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

Nature volume 555, pages 103–106 (2018)Cite this article

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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 unclear1. 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.

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Figure 1: Piezo+ cells are EE precursors in the fly midgut.
Figure 2: Piezo regulates EE differentiation through cytosolic Ca2+.
Figure 3: Cytosolic Ca2+ triggers cell proliferation and EE differentiation through different mechanisms.
Figure 4: Mechanical stress increases cytosolic Ca2+ through Piezo.

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Acknowledgements

We thank R. Binari, W. Song and C. Villalta for technical support; C. Xu, S. Mohr and D. Doupe for comments on the manuscript; and G. Hasan for sharing reagents. This work was supported by the Damon Runyon Cancer Research Foundation (L.H.) and a grant from the National Institutes of Health (R21DA039582). N.P. is an investigator of the Howard Hughes Medical Institute. G.S. and A.D.T.S. are supported by the National Institutes of Health (P01GM103770).

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Authors and Affiliations

  1. Department of Genetics, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Li He & Norbert Perrimon

  2. Department of Physics, Center for Brain Science, Harvard University, Cambridge, 02142, Massachusetts, USA

    Guangwei Si & Aravinthan D. T. Samuel

  3. School of Life Science and Technology, Tongji University, Shanghai, 200092, China

    Jiuhong Huang

  4. Howard Hughes Medical Institute, Boston, 02115, Massachusetts, USA

    Norbert Perrimon

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Contributions

J.H. and L.H. performed the initial GAL4 expression screen in fly gut. L.H. and N.P. designed the experiments. L.H. performed the Piezo-related experiments and analysed the data. G.S. and A.D.T.S. designed and fabricated the microfluidic chip and together with L.H. optimized the experimental conditions. L.H. and N.P. wrote the manuscript with input from all of the authors.

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Correspondence to Li He or Norbert Perrimon.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks L. O’Brien, K. Venkatachalam and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

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 promoter6. b, Schematic of Drosophila Piezo gene structure. GAL4 and the polyA tail were knocked in after the first start codon of Piezo; we refer to this knock-in GAL4 line as Piezo-GAL4. The ten predicted Piezo isoforms share the same N terminus. c–f, Piezo expression pattern in the midgut (Piezo-GAL4, UAS-tdTomato3XHA). Tissue was stained with an anti-haemagglutinin (HA) antibody to enhance the original signal. In addition to the small diploid stem cells, Piezo is also expressed in enterocytes after the cardia and around the copper and iron regions 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 labelled by mCherry (Piezo-GAL4, UAS-mCherry; red) and Esg+ cells labelled by esg-GFP (green). i, Midgut with Piezo+ cells labelled by GFP (Piezo-GAL4, UAS-Piezo-GFP; green). Dl+ stem cells were stained with an anti-Dl antibody (red). Arrowheads denote Piezo cells. j, Midgut expressing Piezo (GFP+, green) in Esg+ cells, with F-actin labelled by UASp-Act5C-mRFP (red). Piezo may form large cytoplasmic aggregates under stressed conditions19, however, in the fly midgut, the GFP-tagged Piezo protein is localized primarily on the plasma membrane under both quiescent and over-proliferation conditions (i, j). k, esg-GFP is used as an indicator of newborn EEs. Under normal physiological conditions, around 2–3% of Esg+ cells are also positive for Pros, suggesting that they are either differentiating or have just differentiated into EEs (denoted by arrowheads). All the newborn EEs are also positive for Piezo. Piezo and Pros double-positive but Esg-negative cells can be found occasionally (yellow arrowhead), most probably reflecting their late stage of differentiation. l, Piezo+ newborn EEs are composed of both tachykinin-positive (Tk+) and Tk− cells, suggesting that Piezo+ cells are precursors for different types of EE. Arrowheads denote cells positive for both Piezo and Pros (left), Piezo and Tk (middle) or Pros and Tk (right). m, Dl+, Su(H)Gbe+ and Piezo+ cells were traced using Dl-GAL4, Su(H)Gbe-GAL4 and Piezo-GAL4. Arrowheads denote Pros (red) and GFP double-positive cells. n, Compared with Dl-GAL4TS, which generates large GFP+ enterocyte clones, Piezo-GAL4TS primarily generates individual GFP+ cells, with the occasional GFP+ enterocyte cell clone (arrowhead). o, To visualize cells with GAL4 activity, which is repressed by the presence of tub-GAL80TS, we incubated flies at 32 °C overnight before analysis. In this panel, two Pros+ cells are GFP-positive but RFP-negative (indicated by arrowheads), suggesting that they are derived from Piezo+ cells and then stop expressing Piezo. All experiments were independently repeated at least twice with similar results. Scale bars, 50 μm (a, h, n); 500 μm (c), 100 μm (d–f), 25 μm (i, j), 20 μm (k–m), 10 μm (o).

