Bmi1 marks gastric stem cells located in the isthmus in mice.

In the mammalian stomach, the isthmus has been considered as a stem cell zone. However, various locations and proliferative activities of gastric stem cells have been reported. We focused here on the stem cell marker Bmi1, a polycomb group protein, aiming to elucidate the characteristics of Bmi1-expressing cells in the stomach and to examine their stem cell potential. We investigated the Bmi1-expressing cell lineage in Bmi1-CreERT; Rosa26-YFP, LacZ or Rosa26-Confetti mice. We examined the in vivo and ex vivo effects of Bmi1-expressing cell ablation by using Bmi1-CreERT; Rosa26-iDTR mice. The Bmi1 lineage was also traced during regeneration after high-dose tamoxifen-, irradiation- and acetic acid-induced mucosal injuries. In the lineage-tracing experiments using low-dose tamoxifen, Bmi1-expressing cells in the isthmus of the gastric antrum and corpus provided progeny bidirectionally, towards both the luminal and basal sides over 6 months. In gastric organoids, Bmi1-expressing cells also provided progeny. Ablation of Bmi1-expressing cells resulted in impaired gastric epithelium in both mouse stomach and organoids. After high-dose tamoxifen-induced gastric mucosal injury, Bmi1-expressing cell lineages expanded and fully occupied all gastric glands of the antrum and the corpus within 7 days after tamoxifen injection. After irradiation- and acetic acid-induced gastric mucosal injuries, Bmi1-expressing cells also contributed to regeneration. In conclusion, Bmi1 is a gastric stem cell marker expressed in the isthmus of the antrum and corpus. Bmi1-expressing cells have stem cell potentials, both under physiological conditions and during regeneration after gastric mucosal injuries. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Role of the homeodomain transcription factor Bapx1 in mouse distal stomach development.

BACKGROUND & AIMS: Expansion and patterning of the endoderm generate a highly ordered, multiorgan digestive system in vertebrate animals. Among distal foregut derivatives, the gastric corpus, antrum, pylorus, and duodenum are distinct structures with sharp boundaries. Some homeodomain transcription factors expressed in gut mesenchyme convey positional information required for anterior-posterior patterning of the digestive tract. Barx1, in particular, controls stomach differentiation and morphogenesis. The Nirenberg and Kim homeobox gene Bapx1 (Nkx3-2) has an established role in skeletal development, but its function in the mammalian gut is less clear. METHODS: We generated a Bapx1(Cre) knock-in allele to fate map Bapx1-expressing cells and evaluate its function in gastrointestinal development. RESULTS: Bapx1-expressing cells populate the gut mesenchyme with a rostral boundary in the hindstomach near the junction of the gastric corpus and antrum. Smooth muscle differentiation and distribution of early regional markers are ostensibly normal in Bapx1(Cre/Cre) gut, but there are distinctive morphologic abnormalities near this rostral Bapx1 domain: the antral segment of the stomach is markedly shortened, and the pyloric constriction is lost. Comparison of expression domains and examination of stomach phenotypes in single and compound Barx1 and Bapx1 mutant mice suggests a hierarchy between these 2 factors; Bapx1 expression is lost in the absence of Barx1. CONCLUSIONS: This study reveals the nonredundant requirement for Bapx1 in distal stomach development, places it within a Barx1-dependent pathway, and illustrates the pervasive influence of gut mesenchyme homeobox genes on endoderm differentiation and digestive organogenesis.

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.

Development of electrical rhythmicity in the murine gastrointestinal tract is specifically encoded in the tunica muscularis.

