GATA4 Controls Epithelial Morphogenesis in the Developing Stomach to Promote Establishment of Glandular Columnar Epithelium.

BACKGROUND & AIMS: The transcription factor GATA4 is broadly expressed in nascent foregut endoderm. As development progresses, GATA4 is lost in the domain giving rise to the stratified squamous epithelium of the esophagus and forestomach (FS), while it is maintained in the domain giving rise to the simple columnar epithelium of the hindstomach (HS). Differential GATA4 expression within these domains coincides with the onset of distinct tissue morphogenetic events, suggesting a role for GATA4 in diversifying foregut endoderm into discrete esophageal/FS and HS epithelial tissues. The goal of this study was to determine how GATA4 regulates differential morphogenesis of the mouse gastric epithelium. METHODS: We used a Gata4 conditional knockout mouse line to eliminate GATA4 in the developing HS and a Gata4 conditional knock-in mouse line to express GATA4 in the developing FS. RESULTS: We found that GATA4-deficient HS epithelium adopted a FS-like fate, and conversely, that GATA4-expressing FS epithelium adopted a HS-like fate. Underlying structural changes in these epithelia were broad changes in gene expression networks attributable to GATA4 directly activating or repressing expression of HS or FS defining transcripts. Our study implicates GATA4 as having a primary role in suppressing an esophageal/FS transcription factor network during HS development to promote columnar epithelium. Moreover, GATA4-dependent phenotypes in developmental mutants reflected changes in gene expression associated with Barrett's esophagus. CONCLUSIONS: This study demonstrates that GATA4 is necessary and sufficient to activate the development of simple columnar epithelium, rather than stratified squamous epithelium, in the embryonic stomach. Moreover, similarities between mutants and Barrett's esophagus suggest that developmental biology can provide insight into human disease mechanisms.

Leptin Distribution in Rat Foetal and Extraembryonic Tissues in Late Gestation: A Physiological View of Amniotic Fluid Leptin.

Prenatal leptin is key to regulating foetal growth and early metabolic programming. The presence of intact leptin in rat foetal (at late gestation) and neonatal (immediately after birth) stomach content and mucosa has been previously described, suggesting that it may act as a regulatory nutrient for the neonate rats, be internalised by the stomach, and play a physiological role early in life, which requires to be further investigated, including its origin. We aimed to study the ontogeny of the presence of leptin in the foetal stomach and key extraembryonic tissues in rats at late gestation (days 18-21). Leptin concentration was determined by enzyme-linked immunosorbent assay, and placental leptin immunolocalisation was analysed by immunohistochemistry. Leptin showed a sudden appearance in the amniotic fluid (AF) at day 20 of gestation, gastric content (swallowed AF), stomach, and umbilical cord, significantly increasing at day 21. Leptin levels in these fluids and tissues were positively correlated. In the placenta, leptin was detectable at all the studied days, but its localisation changed from widespread throughout the placenta at day 18 to well-defined in the labyrinth zone from day 19 onwards. The results support a possible internalisation of AF leptin by the immature stomach of near-term foetuses and suggest that changes in placental leptin localisation might help to explain the sudden appearance of leptin in AF at gestational day 20, with potential physiological significance regarding short-term feeding control and metabolic programming in the developing offspring.

Ontogeny of the digestive tract of Brycon amazonicus (Teleostei, Bryconidae) under culture conditions: from hatching to juvenile stage.

In the present study, the morphological development of the Brycon amazonicus digestive tract is described to provide basic knowledge for nutritional studies and, therefore, increase the survival of this species during larviculture. Samples were collected from hatching up to 25 days of age, measured, processed and observed under a stereomicroscope and light microscopy. Newly hatched larvae presented their digestive tract as a straight tube, dorsal to the yolk sac, lined with a single layer of undifferentiated cells. At 24 h post-hatching (hPH), the buccopharyngeal cavity was open, but the posterior region of the digestive tube remained closed. At 25 hPH, the digestive tube was completely open and could be divided into buccopharyngeal cavity, oesophagus and intestine. At 35 hPH, the intestine presented a dilatation in the proximal region, which had the function of storing food. Differentiation of the stomach started at 83 hPH, and mucous cells were observed in the epithelium. These cells are important in the production of mucus, whose function is to protect the organ against acidity, although the gastric glands began developing only from 171 hPH, when three stomach regions were observed: cardiac, fundic and pyloric. The gastric glands were observed in the cardiac region, indicating that this organ already had digestive functionality. From 243 hPH, the absorption and assimilation of nutrients were already possible but, only from 412 hPH, the digestive tract was completely developed and functional.