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

a, Midguts from flies treated with bleomycin (10 μg ml−1 in 5% sucrose) or the γ-secretase inhibitor DAPT (4 mM in 5% sucrose). Arrowheads denote cells positive for both Piezo and Pros. Most (>95%) Piezo and Pros double-positive cells are also positive for Esg, suggesting that these cells are newborn EEs that still retain the esg-GFP signal. b, Percentage of newborn EEs (Piezo and Pros double-positive cells versus total Pros+ EEs) in fly midguts under control, bleomycin and DAPT treatments. Cells within 200 μm × 200 μm areas, n = 27 (control), n = 25 (bleo), and n = 22 (DAPT), were analysed. c, Midgut with stem cells labelled by esg-GFP (green), Piezo+ cells labelled by RFP (red), and mitotic cells labelled by anti-pH3 (magenta; arrowhead). d, e, Representative images of midguts from flies fed on either control (5% sucrose) or bleomycin (5% sucrose plus 10 μg ml−1 bleomycin) food. Piezo+ enteroendocrine precursor cells are labelled by GFP (green), mitotic cells are labelled by pH3 staining (red). Arrowheads denote mitotic Piezo+ cells. Because all pH3+ cells are Dl+ cells (according to the Dl-lacZ-labelled midgut), we counted all Piezo− pH3+ cells as pH3+ ISCs. Under both control (5% sucrose) and damage (5% sucrose + 10 μg ml−1 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 than Piezo− Dl+ cells. f, Around 50% of pH3+ Piezo+ cells show low levels of Pros staining. In addition, all pH3+ Pros+ cells are positive for Piezo, suggesting that Piezo+ enteroendocrine precursor cells represent more general EE precursor cells than ‘enteroendocrine mother cells’ (EMCs)21. Arrowheads denote mitotic Piezo+ cells. All experiments were independently repeated at least twice with similar results. g, Random GFP+ clones were generated using hsFLP; Ubi-(FRT.Stop)GFP/Piezo-GAL4; UAS-nlsRFP. Flies (3–4 days old) were heat-shocked at 37 °C for 30 min once to induce clones in ISCs. The flies were then 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 (arrowheads), suggesting that Piezo+ cells are generated from ISCs after adulthood. All experiments were independently repeated at least twice with similar results. Data are mean + s.e.m. P values are from a two-tailed Student’s t-test with unequal variance. Scale bars, 50 μm (a, c), 20 μm (d, f) and 25 μm (g).

Source data

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

a, Piezo+ cells are labelled by GFP. Piezo is enriched in adult midgut precursor cells during larval stages. Strong expression of Piezo is also detected in tracheal cells associated with the midgut (yellow arrowhead denotes tracheal cell nucleus). After pupariation, the GFP signal can be detected at low levels in most midgut cells (including enterocytes), but is enriched in a few stem cells and EEs, which presumably are newborn EEs. Pupal gut 72 h after pupa formation is shown, with arrowheads denoting cells positive for both Piezo and Pros. High levels of Piezo are detected in a large number of EEs present in the pupal midgut, suggesting that the association of Piezo expression and EE differentiation is conserved during the pupal stage. b, Live imaging of larval and pupal midguts expressing GCAMP and tdTomato by Dl-GAL4. Arrowheads denote cells with high GCAMP activity. c, d, Midguts from Piezo-null (PiezoKO) flies show no significant defects in EE generation 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 (PiezoKO, larva), n = 23 (PiezoKO, pupa), n = 28 (PiezoKO, young adult). These results indicate that mechanically 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 grows through increases in cell size. It is only during late stages of third instar larval development that the quiescent adult midgut precursor cells start to proliferate and generate both new enterocytes and EEs for pupal gut formation, and most new EEs (~several hundred) are created within a very narrow time window approximately 72–96 h after pupa formation42. 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 involved during developmental stages. e, f, Knockdown of SERCA using esg-GAL4 during larval stages significantly increases EE cell number. Conversely, overexpression of Piezo (PiezoOE) has no significant phenotype. White circle denotes a cluster of extra EE cells. Number of midgut areas quantified: n = 26 (WT), n = 28 (SERCA-i), n = 26 (PiezoOE). All experiments were independently repeated at least twice with similar results. Data are mean + s.e.m. P values are from a two-tailed Student’s t-test with unequal variance. Scale bars, 50 μm.

