Wnt/ß-catenin promotes gastric fundus specification in mice and humans.

Despite the global prevalence of gastric disease, there are few adequate models in which to study the fundus epithelium of the human stomach. We differentiated human pluripotent stem cells (hPSCs) into gastric organoids containing fundic epithelium by first identifying and then recapitulating key events in embryonic fundus development. We found that disruption of Wnt/ß-catenin signalling in mouse embryos led to conversion of fundic to antral epithelium, and that ß-catenin activation in hPSC-derived foregut progenitors promoted the development of human fundic-type gastric organoids (hFGOs). We then used hFGOs to identify temporally distinct roles for multiple signalling pathways in epithelial morphogenesis and differentiation of fundic cell types, including chief cells and functional parietal cells. hFGOs are a powerful model for studying the development of the human fundus and the molecular bases of human gastric physiology and pathophysiology, and also represent a new platform for drug discovery.

Expression of receptors regulating gastric acidity in the developing sheep stomach.

Acid secretion first appears in the stomach during the later stages of fetal development. Gastric acid secretion is regulated by the stimulatory effects of gastrin, histamine, acetylcholine and the inhibitory actions of somatostatin on their respective receptors. A semi-quantitative reverse transcriptase-polymerase chain reaction method for the determination of changes in mRNA expression for these receptors was developed and correlated with known changes in gastric acidity. Glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) was used as a reference and an internal standard. The antrum and fundus from four age groups were assayed: 80 days of gestation, 110 days of gestation, term (145 days) and adult animals. The CCK B/gastrin and the histamine (H(2)) receptor mRNA were significantly lower in samples from the fundus of fetuses, from 80 and 110 days of gestation when compared with the adult fundus. Histamine receptor mRNA in the antrum was also significantly lower in the 80 and 110 days of gestation samples relative to the term fetal antrum. Somatostatin II receptor mRNA levels in the antrum decreased with increasing age with no change in the fundus. These findings suggest that changes in receptor gene expression, may be responsible for the diminished gastric acidity and responsiveness observed in the fetal stomach.

Expression of N-acetylglucosamine residues in developing rat fundic gland cells.

The development of rat fundic gland was studied by immunohistochemistry using a recently developed monoclonal antibody, HIK 1083, at both light and electron microscope levels. Antibody HIK 1083 recognized oligosaccharides with a non-reducing terminal alpha-linked N-acetylglucosamine (GlcNAc) residue. In the developing rat fundic gland, cells expressing alpha-GlcNAc residues were discernible from day 19.5 of gestation and continued to exist till adult. The distribution of the alpha-GlcNAc expressing cells was consistent with that described previously for cells reacting to Griffonia simplicifolia lectin (GSA-II) in all developmental stages. These cells were located at the bottom of the fundic gland when they first appeared. With the elongation and maturation of the gland, these cells moved upwards and were finally restricted in the neck region of the gland. Combining previous reports and the present electron microscopical observations, HIK 1083-positive cells in the adult rat fundic gland are mucous neck cells. The interaction between antibody HIK 1083 and GSA-II lectin was investigated. GSA-II prevented the subsequent binding of HIK 1083, while HIK 1083 did not prevent GSA-II binding to mucous neck cells. Our results suggested that alpha-GlcNAc residues exist in rat fundic gland from day 19.5 of gestation and continue to exist till adult. Cells expressing alpha-GlcNAc residues appeared as typical mucous neck cells from postnatal four weeks.

Ontogeny of the rat parietal cell: analysis using anti-parietal cell antibody and transmission electron microscopy.

We studied the ontogeny of the rat parietal cell using human anti-parietal cell antibody and transmission electron microscopy. In the gastric fundus of the rat, we found that the epithelium changed from stratified to columnar at gestational day 18.5. Gastric pits began to form at gestational day 19.5. Primitive fundic glands appeared at gestational day 20.5. Human anti-parietal cell antibody specifically stained the rat parietal cells. By this immunohistochemical staining, rat parietal cells were identified from gestational day 19.5. At first we observed only a few plump parietal cells sparsely located in the fundic glands. In neonatal rats, the parietal cells increased in number and began to distribute themselves over a wider area of the primitive fundic glands especially in the lower half. As the rats grew, the distribution area of the parietal cells expanded to cover the whole of the glands except for the foveolar region. Parietal cells in the isthmus and neck regions were round and plump, while those in the basal region were slender and polygonal. We found that throughout the development of the fundic glands there were several ultrastructural changes of the parietal cells. In the late fetal period, parietal cells containing lysosomes and secretory granules were observed, but no tubulovesicles were identified. Development of the tubulovesicles was remarkable until one week after birth. The ultrastructure of the parietal cells of the neonate and adult varied, depending on their distribution area. We found that parietal cells in the basal region of the fundic glands which are fully matured cells had wider intracellular secretory canaliculi, while cells in the upper region had narrower canaliculi; this indicates the functional difference between hydrochloric acid secretion in parietal cells of the two regions.

Immunocytochemistry and in situ hybridization studies of pepsinogen C-producing cells in developing rat fundic glands.