1. Interstitial cells of Cajal (ICCs) have been identified as pacemaker cells in the gastrointestinal (GI) tracts of vertebrates. We have studied the development of ICCs in pacemaker regions and the onset of electrical rhythmicity in the gastric antrum, small bowel and proximal colon of the mouse. 2. ICCs, as detected by c-Kit immunofluorescence, were found during embryogenesis in regions of the GI tract that eventually become pacemaker areas. Prior to birth, these cells were organized into well-structured networks, and by the end of the embryonic period the morphology of ICC networks in pacemaker regions appeared very similar to that observed in adult animals. 3. Electrical rhythmicity was recorded prior to birth (by E18) in the proximal GI tract (stomach and jejunum), and this activity developed to adult-like behaviour within a week after birth. In the ileum and proximal colon rhythmicity developed after birth, and adult-like characteristics were apparent within the first week. 4. Post-junctional responses of smooth muscles to neural inputs could be recorded at birth, and stimulation of intrinsic nerves often led to oscillatory activity resembling slow waves for up to several minutes following brief stimuli. Nerve stimulation augmented spontaneous activity in the proximal portions of the GI tract and elicited rhythmic activity temporarily in quiescent tissues of the distal GI tract. 5. ICCs and rhythmicity developed in an apparently normal manner in tissues isolated at birth and placed in organ culture. These data suggest that the tunica muscularis provides a suitable microenvironment for the development of ICCs and rhythmicity without the need for extrinsic stimuli. 6. Treatment of small intestinal tissues taken from embryos at E15 with neutralizing c-Kit antibodies abolished ICC development and the organization of ICCs into networks that typically occurs during the late embryonic period. Treatment of muscles taken from newborn animals with c-Kit antibodies blocked postnatal development of ICCs, disrupted already established and functional ICC networks, and rendered muscles electrically quiescent. 7. In summary, ICC networks develop in the pacemaker regions of the murine GI tract before birth. Development and organization of ICCs of the myenteric plexus region into networks precedes the development of electrical rhythmicity. Post-natal development of electrical rhythmicity is mainly characterized by enhancement of the amplitude and frequency of slow waves. The development of ICCs and electrical rhythmicity persists in vitro. ICCs appear to be necessary for the initiation of electrical rhythmicity. These findings provide further evidence for the pacemaker role of ICCs.

Cytoarchitecture of the bovine pyloric epithelium during early ontogenesis.

The cytoarchitecture of the pyloric gland region in the early ontogeny of the bovine abomasal mucosa was investigated using light and transmission electron microscopic methods. Two cell types are involved in forming the lamina epithelialis in the youngest fetus with a 24 mm crown-rump-length (CRL): the indifferent cell and the endocrine cell. With a CRL of 71 mm, two other cell types occur, the granule-containing cell and the brush cell. The indifferent cell represents the first stem cell, which develops into the secretory granule-containing cell type. This seems to be the secondary stem cell lining the top of the epithelium as well as the base of the primitive epithelial tubes, and it differentiates into the surface mucous cell and the pylorocyte. Endocrine cells appear as open and closed types and represent the most differentiated cells already present in the youngest specimen in this investigation. The most rare cell type, the brush cell, appears in the bovine abomasal ontogeny much earlier than in other mammalian species.

Immunocytochemical localisation of parietal cells and G cells in the developing human stomach.

Previous studies on the distribution of parietal cells and G cells in normal adult stomachs have shown that in about 20% of specimens parietal cells extended to the pylorus. This study aimed to examine the distribution of parietal cells and G cells in the body and antrum of the developing human stomach in relation to anatomical landmarks, using histological and immunocytochemical methods. In all 15 fetal stomachs examined, parietal cells extended to the pylorus and expressed intrinsic factor and hydrogen-potassium-ATPase activity from week 13 of gestation. By contrast, in only one of the five infant stomachs did parietal cells extend to the pylorus: this is identical to the distribution in the adult. G cells developed in the antrum from 18 weeks' gestation and their distribution did not differ between the fetal and infant stomachs. These findings indicate that parietal cells disappear from the antrum of the stomach in the third trimester of pregnancy, but this process fails to occur in approximately 20% of the population.

Visceral targets specify calcitonin gene-related peptide and substance P enrichment in trigeminal afferent projections.