Source data

Extended Data Figure 4 Piezo regulates stem-cell differentiation primarily through Ca2+ signalling, which is upstream of Notch, Ttk69 and the achaete-scute gene complex (AS-C).

a, Phenotypes associated with UAS-GFP (at 25 °C or 32 °C), UAS-PiezoOE together with Stim-i, InsP3R-i and NICD, and UAS-GFP together with Stim-i, Stim-i + Piezo-i, InsP3R-i, NICD, InsP3R overexpression (InsP3ROE), and OraiOE (at 32 °C). Overexpression of Piezo 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 overexpression of Piezo at 25 °C had no significant effects. However, strong overexpression 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, 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 (InsP3ROE 32 °C), n = 27 (OraiOE 32 °C), n = 31 (Stim-i 32 °C), n = 27 (Stim-i, Piezo-i 32 °C), n = 29 (InsP3R-i 32 °C), n = 29 (NICD 32 °C). c, Average number of mitotic cells within the fly midgut from indicated genotypes. Number of midguts analysed: n = 20 (GFP 25 °C), n = 19 (GFP 32 °C), n = 20 (PiezoOE 25 °C), n = 19 (PiezoOE 32 °C), n = 18 (SERCA-i, 32 °C), n = 18 (InsP3ROE 32 °C), n = 24 (OraiOE 32 °C), n = 19 (Stim-i, 32 °C), n = 19 (Stim-i, Piezo-i 32 °C), n = 19 (PiezoOE, Stim-i, 32 °C), n = 18 (InsP3R-i 32 °C), n = 18 (PiezoOE, InsP3R-i 32 °C), n = 17 (NICD 32 °C), n = 17 (PiezOE, NICD 32 °C). d, e, EE production induced by overexpression of Piezo is blocked by RNAi that targets the acheate-scute complex (AS-C) component asense (ase). Number of midgut areas quantified: n = 29 (ctl), n = 30 (ase-i). f, g, Expression of NICD in the presence of SERCA-i significantly reduced both stem-cell proliferation and EE production. Knockdown of ase specifically blocks EE differentiation but not proliferation. Number of midgut areas quantified: n = 27 (ctrl), n = 24 (NICD), n = 25 (ase-i). Even though ttk69 (also known as ttk) and AS-C knockdown affect Piezo- and SERCA-related phenotypes, Ca2+ signalling probably does not directly affect Ttk69 or AS-C; previous studies have shown that Ttk69 and AS-C reduction can convert Notch-high enteroblasts into EEs43, but neither Piezo overexpression nor SERCA knockdown has any effect in enteroblasts. h, MARCM clones of cells homozygous for FRT (control), PiezoKO, Stim-i, PiezoKO + PiezoOE, PiezoKO + PMCA-i, PiezoKO + SERCA-i, PiezoKO + O-fut1-i, and PiezoKO + ttk69-i. Rescue/reversion of the reduction of EEs in Piezo-null clones by increasing levels of 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-fut1, and knocking down EE cell fate repressor Ttk69). All data are from at least two independent replicates and are expressed as mean + s.e.m. P values are from a two-tailed Student’s t-test with unequal variance. Scale bars, 50 μm.

