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.

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.

[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.

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.

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.

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.

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.

The stomach mesenchymal transcription factor Barx1 specifies gastric epithelial identity through inhibition of transient Wnt signaling.

Inductive interactions between gut endoderm and the underlying mesenchyme pattern the developing digestive tract into regions with specific morphology and functions. The molecular mechanisms behind these interactions are largely unknown. Expression of the conserved homeobox gene Barx1 is restricted to the stomach mesenchyme during gut organogenesis. Using recombinant tissue cultures, we show that Barx1 loss in the mesenchyme prevents stomach epithelial differentiation of overlying endoderm and induces intestine-specific genes instead. Additionally, Barx1 null mouse embryos show visceral homeosis, with intestinal gene expression within a highly disorganized gastric epithelium. Barx1 directs mesenchymal cell expression of two secreted Wnt antagonists, sFRP1 and sFRP2, and these factors are sufficient replacements for Barx1 function. Canonical Wnt signaling is prominent in the prospective gastric endoderm prior to epithelial differentiation, and its inhibition by Barx1-dependent signaling permits development of stomach-specific epithelium. These results define a transcriptional and signaling pathway of inductive cell interactions in vertebrate organogenesis.

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.

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.

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.

The ontogeny and developmental physiology of gastric acid secretion.

The production of acid by the stomach is a tightly controlled physiological process that involves neural and hormonal mechanisms and the input of several epithelial cell types. The past several years have seen significant advances in our understanding of the molecular ontogenesis of the stomach and the factors controlling stomach innervation, as well as the differentiation of gastric epithelial cell lineages and their respective hormones/factors that influence acid production. The programmed development of each of these elements is exquisitely regulated and allows human neonates to produce gastric acid; it also helps us define expectations of acid production in preterm infants at all gestational ages.

The expression of hepatocyte nuclear factor-4alpha, a developmental regulator of visceral endoderm, correlates with the intestinal phenotype of gastric adenocarcinomas.

AIMS: Hepatocyte nuclear factor (HNF)-4alpha is a developmental regulator of the visceral endoderm, which is expressed in the embryonic gut and later in the adult intestine and colon. However, adult gastric mucosa does not express HNF-4alpha. We investigated the possible involvement of HNF-4alpha in intestinal metaplasia and the intestinalisation of gastric adenocarcinomas. METHODS: Thirty-five cases of adenocarcinomas and 46 cases of adjacent non-neoplastic mucosa with (22 lesions) or without (24 lesions) intestinal metaplasia were immunostained for HNF-4alpha. The gastric or the intestinal phenotype was also examined using immunohistochemistry for MUC5AC, MUC2, CD10, and gastric-type mucin (GTM). Adenocarcinomas were classified into the gastric type (G-type, 42.9%), the mixed gastric and intestinal type (GI-type, 31.4%), and the intestinal type (I-type, 25.7%). RESULTS: The HNF-4alpha expression was exclusively seen in glandular cells with intestinal metaplasia, which was correlated with MUC2 expression (p<0.05) and inversely correlated with MUC5AC expression (p<0.05). All adenocarcinomas more or less expressed HNF-4alpha, with an intense expression being seen in the I-type (p<0.01) and in well-differentiated adenocarcinomas (p<0.03). CONCLUSIONS: HNF-4alpha expression is associated with the intestinal phenotype of non-neoplastic and neoplastic gastric glandular cells, suggesting a possible involvement in the establishment and/or maintenance of the intestinal phenotype of the gastric mucosa and adenocarcinomas.

Genes preferentially expressed in embryo stomach are predominantly expressed in gastric cancer.

Eight complementary DNA (cDNA) clones highly expressed in fetal rat stomach but not in normal adult rat stomach were isolated after screening 2 x 10(4) independent recombinants from a subtracted cDNA library. The cDNA library was first prepared from RNAs of total stomach at 16 days gestational period, and this cDNA library was subtracted by cDNAs prepared from adult rat total stomach RNA, using a novel PCR-based cDNA subtraction method. Northern blot analysis revealed that as many as six of eight clones thus isolated were overexpressed in at least some of the human or rat gastric cancers. From analysis of partial nucleotide sequence, four cDNA clones were identified as profilin, pro-alpha 1 (1) collagen, nucleolar protein B23.2, and elongation factor 1 alpha subunit. The remaining two clones were derived from novel genes. These novel genes, L-1 and L-2, are developmentally well regulated in the stomach. The present results clearly show that genes expressed preferentially in embryo stomach are most likely to be highly expressed in gastric cancer. The method described here provides us with a rapid method for identification of genes with significantly increased expression in cancer.

