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Cholinergic neurons trigger epithelial Ca2+ currents to heal the gut

Nature volume 623, pages 122–131 (2023)Cite this article

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Abstract

A fundamental and unresolved question in regenerative biology is how tissues return to homeostasis after injury. Answering this question is essential for understanding the aetiology of chronic disorders such as inflammatory bowel diseases and cancer1. We used the Drosophila midgut2 to investigate this and discovered that during regeneration a subpopulation of cholinergic3 neurons triggers Ca2+ currents among intestinal epithelial cells, the enterocytes, to promote return to homeostasis. We found that downregulation of the conserved cholinergic enzyme acetylcholinesterase4 in the gut epithelium enables acetylcholine from specific Egr5 (TNF in mammals)-sensing cholinergic neurons to activate nicotinic receptors in innervated enterocytes. This activation triggers high Ca2+, which spreads in the epithelium through Innexin2–Innexin7 gap junctions6, promoting enterocyte maturation followed by reduction of proliferation and inflammation. Disrupting this process causes chronic injury consisting of ion imbalance, Yki (YAP in humans) activation7, cell death and increase of inflammatory cytokines reminiscent of inflammatory bowel diseases8. Altogether, the conserved cholinergic pathway facilitates epithelial Ca2+ currents that heal the intestinal epithelium. Our findings demonstrate nerve- and bioelectric9-dependent intestinal regeneration and advance our current understanding of how a tissue returns to homeostasis after injury.

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Fig. 1: ACh sensitivity is required for recovery.
Fig. 2: nAChRβ3 is required in ECs for recovery.
Fig. 3: nAChRβ3-mediated Ca2+ promotes recovery.
Fig. 4: ARCEN–EC interactions promote recovery.
Fig. 5: Ca2+ spreads via Inx2–Inx7 gap junctions.

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Data availability

Reagents are available upon request. The snRNA-seq datasets generated in this work are publicly available in the Gene Expression Omnibus (GEO) database under accession code GSE218641, snRNA-seq dataset of gut from OreR female flies during Homeostasis and Recovery. Single-nuclei profiling data from this study can be found at https://www.flyrnai.org/tools/rna_seq_base/web/showProject/39/plot_coord=1/sample_id=all, to allow users to query the expression of any gene of interest. Source data are provided with this paper. All other data are available in figures, Extended Data figures and Supplementary Information files.

Code availability

This study does not use any custom codes for analysis. snRNA-seq datasets were analysed using the standard Seurat pipeline.

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Acknowledgements

We thank S. Mohr and J. Blau for comments on the manuscript. Confocal imaging was conducted at the MicRoN Facility at Harvard Medical School, and we thank P. Montero Llopis for advice. We thank the Cepko laboratory at Harvard Medical School for sharing their vibratome. We also thank M. Levin for discussions and H. Bellen, G. Tanentzapf, K. O’Connor-Giles, X. Yang, C. Potter, T. R. Laverty, G. Rubin, Janelia FlyLight, DSHB, DRSC/TRiP, VDRC and the Bloomington Stock Center for fly lines, antibodies and reagents. We thank F. Wirtz-Peitz, S. G. Tattikota, R. Binari and H. Li for help in this project, and P. Jouandin, P. Saavedra, L. Lane, D. Doupé, J. Bosch, B. Ewen-Campen, L. Liu, C. Xu, M. R. Riddle and T. Huycke for advice and reagents. We thank C. Villalta and Bestgene for fly injections, H. Elliot and M. Cicconet at the Image and Data Analysis Facility (IDAC), and S. Norrelykke at the Image Analysis Collaboratory (IAC) at Harvard Medical School for advice on Imaris; S. Alon for advice on expansion microscopy; and the Biopolymers Facility and Computing facilities and PCMM Flow Cytometry Facility at Harvard Medical School. All illustrations were created using BioRender.com and publication licences have been obtained. During this study, A.P. was a Good Ventures fellow of the Life Science Research Foundation and next was supported by the Center for the Study of Inflammatory Bowel Disease (grant no. DK043351). N.P. is an investigator of the Howard Hughes Medical Institute. Y.H., Yifang Liu and A.C. were supported by grant no. P41GM132087 and grant no. BBSRC-NSF/BIO (grant no. DBI-2035515). Ying Liu was supported by the Finnish Cultural Foundation.

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Authors and Affiliations

  1. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Afroditi Petsakou, Yifang Liu, Ying Liu, Aram Comjean, Yanhui Hu & Norbert Perrimon

  2. Howard Hughes Medical Institute, Boston, MA, USA

    Norbert Perrimon

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  1. Afroditi Petsakou
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Contributions

A.P. and N.P. conceptualized the project. A.P. was responsible for the methods, data acquisition, resources, visualization and imaging. A.P. was responsible for the independent replication of experiments at Harvard Medical School. Ying Liu and A.P. performed the FACS analyses. Yifang Liu, Y.H., A.C. and A.P. analysed the single-nuclei data. A.P. wrote the original draft of the manuscript. A.P. and N.P. reviewed, edited and revised the manuscript.

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Correspondence to Afroditi Petsakou or Norbert Perrimon.