The morphological development of the proventriculus of Dandarawi chick: Light and electron microscopical studies.

This study was carried out on 40 chick embryos collected from incubated eggs of Dandarawi chicken (Gallus gallus domesticus) on the 5th to 19th incubation day (27 to 45 Hamburger and Hamilton, H&H stages). In addition, 15 chicks were collected on the day of hatching (stage 46 H&H), one week and two weeks post-hatching to demonstrate the histological, histochemical, and electron microscopic developmental changes of the proventriculus (of the digestive tract). Histologically, the proventriculus was observed as a narrow tube at 27 H&H stage. It was lined by pseudostratified columnar epithelium through 27-39 H&H stages and from the stage 43 till post-hatching, it was lined by simple columnar epithelium. The Lamina muscularis mucosa could be identified at stage 43. The proventricular glands were detected firstly at stage 31 and branching at stage 35. Histochemically, the surface epithelium and proventricular glands reacted positively to PAS, alcian blue and bromophenol blue from stage 31 till maturity. The glands displayed an apocrine mode of secretion at stage 39 and their cytoplasm contained abundant mitochondria, RER, secretory granules, and lipid droplets. Enteroendocrine cells could be observed among the glandular and surface epithelium at stage 45 H&H. The interstitial tissue contained fibroblasts and telocytes. The telocytes were firstly detected at stage 35 H&H and composed of a cell body and two long cell processes called telopodes. The tunica muscularis differentiated into three layers of smooth muscle fibers at stage 37 H&H. The cellular and stromal organizations of the proventriculus and their relations to the development and function were discussed.

Molecular ontogeny of the stomach in the catshark Scyliorhinus canicula.

The origin of extracellular digestion in metazoans was accompanied by structural and physiological alterations of the gut. These adaptations culminated in the differentiation of a novel digestive structure in jawed vertebrates, the stomach. Specific endoderm/mesenchyme signalling is required for stomach differentiation, involving the growth and transcription factors: 1) Shh and Bmp4, required for stomach outgrowth; 2) Barx1, Sfrps and Sox2, required for gastric epithelium development and 3) Cdx1 and Cdx2, involved in intestinal versus gastric identity. Thus, modulation of endoderm/mesenchyme signalling emerges as a plausible mechanism linked to the origin of the stomach. In order to gain insight into the ancient mechanisms capable of generating this structure in jawed vertebrates, we characterised the development of the gut in the catshark Scyliorhinus canicula. As chondrichthyans, these animals retained plesiomorphic features of jawed vertebrates, including a well-differentiated stomach. We identified a clear molecular regionalization of their embryonic gut, characterised by the expression of barx1 and sox2 in the prospective stomach region and expression of cdx1 and cdx2 in the prospective intestine. Furthermore, we show that gastric gland development occurs close to hatching, accompanied by the onset of gastric proton pump activity. Our findings favour a scenario in which the developmental mechanisms involved in the origin of the stomach were present in the common ancestor of chondrichthyans and osteichthyans.

Trop2 marks transient gastric fetal epithelium and adult regenerating cells after epithelial damage.

Mouse fetal intestinal progenitors lining the epithelium prior to villogenesis grow as spheroids when cultured ex vivo and express the transmembrane glycoprotein Trop2 as a marker. Here, we report the characterization of Trop2-expressing cells from fetal pre-glandular stomach, growing as immortal undifferentiated spheroids, and their relationship with gastric development and regeneration. Trop2(+) cells generating gastric spheroids differed from adult glandular Lgr5(+) stem cells, but appeared highly related to fetal intestinal spheroids. Although they shared a common spheroid signature, intestinal and gastric fetal spheroid-generating cells expressed organ-specific transcription factors and were committed to intestinal and glandular gastric differentiation, respectively. Trop2 expression was transient during glandular stomach development, being lost at the onset of gland formation, whereas it persisted in the squamous forestomach. Undetectable under homeostasis, Trop2 was strongly re-expressed in glands after acute Lgr5(+) stem cell ablation or following indomethacin-induced injury. These highly proliferative reactive adult Trop2(+) cells exhibited a transcriptome displaying similarity with that of gastric embryonic Trop2(+) cells, suggesting that epithelium regeneration in adult stomach glands involves the partial re-expression of a fetal genetic program.

