine precursor cells, which have reduced proliferation ability and are destined to become enteroendocrine cells. Loss of Piezo activity reduces the generation of enteroendocrine cells in the adult midgut. In addition, ectopic expression of Piezo in all stem cells triggers both cell proliferation and enteroendocrine cell differentiation. Both the Piezo mutant and overexpression phenotypes can be rescued by manipulation of cytosolic Ca²⁺ levels, and increases in cytosolic Ca²⁺ resemble the Piezo overexpression phenotype, suggesting that Piezo functions through Ca²⁺ signalling. Further studies suggest that Ca²⁺ signalling promotes stem-cell proliferation and differentiation through separate pathways. Finally, Piezo is required for both mechanical activation of stem cells in a gut expansion assay and the increase of cytosolic Ca²⁺ in response to direct mechanical stimulus in a gut compression assay. Thus, our study demonstrates the existence of a specific group of stem cells in the fly midgut that can directly sense mechanical signals through Piezo.

Extended Data Figure 1.. Piezo expression pattern and Piezo⁺ cell lineage in the fly midgut.

a. Expression pattern of Gal4 (BL59266) driven by the Piezo promoter. b. Schematic of Drosophila Piezo gene structure. Gal4 together with poly-A tail was knocked in after the first start codon of Piezo. The ten predicted Piezo isoforms share the same N-terminus. We refer to this knock-in Gal4 line as Piezo-Gal4[KI]. All Piezo-Gal4 lines used in the manuscript are Piezo-Gal4[KI]. c-f. Piezo expression pattern in the midgut (Piezo-Gal4,UAS-tdTomato3XHA). Tissue was stained with anti-HA antibody to enhance the original signal. In addition to the small diploid stem cells, Piezo is also expressed in ECs after the cardia and around the Cu/Fe region of the midgut. Gal4 activity outside the intestinal epithelium from tracheal cells can also be detected. g. Expression pattern of Piezo mRNA along different sections of the midgut. h. Drosophila midgut with Piezo⁺ cells labeled by PiezoGal4, UAS-mCherryCAAX (Piezo>mCherry) and esg⁺ cells labeled by esg-nlsGFP. i. Midgut with Piezo⁺ cells labeled by Piezo-Gal4, UAS-Piezo-GFP. Dl⁺ stem cells were stained by anti-Delta antibody. Cells positive for Piezo^GFP are indicated by arrowheads. j. Midgut expressing UAS-Piezo-GFP in esg⁺ cells with F-actin labeled by UAS-Act5C-RFP. A recent study has shown that Piezo may form large cytoplasmic aggregates under stressed condition, however, in the fly midgut, the GFP-tagged Piezo protein is localized primarily on the plasma membrane under both quiescent or over-proliferation conditions (i,j). k. esg>GFP is used as an indicator of “newborn” EEs. Under normal physiological condition, around 2–3% of esg⁺ cells stain for Pros, suggesting that they are either differentiating or have just differentiated into EEs (indicated by arrowheads). In addition, all the newborn EEs are also positive for Piezo. Piezo and Pros double positive but esg negative cells can be found occasionally (indicated by yellow arrowhead), most likely reflecting their late stage of differentiation. l. If the newborn EEs are restricted to any specific EE subtype was tested. Tachykinin (Tk) is stained with antibody. Piezo⁺ newborn EEs are composed of both Tk⁺ and Tk⁻ cells, suggesting that Piezo⁺ cells are precursors for different types of EEs. Cells positive for both Piezo and Pros (left panel), Piezo and Tk (middle panel), Pros and Tk (right panel) were indicated by arrowheads. m. Dl⁺, Su(H)⁺ and Piezo⁺ cells were traced using Dl-Gal4, Su(H)-Gal4, and Piezo-Gal4 together with UAS-Flp, Act>FRT>Stop>FRT>nlsGFP. TubGAL80^TS. GAL80^TS was used to suppress the early activity of Gal4 before adulthood. GFP⁺ clones were induced by transient incubation at 32°C. Pros (Red) and GFP double positive cells are indicated by arrowheads. n. Compared with Dl-Gal4^TS, which generate large GFP⁺ EC clones, Piezo-Gal4^TS primarily generates individual GFP⁺ cells with occasional GFP⁺ EC cell clone (indicated by arrowhead). o. To visualize the cells with Gal4 activity, which is repressed by the presence of tubGal80^TS, we incubated the flies at 32°C overnight before analysis. In this figure, two Pros⁺ cells are GFP positive but RFP negative (indicated by arrowheads), suggesting that they are derived from Piezo⁺ cells and stop expressing Piezo. All experiments were independently repeated at least twice with similar results presented in the figures. Scale bar: a, 50 μm; c, 500 μm.; d-f, 100 μm. h, 50 μm; i, j, 25 μm; k,l,m, 20 μm; n, 50 μm; o, 10 μm.