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Extended data figures and tables

Extended Data Fig. 1 Ace is downregulated during recovery.

a, Expression levels of conserved inflammatory cytokines (unpaired-3/upd-3 and eiger/egr) in guts of Ore R flies undergoing DSS-induced repair. Normalized to Homeostasis. n = 3 biologically independent samples per condition. Tukey’s one-way Anova: p = 0.0046 (upd3: Hom. vs Injury & Injury vs Rec. d4), p = 0.0061(upd3: Injury vs Rec. d2), p = 0.0096 (egr: Hom. vs Injury), p = 0.006 (Injury vs Rec. d4). b, Expression levels of markers for progenitor cells (PCs) (escargot, esg), ECs (pdm1), enteroendocrine cells (EEs) (prospero, pros) in guts of Ore R flies undergoing DSS-induced repair. Normalized to Homeostasis. Homeostasis: n = 3 (esg), n = 6 (pdm1), n = 5 (pros) biologically independent samples. Injury: n = 3 (esg), n = 6 (pdm1), n = 6 (pros) biologically independent samples. Rec. d2: n = 3 (esg), n = 7 (pdm1, pros) biologically independent samples. Rec. d4: n = 3 (esg), n = 4 (pdm1, pros) biologically independent samples. Tukey’s one-way Anova: p = 0.0393 (Hom. vs Injury, esg), p = 0.0112 (Injury vs Rec. d4, esg). Dunn’s Kruskal-Wallis test: p = 0.0384 (Hom. vs Injury, pdm1), p = 0.0227 (Injury vs Rec. 4, pdm1), p = 0.0157 (Injury vs Rec. d2, pros). c, Annotated gut cell type clusters of Ore R flies after snRNAseq, visualized with UMAP (n = total of 8073 nuclei). d, Graph depicting the number of gut nuclei recovered per cluster and per condition after snRNAseq of Ore R flies (n = total of 7411 nuclei in gut clusters). e, Dot plot per snRNAseq gut cluster illustrating the average expression (blue color range) and percent of expression (dot size) of marker genes for ECs (pdm1, myo1A, mex1), EEs (pros, piezo, AstA) and for PCs (esg, Notch, Sox21a) [PCs: ISC/enteroblast (EB) and proEC]. (n = total of 7411 gut nuclei). f, Heatmap of significantly upregulated and downregulated genes in EC clusters (S. Table 1–2) after snRNAseq (n = total of 4547 nuclei in EC clusters). Red arrow: Acetylcholinesterase (Ace). *: 0.05 > p > 0.01, **: 0.01 > p > 0.001. Data are presented as mean values ± SEM.

Source data

Extended Data Fig. 2 ACh sensitivity is required for recovery.

a, Violin plot illustrating the mean expression of Ace per condition in EC clusters after snRNAseq of Ore R flies (p = 0.0364). n = 4547 nuclei in EC clusters. Statistics: two-tailed negative binomial exact test, adjusted with Benjamini-Hochberg procedure. Violin plot: median, 1st and 3rd quartile. b, Graph depicting percentage of nuclei expressing Ace per snRNAseq gut cluster and condition (n = 7411 gut nuclei). c, Expression levels of Ace in Ore R guts after 18 h of Ecc15 bacterial infection (yellow) or 2 days after Bleomycin-injury (pink) compared to unchallenged guts (5% sucrose: grey, homeostasis: light grey). n = 3 biologically independent samples per condition. two-tailed t-test: p = 0.0179 (Suc. vs Ecc15), p = 0.0121 (Hom. vs Bleo). d, Validation of ACE overexpression using CRISPR-OE. n = 3 biologically independent samples per genotype. two-tailed t-test: p = 0.0461. e, Mitotic division counts of proliferating ISCs with anti-pH3 from midgut of control (mexTS > dCas9VPR) and flies with conditional Ace overexpression (mexTS > dCas9VPR, gRNA-Ace) in ECs during Recovery 2x (like Fig. 1f). n = 11 guts per genotype examined over 2 independent experiments. two tailed Mann-Whitney test: p = 0.0001. f, pH3+ counts from midgut of control (howTS > dCas9VPR, hmlTS > dCas9VPR) and flies with conditional Ace overexpression in the visceral muscle (howTS > dCas9VPR, gRNA-Ace) and in hemocytes (immune cells, hmlTS > dCas9VPR, gRNA-Ace). Conditions as Fig. 1e. Two-way Anova. howTS>dCas9VPR: n = 15(Hom, Rec.d4), n = 12(Injury) guts; howTS>dCas9VPR,gRNA-Ace: n = 15(Hom, Rec.d4), n = 13 (Injury) guts; hmlTS > dCas9VPR: n = 13(Hom.), n = 12(Injury), n = 10(Rec. d4) guts, hmlTS>dCas9VPR,gRNA-Ace: n = 14(Hom.), n = 12 (Injury), n = 11(Rec. d4) guts, examined over 3 independent experiments. g, pH3+ counts from midgut of control (myo1ATS> dCas9VPR, mexTS > dCas9VPR) and flies with conditional Ace overexpression (myo1ATS> dCas9VPR, gRNA-Ace and mexTS > dCas9VPR, gRNA-Ace) in ECs after 18hrs of 5% sucrose feeding or 18 h of Ecc15 oral infection (29 °C). myo1ATS> dCas9VPR: n = 10(Suc.), n = 23 (Ecc15) guts; myo1ATS>dCas9VPR,gRNA-Ace: n = 11(Suc.), n = 24(Ecc15) guts; mexTS> dCas9VPR: n = 16(Suc.), n = 17(Ecc15) guts; mexTS>dCas9VPR, gRNA-Ace: n = 15(Suc.), n = 16 (Ecc15) guts, examined over 2 independent experiments. Tukey’s one-way Anova. h, pH3+ counts from mexTS > dCas9VPR and mexTS > dCas9VPR, gRNA-Ace guts during Homeostasis, 2 days feeding with Bleomycin (Bleo d2) and 2 days recovery after Bleomycin (Rec-Bleo d2) at 29 °C. mexTS> dCas9VPR: n = 15(Hom.), n = 17(Bleo.), n = 32 (Rec.) guts; mexTS>dCas9VPR, gRNA-Ace: n = 16(Hom.), n = 17 (Bleo.), n = 29 (Rec.) guts, examined over 3 independent experiments. Tukey’s one-way Anova. i, Relative fluorescence intensity (ΔF/F0) per frame (5 s per frame) and per genotype of individual guts as described in Fig. 1g. *: 0.05>p > 0.01, ***: p < 0.001. Data are presented as mean values ± SEM.