The winding road to understanding the neonatal origins of inflammatory gastrointestinal disorders.

Beginning with the observation that birth weight correlates with increased risk of cardiovascular disease, the concept of neonatal programming, that the environmental influence on fetal and neonatal development results in modification of the risk profile for adult disease, has begun to emerge as an important component to understanding the origin of chronic diseases of many different organ systems. Until recently, the gastrointestinal system has not been considered. Our understanding of the pathogenesis of many intestinal inflammatory disorders is still incomplete; however, a brief review of what is known reveals several opportunities for the early intraluminal environment to affect the development of the intestinal immune system. Early clinical observations such as the increased risk of celiac disease observed in those born by cesarean section and the protective effect of breast-feeding against inflammatory bowel disease and celiac disease support the role of neonatal programming in the development of chronic inflammatory gastrointestinal disease. Additional, more robust clinical studies are needed to confirm this role. Furthermore, examination of the possible mechanisms of immune phenotype modification is necessary.

Expression pattern of the homeotic gene Bapx1 during early chick gastrointestinal tract development.

Regulation of the Bone Morphogenetic Protein (BMP) signaling pathway is essential for the normal development of vertebrate gastrointestinal (GI) tract, but also for the differentiation of the digestive mesenchymal layer into smooth muscles and submucosal layer. Different studies demonstrated that Bapx1 (for bagpipe homeobox homolog 1) negatively regulates the BMP pathway, but its precise expression pattern during the development and the differentiation of the GI tract mesenchyme actually remains to be examined. Here, we present the spatio-temporal expression profile of Bapx1 in the chick GI tract. We show that Bapx1 is first expressed in the undifferentiated mesenchyme of the gizzard and the colon. After the differentiation of the digestive mesenchyme, we found Bapx1 strongly expressed in the gizzard smooth muscle and in the submucosa layer of the colon. This expression pattern provides new insights into the roles of Bapx1 during the regionalization of the GI tract and the differentiation of the digestive mesenchyme of the colon and the stomach.

The distribution of gastrin, somatostatin and glucagon immunoreactive (IR) cells in ostrich stomach during the pre- and post-hatching period.

Peptides of the gastrointestinal tract play a significant role in the digestive processes and the development of the body; therefore, it is important to have an understanding of location and distribution of gastrin, somatostatin and glucagon immunoreactive (IR) cells in the stomach mucosa of growing birds. For this purpose, 6 embryos and 37 chicks from an ostrich farm in Latvia were used. Tissue samples were collected from the proventriculus - superficial and deep glandular region and from the ventriculus - side wall and pyloric region. The number of cells was determined in 10 mucosal fields of each tissue sample. For statistical analysis, the one-way anova method was used. Gastrin IR cells regarding the stomach mucosa were found only in the pyloric region. Somatostatin IR cells were most densely located in the pyloric region too, but some cells were also discovered in the mucosa of proventriculus and ventriculus. Glucagon IR cells were found in the epithelium of the deep glands of the proventriculus and only some cells of the superficial glands of the proventriculus, and the ventriculus side wall mucosa. Gastrin and somatostatin IR cells were present in a comparatively large quantity in the ostrich chicks' ventriculus - pyloric region yet not long before hatching. They were located deep in the mucosa of pyloric glands, and their number tended to increase with birds advancing in age.

[Morphological features of the esophagogastric transition zone at fetuses and newborns].