Extended Data Figure 2.. Piezo⁺/EP cells are ISC-derived EE precursors with reduced mitotic ability.

a. Midguts from flies treated with Bleomycin (10 μg/ml Bleo in 5% sucrose) or the γ-secretase inhibitor DAPT (4 mM DAPT in 5% sucrose). Cells that are positive for both Piezo and Pros are indicated by arrowheads. Note that the majority (>95%) of Piezo and Pros double positive cells are also positive for esg, suggesting that these cells are “newborn” EEs that still retain esg-GFP signal. b. Percentage of the newborn EEs (Piezo and Pros double positive cells vs. total Pros⁺ EEs) in fly midguts under control, Bleomycin, and DAPT treatments. Cells within 200 μm X 200 μm areas, n=27 (control), n=25 (Bleo), and n=22 (DAPT), were analyzed. c. Midgut with stem cells labeled by esg-GFP (Green), Piezo⁺ cells labeled by Piezo-Gal4>nlsRFP (Red), and mitotic cells labeled by anti-phospho-Histone H3 (pH3) (Magenta). pH3⁺ mitotic cell is indicated by arrowhead. d,e. Representative images of midguts from flies feed on either control (5% sucrose) or Bleomycin (5% sucrose plus 10ug/ml Bleomycin) food. Piezo⁺ EP cells are labeled by Piezo-Gal4>nlsGFP (Green), mitotic cells are labeled by pH3 staining (Red). Mitotic Piezo⁺ cells are indicated by arrowheads. Since all pH3⁺ cells are Dl⁺ cells (according to Dl-lacZ labeled midgut), we counted all Piezo negative pH3⁺ cells as pH3⁺ ISCs. Under both control (5% sucrose) and damage (5% sucrose + 10 μg/ml Bleomycin) conditions, only around 8–10% of the pH3⁺ cells are Piezo⁺ (~40% of total Dl⁺ cells), suggesting that Piezo⁺ cells are significantly less mitotically active compared to Piezo- Dl⁺ cells. f. If the pH3⁺ Piezo⁺ cells are also Pros⁺ as previously described “enteroendocrine mother cell (EMC)” was tested. Around 50% of these pH3⁺ Piezo⁺ cells show low levels of Pros staining. Meanwhile, all the pH3⁺ Pros⁺ cells are positive for Piezo, suggesting that Piezo⁺ EP cells represent more general EE precursor cells compared to EMCs. Mitotic Piezo⁺ cells are indicated by arrowheads. All experiments were independently repeated at least twice with similar results presented in the figures. g. Random GFP⁺ clones were generated using hsFlp; Ubi-(FRT.Stop)GFP/Piezo-Gal4; UAS-nlsRFP. 3–4 days old flies were heat shocked at 37°C for 30 min once to induce clones in ISCs. Then these flies were kept at 25°C for 2 weeks before analysis. Within each GFP⁺ clone, which is derived from ISCs, there are typically 1–2 Piezo⁺ cells in the cluster (indicated by arrowheads), suggesting that Piezo⁺ cells are generated from ISCs after adulthood. All experiments were independently repeated at least twice with similar results presented in the figures. Data are expressed as mean + s.e.m. P-values are calculated from two-tailed Student t-test with unequal variance. Scale bar: a,c, 50 μm; d,f 20 μm; g, 25 μm.