Source data

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

a, Fly midguts of each indicated genotype/condition were analysed after incubation for 5 and 10 days at 32 °C. Esg+ cells (GFP+, green) and EE cells (Pros+, red). Representative images from two independent replicates. 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 (StimOE, n = 15 (5 days), n = 17 (10 days), SERCA-i (n = 18 (5 days), n = 16 (10 days)), PiezoOE (n = 17 (5 days), n = 18 (10 days)), PMCA-i (n = 15 (5 days), n = 15 (10 days)), and flies fed bleomycin-containing food (regular food + 10 μg ml−1 bleomycin, n = 15 (5 days), n = 13 (10 days)). c, Quantification of Pros+ EE cell number from 10,000-μm2 regions: n = 31 (5 days), n = 30 (10 days) (control); n = 30 (5 days), n = 32 (10 days) (StimOE); n = 30 (5 days), n = 30 (10 days) (SERCA-i); n = 31 (5 days), n = 32 (10 days) (PiezoOE); n = 32 (5 days), n = 31 (10 days) (PMCA-i); n = 29 (5 days), n = 28 (10 days) (Bleo+). Bleomycin treatment or PMCA-i significantly reduced the number of EEs. This reduction is primarily due to increased turnover of EEs, as blocking cell mitosis for 5 days had no significant effect on EE cell number (Extended Data Fig. 7). The differences between stem-cell proliferation and EE differentiation may be 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 10 μg ml−1 bleomycin) treatment. Representative images from two independent replicates. 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 a 10,000-μm2 area, except for pH3, which is quantified from the whole midgut. All data are mean + s.e.m. P values are from a two-tailed Student’s t-test with unequal variance. Scale bars, 50 μm.

Source data

Extended Data Figure 6 Piezo overexpression increases cytosolic Ca2+ levels, which further triggers proliferation of ISCs but not enteroblasts.

a, Overexpression of Piezo in Esg+ (GFP+, green) cells at 32 °C causes an increase in cytosolic Ca2+ (indicated by the red fluorescent calcium reporter RGECO) compared to control (esg-GAL4/UAS-GFP, UAS-RGECO). Representative images from three short time-lapse imagings of cultured fly midguts. Scale bar, 50 μm. b, Typical traces of Ca2+ oscillations in Esg+ cells of midgut from either control or PiezoOE flies from three independent replicates. c, Ca2+ oscillation frequency of Esg+ cells from either control or Piezo overexpression midguts. Data are from 27 cells from three replicates for each condition. d, Average RGECO signal intensity in all GFP+ cells (blue) and percentage of Ca2+-positive cells (signal higher than 3× the s.d. of background) compared to total GFP+ cells (orange). Signal intensities were calculated from 10,000-μm2 regions: n = 17 (control), n = 22 (PiezoOE) from three independent experiments. e, Bleomycin (10 μg ml−1) (5 days of treatment) triggers a significant increase in Esg+ cells and EE cells in both wild-type and PiezoKO flies. Representative images from three independent replicates are shown. f, Images of live midguts from wild-type and PiezoKO flies. Flies were fed on food containing bleomycin for 3 days before imaging. g, h, Traces of Ca2+ oscillations in Dl+ stem cells from wild-type and Piezo mutant flies fed on bleomycin for 4–5 days. Bleomycin treatment causes some stem cells to maintain constant high Ca2+ levels, whereas others show reduced oscillation frequency but an increased average GCaMP/RFP intensity (G/R) ratio. These data show that tissue damage by bleomycin triggers stem-cell proliferation, EE production and an increase in cytosolic Ca2+, independently of Piezo. Thirty cells from n = 4 (control), n = 4 (Bleo+), and n = 5 (PiezoKO and Bleo+) independent guts are plotted. i, Overexpression of PiezoOE in Esg+ cells (32 °C) increases the proportion of Dl+ cells (labelled by Dl-lacZ; red) within the Esg+ population. j, Piezo overexpression promotes the Dl+/Esg+ cell ratio. Ratio between Dl+ and Esg+ cells within 10,000-μm2 regions: n = 21 (control) and n = 22 (PiezoOE) from two independent replicates, are analysed. k, l, Overexpression of Piezo or knockdown of SERCA in Su(H)Gbe+ enteroblast 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 (PiezoOE). Data are mean + s.e.m. P values are from a two-tailed Student’s t-test with unequal variance. Scale bars, 50 μm (a, e, f, k) and 20 μm (i).