Source data

Extended Data Fig. 3 nAChRβ3 is required in ECs for recovery.

a, nAcRβ3RNAi validation. n = 6 (control), n = 3 (nAChRβ3RNAi, nAChRβ3RNAi-2) biologically independent samples. Statistics: Dunnett’s one-way Anova. b, pH3+ counts from control (myo1ATS>+) guts and when nAChRβ3 is reduced in ECs (myo1ATS>nAChRβ3RNAi-2). Recovery d7: 7 days standard food (29 °C) after 4 days DSS-feeding (23 °C). Homeostasis d7: 7 days standard food (29 °C). p = 0.0496 (Dunn’s Kruskal-Wallis test). myo1ATS>+: n = 23(Hom.), n = 20(Rec. d7) guts; myo1ATS>nAChRβ3RNAi-2: n = 23(Hom.), n = 17(Rec. d7) guts, examined over 3 independent experiments. c, pH3+ counts from mexTS>+ and mexTS>nAChRβ3RNAi guts. Recovery d14: 14 days standard food (29 °C) after 4 days of DSS-feeding (23 °C). Homeostasis d14: 14 days standard food (29 °C). p = 0.0007(Kruskal-Wallis test). mexTS>+ : n = 16(Hom.), n = 20(Rec. d14) guts; mexTS>nAChRβ3RNAi: n = 16(Hom.), n = 19(Rec. d14) examined over 3 independent experiments. d, pH3+ counts from myo1ATS>+ and myo1ATS >nAChRβ3RNAi guts after Ecc15 oral infection and after 5% sucrose feeding. Conditions like Extended Data Fig. 2g. p = 0.0099 (Dunn’s Kruskal-Wallis test). myo1ATS>+ : n = 17 (Suc.), n = 19 (Ecc15) guts; myo1ATS >nAChRβ3RNAi n = 15 (Suc.), n = 26 (Ecc15) guts examined over 3 independent experiments. e, Representative color-coded sequential frames before (fr20) and after (fr200) ACh administration from control (mexTS > GCAM7c) and mexTS > GCAM7c +nAChRβ3RNAi midguts (as Fig. 1g). scale bar: 25 μm. Accompanying graph: average relative fluorescence intensity (ΔF/F0) per frame (5 s per frame) and per genotype. n = 4 (control), n = 5 (nAChRβ3RNAi) guts examined over 2 independent experiments (two-way Anova). Individual ΔF/F0 per gut on Extended Data Fig. 3j. f, pH3+ counts from control and when reducing nAcRβ3 in progenitor cells (PCs) (esgTS>nAChRβ3RNAi), enteroblasts (EBs) (su(H)GBETS>nAChRβ3RNAi), enteroendocrine cells (EEs) (prosTS> nAChRβ3RNAi), visceral muscle (howTS > nAChRβ3RNAi) and hemocytes (hmlTS> nAChRβ3RNAi). Conditions as Fig. 1e. Statistics: Sidak’s two-way Anova. esgTS>+ : n = 16 (Hom., Injury) n = 17 (Rec.) guts; esgTS>nAChRβ3RNAi: n = 14(Hom.), n = 20(Injury), n = 18(Rec) guts, over 2 independent experiments. Su(H)GBETS>+ : n = 16(Hom.), n = 15 (Injury), n = 13 (Rec.) guts; Su(H)GBETS>nAChRβ3RNAi: n = 11(Hom., Injury), n = 15(Rec) guts examined over 2 independent experiments. prosTS>+ : n = 10 (Hom.), n = 15(Injury), n = 12(Rec.) guts; prosTS>nAChRβ3RNAi: n = 10(Hom.), n = 9(Injury), n = 13(Rec) guts examined over 2 independent experiments. howTS>+ : n = 20(Hom.), n = 13(Injury), n = 16(Rec.) guts; howTS>nAChRβ3RNAi: n = 19(Hom.), n = 13(Injury), n = 16(Rec) guts examined over 2 independent experiments. hmlTS>+ : n = 12(Hom.), n = 13(Injury), n = 14(Rec.) guts; hmlTS>nAChRβ3RNAi: n = 12(Hom.), n = 14(Injury), n = 13(Rec) guts examined over 2 independent experiments. g, Graph depicting percentage of nuclei expressing nAChRβ3 per snRNAseq gut cluster (n = 7411 gut nuclei). h, nAChRβ3-flag validation: Midgut expressing nAChRβ3-flag (mexTS> +/nAChRβ3-flag) and when knocking down nAChRβ3 in ECs (mexTS>nAChRβ3RNAi/nAChRβ3-flag). anti-Flag: nAChRβ3-flag (yellow). DAPI: nuclei (blue). Images are representative of 2 independent experiments with similar results. scale bar: 25 μm. i, nAChRβ3 expression levels in Ore R guts. Tukey’s one-way Anova: p = 0.0104(Hom. vs Rec. d1), p = 0.0085(Rec. d1 vs Rec. d8). n = 6 (Hom.), n = 3(Rec. d1, Rec. d8) biologically independent samples. j-k, Relative fluorescence intensity (ΔF/F0) per frame (5 s per frame) and genotype of each gut as described in Extended Data Fig. 3e and Fig. 2g, respectively. *: 0.05> p > 0.01, **: 0.01 < p < 0.001, ***: p < 0.001. Data are presented as mean values ± SEM.