Extended Data Figure 3.. Expression and function of Piezo in larval and pupal midguts.

a. Expression pattern of Piezo in larval and pupal midguts. Piezo⁺ cells are labeled by Piezo-Gal4>nlsGFP. Piezo is enriched in adult midgut precursor cells (AMPs) during larval stages. Strong expression of Piezo is also detected in tracheal cells associated with the midgut (the nucleus of tracheal cells are indicated by yellow arrowhead). After pupariation, the GFP signal can be detected at low level in most midgut cells (including ECs), but enriched in a few stem cells and EEs, which presumably are newborn EEs. Pupal gut 72 hours after pupae formation (APF) is shown with cells positive for both Piezo and Pros are indicated by arrowheads in Zoom1 and Zoom 2. Importantly, high Piezo level is detected in a large number of EEs present in the mid-section of the pupal gut, suggesting that the association of Piezo expression and EE differentiation is probably conserved during the pupal stage. b. Live imaging of larval and pupal midguts expressing GCAMP and mcd8RFP by Dl-Gal4. Cells with high GCAMP activity are indicated by arrowheads. c,d. Midguts from Piezo null flies show no significant EE generation defects during larval, pupal, or early adult stages (1–2 Days after eclosion). Number of midgut areas quantified: n=24 (WT, larva), n=23 (WT, pupa), n=28 (WT, young adult), n=23 (Piezo^KO, larva), n=23 (Piezo^KO, pupa), n=28 (Piezo^KO, young adult). These results indicate that mechanical controlled Piezo activation is not the major mechanism for EE production during early development. Unlike the adult midgut, the larval midgut does not regenerate through mitosis and only grow through increases in cell size. It is only during late stages of 3rd instar larval development that the quiescent AMPs start to proliferate and generate both new ECs and EEs for pupal gut formation, and the majority of new EEs (~ several hundred) are created within a very narrow time window ~ 72–96 hr APF (after pupae formation). Therefore, the generation of EEs is 15–30 times faster at that stage than during the adult stage under physiological condition, suggesting that a different mechanism that stimulates strong acute EE differentiation is probably involved during developmental stages. e,f. Knocking down SERCA using esg-Gal4 during larval stages significantly increases EE cell number. Meanwhile, overexpression of Piezo-GFP has no significant phenotype. A cluster of extra EE cells are indicated by write circle. Number of midgut areas quantified: n=26 (WT), n=28 (Serca^RNAi), n=26 (Piezo^GFP). All experiments were independently repeated at least twice with similar results presented in the figures. Data are expressed as mean + s.e.m. P-values are calculated from two-tailed Student t-test with unequal variance. Scale bar: 50 μm.

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 μm 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 EEs, 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.

Extended Data Figure 5.. Prolonged increase of stem cell proliferation may reduce EE cell number.

a. Fly midguts of each indicated genotype/condition were analyzed after 5 and 10 days incubations at 32°C. esg⁺ cells and EE cells were labeled by esg>GFP and anti-Pros staining. Representative images from two independent replicates were shown. b. Quantification of mitosis (pH3+ cell number) of midguts from flies expressing GFP only (control, n=16/5 days, n=16/10 days), full-length Stim (Stim^OE, n=15/5 days, n=17/10 days), Serca^RNAi (Serca-i, n=18/5 days, n=16/10 days), Piezo^GFP (n=17/5 days, n=18/10 days), PMCA^RNAi (PMCA-i, n=15/5 days, n=15/10 days), and flies fed Bleo⁺ containing food (regular food + 10ug/ml Bleomycin, n=15/5 days, n=13/10 days). c. Quantification of Pros⁺ EE cell number from 10,000 μm regions: n=31/5 days, n=30/10 days (control); n=30/5 days, n=32/10 days (Stim^OE); n=30/5 days, n=30/10 days (Serca^RNAi); n=31/5 days, n=32/10 days (Piezo^GFP); n=32/5 days, n=31/10 days (PMCA^RNAi); n=29/5 days, n=28/10 days (Bleo⁺). Bleomycin treatment or PMCA^RNAi significantly reduced the number of EEs. This reduction is primarily due to increased turn-over of EEs, since blocking cell mitosis for 5 days had no significant effect on EE cell number (Extended Data Figure 7). The differences between stem cell proliferation and EE differentiation may due to a different level of cytosolic Ca²⁺ increase and the Ca²⁺ depletion in the ER store. d. Change of Piezo⁺ cells and EEs after 5 and 10 days of control (5% sucrose) or Bleomycin (5% sucrose plus 10ug/ml Bleomycin) treatment. Representative images from two independent replicates were shown. e. Quantification of Piezo⁺ cells and EEs from 10–15 midguts for each condition. Both Piezo⁺ cells and EEs number increased after 5 days of Bleomycin treatment and significantly decreased after 10 days of treatment. Cell numbers were quantified within 10,000 μm area, except for pH3, which is quantified from the whole midgut. All data are expressed as mean + s.e.m. values. P-values are calculated from two-tailed Student t-test with unequal variance. Scale bar, 50 μm.

