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.

A bacterial filter protects and structures the gut microbiome of an insect.

Associations with symbionts within the gut lumen of hosts are particularly prone to disruption due to the constant influx of ingested food and non-symbiotic microbes, yet we know little about how partner fidelity is maintained. Here we describe for the first time the existence of a gut morphological filter capable of protecting an animal gut microbiome from disruption. The proventriculus, a valve located between the crop and midgut of insects, functions as a micro-pore filter in the Sonoran Desert turtle ant (Cephalotes rohweri), blocking the entry of bacteria and particles ⩾0.2 µm into the midgut and hindgut while allowing passage of dissolved nutrients. Initial establishment of symbiotic gut bacteria occurs within the first few hours after pupation via oral-rectal trophallaxis, before the proventricular filter develops. Cephalotes ants are remarkable for having maintained a consistent core gut microbiome over evolutionary time and this partner fidelity is likely enabled by the proventricular filtering mechanism. In addition, the structure and function of the cephalotine proventriculus offers a new perspective on organismal resistance to pathogenic microbes, structuring of gut microbial communities, and development and maintenance of host-microbe fidelity both during the animal life cycle and over evolutionary time.

Microstructure Features of Proventriculus and Ultrastructure of the Gastric Gland Cells in Chinese Taihe Black-bone Silky Fowl (Gallus gallus domesticus Brisson).

To investigate microstructure of proventriculus and ultrastructure of the gastric gland cells from Chinese Taihe black-bone silky fowl (BSF), the proventriculus of 4-week-old BSF was sampled. Conventional histological and transmission electron microscope (TEM) methods were used in this study. The wall of the Taihe BSF proventriculus was consisted of four layers, the mucous, submucosa, muscularis externa and the serosa as others birds. The muscularis externa of the birds' proventriculus contained three layers. Much of the melanin was present in loose connective tissue of lamina propria, submucosa, and muscularis externa unlike others. In addition, the ultrastructure of the gastric gland cells was observed by TEM. There was only one kind of gland cell, for example oxynticopeptic cell in proventriculus of Taihe BSF. The oxynticopeptic cells contained numerous mitochondria, cisternae of rough endoplasmic reticulum (CRER), intracellular canaliculi (IC) that secrete hydrochloric acid and small amounts of pepsinogen granules. The rough endoplasmic reticulum (RER) was irregular cisternae with ribosomes and surrounded tightly the mitochondria along their configuration. The electron-dense pepsinogen granules were round with various sizes. The neighbouring oxynticopeptic cells were closed up with tight junction and gap junction. The inter-space between the neighbouring oxynticopeptic cells was stenosis or was filled with electron-dense extracellular substance. In conclusion, the gastric gland cells of Chinese Taihe BSF proventriculus were only oxynticopeptic cells that secrete hydrochloric acid and pepsinogen, but no parietal cells and chief cells of mammal. The gastric gland cells of proventriculus were underdeveloped compared with those of mammals.

The Drosophila larva as a tool to study gut-associated macrophages: PI3K regulates a discrete hemocyte population at the proventriculus.

Immune cells not only patrol the body in the circulation but also importantly, associate with specific tissues, such as the intestinal epithelium. The complex interactions between immune cells and their target tissues are difficult to study and simple, genetically tractable models are lacking. Here, we present the first thorough characterization of gut-associated macrophages in Drosophila larvae. We analyze their gene expression, morphology, development and lineage and importantly, demonstrate that they are functional (phagocytic) macrophages. We test their regulation by phosphoinositide 3-kinase (PI3K) and show evidence that this pathway regulates the population size of gut hemocytes and their phagocytic activity, reminiscent of recent findings in mammalian colitis models. Our data suggest that PI3K signaling modifies the adhesive properties of hemocytes, a possible mechanism for gut-hemocyte regulation. These results demonstrate the potential of the Drosophila larva as a simple tool to uncover mechanisms regulating recruitment and maintenance of innate immune cells at their target tissues.

Ontogeny of ghrelin mRNA expression and identification of ghrelin-immunopositive cells in the gastrointestinal tract of the Peking duck, Anas platyrhynchos.

Ghrelin is an acylated peptide and an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), and stimulates growth hormone release and food intake in mammals. Peking duck is a very fast growing species of poultry. Although the sequence and structure of ghrelin have recently been determined, the expression of ghrelin in Peking duck has not been studied. Here, we investigated the tissue expression and distribution of ghrelin by RT-PCR and immunohistochemistry, respectively, in Peking duck at different stages of development. Ghrelin mRNA expression was mainly detected in the proventriculus and proventriculus-gizzard junction. It was first expressed, but weakly, on embryonic day 14 (E14); the expression increased by embryonic day 21 (E21), and was maintained at high levels between post-hatching-day 1 (P1) and post-hatching-day 60 (P60). Weak expression of ghrelin mRNA was also found in the gizzard and duodenum. In the gastrointestinal tract of growing Peking duck in P60, the largest number of ghrelin-ip cells was detected in the epithelium of the compound tubular glands in the proventriculus and the next largest number was in the proventriculus-gizzard junction. Very few ghrelin-ip cells were located in the epithelium of the simple tubular glands adjacent to the gizzard. No ghrelin-ip cells were observed elsewhere in the gastrointestinal tract. Ghrelin-ip cells were found in embryos as early as day E21; at the same time, the compound tubular glands in the proventriculus had formed. The numbers of ghrelin-ip cells on P1 were similar to those of E21 embryos. However, on P60, high numbers of strongly stained ghrelin-ip cells were found to be scattered in the epithelium of the compound tubular glands in the proventriculus. The density of ghrelin-ip cells (cells/mm(2)) in the proventriculus on P60 was significantly greater than those of P1 and E21 embryos. These results demonstrate that ghrelin is expressed in the Peking duck gastrointestinal tract, especially in the proventriculus, from mid-late-stage embryos to growing period and suggested an involvement of ghrelin in the development and biology of the gastrointestinal tract of the Peking duck.