Source data

Extended Data Fig. 4 nAChRβ3 promotes EC maturation.

a, Representative image of posterior midgut from control (mexTS>+/Egr-GFP) flies and when nAChRβ3 is reduced in ECs (mexTS>nAChRβ3RNAi/ Egr-GFP) together with the protein trap Egr-GFP (anti-GFP, green). Accompanying graph: fluorescence fold change per image. n = 10 (control), n = 9 (nAChRβ3RNAi) guts examined over 2 independent experiments. Statistics: two-tailed Mann-Whitney test, p = 0.0057. Conditions as Fig. 1e. scale bar: 50 μm. b, Representative images of posterior midguts from control (mexTS>+/Vn-LacZ) and when nAChRβ3 is reduced in ECs (mexTS> nAChRβ3RNAi/Vn-LacZ) together with the Vn-LacZ reporter (anti-β-gal, white). Accompanying graph: fluorescence fold change per image. n = 10 (control), n = 9 (nAChRβ3RNAi) guts examined over 2 independent experiments. Conditions as Fig. 1e. Statistics: two-tailed t-test, p = 0.0279. scale bar: 50 μm. c, Representative images of posterior midguts from myo1ATS>+ and myo1ATS> nAChRβ3RNAi flies stained with anti-Dcp1: cell death (pink). Conditions as Fig. 1e. Accompanying graph: Dcp1+ cells per image. n = 8 (control), n = 11 (nAChRβ3RNAi) guts examined over 2 independent experiments. Statistics: two-tailed Mann-Whitney test. scale bar: 50 μm. d, Representative images of posterior midguts from control (myo1ATS>+/Diap1-LacZ) and when knocking down nAChRβ3 in ECs (myo1ATS> nAChRβ3RNAi/Diap1-LacZ) together with Diap1-LacZ (Yki target gene, anti-β-gal: white). Conditions as Fig. 1e. Accompanying graph: fluorescence fold change per image. n = 7 (control), n = 8 (nAChRβ3RNAi) guts examined over 2 independent experiments. Statistics: two-tailed t-test. scale bar: 50 μm. e, Representative images of posterior midgut from control (myo1ATS>+/Ex-LacZ) and when nAChRβ3 is reduced in ECs (myo1ATS> nAChRβ3RNAi/Ex-LacZ) together with Ex-LacZ (Yki target gene, anti-β-gal: white). Conditions as Fig. 1e. Boxplot: fluorescence fold change per image (median, 1st and 3rd quartile, whiskers: minimum maximum values). n = 11 guts per genotype examined over 2 independent experiments. Statistics: two-tailed Mann-Whitney test, p = 0.0014. scale bar: 50 μm. f-g, Representative images from posterior midgut of mexTS>+ and mexTS> nAChRβ3RNAi flies stained with anti-pdm1: EC (yellow). Conditions as Fig. 1e. Boxplot: pdm1+ cells over total nuclei per image (median, 1st and 3rd quartile, whiskers: minimum maximum values). n = 15 (control), n = 18 (nAChRβ3RNAi) guts examined over 3 independent experiments. Statistics: two-tailed t-test. scale bars: 50 μm(f), 100 μm(g). h, Pros expression levels. n = 3 biologically independent samples per genotype. Statistics: two-tailed t-test. i, Representative images of posterior midgut assayed with MQAE dye (intracellular Cl- via diffusion-limited collisional quenching, yellow) and SodiumGreen dye (intracellular Na+, green) from control myo1ATS>+ and myo1ATS>nAChRβ3RNAi flies. PI: nuclei (Propidium Iodide, red). See Methods for conditions. Accompanying graphs: fluorescence fold change per image. n = 9 (control/MQAE), n = 7 (nAChRβ3RNAi/MQAE) guts, n = 7 (SodiumGreen per genotype) guts examined over 2 independent experiments per dye. Statistics: p = 0.0115 (two tailed Mann-Whitney test/ MQAE), p = 0.0103 (two-tailed t-test/SodiumGreen). scale bar: 20 μm. j, Representative images from posterior midgut with progenitor cells expressing NFAT-CaLexA (esgTS>NFAT-CaLexA). anti-GFP: Ca2+ (green). NFAT-CaLexA was expressed for 2 days (29 °C) per condition. Accompanying graph: fluorescence fold change per image. n = 8 (Hom., Injury), n = 10 (Rec. d2) guts examined over 2 independent experiments (Tukey’s one-way Anova). scale bar: 50 μm. *: 0.05 > p > 0.01, **: 0.01 < p < 0.001, ***: p < 0.001. n.s.: non-significant. Data are presented as mean values ± SEM.

Source data

Extended Data Fig. 5 nAChRβ3-mediated Ca2 promotes recovery.