Extended Data Figure 6.. Piezo over-expression increases cytosolic Ca²⁺ level which further triggers proliferation of ISCs but not EBs.

a. Overexpression of Piezo^GFP in esg+ cells (esg>Gal4/UAS-Piezo^GFP; UAS-RGECO) at 32°C causes an increase in cytosolic Ca²⁺ (indicated by the calcium reporter RGECO) compared to control (esg>Gal4/UAS-GFP; UAS-RGECO). Representative images from three short time-lapse imaging of cultured fly midguts were shown. Scale bar: 50 μm. b. Typical traces of Ca²⁺ oscillations in esg⁺ cells of midgut from either control or Piezo^GFP flies from three independent replicates. c. Ca²⁺ oscillation frequency of esg⁺ cells from either control or Piezo^GFP midguts. Data of 27 cells from three replicates for each condition were shown. d. Statistics for average RGECO signal intensity in all GFP⁺ cells (blue) and percentage of Ca²⁺ positive cells (signal higher than 3X s.d. of background) compared to total GFP⁺ cells (orange). Signal intensities were calculated from 10,000 μm² regions: n=17 (control), n=22 (Piezo^GFP) from three independent experiments. e, Bleomycin (Bleo, 10ug/ml) (5 days treatment) triggers a significant increase of esg⁺ cell and EE cells in both WT and Piezo^KO flies. Represented images from three independent replicates were shown. f, Images of live midguts from WT and Piezo^KO flies. Both flies were fed on food containing Bleomycin for 3 days before imaging. g,h. Traces of Ca²⁺ oscillations in Dl⁺ stem cells from WT and Piezo mutant flies fed on Bleomycin for 4–5 days. Bleomycin treatment causes some stem cells to maintain constant high Ca²⁺ levels, while others show reduced oscillation frequency but increased average GCaMP/RFP intensity ratio (G/R ratio). These data show that tissue damage by Bleomycin triggers stem cell proliferation, EE production, and an increase of cytosolic Ca²⁺, independent of Piezo. 30 cells from n=4 (control), n=4 (Bleo⁺), and n=5 (Piezo^KO + Bleo⁺) independent guts were plotted. i. Overexpression of Piezo^GFP in esg⁺ cells (32°C) increases the ratio of Dl⁺ cells (labeled by Dl-lacZ) within the esg⁺ population. j. Piezo overexpression promotes Dl⁺ stem cells ratio in esg⁺ cells. Ratio between Dl⁺ and esg⁺ cells within 10,000 μm regions: n=21 (control) and n=22 (Piezo^GFP) from two independent replicates, are analyzed. k,l. Overexpressing Piezo or knocking down Serca in Su(H)Gbe⁺ EB cells showed no significant phenotype, suggesting that their effect may be blocked by high Notch activity. Number of midgut areas quantified: n=18 (control), n=20 (Serca-i), n=16 (Piezo^GFP). Data are expressed as mean + s.e.m. values. P-values are calculated from two-tailed Student t-test with unequal variance. Scale bar: a,e,f, 50 μm; i, 20 μm; k, 50 μm.

