Notch1 and Notch2 receptors regulate mouse and human gastric antral epithelial cell homoeostasis - PubMed (original) (raw)

. 2017 Jun;66(6):1001-1011.

doi: 10.1136/gutjnl-2015-310811. Epub 2016 Mar 1.

Elise S Demitrack 1, Theresa M Keeley 1, Andrew Tam 1, Nilsa La Cunza 1, Priya H Dedhia 2, Jason R Spence 3, Diane M Simeone 1 2, Ichiko Saotome 4, Angeliki Louvi 4, Christian W Siebel 5, Linda C Samuelson 1 3

Affiliations

Notch1 and Notch2 receptors regulate mouse and human gastric antral epithelial cell homoeostasis

Gail B Gifford et al. Gut. 2017 Jun.

Abstract

Objective: We tested the ability of Notch pathway receptors Notch1 and Notch2 to regulate stem and epithelial cell homoeostasis in mouse and human gastric antral tissue.

Design: Mice were treated with the pan-Notch inhibitor dibenzazepine (DBZ) or inhibitory antibodies targeting Notch1 and/or Notch2. Epithelial proliferation, apoptosis and cellular differentiation were measured by histological and molecular approaches. Organoids were established from mouse and human antral glands; growth and differentiation were measured after treatment with Notch inhibitors.

Results: Notch1 and Notch2 are the predominant Notch receptors expressed in mouse and human antral tissue and organoid cultures. Combined inhibition of Notch1 and Notch2 in adult mice led to decreased epithelial cell proliferation, including reduced proliferation of LGR5 stem cells, and increased apoptosis, similar to the response to global Notch inhibition with DBZ. Less pronounced effects were observed after inhibition of individual receptors. Notch pathway inhibition with DBZ or combined inhibition of Notch1 and Notch2 led to increased differentiation of all gastric antral lineages, with remodelling of cells to express secretory products normally associated with other regions of the GI tract, including intestine. Analysis of mouse and human organoids showed that Notch signalling through Notch1 and Notch2 is intrinsic to the epithelium and required for organoid growth.

Conclusions: Notch signalling is required to maintain gastric antral stem cells. Notch1 and Notch2 are the primary Notch receptors regulating epithelial cell homoeostasis in mouse and human stomach.

Keywords: EPITHELIAL DIFFERENTIATION; EPITHELIAL PROLIFERATION; GASTRIC EPITHELIAL CELL FUNCTION; STEM CELLS.

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Conflict of interest statement

Competing interests CWS is an employee of Genentech and owns shares of Roche.

Figures

Figure 1

Figure 1

Notch signalling in gastric antral cells. (A–D) Antral glands isolated from CBF:H2B-Venus mice (green, nuclear) costained for (B) chromogranin A (CHGA) (red), (C) doublecortin like kinase 1 (DCLK1) (red), or (D) Lgr5-GFP (green, cytoplasmic) and DAPI (blue). Insets show higher-powered views for each channel. (E) Notch receptor expression was determined by qRT-PCR analysis of total RNA isolated from full-thickness antral tissue or antral glands (mean±SEM; n=3–5 mice). ND, not detected. (F) Confocal imaging of paraffin sections from N1-Cre;Tom and N2-Cre;Tom mice costained for red fluorescent protein (RFP) (red), E-cadherin (green) and DAPI (blue) 3 days or 2 weeks after tamoxifen activation of Cre recombinase. Arrows and arrowheads indicate epithelial and non-epithelial Tomato expression, respectively. (G) Frozen tissue sections from CBF: H2B-Venus mice costained for the endothelial marker Platelet/Endothelial Cell Adhesion Molecule 1 (PECAM-1) (left, red) or the smooth muscle cell marker α-smooth muscle actin (SMA) (right, red). Costained cells in boxed regions are marked (arrowheads) in higher-powered insets. Scale bar: 50 μm.

Figure 2

Figure 2

Notch1 and Notch2 regulate antral cell proliferation. (A, B and D–G) Proliferating cells were detected in (A) vehicle, (B) dibenzazepine (DBZ), (D) Gd antibody control (αGd), (E) αN1+αN2, (F) αN1 and (G) αN2 tissues by staining for EdU (green) with DAPI (red). (C and H) Morphometric quantification of EdU cells (mean±SEM; n=5–7 mice). (I and J) Proliferating LGR5 stem cells were visualised in treated Lgr5-GFP mice by GFP (green) and Ki67 (red) costaining (arrow) with DAPI (blue) and quantified by morphometrics (mean±SEM; n=5–7 mice; 40–100 glands/mouse). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 versus vehicle using Students’t test or versus αGd using one-way ANOVA. Scale bar: 50 μm (A, B and D–G) or 25 μm (I).