a, pH3+ counts of control (mexTS>+), guts with Orai (Ca2+ channel) overexpressed in ECs (mexTS>Orai), nAChRβ3 reduced in ECs (mexTS>nAChRβ3RNAi-2) and combined (mexATS>nAChRβ3RNAi-2+ Orai) during Recovery d4 (as Fig. 1e). n = 15 (mexTS>+), n = 19 (mexTS>nAChRβ3RNAi-2), n = 16 (mexTS>Orai), n = 11(mexATS>nAChRβ3RNAi-2+ Orai) guts examined over 2 independent experiments. Statistics: Dunn’s Kruskal-Wallis test, p = 0.013 (nAChRβ3RNAi-2 vs nAChRβ3RNAi-2+ Orai). b, Representative images of posterior midgut of flies as in Extended Data Fig. 5a. anti-pdm1: ECs (grey). Boxplot: pdm1+ cells over total nuclei per image (median, 1st and 3rd quartile, whiskers: minimum maximum values). n = 10 (control), n = 9 (nAChRβ3RNAi-2, Orai nAChRβ3RNAi-2+ Orai guts) guts examined over 2 independent experiments. Statistics: Tukey’s one-way Anova, p = 0.0048 (nAChRβ3RNAi-2 vs Orai), p = 0.0144 (nAChRβ3RNAi-2 vs nAChRβ3RNAi-2+ Orai). scale bar: 25 μm. c, Representative images of posterior midguts assayed with MQAE (like Extended Data Fig. 4i) of myo1ATS>+, myo1ATS>Orai, myo1ATS>nAChRβ3RNAi-2 and myo1ATS>nAChRβ3RNAi-2 + Orai flies. Accompanying graph: fluorescence fold change per image. n = 6 (myo1ATS>+), n = 6 (myo1ATS>nAChRβ3RNAi-2), n = 7(myo1ATS>Orai), n = 7 (myo1ATS>nAChRβ3RNAi-2 +Orai) guts examined over 2 independent experiments. Statistics: Tukey’s one-way Anova, p = 0.0002 (control vs nAChRβ3RNAi-2), p = 0.0084 (Orai vs nAChRβ3RNAi-2), p = 0.0441 (nAChRβ3RNAi-2 vs Orai + nAChRβ3RNAi-2). scale bar: 25 μm. d, pH3+ counts from control (mexTS>+) and flies overexpressing parvalbumin in ECs (mexTS>PV). Conditions as Fig. 1e. Control: n = 14 (Hom.), n = 37 (Rec.d4) guts, mexTS>PV: n = 17 (Hom.), n = 30 (Rec.d4) guts examined over 3 independent experiments. Statistics: Dunn’s Kruskal-Wallis test. e, Validation of UAS-nAcRβ3. n = 6 (control), n = 3 (myo1ATS> nAcRβ3) biologically independent samples. Statistics: two-tailed Mann-Whitney test, p = 0.0357. f, Validation of a’: mexLexA (mexLexA::GAD), scale bar: 200 μm. Image is representative of 2 independent experiments with similar results; b’: LexAopnAChRβ3. n = 3 biologically independent samples per genotype. Statistics: two-tailed t-test, p = 0.0012. g, a’: Representative color-coded sequential frames of mexTS>GCAM7c and mexTS>GCAM7c+nAChRβ3 guts before (t15) and after (t150) nicotine administration. b’: average relative fluorescence intensity (ΔF/F0) per frame (5 s per frame) and genotype. Conditions: 2 days standard food (29 °C). N = 6 guts per genotype examined over 2 independent experiments. c’: relative fluorescence intensity of each gut. Statistics: two-way Anova. scale bar: 25 μm. h, Representative images of posterior midguts from control (mexLexATS>+/upd3>GFP) and when nAChRβ3 is overexpressed in ECs (mexLexATS> LexAopnAChRβ3 /upd3>GFP) together with upd3-Gal4 driving UAS-GFP (anti-GFP, white). Conditions as Fig. 3g. Accompanying graph: fluorescence fold change per image. n = 8 (control), n = 9 (mexLexATS> LexAopnAChRβ3) guts examined over 2 independent experiments. Statistics: two-tailed Mann-Whitney test, p = 0.0002. scale bar: 100 μm. i, Representative images of posterior midguts from mexLexATS>+/Egr-GFP and mexLexATS>LexAopnAChRβ3/Egr-GFP flies (anti-GFP, green). Conditions as Fig. 3g. Accompanying graph: fluorescence fold change per image. n = 9 guts per genotype examined over 2 independent experiments. Statistics: two-tailed t-test, p = 0.0006. scale bar: 25 μm. j, Representative images of posterior midguts from control (mexTS>+) and mexTS>nAChRβ3 flies stained with anti-Dcp1(pink). Conditions like Fig. 3g. Accompanying graph: Dcp1+ cells per image. n = 9 (control), n = 13 (mexTS>nAChRβ3) guts per genotype examined over 2 independent experiments. Statistics: two-tailed Mann-Whitney test, p = 0.0064. scale bar: 100 μm. DAPI: nuclei (blue). PI: nuclei (Propidium iodide, red). *: 0.05>p > 0.01, **: 0.01<p < 0.001, ***: p < 0.001. Data are presented as mean values ± SEM.

Source data

Extended Data Fig. 6 R49E06-neurons innervate the gut.