Extended Data Figure 7.. Cytosolic Ca²⁺ triggers ISC proliferation and EP differentiation into EEs.

a. Image of chamber used for optogenetic activation of ChR. b,c. Flies expressing GFP only in Dl⁺ stem cells or Piezo⁺ EP (EE precursor) cells were treated under either dark or light + ATR condition for two weeks like the flies expressing ChR. No significant phenotype was induced by the treatment alone. Number of midgut areas quantified: n=29 (Dl, Dark), n=33 (Dl, light+ATR), n=31 (Piezo, Dark), n=34 (Piezo, light+ATR). Representative results from two independent replicates are shown. d. Mitosis quantification of midgut from indicated genotype/condition. Activating ChR in Dl⁺ cells significantly promotes stem cell proliferation. Only a mild increase of mitosis was detected in ChR active Piezo⁺ EP cells, suggesting that the primary effect of Ca²⁺ in EP cells is to promote differentiation. Data are collected from 30 guts (Dl>ChR); 30 guts (Piezo>ChR); 29 guts (Dl); guts (Piezo) from two independent replicates. pH3⁺ cell number is quantified from the whole midgut. e,f. Activation of CsChrimson in Dl⁺ stem cells with both Stim and InsP3R knocked-down shows reduced increase of stem cells and EEs compared to WT stem cells. Flies were raised at 18°C and shifted to 25°C during the experiment. Cell numbers are quantified within 10,000 μm area from 29 regions (dark) and 31 regions (light + ATR) from two independent replicates. g. Overexpression of Piezo in esg⁺ cells increases MAPK pathway activity. Phosphorylation of extracellular signal-regulated kinase (dpErk) is significantly increased in Piezo-overexpressing cells. Representative images from two independent experiments are shown. h,i. Knocking down Ras significantly reduces stem cell proliferation caused by Piezo overexpression, but does not block Piezo triggered EE differentiation. Flies were kept at 32°C for 4–5 days before analysis. esg⁺ and EE cell number were quantified from n=29 (control) and n=30 (Piezo^GFP) midguts areas from two independent experiments. “Newborn” EEs, that are positive for both esg and Pros, are indicated by arrowheads. j,k. Knocking-down Yorkie using Yki^RNAi completely blocks stem cell proliferation but not the increase of EE cells induced by either Piezo overexpression or Serca knock-down. In addition, knocking-down Serca together with Yorkie also significantly reduced stem cell number, suggesting a depletion of stem cells caused by constant EE differentiation. Cell numbers were quantified within 30 midgut areas for each genotype. l. Midguts from flies fed on control (5% sucrose), Thap (5% sucrose + 0.5μM Thapsigargin), Thap+Tram (5% sucrose + 0.5μM Thapsigargin + 10μM Trametinib), and Tram (5% sucrose + 5μM Trametinib) for 4 days. Representative images from 3 independent experiments are shown. The increase of cytosolic Ca²⁺ by Thap promotes stem cell proliferation, EP (enteroendocrine precursor/Piezo⁺ cell) production, and EE differentiation. Newborn EEs, which are positive for esg, Piezo and Pros, are indicated by white arrowheads. m. Data are collected from Quantification of mitotic cells from n=15 (control), n=16 (Thap), n=17 (Thap + Tram), and n=16 (Tram) midguts. Thap treatment triggers a significant increase in mitosis, which is largely reduced by the mitogen-activated protein kinase (MAP kinase) inhibitor Tram. n. Percentage of Piezo⁺ cells within esg⁺ cell population. Number of areas quantified: n=29 (Ctl), n=31 (Thap), n=32 (Thap+Tram), n=29 (Tram). o. Representative Ca²⁺ images of live midgut from control, Thap, and Thap+Tram treated flies. Similar results are collected from 4 independent guts for each condition. p,q. Thap treatment caused a reduction of oscillation frequency but an increase of average GCaMP/RFP ratio (G/R ratio). The increase of cytosolic Ca²⁺ by Thap is not affected by MAPK inhibition. Data are collected from 29 Cells from 3 independent guts for each condition. All data are expressed as mean + s.e.m. values (shown in red). P-values are calculated from two-tailed Student t-test with unequal variance. Scale bar: 50 μm.