Morphological research of the esophagogastric transition mucosa at 35 fetuses and newborns was done. The esophagogastric transition was lined by high columnar epithelium and mucos glands. At fetuses of 22-24 week gestational age studied zone didn't have any glands. Histochemical features of the epithelium, particularly MUC5AC positive staining, corresponded to cardial type of the Barrett esophagus, defined at adults. We have revealed that mucosa of the esophagogastric transition has gastric origin and arises before birth. We found out the islets of columnar epithelium on the surface of the laminated pavement epithelium, indicated about its uneven development up to the birth. The sites of immature epithelium could be considered as transformation zones both of laminated pavement epithelium or columnar one.

Pregnancy-associated glycoprotein, chymosin and pepsinogen immunoreactivity of proteins extracted from fetal gastric tissue in bovine species.

The objective of this work was to investigate the expression of gastric aspartic proteinases in fundic and pyloric mucosa removed from bovine fetuses. For this purpose, fractions issued from classical biochemical protocols were analyzed by proteolytic method, by PAG-RIA and by Western blot with the use of antisera raised against both pepsinogens and PAG. A strong reaction of proteins extracted from the fundic mucosa collected at the beginning of pregnancy was revealed with both anti-bPAG-I and anti-bPAG-II antisera, suggesting the expression of pepsinogen F in bovine species. Concerning pyloric mucosa, the analysis by Western blot highlighted a very strong immunoreaction with the anti-bovine chymosin serum. Amino-terminal sequencing allowed to identify bovine fetuin and albumin in fundic extracts, chymosin in the pyloric mucosa extracts, as well as some unknown proteins in both mucosa. Despite no N-terminal microsequence corresponding to the hypothetical pepsinogen F could be identified, it cannot be excluded that an existing bovine pepsinogen F-like molecule could be degraded during the purification procedure or that co-purified proteins could be responsible for masking its N-terminal microsequence.

Ontogenesis of the omasum: a comparative analysis of the Merino sheep and Iberian red deer.

The aim of this study is to describe differences in the ontogenesis of the omasum in sheep (domestic ruminant) and deer (wild ruminant). A total of 50 embryos and fetuses of Merino sheep and 50 Iberian deer were used, from the first stages of prenatal life until birth. For the study, the animals were divided into five experimental groups according to the most relevant histological characteristics. The appearance of the omasum from the primitive gastric tube was earlier in sheep (22% gestation, 33 days) than in deer (25% gestation, 66 days). In both cases it displayed a primitive epithelium of a stratified, cylindrical, non-ciliary type. The appearance of four laminae of different sizes was always earlier in sheep than deer. At around 36% gestation in sheep (53 days) and 36% (97 days) in deer, the omasum consisted of 4 clearly-differentiated layers: mucosa (with epithelial layer and lamina propria), submucosa, tunica muscularis and serosa. The temporal order of appearance of the four order laminae and omasal papillae was always earlier in sheep than deer. The tegumentary mucosa of the omasum was without secretion capability in the first embryonic phases. From 67 days (26% gestation) the neutral mucopolysaccharides appeared in deer and at 46 days (30% gestation) in sheep. In both cases they continued to decrease until birth, this decrease being more pronounced in deer. Finally, the presence of neuroendocrine and glial cells was detected in deer at earlier stages than in sheep.

Epithelial BMP signaling is required for proper specification of epithelial cell lineages and gastric endocrine cells.

Bone morphogenetic protein (BMP) signaling within the gastrointestinal tract is complex. BMP ligands and their receptors are expressed in both epithelial and mesenchymal compartments, suggesting bidirectional signaling between these two entities. Despite an increasing interest in BMP signaling in gut physiology and pathologies, the distinct contribution of BMP signaling in the epithelium vs. the mesenchyme in gastrointestinal homeostasis remains to be established. We aimed to investigate the role of epithelial BMP signaling in gastric organogenesis, gland morphogenesis, and maintenance of epithelial cell functions. Using the Cre/loxP system, we generated a mouse model with an early deletion during development of BMP receptor 1A (Bmpr1a) exclusively in the foregut endoderm. Bmpr1a(ΔGEC) mice showed no severe abnormalities in gastric organogenesis, gland epithelial proliferation, or morphogenesis, suggesting only a minor role for epithelial BMP signaling in these processes. However, early loss of BMP signaling in foregut endoderm did impact on gastric patterning, leading to an anteriorization of the stomach. In addition, numbers of parietal cells were reduced in Bmpr1a(ΔGEC) mice. Epithelial BMP deletion significantly increased the numbers of chromogranin A-, ghrelin-, somatostatin-, gastrin-, and serotonin-expressing gastric endocrine cells. Cancer never developed in young adult (<100 days) Bmpr1a-inactivated mice although a marker of spasmolytic polypeptide-expressing metaplasia was upregulated. Using this model, we have uncovered that BMP signaling negatively regulates the proliferation and commitment of endocrine precursor cells. Our data also indicate that loss of BMP signaling in epithelial gastric cells alone is not sufficient to induce gastric neoplasia.