Proventriculus of Cephalotes ants: a structural and comparative analysis.

The use of optic microscopy and scanning electron microscopy propitiated the comparative examination of the structure of the proventriculus bulb of Cephalotes atratus, Cephalotes clypeatus and Cephalotes pusillus. This portion of the digestive tract possesses highly sclerotized projections which act in the selection of victuals. This structure is of importance to phylogeny studies and thus was compared with others known species of ants in a dendrogram adapted from Eisner (1957). We did not detect differences among the three studied species in the nature of histochemistry and ultra morphology.

Correlation between Musashi-1 and c-hairy-1 expression and cell proliferation activity in the developing intestine and stomach of both chicken and mouse.

Musashi-1 (Msi-1) is an RNA-binding protein that plays key roles in the maintenance of neural stem cell states and in their differentiation into neural cells. Msi-1 has also been proposed as a candidate marker gene of mammalian intestinal stem cells and their immediate lineages. In this study, we examined Msi-1 expression in the small intestine and the stomach of both chicken and mouse during embryonic, fetal and postnatal development. In addition, we analyzed the expression of c-hairy-1, a chicken homologue of mouse Hes1, and assessed the proliferative activity of the cells expressing both of these factors. Significantly, during the development of these digestive organs in both species Msi-1 expression showed dynamic changes, suggesting that it is important for digestive organ development, particularly for epithelial differentiation. Based on our observations of the expression patterns of Msi-1 and c-hairy-1 in the adult small intestine, we speculate that Msi-1 is also a stem cell marker of the chicken small intestinal epithelium.

Analysis of transcription regulatory regions of embryonic chicken pepsinogen (ECPg) gene.

Genes encoding pepsinogens, zymogens of digestive enzyme pepsins, are expressed specifically in the gland epithelial cells of the vertebrate stomach, and their expression is also developmentally regulated, therefore providing a good model for the analysis of transcriptional regulation of genes. In the development of chicken embryonic stomach, the epithelium invaginates into the mesenchyme and forms glands and gland epithelial cells then begin to express embryonic chicken pepsinogen (ECPg) gene. It has been shown that cGATA5 binds directly GATA binding sites located within 1.1-kbp upstream of ECPg gene and activates its transcription. To find more precisely the sequences necessary for ECPg gene transcription, we carried out deletion and mutation analysis with 1.1-kbp upstream region. The results suggest that binding of GATA factor to three GATA binding sites within the upstream region -656 to -419 synergistically regulates ECPg expression in the gland epithelial cells.

Existence of ghrelin-immunopositive and -expressing cells in the proventriculus of the hatching and adult chicken.

Ghrelin was isolated from the rat stomach as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) and has been found in the gastrointestinal tract of many vertebrates. Although the sequence and structure of chicken ghrelin has recently been determined, morphological characteristics of ghrelin cells in the chicken gastrointestinal tract are still obscure. In this study, we investigated ghrelin expression and distribution of ghrelin-producing cells in the hatching and adult chicken gastrointestinal tract by RT-PCR, immunohistochemistry and in situ hybridization. Ghrelin mRNA expression was observed mainly in the proventriculus in the hatching chicken and in the proventriculus, pylorus and duodenum of the adult chicken by RT-PCR. Ghrelin-immunopositive (ghrelin-ip) cells in the proventriculus were located at the mucosal layer but not in the myenteric plexus or smooth muscle layer. The number of ghrelin-ip cells in the adult chicken was greater than that in the hatching chicken. Interestingly, in the adult chicken, the number of ghrelin-ip cells were almost the same as that of ghrelin mRNA-expressing (ghrelin-ex) cells; however, in the hatching chicken, the number of ghrelin-ex cells was greater than that of ghrelin-ip cells. These results clearly demonstrate that ghrelin-producing cells exist in the chicken gastrointestinal tract, especially in the proventriculus, from hatching to adult stages of development, as well as in mammals.

Application of efficient and specific gene transfer systems and organ culture techniques for the elucidation of mechanisms of epithelial-mesenchymal interaction in the developing gut.