Extended Data Figure 8.. Over-feeding triggers stem cell proliferation and EE increase.

a. Schematic illustration of fly midguts from control (5% sucrose) or MC (5% sucrose + 10% Methylcellulose) fed flies. b. “Smurf” assay of flies fed on both control and MC food shows no damage of gut integrity. Two independent replicates showed similar results. c,d. Image of a midgut feed on MC food. The cell proliferation phenotype is associated with midgut diameter increase but not food content. Data are collected from 23 midgut areas from two independent experiments for each condition. e,f. Midguts from flies fed on MC food with no increase of gut diameter shows no phenotype compared with control. Data are collected from 31 regions (control) and 28 regions (MC feed) from three independent experiments. g,h. Feeding-induced cell proliferation produces more Piezo⁺ cells, which differentiate into EEs. All newborn EEs are indicated by white arrowheads. Data are collected from 27 areas from 2 independent experiments for each condition. i,j, Feeding-induced midgut enlargement triggers a significant increase in EP/Piezo⁺ cell number. Data are collected from n=30 (control) and n=32 (MC feed) midgut areas from two independent replicates. k,l. Feeding-trigged stem cell proliferation and EE increase are blocked in Piezo null mutant. Data are collected from n=27 (control) and n=32 (Piezo^KO) midgut areas from two independent replicates. P-values for both esg and EE are smaller than 0.001. m. Linage tracing experiment (using Piezo-Gal4) under overfed condition shows a significant increase in cell number (2–3) in the same cluster compared to tracing result under control condition, suggesting that either more Piezo cells were created from ISCs or more Piezo⁺ cells divide to create more progeny. Cells positive for both GFP and Pros are indicated by arrowheads. n, Images of live midguts from the following conditions/genotypes: control, MC fed without midgut diameter increase (normal size), MC fed with enlarged midgut diameter, MC fed with Piezo^RNAi and enlarged midgut diameter, and MC fed with InsP3R^RNAi + Stim^RNAi and enlarged midgut diameter. o. Representative traces of Ca²⁺ oscillations in Dl⁺ stem cells of flies from indicated treatment/genotypes. Data are collected from 3 independent experiments for each genotype/condition. p,q. Ca²⁺ oscillation frequency and GCaMP/RFP intensity ratio of 30 cells from 3 individual guts for each genotype are plotted. Mean  ± s.e.m. is displayed in red. Enlarged midgut fed on MC food shows reduced Ca²⁺ oscillation frequency but increased average cytosolic Ca²⁺ level. MC food alone does not trigger any significant change of Ca²⁺ activity. Knocking-down either Piezo or both Stim and InsP3R blocks this feeding-induced increase of cytosolic Ca²⁺. Knocking-down InsP3R or Stim alone has no significant effect on cytosolic Ca²⁺ (Data not shown), which is probably due to the reduced expression level of Dl-Gal4 compared with esg-Gal4. The change of Ca²⁺ activity in MC-fed enlarged midguts is similar to some cells in the Bleomycin damaged midguts (Extended Data Fig. 6 f,g). However, the majority of cells from MC-fed enlarged midguts 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. 6 f,g). Data are indicated as mean + s.e.m. values. P-values are calculated from two-tailed Student t-test with unequal variance. Scale bar: e,i,k,n, 50 μm; g, 25 μm; m, 10 μm.

Extended Data Figure 9.. Direct mechanical activation of the Piezo channel triggers an increase of cytosolic calcium in stem cells.