Notch signaling in stomach epithelial stem cell homeostasis.

The mammalian adult gastric epithelium self-renews continually through the activity of stem cells located in the isthmus of individual gland units. Mechanisms facilitating stomach stem and progenitor cell homeostasis are unknown. Here, we show that Notch signaling occurs in the mouse stomach epithelium during development and becomes restricted mainly to the isthmus in adult glands, akin to its known localization in the proliferative compartment of intestinal villi. Using genetic and chemical inhibition, we demonstrate that Notch signaling is required to maintain the gastric stem cell compartment. Activation of Notch signaling in lineage-committed stomach epithelial cells is sufficient to induce dedifferentiation into stem and/or multipotential progenitors that populate the mucosa with all major cell types. Prolonged Notch activation within dedifferentiated parietal cells eventually enhances cell proliferation and induces adenomas that show focal Wnt signaling. In contrast, Notch activation within native antral stomach stem cells does not affect cell proliferation. These results establish a role for Notch activity in the foregut and highlight the importance of cellular context in gastric tumorigenesis.

Controversies of cardiac glands in the proximal stomach: a critical review.

Cardiac glands (CG), along with oxyntocardiac glands, in a normal human constitute cardiac mucosa (CM) that is positioned in the proximal stomach with a length of 10-30 mm, according to traditional teaching. This doctrine has been recently challenged. On the basis of studies on autopsy and biopsy materials in the esophagogastric junction region, some investigators have reported the presence of CG in only 50% of the general US population. They believed that CG were an acquired, metaplastic lesion as a result of gastroesophageal reflux disease. Subsequent recent study results from other research groups showed the presence of CG in the proximal stomach in embryos, fetuses, pediatric, and adult patients in most Europeans and Americans, and almost all Japanese and Chinese patients. These new data showed the following important findings: (i) CG are confirmed to be congenital in the proximal stomach; (ii) the length of CM is much shorter, approximately 5 mm in Caucasians in Europe and North America, and approximately 13 mm in Japanese and probably also in Chinese; (iii) CG are also present in the distal superficial esophagus underneath squamous mucosa in almost all Japanese and Chinese patients, but not so common in Caucasians in Europe, and not clear in Caucasians in North America. The recent data indicate a clear difference in the distribution of CG in the proximal stomach among different ethnic populations, and might explain different disease pathogenesis mechanisms among various ethnic patient groups.

The gastric mucosa development and differentiation.

The development and differentiation of the gastric mucosa are controlled by a complex interplay of signaling proteins and transcriptional regulators. This process is complicated by the fact that the stomach is derived from two germ layers, the endoderm and the mesoderm, with the first giving rise to the mature epithelium and the latter contributing the smooth muscle required for peristalsis. Reciprocal epithelial-mesenchymal interactions dictate the formation of the stomach during fetal development, and also contribute to its continuous regeneration and differentiation throughout adult life. In this chapter, we discuss the discoveries that have been made in different model systems, from zebrafish to human, which show that the Hedgehog, Wnt, Notch, bone morphogenetic protein, and fibroblast growth factor (FGF) signaling systems play essential roles during various stages of stomach development.

Gastric mucosal lymphoid follicles: histology, distribution, frequency, and etiologic features.

