The major signaling pathways regulating gastric stem cells are unknown. Here we report that Notch signaling is essential for homeostasis of LGR5 + antral stem cells. Pathway inhibition reduced prolife-

The Notch signaling pathway regulates intestinal epithelial cell homeostasis, including stem cell maintenance, progenitor cell proliferation and differentiation. Notch1 and Notch2 receptors are expressed in the epithelium, but individual contributions to these functions are unclear. We used genetic deletion to define receptor roles on stem cell function, cell proliferation/differentiation, and repair after injury. Loss of Notch1 induced a transient secretory cell hyperplasia that spontaneously resolved over time. In contrast, deletion of Notch2 had no secretory cell effect. Compound deletions of Notch1 and Notch2 resulted in a more severe secretory cell hyperplasia than deletion of Notch1 alone. Furthermore, only double deletion of Notch1 and Notch2 decreased cell proliferation, suggesting a low threshold for maintenance of proliferation compared to differentiation. Stem cells were affected by deletion of Notch1, with reduced expression of Olfm4 and fewer LGR5+ stem cells. Deletion of Notch2 had no apparent affect on stem cell homeostasis. However, we observed impaired crypt regeneration after radiation in both Notch1- and Notch2-deleted intestine, suggesting that higher Notch activity is required post-injury. These findings suggest that Notch1 is the primary receptor regulating intestinal stem cell function and that Notch1 and Notch2 together regulate epithelial cell proliferation, cell fate determination, and post-injury regeneration.

The gastrointestinal (GI) tract epithelium is continuously replenished by actively cycling stem and progenitor cells. These cell compartments are regulated to balance proliferation and stem cell renewal with differentiation into the various mature cell types to maintain tissue homeostasis. In this topical review we focus on the role of the Notch signalling pathway to regulate GI stem cell function in adult small intestine and stomach. We first present the current view of stem and progenitor cell populations in these tissues and then summarize the studies that have established the Notch pathway as a key regulator of gastric and intestinal stem cell function. Notch signalling has been shown to be a niche factor required for maintenance of GI stem cells in both tissues. In addition, Notch has been described to regulate epithelial cell differentiation. Recent studies have revealed key similarities and differences in how Notch regulates stem cell function in the stomach compared to intestine. We summarize the literature regarding Notch regulation of GI stem cell proliferation and differentiation, highlighting tissue‐specific functions to compare and contrast Notch in the stomach and intestine.

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.

Cyclooxygenase-1 (Cox-1) contributes to gastric defense of healthy tissue, but the role in the protection of the gastric epithelium after minor, acute damage has been difficult to study in vivo. Using 710-nm two-photon light absorption to create microscopic gastric damage in anesthetized mice with the gastric mucosal surface surgically exposed and perfused on the microscope stage, the acute response of surface cells to injury could be monitored using in vivo microscopy within seconds after injury. Using exogenous (Cl-NERF) and endogenous fluorophores, extracellular pH and cell death were monitored in real time during the entire damage and repair cycle. Two-photon damage was initiated by scanning approximately 200 microm(2) of gastric surface cells with high laser intensity, causing rapid bleaching of NAD(P)H fluorescence in optically targeted cells. In both Cox-1(+/-) and Cox-1(-/-) mice, a similar initial damage area expanded to include bystander epithelial cells over the next 2-5 min, with larger maximal damage noted in Cox-1(-/-) mice. The maximal damage size seen in Cox-1(-/-) mice could be reduced by exogenous dimethyl-PGE(2). All damaged cells exfoliated, and the underlying epithelium was coincidently repaired over a time interval that was briefer in Cox-1(+/-) (12 +/- 2 min, n = 12) than in Cox-1(-/-) (24 +/- 4 min, n = 14) mice. Directly after damage, pH increased transiently in the juxtamucosal layer (maximal at 3-6 min). A smaller peak pH change was noted in Cox-1(-/-) mice (DeltapH = 0.3 +/- 0.04) than in Cox-1(+/-) mice (DeltapH = 0.6 +/- 0.2). Recovery to normal surface pH took longer in Cox-1(-/-) mice (27 +/- 5 min) than in Cox-1(+/-) mice (12 +/- 1 min). In conclusion, constitutive loss of Cox-1 leaves the gastric mucosa more prone to damage and slowed repair of microlesions.