a. Image of the microfluidic chip used for the ex vivo mechanical trigger experiment. b-c. Design of the channels on the microfluidic chip. Compressed air was delivered through left and right channels and controlled by a manual gauge. Dissected fly midguts were loaded into the main channel (center) from an inlet at the bottom. d. During each compression cycle, the midgut was squeezed to achieve ~30–35% reduction in diameter from both sides. The switching time between compression and relaxation is ~1 s. e. Representative samples of ex vivo mechanical trigger experiment. Time 0 s and 40 s were taken immediately before and after compression. The total compression time is 40 s. Transmission light (up panel) and GCaMP6s signal (bottom panel) are shown. Compared to control, loss of Piezo significantly blocked activation of stem cells by mechanical compression. f. Plots of activated cells numbers during one triggering cycle (50 s) for control (n=12) and Piezo^KO (n=15) fly midguts. Data were collected from 4–5 individual midguts. All GCaMP positive cells (brighter than the 5 folds of background signal) within the field were counted. Periods of compression and relaxation are indicated by green and yellow colors, respectively. g. Averaged response curves of multiple compression cycles (n=12 for control and n=10 for PiezoKO) from control (blue) and PiezoKO (orange) midguts. h. Typical traces of Ca²⁺ activities in WT stem cells that respond to the mechanical stimulus. Data is represented in curve plot (first panel) and heatmap plot (second panel), respectively. Compression period is from 0 to 40 seconds (indicated by black box). Typical traces of Ca²⁺ activities with indicated genotypes. Stem cells with Piezo knockdown or mutant do not respond to the mechanical stimulus. Knocking-down Serca causes a constant high cytosolic Ca²⁺. Knocking-down both Stim and InsP3R significantly reduces random Ca²⁺ activities and largely blocks mechanically triggered a Ca²⁺ increase. Data are collected from 3 independent experiments for each genotype/condition. i. Images of cultured midguts from control, Piezo^RNAi, Piezo^KO, Serca^RNAi, InsP3R^RNAi + Stim^RNAi flies. j. Typical traces of Ca²⁺ activities in stem cells of indicated genotypes. Data are collected from 3 independent guts for each genotype/condition. k,l. Ca²⁺ oscillation frequency and GCaMP/RFP intensity ratio (G/R ratio) in cells from 35 cells (control), 35 cells (Piezo^RNAi), 34 cells (Piezo^KO), 36 cells (Serca^RNAi), 33 cells (InsP3R^RNAi + Stim^RNAi) from 3 independent experiment for each condition/genotype. Neither Piezo^RNAi nor Piezo^KO significantly affect Ca²⁺ activities. Knocking-down Serca induces a constant increase of cytosolic Ca²⁺ in most cells. Knocking-down both InsP3R and Stim stem cells significantly reduces their Ca²⁺ activities. Our data indicate that mechanical stresses generated during food digestion may activate Piezo and promote EE generation in vivo. However, we note that the time-scale between our ex vivo mechanical activation and in vivo cell proliferation and differentiation experiment is very different, especially as the in vivo property of Piezo-mediated Ca²⁺ activity in EP cells is unknown. According to our observations, only a small percentage (<5%) of Piezo⁺ cells become EEs every day under normal condition (interpreted from Piezo/Pros double positive cell number). Therefore, it is possible that either Piezo in vivo is difficult to activate through physiological level mechanical stimulus or that long-term cumulative Piezo activation is required to trigger EEs differentiation. Mean  ± s.e.m. is displayed in red. P-values are calculated from two-tailed Student t-test with unequal variance. Scale bar: 50 μm.

Extended Data Figure 10.. Model.

a. Under normal conditions, Piezo⁺ cells, which we refer to as endocrine precursor (EP) cells, are unipotent stem cells that are mitotically quiescent and have a predetermined EE cell fate. In the presence of mechanical stimulation, the Piezo channel is activated and leads to an increase of cytosolic Ca²⁺ in Piezo⁺ EP cells. Ca²⁺ increase in EP cells triggers strong ell differentiation into EEs, which is probably mediated through inhibition of Notch activity and consequent increase of Sc/Ase transcription activity. b. The presence of food in the intestine triggers an elevated mechanical stress during food transport and visceral muscle contraction. Our results suggest that mechanical signaling activates the mechanosensitive channel Piezo in quiescent EP cells, leads to an increase in cytosolic Ca²⁺ level, which maintains the basal level EE cell production under-physiological condition and promotes fast EE generation under abnormal fed condition. We hypothesize that, as a key regulator of midgut function, EE cells might secrete hormones to enhance different long-term gastric functions including appetite, digestion, nutrient absorption, or gastric emptying.