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Extended Data Figure 7 Cytosolic Ca2+ triggers ISC proliferation and enteroendocrine precursor 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+ enteroendocrine precursor (EE precursor) cells were treated under either dark or light + ATR conditions for 2 weeks, as per 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 in mitosis was detected in ChR-active Piezo+ enteroendocrine precursor cells, suggesting that the primary effect of Ca2+ in enteroendocrine precursor cells is to promote differentiation. Data are 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 the channelrhodopsin CsChrimson in Dl+ stem cells with both Stim and InsP3R knocked down causes a reduced increase in stem cells and EEs compared to wild-type stem cells. Flies were raised at 18 °C and shifted to 25 °C during the experiment. Cell numbers are quantified within a 10,000-μm2 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 dpErk is significantly increased in Piezo-overexpressing cells. Representative images from two independent experiments are shown. h, i, Knockdown of 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 numbers were quantified from n = 29 (control) and n = 30 (PiezoOE) midgut areas from two independent experiments. Arrowheads denote newborn EEs (positive for both Esg and Pros). j, k, Knockdown of yorkie using yki-i completely blocks stem-cell proliferation but not the increase of EE cells induced by either Piezo overexpression or SERCA knockdown. In addition, knockdown of SERCA together with yki also significantly reduced stem-cell number, suggesting a depletion of stem cells caused by constant EE differentiation. Cell numbers were quantified from 30 midgut areas for each genotype. l, Midguts from flies fed on control (5% sucrose), thapsigargin (5% sucrose, 0.5 μM thapsigargin; Thap), thapsigargin + trametinib (5% sucrose, 0.5 μM thapsigargin, 10 μM trametinib; Thap + Tram), and trametinib (5% sucrose, 5 μM trametinib; Tram) for 4 days. Representative images from three independent experiments are shown. The increase of cytosolic Ca2+ by thapsigargin promotes stem-cell proliferation, enteroendocrine precursor (Piezo+ cell) production, and EE differentiation. White arrowheads denote newborn EEs (positive for Esg, Piezo and Pros). m, Quantification of mitotic cells from n = 15 (control), n = 16 (Thap), n = 17 (Thap + Tram), and n = 16 (Tram) midguts. Thapsigargin treatment triggers a significant increase in mitosis, which is largely reduced by the MAPK inhibitor trametinib. n, Percentage of Piezo+ cells within the 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, thapsigargin-treated, and thapsigargin plus trametinib-treated flies. Similar results were collected from 4 independent guts for each condition. p, q, Thapsigargin treatment caused a reduction in oscillation frequency but an increase in the average GCaMP/RFP (G/R) ratio. The increase in cytosolic Ca2+ by thapsigargin treatment is not affected by MAPK inhibition. Data are from 29 cells from 3 independent guts for each condition. Data are mean + s.e.m. (shown in red). P values are from a two-tailed Student’s t-test with unequal variance. Scale bars, 50 μm.

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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 (PiezoKO) mutant. Data are from n = 27 (control) and n = 32 (PiezoKO) 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).

Source data

Extended Data Figure 9 Direct mechanical activation of the Piezo channel triggers an increase in cytosolic Ca2+ 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 the left and right channels and controlled by a manual gauge. Dissected fly midguts were loaded into the main channel (centre) from an inlet at the bottom. d, During each compression cycle, the midgut was squeezed to achieve an approximately 30–35% reduction in diameter from both sides. The switching time between compression and relaxation is approximately 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 (top) and GCaMP6s signal (bottom) are shown. Compared to control, the 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 PiezoKO (n = 15) fly midguts. Data were from 4–5 individual midguts. All GCaMP-positive cells (5-fold brighter than the midgut autofluorescence signal) within the field were counted. Periods of compression and relaxation are indicated by green and yellow colours, 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 wild-type stem cells that respond to the mechanical stimulus. Data are represented in curve plot (first panel) and heat-map plot (second panel). The compression period is from 0 to 40 s (black box). Typical traces of Ca2+ activities with indicated genotypes. Stem cells with Piezo knockdown or mutant do not respond to the mechanical stimulus. Knockdown of SERCA causes a constant high cytosolic Ca2+. Knockdown of both Stim and InsP3R significantly reduces random Ca2+ activities and largely blocks the mechanically triggered Ca2+ increase. Data are from three independent experiments for each genotype/condition. i, Images of cultured midguts from control, Piezo-i, PiezoKO, SERCA-i, and InsP3R-i + Stim-i flies. j, Typical traces of Ca2+ activities in stem cells of indicated genotypes. Data are from three independent guts for each genotype/condition. k, l, Ca2+ oscillation frequency and GCaMP/RFP (G/R) intensity ratio in n = 35 cells (control), n = 35 cells (Piezo-i), n = 34 cells (PiezoKO), n = 36 cells (SERCA-i), n = 33 cells (InsP3R-i + Stim-i) from three independent experiment for each condition/genotype. Neither Piezo-i nor PiezoKO significantly affect Ca2+ activities. Knockdown of SERCA induces a constant increase of cytosolic Ca2+ in most cells. Knockdown of 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, the timescale 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 enteroendocrine precursor cells is unknown. According to our observations, only a small percentage (<5%) of Piezo+ cells become EEs every day under normal conditions (interpreted from the Piezo and Pros double-positive cell number). Therefore, it is possible that either Piezo is difficult to activate in vivo by physiological levels of mechanical stimuli, or long-term cumulative Piezo activation is required to trigger EE differentiation. Mean ± s.e.m. is displayed in red. P values are from a two-tailed Student’s t-test with unequal variance. Scale bars, 50 μm.