a, pH3+ counts from control and from flies with conditional reduction of ChAT (Choline Acetyltransferase) in enteroendocrine cells (EEs) (prosTS>), progenitor cells (PCs) (esgTS>), ECs (myo1ATS>), hemocytes (hmlTS>), visceral muscle (howTS>). Conditions as Fig. 1e. prosTS>+: n = 20 (Hom.), n = 9 (Injury), n = 18 (Rec.) guts, prosTS>ChATRNAi: n = 20 (Hom.), n = 9 (Injury), n = 16 (Rec.) guts; esgTS>+: n = 13 (per condition) guts, esgTS>ChATRNAi: n = 15 (Hom.), n = 12 (Injury), n = 15 (Rec.) guts; myo1ATS>+: n = 11(Hom.), n = 10 (Injury), n = 12 (Rec.) guts, myo1ATS>ChATRNAi: n = 12 (Hom.), n = 10 (Injury), n = 11 (Rec.) guts; hmlTS>+: n = 13 (Hom.), n = 11 (Injury), n = 10 (Rec.) guts, hmlTS>ChATRNAi: n = 12 (Hom.), n = 11 (Injury), n = 11 (Rec.) guts; howTS>+: n = 11(Hom.), n = 10 (Injury), n = 15 (Rec.) guts, howTS>ChATRNAi: n = 12 (Hom.), n = 11 (Injury), n = 15 (Rec.) guts examined over two independent experiments per cell type. Statistics: Sidak’s two-way Anova. b, pH3+ counts from control (esg>+) and from flies without EEs (esg>scRNAi). n = 12 (control), n = 10 (esg>scRNAi) guts examined over 2 independent experiments. Statistics: two-tailed t-test. c, VNC (adult Ventral Nerve Cord), brain, and gut from R49E06>mCD8GFP and R49E06>2xEGFP flies. Images are representative of 3 independent experiments with similar results. anti-GFP: green. scale bar: 100 μm. d, Posterior VNC from R49E06>mCD8GFP flies and together with the cholinergic repressor R49E06>mCD8GFP/ChAT-Gal80. anti-GFP: green. scale bar: 100 μm. Images are representative of 2 independent experiments with similar results. e-f, Z-stack (e) and single Z-planes (f) from posterior VNC of R49E06>6xmCherry. anti-DsRed: magenta, anti-ChAT: yellow, numbered squares: ChAT+R49E06-neurons. scale bar: 50 μm(e), 10 μm (f). Image (e) is representative of 2 independent experiments with similar results. g, Posterior VNC from R49E06>6xmCherry. anti-pros: magenta, anti-DsRed: cyan. Ventral and dorsal from same stack. scale bar: 100 μm. Images are representative of 2 independent experiments with similar results. h, R49E06-innervations (anti-GFP, green) in midguts of R49E06>mCD8GFP flies; a’: R4, Homeostasis, b’: R5, Recovery d2. Red square: domain of R49E06-innervation in c. c’: R49E06-innervation (grey line), anti-ChAT: yellow. scale bar: 20 μm(h-a’), 10 μm (h-b’), 2 μm (h-c’). Images are representative of 2 independent experiments with similar results per condition. i, a’: innervated R4, b’: R5 from R49E06>mCD8GFP flies during Homeostasis. anti-GFP: R49E06-innervations (green), anti-Syt1: synaptic-vesicle marker (magenta). Yellow dots: Syt1+ boutons across innervations. scale bar: 4 μm. Images are representative of 2 independent experiments with similar results. j, R49E06TS>syt1HA fly abdomen during Recovery d2. Yellow arrows: R49E06-innervations (anti-HA, magenta). scale bar: 50 μm. Images are representative of 3 independent experiments with similar results. k, Fly abdomen with DenMarK expressed in R49E06-projections (R49E06TS>DenMark) during Recovery d2 (as Fig. 4a). anti-DsRed: DenMark (yellow). White arrow: DenMark-expressing ARCEN-projections, Dotted red line: gut. scale bar: 100 μm. Images are representative of 2 independent experiments with similar results. l, R5 from R49E06TS>syt1HA+mexLexA>6xLexAopGFP (a’) and R49E06TS>syt1HA+mexLexA>LexAopGFP (b’) flies during Recovery d2. Yellow arrowheads: R49E06-innervations carrying the presynaptic marker syt1HA (anti-HA, magenta) near ECs (anti-GFP, green). Images are representative of 3 independent experiments with similar results. scale bar: 10 μm. Phalloidin: muscle (blue). DAPI: nuclei (blue or white); n.s.: non-significant. Data are presented as mean values ± SEM.

Source data

Extended Data Fig. 7 Egr-ARCENs signaling promotes recovery.