Figure 3

Figure 3

Enhanced apoptosis after Notch inhibition. (A, B and E–H) Apoptosis was measured by costaining for cleaved caspase-3 (CC3) (green) and E-cadherin (Ecad) (red) with DAPI (blue) in (A) vehicle, (B) dibenzazepine (DBZ), (E) αGd, (F) αN1+αN2, (G) αN1 and (H) αN2-treated mice. (C and D) Morphometric quantification of apoptotic CC3/E-cad costained cells (arrows) (mean±SEM; n=5–7 mice). Scale bar: 20 μm.

Figure 4

Figure 4

Notch1 and Notch2 regulate mouse antral organoid growth. (A) Cells undergoing Notch signalling (green) were observed in antral organoids established from the CBF:H2B-Venus Notch reporter mouse strain. (B) Gene expression of Notch receptors determined by qRT-PCR analysis of antral organoids (mean±SEM n=3 independent organoid lines). ND, not detected. Morphology (C, D and F–I) and quantitative measure of organoid size (E and J) following 5 days of treatment with (C) vehicle, (D) N-(_N_-(3,5-Difluorophenacetyl-L-alanyl))-(S)-phenylglycine t-butyl ester (DAPT), (F) αGd, (G) αN1+αN2, (H) αN1 or (I) αN2 (mean±SEM; n=3 lines). Scale bar: 50 μm (A) or 250 μm (C, D and F–I).

Figure 5

Figure 5

Notch1 and Notch2 regulate antral endocrine cell differentiation. (A, B and F–I) Gastrin immunostaining (green) of paraffin sections from mice treated with (A) vehicle, (B) dibenzazepine (DBZ), (F) αGd, (G) αN1+αN2, (H) αN1, and (I) αN2 with DAPI (red). (C and J) Gastrin cells were quantitated by morphometrics (mean±SEM, n=4–5). Gene expression analysis of gastrin (Gast) and chromogranin A (Chga) in (D, E, K and L) full-thickness tissue or (M and N) organoids (mean±SEM; n=5–7 mice or 3 independent organoid lines). Scale bar: 50 μm.

Figure 6

Figure 6

Notch1 and Notch2 regulate surface mucous cell differentiation. (A, B and D–G) Immunostaining for the surface mucous cell marker CLCA1 in (A) vehicle, (B) dibenzazepine (DBZ), (D) αGd, (E) αN1+αN2, (F) αN1 and (G) αN2-treated mice. (C and H) qRT-PCR analysis of Clca1 expression (mean±SEM; n=5–7 mice). (I and J) Transmission electron microscope images of surface mucous cells from (I) vehicle or ( J) DBZ-treated mice. Scale bar: 50 μm (A, B and D–G) or 2 μm (I and J).

Figure 7

Figure 7

Notch inhibition remodels cells at the antral gland base. (A, B and E–H) Costaining with GSII lectin (green) and an anti-gastric intrinsic factor (anti-GIF) antibody (red) with DAPI (blue) on (A) vehicle, (B) dibenzazepine (DBZ), (E) αGd, (F) αN1+αN2, (G) αN1 and (H) αN2-treated mice. (C, D and I–L) Gene expression analysis of trefoil factor 2 (Tff2) and Gif by qRT-PCR analysis of full-thickness tissue or organoids (mean±SEM; n=4–7 mice). (M and N) Transmission electron microscope images of gland base cells from (M) vehicle or (N) DBZ-treated mice. Scale bar: 50 μm (A, B and E–H) or 2 μm (M and N).

Figure 8

Figure 8

Increased expression of gastric corpus and intestinal cell markers in dibenzazepine (DBZ)-treated antral tissue. (A–C) Immunostaining and gene expression analysis of histidine decarboxylase (HDC). (D–F) Immunostaining and gene expression analysis of Muc2. (G–J) Gene expression analysis of trefoil factor 3 (Tff3), matrix metalloproteinase-7 (Mmp7), cryptdin, and regenerating islet-derived 3 γ (Reg3γ) in antral RNA isolated from vehicle or DBZ-treated mice (mean±SEM; n=3–6 mice).

Figure 9

Figure 9

Human antral organoid growth is regulated by Notch1 and Notch2. (A and B) Notch receptor mRNA abundance was measured by qRT-PCR in (A) full-thickness human tissue or (B) antral organoids (mean±SEM, n=3 patients or patient-derived organoid lines). See online supplementary figures S5 and S6, table S3 for detailed information on human tissues. Morphology (C, D and F–I) and quantitative measure of organoid size (E and J) following treatment with (C) vehicle, (D) N-(_N_-(3,5-Difluorophenacetyl-L-alanyl))-(S)-phenylglycine t-butyl ester (DAPT), (F) αGd, (G) αN1+αN2, (H) αN1 or (I) αN2 (mean±SEM, n=3 independent patient-derived organoid lines). Scale bar: 250 μm. ND, not detected.

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