Figure 1.. Piezo⁺ cells are EE precursors in the fly midgut.

a. Piezo⁺ cells are esg-GFP and Dl-lacZ positive, but Su(H)Gbe-lacZ negative. b. Percentage of Piezo⁺ cells in esg⁺ cells. For Dl⁺: n=238 (Dl⁺), n=457 (esg⁺). For Piezo⁺: n=151 (Piezo⁺), n=682 (esg⁺). c. “newborn” EEs (arrowheads) are Piezo⁺. d. Piezo⁺ cells (RFP⁺) generate GFP⁺ EEs (arrowhead). e. Statistics of GFP⁺ ECs and EEs using Piezo-Gal4, Su(H)Gbe-Gal4, and Dl-Gal4. Number of cells analyzed: n=561 (Dl), n=432 (Su(H)), n=90 (Piezo). f. Bleomycin and DAPT treatment increase esg⁺, EP and EE cell numbers. Areas quantified: n=23 (Ctl), n=21 (Bleo), n=32 (DAPT). g,h. Elimination of Piezo⁺ cells by conditional expression of Rpr. Areas quantified: n=27 (Ctl), n=29 (Rpr), n=27 (Recover). i. pH3 staining of mitotic EPs (arrowhead). Data are expressed as mean + s.e.m. P-values are from two-tailed t-test. Scale bar: a, 20 μm; c,d,i, 10 μm; g, 50 μm.

Figure 2.. Piezo regulates EE differentiation through cytosolic Ca²⁺.

a,b. Midgut of flies homozygous for Piezo^KO shows reduced EE generation after 30 days after eclosion. Areas quantified: n=32 (WT 5 days), n=32 (WT 30 days), n=35 (Piezo^KO 5 days), n=32 (Piezo^KO 30 days). c,d. MARCM clones of cells with indicated genotypes (arrowheads indicate the GFP⁺ EEs). Ratio of EEs in the clone (normalized to control) is quantified. Number of clones quantified: n=32 (FRT), n=35 (Piezo^KO), n=26 (Stim-i), n=28 (Piezo^KO, Piezo^OE), n=31 (Piezo^KO, Pmca-i), n=35 (Piezo^KO, Serca-i), n=28 (Piezo^KO, O-fut-i). e. esg⁺ and EE cell numbers were quantified in midgut expressing indicated genes using esg-Gal4. Number of areas quantified: n=22 (Ctl), n=28 (Piezo^OE), n=23 (Serca-i), n=21 (Piezo^OE, Stim-i), n=24 (Piezo^OE, InsP3R-i), n=26 (Piezo^OE, N-ICD). Data are expressed as mean + s.e.m. P-values are from two-tailed t-test. Scale bar: a, 50 μm; c, 25 μm.

Figure 3.. Cytosolic Ca²⁺ triggers cell proliferation and EE differentiation through different mechanisms.

a,b. Increase of cytosolic Ca²⁺ by channelrhodopsin (ChR) in Dl⁺ and Piezo⁺ EP cells. Dl⁺, Piezo⁺, and EE cell numbers are quantified. Number of areas quantified: n=28 (Dark, Dl-Gal4), n=30 (Light+ATR, Dl-Gal4), n=30 (Dark, Piezo-Gal4), n=31 (Light+ATR, Piezo-Gal4). c,d. Midguts of Thapsigargin (Thap) and Trametinib (Tram) treated flies. Number of areas quantified: n=29 (Ctl), n=31 (Thap), n=32 (Thap+Tram), n=29 (Tram). Data are expressed as mean + s.e.m. P-values are from two-tailed t-test. Scale bar, 50 μm.

Figure 4.. Mechanical stress increases cytosolic Ca²⁺ through Piezo.

a. Flies fed on methylcellulose (MC) containing food. b,c. MC feeding increases esg⁺ and EE cell numbers in the midguts, which is blocked by Piezo^RNAi and Stim^RNAi. Number of areas quantified: n=25 (Ctl), n=23 (MC), n=20 (MC+Piezo-i), n=25 (MC+Stim-i). d. An illustrated microfluidic channel that holds and compresses the midgut for ex vivo mechanical trigger experiment. e. Representative example of 3 cycles of consecutive mechanical activation. Number of calcium⁺ cells is plotted over time. Green: compression period. Yellow: relaxation period. f. Average GCaMP activity during compression from control, Piezo^KO, Piezo^RNAi, Serca^RNAi, and Stim^RNAi + InsP3R^RNAi flies. g. Model for mechanical regulation of EP differentiation in the fly midgut. Ca²⁺ plays different roles in ISCs (proliferation) and EPs (differentiation). Data are expressed as mean + s.e.m. P-values are from two-tailed t-test. Scale bar: a. 10 mm, b. 50 μm.