a-b, Pros, upd3, vn, egr expression levels from control (R49E06TS>+) guts and guts after knocking down ChAT in R49E06-neurons (R49E06TS>ChATRNAi). Conditions as Fig. 1e. a: n = 3 biologically independent samples. b: n = 4 biologically independent samples. Normalized to R49E06TS>+. Statistics: (a) two-tailed t-test, (b) Sidak’s two-way Anova: p = 0.0038 (upd3), p = 0.0412 (vn), p = 0.0298(egr). c, pH3+ counts from R49E06TS>+ (control), R49E06TS>ChATRNAi flies and flies co-expressing the VNC repressor (R49E06TS>ChATRNAi/Tsh-Gal80). n = 21 (control), n = 24 (R49E06TS>ChATRNAi, R49E06TS>ChATRNAi/Tsh-Gal80) guts examined over 3 independent experiments. Statistics: Dunn’s Kruskal-Wallis test: p = 0.0004. Conditions as Fig. 1e. d, pH3+ counts from R49E06TS>+ and R49E06TS>ChATRNAi flies after oral Ecc15 infection or 5% sucrose feeding (as Extended Data Fig. 2g). n = 27 (R49E06TS>+ per condition), n = 24 (R49E06TS>ChATRNAi, sucrose), n = 32 (R49E06TS>ChATRNAi, Ecc15) guts examined over 3 independent experiments. Statistics: Dunn’s Kruskal-Wallis test: p = 0.013. e, Experimental schematic and pH3+ counts, from control (ARCENs>+) files, flies with 6 h thermo-activation of ARCENs with theTrpA1 channel (ARCENs>TrpA1) and when cholinergic neurons are inhibited (ARCENs>TrpA1/ChAT-Gal80). n = 28 (control), n = 29 (ARCENs>TrpA1), n = 12 (ARCENs>TrpA1/ChAT-Gal80) guts examined over 2 independent experiments. Dunn’s Kruskal-Wallis test: p = 0.0005(control vs TrpA1), p = 0.0069 (TrpA1 vs TrpA1/ChAT-Gal80). f, upd3, vn, egr expression levels from ARCENs>+ (control) and ARCENs>TrpA1 guts. Conditions as Extended Data Fig. 7e. n = 3 biologically independent samples per genotype. Normalized to ARCENs>+. Sidak’s two-way Anova: p = 0.0114 (upd3), p = 0.0109 (vn), p = 0.0461(egr). g, Validation of R49E06QF. a’: gut (scale bar: 200 μm), b’: posterior VNC (scale bar 50 μm). Images are representative of 2 independent experiments with similar results. h, pH3+ counts from control (ARCENsTS>+) flies, flies with TNF receptor wgn reduced in ARECNs (ARCENsTS>wgnRNAi-2) and flies co-expressing the VNC repressor (ARCENsTS>wgnRNAi-2/Tsh-Gal80). Conditions as Fig. 1e. n = 21 (control), n = 22 (ARCENsTS>wgnRNAi-2), n = 25(ARCENsTS>wgnRNAi-2/Tsh-Gal80) guts examined over 3 independent experiments. Dunn’s Kruskal-Wallis: p = 0.0004(control vs wgnRNAi-2), p = 0.0105(wgnRNAi-2 vs wgnRNAi-2 /Tsh-Gal80). i, pH3+ from control (LucRNAi) and flies with egr reduced (egrRNAi) in progenitor cells (PCs) (esgTS>), ECs (mexTS>) and hemocytes (hmlTS>). Conditions as Fig. 1e. esgTS: n = 13 (control), n = 14 (egrRNAi) guts examined over 2 independent experiments (two-tailed Mann-Whitney test). mexTS: n = 12 (control), n = 13 (egrRNAi) guts examined over 2 independent experiments (two-tailed Mann-Whitney test). hmlTS: n = 14 (control), n = 13 (egrRNAi) guts examined over 2 independent experiments (two-tailed Mann-Whitney test, p = 0.028). esg+mexTS: n = 14 guts per genotype examined over 2 independent experiments (two-tailed Mann-Whitney test). mex+hmlTS: n = 16 (control), n = 13 (egrRNAi) guts examined over 2 independent experiments (two-tailed t-test). esg+hmlTS: n = 21 (control), n = 19 (egrRNAi) guts examined over 3 independent experiments (two-tailed Mann-Whitney test). esg+mex+hmlTS: n = 16 (control), n = 19 (egrRNAi) guts examined over 3 independent experiments (two-tailed Mann-Whitney test). j, pH3+ counts from esg+hml+mexTS>LucRNAi (control) and esg+hml+mexTS>egrRNAi flies. Conditions as Fig. 1e. control: n = 23 (Hom.), n = 22 (Injury), n = 40 (Rec.) guts; egrRNAi: n = 24 (Hom.), n = 22 (Injury), n = 39 (Rec.) guts examined over 3 independent experiments. Statistics: Sidak’s two-way Anova. k, a’: Schematic of Egr-GFP bound to extracellular morphotrap (VHHaGFP). b’-d’: Images from the abdomen (b’,d’) and thorax (c’) of flies expressing the morhotrap in ARCENs while expressing Egr-GFP (ARCENsTS>morphotrap/Egr-GFP) and of control flies without the morphotrap (ARCENsTS>Cherry/Egr-GFP). white dotted lines: ARCEN projections. red lines: midgut (b’,d’: posterior, c’: anterior). Sectioning as Fig. 4a. anti-DsRed: Morphotrap and Cherry (magenta). Anti-GFP: Egr (green), Phalloidin: muscle (blue), DAPI: nuclei (blue). Images are representative of 2 independent experiments with similar results. scale bar 50 μm. n.s.: non-significant, *: 0.05 > p > 0.01, **: 0.01 < p < 0.001, ***: p < 0.001. Data are presented as mean values ± SEM.

Source data

Extended Data Fig. 8 Ca2+ spreads in ECs via gap junctions.

a, Gut image with a subpopulation of ECs (spECs) expressing GFP (spECs > 6xGFP) that are located in R4c (between R4 and R5). anti-GFP: green. Red dotted square: area imaged in Extended Data Fig. 8b. scale bar 100 μm. Image is representative of 2 independent experiments with similar results. b, a’: Posterior midgut images of flies expressing CsChrimson in spECs (spEC>CsChrimson, yellow) while expressing GCAMP7c in all ECs (mexLexA >LexAopGCAMP7c, grey). b’: Color-coded sequential frames from spEC>CsChrimson+mexLexA > LexAopGCaMP7c gut prior (t30) and during (t60) CsChrimson-activation. Lower panels: Heptanol addition (gap junction blocker). c’: Fluorescence intensity (ΔF/F0) of neighboring ECs (non-expressing CsChrimson) per frame (~3 sec/frame) per condition. Upper graph: average ΔF/F0 per condition. Lower graph: individual ΔF/F0 per gut. n = 10 (Neighboring ECs), n = 9 (Neighboring ECs+Heptanol) guts examined over 3 independent experiments. Statistics: two-way Anova. yellow dots: CsChrimson-expressing ECs (spECs). scale bar 20 μm. c, Mean expression of each Innexin (gap junction components) in all snRNAseq EC clusters per condition (n = 4547 nuclei in EC clusters). Boxplot: median, 1st and 3rd quartile, whiskers: minimum maximum values. d, Graph depicting Inx2 and Inx7 mean expression per snRNAseq gut cluster and condition (n = 7411 gut nuclei). e, Graph depicting the percentage of nuclei expressing Inx7 and Inx2 per snRNAseq gut cluster and condition (n = 7411 gut nuclei). f, Posterior midgut expressing GFP in ECs (mex>2xGFP). anti-GFP: green, anti-Inx2: magenta. scale bar 20 μm. Image is representative of 2 independent experiments with similar results. g, Posterior midgut of control (mexTS>+), and when conditionally knocking down Inx2 and Inx7 in ECs (mexTS>Inx2RNAi and mexTS>Inx7RNAi). Conditions: 2 days standard food (29 °C). anti-Inx2: grey. Images are representative of 2 independent experiments with similar results. scale bar 20 μm. h, Validation of Inx7 RNAi. n = 3 biologically independent samples per genotype. Statistics: two-tailed t-test (p = 0.0188). i, pH3+ counts from spECs>CsChrimson and spECs>CsChrimson+ Inx2RNAi flies without or with 7hr opto-activation (red square) during Recovery d1. No light: n = 17(spECs>CsChrimson), n = 18 (spECs>CsChrimson+ Inx2RNAi) guts; Red light: n = 18(spECs>CsChrimson), n = 22 (spECs>CsChrimson+ Inx2RNAi) guts, examined over 3 independent experiments (Tukey’s two-way Anova). j, Posterior midgut images from mexTS>+, mexTS>nAcRβ3, mexTS>Inx2RNAi and mexTS> nAcRβ3+Inx2RNAi flies. scale bar 20μm. Conditions as Fig. 5f. anti-pdm1: ECs (grey). Accompanying boxplot: pdm1+ ratio (median, 1st and 3rd quartile, whiskers: minimum maximum values). n = 7 (mexTS>nAcRβ3), n = 6 (mexTS>+, mexTS>Inx2RNAi, mexTS> nAcRβ3+Inx2RNAi) guts, examined over 2 independent experiments. Tukey’s two-way Anova: p = 0.0392 (mexTS>+ vs mexTS>nAcRβ3), p = 0.0307 (mexTS>+ vs mexTS>Inx2RNAi), p = 0.0003 (mexTS>nAcRβ3 vs mexTS> nAcRβ3+Inx2RNAi). k, Relative fluorescence intensity (ΔF/F0) per frame (3 s per frame) per genotype and per condition of individual guts as described in Fig. 5g. n = 6(Hom.), n = 9(Rec.),n = 8(Inx2RNAi) guts examined over 2 independent experiments. DAPI: blue (nuclei). *: 0.05>p > 0.01, **: 0.01<p < 0.001, ***: p < 0.001. Data are presented as mean values ± SEM.