Rat trigeminal ganglion projections to a visceral target (intracranial blood vessels) are enriched in calcitonin gene-related peptide (CGRP) and substance P (Sub P) compared to trigeminal ganglion projections to a cutaneous target (the forehead skin). We asked if transplants of a novel visceral target (fetal stomach antrum tissue) into the path of the neonatal rat trigeminal frontal nerve projection to forehead skin would induce neuronal CGRP and Sub P enrichment. By postnatal day (P) 25, the percentage of nerves containing CGRP increased from 14-15% in the control trigeminal projection to forehead skin to 20-31% (in different experiments) in the trigeminal projection to transplanted stomach antrum. The percentage of Sub P-containing neurons increased from 10% in the control forehead skin projection to 22% in the trigeminal projection to stomach transplants over the same time period. The number of neurons in the trigeminal frontal nerve projection to stomach antrum transplants was not significantly different from the number of frontal neurons projecting to control forehead skin. We suggest that respecification of trigeminal neurons to the CGRP and Sub P phenotype, not selective survival of CGRP- and Sub P-positive afferents, is the mechanism by which stomach antrum induces enrichment of CGRP and Sub P. A subpopulation of rat trigeminal neurons with cutaneous forehead skin projections also sends a transient axon collateral projection to a visceral target (the cerebral arteries) during early postnatal development. Postnatal maintenance of an axonal projection to a cutaneous target (forehead skin) may be incompatible with a neuron also maintaining a visceral collateral to the cerebral arteries.(ABSTRACT TRUNCATED AT 250 WORDS)

Fetal development of the pyloric muscle.

The fetal development of the pyloric muscle was studied in five human embryos (crown-rump length 5 to 31 mm) and in ten fetuses aged 3 to 9 months. Samples of pyloric muscle were obtained during operation for pyloric stenosis in two infants aged six weeks. Anatomo-radiologic, morphologic and immunohistochemical studies were made on this material, from which it emerged that the pylorus is identifiable by means of specific markers from the 40th day. Its two-layered muscular structure is described in detail. The mechanism of sphincteric function is reviewed. This study assumes clinical importance in the context of the etiopathogenesis of hypertrophic stenosis of the pylorus.

Immunohistochemical and stereological study of neuroendocrine cells in human antrum during the perinatal period.

A correlative immunohistochemical and stereological study of neuroendocrine cells (NEC) was carried out in the antrum of twenty human fetuses with gestational ages from 18 to 42 weeks and of two specimens postnatally. Neuron-specific enolase (NSE) as a common marker of neurons and NEC, as well as gastrin (G-) and somatostatin (D-) immunoreactive cells served for evaluation of volume density, which proved to be the most convenient method for quantitative analysis of NEC. It was observed that a considerable frequency of NEC appeared at 23-24 weeks of gestation (8% of NSE- and 6% of G- cells) and coincided with the adult pattern of intramural innervation. After a repeated increase of NEC in the 26-week-old fetus, the frequency of NEC remained persistant during the perinatal period (10-12% of NSE- and 7-8% of G- cells). An exception was a specimen with a prolonged pregnancy (42 weeks) in which the percentage of NSE- (17%) and G- (10%) cells was almost the same as at 6 weeks postnatally. The maximal quantitative difference of NEC was noted between 6- and 8-week specimens postnatally, e.g. 9% to 22% of G- cells, respectively. Observations obtained by NSE and S-100 protein were also demonstrated in lymphoid cells of gut associated and mesenteric lymphoid tissue.

Immunohistochemical studies on the ontogenesis of some endocrine cells in the chicken antrum and duodenum.