Demonstration of glucagon in the stomach of human fetuses.

Electron microscopy using antiglucagon or antiglicentin antisera and the protein A-gold (pAg) technique revealed a population of immunoreactive cells in the gastric mucosa of human fetuses. The secretory granules of these cells showed the same ultrastructural characteristics and the same labeling pattern as pancreatic alpha-granules. These data demonstrate that the stomach of human fetuses contains cells with secretory granules indistinguishable by morphologic and immunocytochemical criteria from the pancreatic A-cells.

On the origin of cardiac mucosa: a histological and immunohistochemical study of cytokeratin expression patterns in the developing esophagogastric junction region and stomach.

AIM: To examine the fetal and neonatal esophagogastric junction region (EGJ) histologically for the presence of an equivalent to adult cardiac mucosa (CM); to study the expression patterns of all cytokeratins (CK) relevant to the EGJ during gestation; to compare the CK profile of the gestational and the adult EGJ; and to determine the degree of development in the adult EGJ histology and CK profile during gestation. METHODS: Forty-eight fetal autopsy specimens of the EGJ were step-sectioned and stained with hematoxylin and eosin (H and E) to select sections showing the mucosal lining. Immunohistochemistry for CK5, 7, 8, 13, 18, 19, and 20 was performed. Antibody staining was then graded for location, intensity, and degree. RESULTS: The distal esophagus was lined by simple columnar epithelium from 12-wk gestational age (GA). The proximal part of this segment consisted of mucus-producing epithelium, devoid of parietal cells. CK5 and 13 were present exclusively in multilayered epithelia and CK8, 18, and 19 predominantly in simple columnar epithelium. There were no differences in the frequencies of the co-ordinate CK7+/20+ and the CK7-/20- immunophenotypes between different locations. The prevalence of the CK7+/20- immunophenotype decreased, and that of the CK7-/20+ immunophenotype increased significantly from the distal esophagus to the distal stomach. CONCLUSION: Fetal columnar-lined lower esophagus (fetal CLE) may be the equivalent and precursor of the short segments of columnar epithelium found in the distal esophagus of some normal adult subjects. Esophageal simple columnar epithelium without parietal cells (ESN) may be the precursor of adult CM. The similarities between the fetal and adult EGJ and stomach CK expression patterns support the conclusion that adult CM has an identifiable precursor in the fetus. This would then indicate that at least a part of the adult CM has a congenital origin.

The role of capsulin in the morphogenesis and differentiation of fetal rat gastric mucosa.

The signals that guide the morphogenesis and differentiation of rat fetal gastric mucosa remain largely unknown. We have investigated the role of capsulin in pit/gland formation and epithelial cell differentiation in cultured stomach tissue. Embryonic day 16.5 (E 16.5) stomach tissue cultured for three days in the presence of 1 microM hydrocortisone underwent dramatic transformation, from undifferentiated, stratified cells to differentiated epithelia composed of polarised columnar cells with mucous cells and pit/glands. In the presence of capsulin antisense oligonucleotides directed against capsulin mRNA, tissues do not undergo further development. Significantly, both mucous granules and pit/gland formation were inhibited compared to capsulin sense/scrambled oligonucleotide treated controls. However, in tissues treated with specific anti-rat HGF-antiserum to neutralise secreted HGF, pit/gland formation was inhibited, but the number of mucous granules remained unchanged compared to controls treated with non-specific antiserum (mouse monoclonal cytokeratin 8 antiserum). This data suggests that capsulin may have a role in the morphogenesis of pit/glands and mucin granule formation in the developing rat gastric mucosa. We discuss the possibility that this role of capsulin may be partly mediated through the actions of HGF.

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.