Source data

Extended Data Fig. 9 ARCENs trigger nAchRβ3-mediated Ca2+ currents in ECs to promote intestinal epithelial recovery after injury.

Model: During recovery, ECs become sensitive (Ace reduction) and receptive (nAChRβ3 increase) to ACh while ARCEN-innervations strengthen their Syt1+ boutons in an Egr-dependent manner. Cholinergic signaling from ARCENs to ECs triggers nAChR-mediated Ca2+ currents that propagate across more ECs via Inx2–Inx7 gap junctions to advance EC maturation, ion balance and transition to homeostasis. Illustration generated with BioRender.com.

Supplementary information

Supplementary Information

Supplementary methods and full genotypes per figure and Extended Data figure.

Reporting Summary

Supplementary Figure 1

Sorting strategy. Flow cytometric sorting of nuclei from fly guts during Homeostasis and Recovery d2. Nuclei were stained with fluorescent DNA dye DRAQ7. Several nuclei populations indicate polyploid gut cells. n = 70 guts per sample.

Supplementary Tables 1–2

Supplementary Table 1. Top 20 upregulated genes in EC clusters during Homeostasis. Average expression, fold change and P value of the 20 most significantly upregulated genes in EC clusters during Homeostasis as shown in Extended Data Fig. 1f. Statistics: two-tailed negative binomial exact test, adjusted with Benjamini–Hochberg procedure. Supplementary Table 2. Top 20 upregulated genes in EC clusters during Recovery d2. Average expression, fold change and P value of the 20 most significantly upregulated genes in EC clusters during Recovery d2 as shown in Extended Data Fig. 1f. Statistics: two-tailed negative binomial exact test, adjusted with Benjamini–Hochberg procedure.

Supplementary Video 1

Cholinergic sensitivity assay during Homeostasis. Supplementary Videos 1–3 depict cholinergic sensitivity assay in ECs of the posterior midgut as described in Fig. 1g. Supplementary Video 1. mexTS>GCaMP7c during Homeostasis. Supplementary Video 2. mexTS>GCaMP7c during Recovery d2. Supplementary Video 3. mexTS>GCaMP7c + dCas9VPR, gRNA-Ace during Recovery d2.

Supplementary Video 2

Cholinergic sensitivity assay during Recovery d2.

Supplementary Video 3

Cholinergic sensitivity assay and Ace upregulation during Recovery d2.

Supplementary Video 4

Nicotinic assay during Homeostasis. Supplementary Videos 4–6 depict nicotinic sensitivity assay in ECs of the posterior midgut as described in Fig. 2f,g. Supplementary Video 4. mexTS>GCaMP7c during Homeostasis. Supplementary Video 5. mexTS>GCaMP7c during Recovery d2. Supplementary Video 6. mexTS>GCaMP7c + nAChRβ3RNAi during Recovery d2.

Supplementary Video 5

Nicotinic assay during Recovery d2.

Supplementary Video 6

Nicotinic assay and nAChRβ3 reduction during Recovery d2.

Supplementary Video 7

R49E06-axonal projections near ECs. Video of R49E06 innervations at R4 in R49E06TS>syt1HA + mexLexA>6xLexAopGFP fly during Recovery d2 as described in Fig. 4b.

Supplementary Video 8

Inducing Ca2+ currents in ECs. This video depicts Ca2+ propagation in ECs during CsChrimson-activation as described in Fig. 5a from spECs>CsChrimson/mexLexA>LexAopGCaMP7c flies.

Supplementary Video 9

Nicotinic assay in ECs during Recovery d2. Supplementary Videos 9 and 10 depict nicotinic sensitivity assay in ECs of the posterior midgut while reducing Inx2 as described in Fig. 5g. Supplementary Video 9. mexTS>GCaMP7c during Recovery d2. Supplementary Video 10. mexTS>GCaMP7c + Inx2RNAi during Recovery d2.

Supplementary Video 10

Nicotinic assay and Inx2 reduction during Recovery d2.

Source data

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Petsakou, A., Liu, Y., Liu, Y. et al. Cholinergic neurons trigger epithelial Ca2+ currents to heal the gut. Nature 623, 122–131 (2023). https://doi.org/10.1038/s41586-023-06627-y

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