The time of appearance, morphology and topographic distribution of gastrin/CCK-, somatostatin-, 5HT-, and bombesin-like immunoreactive cells during embryonic and postnatal development were studied in chicken antrum and duodenum with immunohistochemical methods. Gastrin/CCK-like cells appeared on or about the 11th day in the antrum and duodenum, somatostatin-like cells around the 12th day in the antrum and the 11th day in the duodenum, bombesin-like cells appeared only in the antrum and only at hatching. In the early stages of development all the immunoreactive cells were localized in the surface epithelium, descending deeper into the glands as these form, although some cells could always be seen in the surface epithelium. Around the 17th day the number of gastrin/CCK-like cells and somatostatin-like cells in the antrum increases, while 5HT-like already become more numerous in the duodenum from the 13th day onwards. Two territories were recognized in the antrum of the adult: the first was near the duodenum where gastrin/CCK-like and somatostatin-like cells, often in close contact, were very numerous; the other territory was near the gizzard where bombesin-like cells were more numerous. Both regions contained 5HT-like cells in smaller number. In adult duodenum, 5HT-like cells were the most numerous, while somatostatin-like cells and gastrin/CCK-like cells, found in more superficial locations, were more scanty.

Ultrastructure and localization of substance P and met-enkephalin immunoreactivity in the human fetal gastric antrum.

The ultrastructural localization and relations of substance P- and met-enkephalin-labeled neuronal structures were examined in the wall of the human gastric antrum during early fetal life. By 14-16 weeks of gestation, clearly discernable neural plexuses and a well developed external muscle coat were present. In the submucous coat, neural plexuses varied from immature forms consisting of 1-4 neurites partially enveloped by Schwann cell processes to more mature plexuses where neurons were completely enclosed by Schwann cell processes. Neuronal profiles with substance P- and met-enkephalin-like immunoreactivities were observed in the submucous plexus. In the myenteric plexus met-enkephalin-like immunoreactivity was seen within cell bodies and neurites. By contrast, although substance P-like immunoreactivity was observed in neurites in the myenteric plexus, no substance P-labeled somata could be identified. Unlabeled terminals were seen in contact with both unlabeled dendrites and met-enkephalinergic neurons. An increase in electron density was observed at the sites of contact. These structures probably represent early stages in the development of synaptic specializations. In addition, met-enkephalin-labeled varicosities were seen in apposition to smooth muscle cells of the circular muscle coat. This suggests that antral smooth muscle cells are directly innervated by met-enkephalin neurons.

Developmental changes of the contractile activity and the drug sensitivity of smooth muscle of the alimentary tract of guinea pig.

The mechanical activity of the smooth muscle of guinea pig alimentary tract changed during fetal and postnatal development. At the F20 (fetus, body weight 20 g) stage of the fetus, the antrum, small intestinal and colonic preparations showed no spontaneous activity. After this stage, spontaneous activity occurred but the patterns of contractile activity of various regions of the alimentary tract differed from those of the adult. However, at later fetal stages (F50 or later), the mechanical patterns of various regions, except the lower region of colon, were similar to adult patterns. An excitatory response to acetylcholine was observed in all regions at the F20 stage but catecholamines were not effective at this stage. Responses to catecholamines appeared later than those to acetylcholine but they were not constant. During development of the colon, the excitatory effect of adrenaline was significant and the effect was blocked by phenoxybenzamine, and isoprenaline inhibited the spontaneous activity. However, sympathetic nerve stimulation at F70 inhibited the mechanical activity and propranolol blocked the inhibitory action of sympathetic nerves. These results indicate the co-existence of alpha-excitatory and beta-inhibitory receptors on the developing colonic smooth muscle cell membrane.

Gastrin in the human fetus. Distribution and molecular forms of gastrin in the antro-pyloric gland area, duodenum and pancreas.

The ontogeny of human fetal gastrin has been investigated by immunocytochemistry and radioimmunochemistry. Gastrin and gastrin cells are shown to appear later in the antral than in the duodenal mucosa. At the ages studied (11--22 weeks of gestation) pancreatic gastrin could not be detected. Gastrin component III was found to predominate in the antrum, whereas component II was quantitatively very important in the duodenum. Circumstantial evidence suggests that fetal gastrin is delivered to the blood, and that it may exert trophic functions in the fetus.