Fig 1: NRG1 enhances crypt budding and protects intestinal organoids from irradiation-induced damage. (A) Outline of the experiment, representative images and quantification of the small intestinal organoid area on day 5 in culture. NR, NOGGIN and RSPO1. Scale bars: 100 µm. (B) Percentage of organoids with zero (no crypts) or at least three (3+ crypts) de novo crypt budding events on day 4 in culture. (C) Approximately 5000 single EGFP+ cells dissociated from Lgr5-CreERT2;IRES-EGFP mouse small intestinal epithelium were plated per well and cultured with EGF, EREG or NRG1. Representative images and quantification of organoid formation capacity at day 5 post plating from three independent experiments are shown. Scale bars: 100 µm. (D) Organoid budding frequency at day 5 after plating. Three parallel wells per condition were analyzed and a representative of three independent experiments is shown. (E) Organoids were cultured for 3 days in ENR, dissociated into single cells and replated in EGF, EREG or NRG1 (100 ng/ml)-containing media. Representative images of two independent experiments of day 2 and day 5 culture from each condition are shown. Scale bars: 200 µm. (F) Luciferase (LUC)-based viability assay of irradiated small intestinal organoids grown in the presence of EGF, EREG and NRG1 (E/E/N; 100 ng/ml). Left graph: viability of non-irradiated control organoids on day 5. Right graph: viability of organoids 24 h after 5 Gy ? irradiation relative to the control (non-irradiated) organoids. A representative of three independent experiments is shown. Asterisks denote a statistically significant difference (*P<0.05; one-way ANOVA with Tukey's post hoc test). Bar graphs (A-D,F) depict the mean, error bars represent s.d.
Fig 2: NRG1 induces a fetal/regenerative transcriptional program in the intestinal epithelium. (A-F) RNA sequencing of small intestinal organoids grown with EGF, EREG or NRG1. (A) Outline of the experiment. RNA was isolated at day 5 and submitted for sequencing. E/E/N, EGF/EREG/NRG1 (100 ng/ml). (B) Representative images of organoids treated with EGF, EREG and NRG1 for 24 h. Scale bars: 100 µm. (C) Principal component analysis (PCA) plot of EGF-, EREG- and NRG1-treated organoid transcriptomes. (D) Unsupervised hierarchical clustering heatmap of the individual samples sequenced. The top 20 genes driving the differences are shown. (E) GSEA of the log2FC-ranked gene list of NRG1- versus EGF-treated organoids. (F) Gene expression shown as log2FC of the indicated genes in NRG1- compared to EGF-treated organoids. Only genes with adjusted P-value<0.1 are shown. (G,H) Expression of Areg, Ereg and Olfm4 in small intestinal organoids for 4 days with EGF, NRG1 or a pulse of NRG1 followed by EGF. (G) Experimental outline. (H) Expression level of indicated genes at day 4. Data are combined from two independent experiments. (I,J) Number of Lgr5-EGFP cells in small intestinal organoids treated with EGF, EREG or NRG1. (I) Experimental setup and representative plots of the flow cytometry analysis. (J) Relative amounts of Lgr5-EGFP cells in organoids treated with EGF, EREG, or NRG1 from four independent experiments. Ctrl, no EGF ligand added. Asterisks indicate statistically significant differences (*P<0.05; measured by one-way ANOVA with Tukey's post hoc test). Bar graphs (H,J) depict the mean, error bars represent s.d.