Mucosal lymphoid follicles reportedly do not occur in normal gastric mucosa or are rare. Detection of many primary and occasional secondary lymphoid follicles in the otherwise normal mucosa of a patient with multiple gastric sarcomas prompted study of the lesions of 84 patients--30 males and 54 females--and 2 fetuses. The conditions in the group were intramural tumors (27 cases), intramucosal lesions (16 cases), and putatively normal mucosa (43 cases). Overall, 229 sections totaling 567 cm of mucosa were examined and the lymphoid follicles counted. Follicles were found in 174 sections of 76 patients (88%) and in all categories examined except the fetuses. There were 773 follicles in fundal mucosa and 43 in antral mucosa (average, 1 follicle/7 mm of fundal mucosa and 1 follicle/21 mm of antral mucosa). The follicles were most frequent in the patients with multiple stromal sarcomas (1 follicle/8 mm of mucosa) and least frequent in pediatric patients.

Different thymosin Beta 4 immunoreactivity in foetal and adult gastrointestinal tract.

BACKGROUND: Thymosin beta 4 (Tbeta(4)) is a member of beta-thymosins, a family of peptides that play essential roles in many cellular functions. A recent study from our group suggested a role for Tbeta(4) in the development of human salivary glands. The aim of this study was to analyze the expression of Tbeta(4) in the human gut during development, and in the adult. METHODOLOGY/PRINCIPAL FINDINGS: Immunolocalization of Tbeta(4) was studied in autoptic samples of tongue, oesophagus, stomach, ileum, colon, liver and pancreas obtained from two human foetuses and two adults. Tbeta(4) appeared unevenly distributed, with marked differences between foetuses and adults. In the stomach, superficial epithelium was positive in foetuses and negative in adults. Ileal enterocytes were strongly positive in the adult and weakly positive in the foetuses. An increase in reactivity for Tbeta(4) was observed in superficial colon epithelium of adults as compared with the foetuses. Striking differences were found between foetal and adult liver: the former showed a very low reactivity for Tbeta(4) while in the adult we observed a strong reactivity in the vast majority of the hepatocytes. A peculiar pattern was found in the pancreas, with the strongest reactivity observed in foetal and adult islet cells. SIGNIFICANCE: Our data show a strong expression of Tbeta(4) in the human gut and in endocrine pancreas during development. The observed differential expression of Tbeta(4) suggests specific roles of the peptide in the gut of foetuses and adults. The observed heterogeneity of Tbeta(4) expression in the foetal life, ranging from a very rare detection in liver cells up to a diffuse reactivity in endocrine pancreas, should be taken into account when the role of Tbeta(4) in the development of human embryo is assessed. Future studies are needed to shed light on the link between Tbeta(4) and organogenesis.

Six2 activity is required for the formation of the mammalian pyloric sphincter.

The functional activity of Six2, a member of the so/Six family of homeodomain-containing transcription factors, is required during mammalian kidney organogenesis. We have now determined that Six2 activity is also necessary for the formation of the pyloric sphincter, the functional gate at the stomach-duodenum junction that inhibits duodenogastric reflux. Our data reveal that several genes known to be important for pyloric sphincter formation in the chick (e.g., Bmp4, Bmpr1b, Nkx2.5, Sox9, and Gremlin) also appear to be required for the formation of this structure in mammals. Thus, we propose that Six2 activity regulates this gene network during the genesis of the pyloric sphincter in the mouse.

Impairment of gastric acid secretion and increase of embryonic lethality in Foxq1-deficient mice.

The mouse Foxq1 gene, also known as Hfh1, encodes a winged helix/forkhead transcription factor. In adult mice, Foxq1 is highly expressed in kidney and stomach. Here, we report that Foxq1 is expressed during prenatal and postnatal stomach development and the transcripts are restricted to acid secreting parietal cells. Mice homozygous for a deletion of the Foxq1 locus on a 129/Sv x C57BL/6J hybrid genetic background display variable phenotypes consistent with requirement of the gene during embryogenesis. Approximately 50% of Foxq1-/- embryos die in utero. Surviving homozygous mutants are normal and fertile, and have a silky shiny coat. Although the parietal cell development is not affected in the absence of Foxq1, there is a lack of gastric acid secretion in response to various secretagogue stimuli. Ultrastructural analysis suggests that the gastric acid secretion defect in Foxq1-deficient mice might be due to impairment in the fusion of cytoplasmic tubulovesicles to the apical membrane of secretory canaliculi.