The ontogeny of pepsinogen C-producing cells in rat fundic glands was studied by means of light and electron microscopy using an antiserum raised against a synthetic peptide based on rat pepsinogen C. To confirm the immunocytochemistry results, the expression of rat pepsinogen C messenger RNA (mRNA) in the fundic gland was also examined by in situ hybridization using a digoxigenin-labeled RNA probe. In adult rats, pepsinogen C was produced by chief cells, mucous neck cells, and intermediate mucopeptic cells. Pepsinogen C-producing cells appeared in embryos as early as 18.5 days' gestation. The development of these cells could be classified into four stages: (1) 18.5 days' gestation to 0.5 days after birth; (2) 0.5 days to 2 weeks after birth; (3) 3-4 weeks after birth; (4) 4-8 weeks after birth. In embryos and young animals, pepsinogen C-producing cells were mucopeptic cells. By 4 weeks after birth, mucous neck cells could be distinguished morphologically. The maturation stages of the chief cells could be traced by electron microscopy along the longitudinal axis of the rat fundic gland by double-staining with anti-pepsinogen C antibody and periodic acid-thiocarbohydrazide-silver proteinate. Positive reactions for pepsinogen C and pepsinogen C mRNA expression were detected in mucous neck cells. Therefore, we conclude that mucous neck cells are precursor cells of chief cells. Mucous neck cells, intermediate cells, and chief cells are in the same differentiating cell lineage.

Ontogeny of proliferative cells in the rat fundic gland.

The ontogeny of proliferative cells in the rat fundic gland was studied using bromodeoxyuridine (BrdU) immunohistochemistry from day 17.5 of gestation to 8 weeks after birth. This ontogenic process is divided into 4 stages. (1) The late fetal period extending to 0 day of birth: Proliferative cells were scattered throughout all levels of the stratified epithelium in the earliest stage (day 17.5-18.5 of gestation). With the appearance of a primitive gastric pit at day 19.5 of gestation, proliferative cells were more numerous at the base of the fundic gland. Proliferative cells were concentrated in the gland base and were rarely seen in the epithelial surface from day 21.5 of gestation onwards. (2) One day to 2 weeks after birth: As fundic gland growth proceeded, proliferative cells remained concentrated in the gland base. (3) Two to 4 weeks after birth: Proliferative cells left the gland base and moved upward to reach the adult location in the isthmus. (4) Four to 8 weeks after birth: The development of the fundic gland was complete and proliferative cells remained in a narrow proliferative zone in the isthmus.

Ontogeny of sulphated glycoconjugate-producing cells in the rat fundic gland.

The ontogeny of sulphated glycoconjugate-producing cells in the rat fundic gland has been studied using high iron diamine (HID), Alcian Blue (AB) at pH 1.0, high iron diamine in combination with Alcian Blue at pH 2.5 (HID-AB), cationic colloidal gold (CCG) at pH 1.0 under light microscopy and CCG (1.0), HID-thiocarbohydrazide (TCH)-silver proteinate (SP)-physical development (PD) under electron microscopy. From day 19.5 of gestation, sulphated glycoconjugate-producing cells were discernible under both light and electron microscopy. The development of such cells can be classified into four stages: (1) a prenatal period from day 19.5 of gestation extending to 0.5 days after birth; (2) 1 day to 2 weeks after birth; (3) 2 to 4 weeks after birth; and (4) the final period from 4 to 8 weeks after birth. Glycoconjugate-producing cells reached maturity by 4 weeks after birth. Our results indicated that glycoconjugate-producing cells were cells along the wall of foveolar lumen, but not those covering the gastric mucosa surface. Our results also suggested that the trans to transmost Golgi apparatus lamellae were the sites of sulphation in the developing rat stomach.

Developmental changes in gastric fundus smooth muscle contractility and involvement of extracellular calcium in fetal and adult guinea pigs.

Delayed gastric emptying is a common problem in preterm infants. The factors underlying this gastroparesis remain unsettled but may involve immaturity of smooth muscle contraction. The present study was designed to test this hypothesis. Muscle strips from the gastric fundus of fetal and adult guinea pigs were studied in vitro for their contractile response to receptor activation (acetylcholine and bethanechol) and membrane depolarization (potassium chloride). The dose-response curves were analyzed for differences in active force development (kg/cm2). The role of extracellular calcium (Ca2+) in the contractile responses was determined by contracting the tissues in a zero-Ca2+ physiologic saline solution and in the presence of nifedipine, a voltage-dependent Ca2+ channel blocker. The results demonstrate the following: 1) tissues from adult animals developed significantly more active force when tested with acetylcholine, bethanechol, and potassium chloride; 2) tissues from the fetal animals were relatively unresponsive to contraction with potassium chloride compared with the adult; and 3) both nifedipine and incubation in a zero-Ca2+ physiologic saline solution had a significantly greater inhibitory effect on the contractions of adult than fetal muscle strips. Our data indicate that smooth muscle in the gastric fundus develops increasing force with maturation. The increased contractility in the adult fundus appears to be due to an increased involvement of extracellular calcium influx, in part through voltage-dependent Ca2+ channels.

Immunocytochemical study of pepsinogen 1-producing cells in the fundic mucosa of the stomach in developing mice.

Development and maturation of pepsinogen 1-producing cells were studied in the gastric fundic mucosa of the mouse by means of light- and electron-microscopic immunocytochemistry using rabbit anti-rat pepsinogen 1-serum. In the adult mouse, secretory granules in mucous neck cells, transitional mucous neck/chief cells and chief cells are immunolabeled. The numerical density of gold particles on zymogen granules is not significantly altered among different stages of maturation of chief cells. In addition, rough endoplasmic reticulum and Golgi complex of these cell types show a weak labeling. In mice from day 16 of gestation to postnatal day 14, mucous neck cells and chief cells cannot be distinguished, but only one type of pepsinogen 1-producing cell, called 'primitive chief cell', is identified in the fundic gland. The intensity of immunoreactivity of secretory granules in primitive chief cells is uniform within an individual cells but varies greatly among different cells. The majority of primitive chief cells contains weakly labeled granules regardless of the maturation stage of cells or of animals. On postnatal day 21, mucous neck, transitional and chief cells are distinguishable, and secretory granules in these cells are intensely immunolabeled as in the adult. These results suggest that pepsinogen 1-production rapidly increases with differentiation of mucous neck and chief cells.