Fig 3: EGF family ligands EREG and NRG1 are expressed in mouse adult GI fibroblasts. (A) Comparison of transcript levels of the indicated genes in different cell types of the mouse small intestine. Non-Foxl1 lineage stromal cells (Stroma), sorted Foxl1-lineage cells representing subepithelial fibroblasts (Foxl1 cells), intestinal epithelium stem cells expressing Lgr5 (Stem cells) and non-stem-cell epithelial cells (Epithelium) identified by Shoshkes-Carmel et al. (2018) (GSE94072). FPKM, fragments per kilobase of transcript per million mapped reads. See also Fig. S1A-C. (B) Uniform manifold approximation and projection (UMAP) plot (left) of mouse colon mesenchymal cell clusters identified from Roulis et al. (2020) (GSE142431) and dot plot (right) depicting known markers for fibroblast subpopulations as well as Ereg and Nrg1. Endoth, endothelial cells; LymphEndoth, lymphatic endothelial cells; Myofibr, myofibroblasts. See also Fig. S1E. (C) Representative images of primary small intestinal fibroblasts (Fib) and small intestinal epithelial organoids (Epi) (left) and mRNA expression levels of the indicated genes (right). Primary intestinal fibroblasts, n=7 independent mice; intestinal epithelial organoids, n=4 independent mice. Bar graphs depict the mean, error bars represent s.d. Asterisks indicate statistical significance (*P<0.05, two-tailed unpaired t-test). Scale bars: 100 µm. (D) Immunofluorescence staining of PDGFRA and NRG1 in homeostatic mouse jejunum and distal colon. Yellow arrowheads mark examples of cells with expression of PDGFRA in the membrane and NRG1 in the cytoplasm. Scale bars: 50 µm.
Fig 4: NRG1 induces alterations in the epithelial actin cytoskeleton. (A) GSEA of GO Biological Process (GO_BP) gene sets enriched in NRG1-treated small intestinal organoids. The top 20 enriched datasets are shown, and the cytoskeleton and wound healing-related datasets are highlighted in red. (B) GSEA plots of the NRG1 treatment-enriched GO_BP gene sets ‘Actin filament-based process’ and ‘Response to wounding’. (C) Representative images of small intestinal organoid morphology under a light microscope when cultured with EGF or NRG1. (D) Confocal microscopy analysis of apical F-actin in organoids grown for 48 h in the presence of EGF or NRG1. (E) Representative figure of F-actin staining in the distal colon from control and irradiated mice. (F) Effect of ROCK inhibition to small intestinal organoid growth. Representative images and quantification of the organoid area in the indicated conditions are from three independent experiments. Y, 10 µM Y-27632. Scale bars: 100 µm (C,F); 50 µm (D,E). The asterisk indicates a statistically significant difference (*P<0.05; measured by one-way ANOVA with Tukey's post hoc test). Mean and standard deviation are shown.
Fig 5: Ereg and Nrg1 gene expression is induced during intestinal regeneration. (A) Split violin plots visualizing the expression levels of the indicated genes in fibroblast clusters identified by Kinchen et al. (2018) (GSE114374). Expression levels in normal colon fibroblasts are shown in green, and expression levels in DSS-induced inflammation-related fibroblasts in red. (B,C) qRT-PCR analysis of the indicated genes from isolated jejunum (B) and colon (C) from control mice and from mice 3, 24, and 72 h post 11 Gy WBI; n=3-4 mice per time point. Asterisks indicate statistical significance (*P<0.05, one-way ANOVA with Tukey's post hoc test). (D) Immunofluorescence staining of PDGFRA and NRG1 in regenerating (4 days post irradiation) mouse jejunum and colon. White dotted lines indicate the border between the stroma and the epithelium; yellow arrowheads indicate colocalization of NRG1 and PDGFRA. Scale bars: 50 µm. (E,F) qRT-PCR analysis of the indicated genes in primary intestinal fibroblasts with 24-h (E) and 72-h (F) treatments of EGF, EREG, NRG1 (100 ng/ml), LPS (1 µg/ml), TNFA, IFNG (10 ng/ml) and IL-1B (20 ng/ml). Data were derived from two to four independent mice with at least two technical replicates per mouse. Asterisks denote statistically significant (*P<0.05) differences from non-treated fibroblasts (one-way ANOVA with Tukey's post hoc test). Bar graphs (B,C,E,F) depict the mean, and error bars represent s.d.
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