Here we demonstrate that the Notch signaling pathway is essential for proliferation of stem cells in the mouse and human gastric corpus. We identify NOTCH1 and NOTCH2 as the predominant Notch receptors expressed in both mouse and human corpus and show that both receptors are required for corpus stem cell proliferation. We show that chronic Notch activation in corpus stem cells induces hyperproliferation and tissue hypertrophy, suggesting that Notch may drive gastric tumorigenesis.

The gastric epithelium is sustained by a population of stem cells that replenish the various mature epithelial lineages throughout adulthood. Regulation of stem and progenitor cell proliferation occurs via basic developmental signaling pathways, including the Notch pathway, which recently was described to promote gastric stem cell proliferation in both mice and human beings. Current cancer theory proposes that adult stem cells that maintain gastrointestinal tissues accumulate mutations that promote cancerous growth, and that basic signaling pathways, such as Notch, which stimulate stem cell proliferation, can promote tumorigenesis. Accordingly, constitutive Notch activation leads to unchecked cellular proliferation and gastric tumors in genetic mouse models. Furthermore, there is emerging evidence suggesting that the Notch pathway may be activated in some human gastric cancers, supporting a potential role for Notch in gastric tumorigenesis. In this review, we first summarize the current understanding of gastric stem cells defined by genetic mouse studies, followed by discussion of the literature regarding Notch pathway regulation of gastric stem cell function in the mouse and human beings. Notch action to maintain gastric epithelial cell homeostasis and the cellular consequences of dysregulated signaling to promote tumorigenesis are discussed, including studies associating Notch activation with human gastric cancer. Finally, we compare and contrast Notch function in the stomach with other gastrointestinal tissues, including the intestine, to highlight the sensitivity of the stomach to Notch-induced tumors.

Gastric surface pH (pH(o)) transiently increases in response to focal epithelial damage. The sources of that increase, either from paracellular leakage of interstitial fluid or transcellular acid/base fluxes, have not been determined. Using in vivo microscopy approaches we measured pH(o) with Cl-NERF, tissue permeability with intravenous fluorescent-dextrans to label interstitial fluid (paracellular leakage), and gastric epithelial intracellular pH (pH(i)) with SNARF-5F (cellular acid/base fluxes). In response to two-photon photodamage, we found that cell-impermeant dyes entered damaged cells from luminal or tissue compartments, suggesting a possible slow transcellular, but not paracellular, route for increased permeability after damage. Regarding cytosolic acid/base status, we found that damaged cells acidified (6.63 +/- 0.03) after photodamage, compared with healthy surface cells both near (7.12 +/- 0.06) and far (7.07 +/- 0.04) from damage (P < 0.05). This damaged cell acidification was further attenuated with 20 muM intravenous EIPA (6.34 +/- 0.05, P < 0.05) but unchanged by addition of 0.5 mM luminal H(2)DIDS (6.64 +/- 0.08, P > 0.05). Raising luminal pH did not realkalinize damaged cells, suggesting that the mechanism of acidification is not attributable to leakiness to luminal protons. Inhibition of apical HCO(3)(-) secretion with 0.5 mM luminal H(2)DIDS or genetic deletion of the solute-like carrier 26A9 (SLC26A9) Cl(-)/HCO(3)(-) exchanger blocked the pH(o) increase normally observed in control animals but did not compromise repair of damaged tissue. Addition of exogenous PGE(2) significantly increased pH(o) in wild-type, but not SLC26A9 knockout, animals, suggesting that prostaglandin-stimulated HCO(3)(-) secretion is fully mediated by SLC26A9. We conclude that cellular HCO(3)(-) secretion, likely through SLC26A9, is the dominant mechanism whereby surface pH transiently increases in response to photodamage.