Extended Data Figure 10 A model of Piezo activation and downstream signalling.

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 in cytosolic Ca2+ in Piezo+ enteroendocrine precursor cells. Ca2+ increases in enteroendocrine precursor cells trigger strong cell differentiation into EEs, which is probably mediated by inhibition of Notch activity and consequent increase of AS-C transcription activity. b, The presence of food in the intestine triggers increased mechanical stress during food transport and visceral muscle contraction. Our results suggest that mechanical signalling activates the mechanosensitive channel Piezo in quiescent enteroendocrine precursor cells and leads to an increase in cytosolic Ca2+ levels, which maintain the basal level EE cell production under physiological conditions and promote fast EE generation under abnormally fed conditions. 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.

Supplementary information

Life Sciences Reporting Summary (PDF 86 kb)

Supplementary Table 1

This file contains a list of complete genotypes for the figures. (XLSX 12 kb)

Supplementary Table 2

This file contains a list of primers used for TR-qPCR. (XLSX 9 kb)

Cytosolic Ca2+ activities in control fly midguts

Guts from flies incubated for 4-5 days at 32 °C were dissected and imaged in live imaging medium. Z-stack images were acquired with 20 sec interval. A maximal intensity z-projection is shown in the video. Genotype of the sample: esg-Gal4, tub-Gal80ts, UAS-GFP; UAS-IVS-NES-jRGECO. (AVI 4880 kb)

Cytosolic Ca2+ activities in Piezo over-expressing fly midguts

Guts from flies incubated for 4-5 days at 32 °C were dissected and imaged in live imaging medium. Z-stack images were acquired with 20 sec interval. A maximal intensity z-projection is shown in the video. Genotype of the sample: esg-Gal4, tub-Gal80ts, UAS-GFP; UAS-GFP-Piezo/UAS-IVS-NES-jRGECO. (AVI 5080 kb)

Cytosolic Ca2+ activities in midguts of flies treated with a Serca inhibitor

Adult flies were kept on control food (5% sucrose), Thap food (5% sucrose + 0.5µM Thapsigargin), or Thap+Tram food (5% sucrose + 0.5µM Thapsigargin + 10µM Trametinib) for 4 days before imaging. Guts from flies with the indicated genotypes were dissected in live imaging medium. Z-stack images were acquired with 20 sec interval. A maximal intensity z-projection is shown in the video. Genotype of the sample: UAS- IVS-GCaMP6s / UAS-tdTomato, Dl-Gal4. (AVI 2348 kb)

Cytosolic Ca2+ activities in midguts of flies feed on MC food

Adult flies with indicated genotypes were fed on control (5% sucrose) or MC (5% sucrose + 10% Methylcellulose) food for 4-5 days. Midguts were dissected in live imaging medium. Z-stack images were acquired with 20 sec interval. A maximal intensity z-projection is shown in the video. Genotypes of the samples: Control, MC Fed (normal), MC Fed: UAS- IVS-GCaMP6s / UAS-tdTomato; Dl-Gal4. MC Fed, Piezo-i: UAS- IVS-GCaMP6s / UAS-tdTomato; Dl-Gal4/UAS-PiezoRNAi. MC Fed, Stim-i, IP3R-i: tubGal80ts/+; UAS- IVS-GCaMP6s / UAS-InsP3RRNAi; UAS-tdTomato, Dl-Gal4/ UAS- StimRNAi. (AVI 2692 kb)

Cytosolic Ca2+ activities in WT and mutant of flies

Guts from flies incubated for 5-7 days at 32 °C were dissected and imaged in live imaging medium. Z-stack images were acquired with 20 sec interval. A maximal intensity z-projection is shown in the video. Genotypes of the samples: UAS- IVS-GCaMP6s / UAS-tdTomato; Dl-Gal4. UAS- IVS-GCaMP6s / UAS-tdTomato; Dl-Gal4/UAS-PiezoRNAi. UAS- IVS-GCaMP6s, PiezoKO / PiezoKO; UAS-tdTomato /Dl-Gal4. tubGal80ts/+; UAS- IVS-GCaMP6s / UAS-tdTomato; Dl-Gal4/UAS-SercaRNAi. tubGal80ts/+; UAS- IVS-GCaMP6s / UAS- StimRNAi; UAS-tdTomato, Dl-Gal4/UAS-InsP3RRNAi. (AVI 2180 kb)

Cytosolic Ca2+ activities in control fly midguts under mechanical compression.

Guts from flies incubated for 5-7 days at 32 °C were dissected and imaged in live imaging medium. For each cycle, the gut was compressed for ~40 sec. Z-stack images were acquired with 2 sec interval. A maximal intensity z-projection is shown in the video. Genotype of the sample: UAS- IVS-GCaMP6s / UAS-tdTomato, Dl-Gal4. (AVI 1673 kb)

Cytosolic Ca2+ activities in PiezoRNAi fly midguts under mechanical compression.

Guts from flies incubated for 5-7 days at 32 °C were dissected and imaged in live imaging medium. For each cycle, the gut was compressed for ~40 sec. Z-stack images were acquired with 2 sec interval. A maximal intensity z-projection is shown in the video. Genotype of the sample: UAS- IVS-GCaMP6s / UAS-tdTomato, Dl-Gal4/ UAS-PiezoRNAi. (AVI 1655 kb)

Cytosolic Ca2+ activities in PiezoKO fly midguts under mechanical compression.

Guts from flies incubated for 5-7 days at 32 °C were dissected and imaged in live imaging medium. For each cycle, the gut was compressed for ~40 sec. Z-stack images were acquired with 2 sec interval. A maximal intensity z-projection is shown in the video. Genotype of the sample: UAS- IVS-GCaMP6s, PiezoKO / PiezoKO; UAS-tdTomato /Dl-Gal4. (AVI 2487 kb)

Cytosolic Ca2+ activities in SercaRNAi fly midguts under mechanical compression

Guts from flies incubated for 5-7 days at 32 °C were dissected and imaged in live imaging medium. For each cycle, the gut was compressed for ~40 sec. Z-stack images were acquired with 2 sec interval. A maximal intensity z-projection is shown in the video. Genotype of the sample: tubGal80ts/+; UAS- IVS-GCaMP6s / UAS-tdTomato; Dl-Gal4/UAS-SercaRNAi. (AVI 1490 kb)

Cytosolic Ca2+ activities in StimRNAi + InsP3RRNAi fly midguts under mechanical compression

Guts from flies incubated for 5-7 days at 32 °C were dissected and imaged in live imaging medium. For each cycle, the gut was compressed for ~40 sec. Z-stack images were acquired with 2 sec interval. A maximal intensity z-projection is shown in the video. Genotype of the sample: tubGal80ts/+; UAS- IVS-GCaMP6s / UAS- StimRNAi; UAS-tdTomato, Dl-Gal4/UAS-InsP3RRNAi. (AVI 1330 kb)

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He, L., Si, G., Huang, J. et al. Mechanical regulation of stem-cell differentiation by the stretch-activated Piezo channel. Nature 555, 103–106 (2018). https://doi.org/10.1038/nature25744

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