Epithelial-mesenchymal interactions are very important in the development of the vertebrate gut. In the avian embryonic stomach (proventriculus), expression of embryonic chick pepsinogen (ECPg) gene, which is specific to developing glandular cells in stomach epithelium, is regulated by mesenchymal influence. Molecular mechanisms of tissue-specific transcriptional regulation of the ECPg gene and the molecular nature of the mesenchymal signals were analyzed using a combination of the classic organ culture system and gene transfer strategies. In the present review, three methods for the introduction of DNA into tissues are described: lipofection, electroporation and retroviral infection, and characteristics of each system are discussed.

Distribution and developmental change of lymphoid tissues in the chicken proventriculus.

In the chicken proventricular mucosa, aggregations of lymphocytes were localized in three different sites of the lamina propria, namely, underneath the surface epithelium, near the duct orifice of the deep proventricular gland, and in the gland tissue itself. In the lymphoid masses underneath the surface epithelium and in those near the duct orifice, CD4+ T lymphocytes and TCR2+ T lymphocytes occupied their central part, and B lymphocytes were localized in the periphery. CD8+ T lymphocytes and TCR1+ lymphocytes were evenly distributed in the masses. Infiltration of lymphocytes into these sites was first observed on the 20th embryonic day. At 1 week after hatching, CD3+ lymphocytes began to occupy the central area of the masses and His-C1+ B lymphocytes tended to be located in the periphery. Ultrastructurally, M cells were found neither in the epithelium of the mucosa nor in that of the excretory duct close to the lymphoid masses. In the deep proventricular gland, the lymphoid masses had a germinal center consisting of B lymphocytes, surrounded by the T lymphocyte-rich periphery. These masses were first recognized at the 3rd post-hatching week, presumably being formed against possible antigens invading into the lumen of the proventricular gland. On the other hand, the lymphoid masses beneath the surface epithelium and those near the duct orifice existing before the hatching period were considered to be prepared to establish the local mucosal immune barriers against the expectant antigenic invasion.

BMPs are necessary for stomach gland formation in the chicken embryo: a study using virally induced BMP-2 and Noggin expression.

Epithelial-mesenchymal interactions are necessary for the normal development of various digestive organs. In chicken proventriculus (glandular stomach), morphogenesis and differentiation of the epithelium depend upon the inductive signals coming from underlying mesenchyme. However, the nature of such signals is still unclear despite extensive analyses carried out using experimental tissue recombinations. In this study we have examined the possible involvement of bone morphogenetic proteins (BMPs) in the formation of stomach glands in the chicken embryo. Analysis of the expression patterns of BMP-2, -4 and -7 showed that these BMPs were present in the proventricular mesenchyme prior to the initiation of the proventricular gland formation. BMP-2 expression, in particular, was restricted to the proventriculus among anterior digestive organs. Virus-mediated BMP-2 overexpression resulted in an increase in the number of glands formed. Moreover, ectopic expression of Noggin, which antagonizes the effect of BMPs, in the proventricular mesenchyme or epithelium, led to the complete inhibition of gland formation, indicating that BMP signals are necessary for the proventricular gland formation. These findings suggest that BMPs are of prime importance as mesenchymal signals for inducing proventricular glands.

Drosophila endoderm development requires a novel homeobox gene which is a target of Wingless and Dpp signalling.

We have identified and cloned a novel type of homeobox gene that is composed of two homeodomains and is expressed in the Drosophila endoderm. Mutant analysis reveals that its activity is required at the foregut/midgut boundary for the development of the proventriculus. This organ regulates food passage from the foregut into the midgut and forms by the infolding of ectoderm and endoderm-derived tissues. The endodermal outer wall structure of the proventriculus is collapsed in the mutants leading to a failure of the ectodermal part to invaginate and build a functional multilayered organ. Lack-of-function and gain-of-function experiments show that the expression of this homeobox gene in the proventriculus endoderm is induced in response to Wingless activity emanating from the ectoderm/endoderm boundary whereas its expression in the central midgut is controlled by Dpp and Wingless signalling emanating from the overlying visceral mesoderm.

Histochemical characterization of the mucins of the epithelial cells in the chick embryo proventriculus.

The histochemical characteristics of the epithelial mucins, which are produced by the lining epithelium, the superficial glands and the compound glands of the proventriculus were investigated in the chick embryo from the 7th day of incubation to hatching. Results showed that the time of appearance of the carboxylated, sulphated and neutral mucosubstances differed at the three investigated sites. From the 13th to the 19th day of incubation, intense production of the various mucins was detected at the three sites. However, the modality of intracytoplasmatic storage and the production of mucins in both the cells of the lining epithelium and the superficial glands appeared to differ from those observed in the cells of the compound glands. At the 20th and 21st day, the mucins were primarily produced by the lining epithelium and the superficial glands, whereas production diminished in the compound glands. Furthermore, from the 7th to the 17th day of incubation, the cells of the principal ducts of the compound glands showed the same histochemical characteristics and modality of production as those of the glandular cells of the compound glands. On the other hand, from the 18th day to hatching, these cells assumed the same characteristics as those of the lining epithelium and of the superficial glands.

Ultraestructure of oxynticopeptic cell of the deep proventricular gland in the red-capped cardinal: paroaria gularis gularis, Linnaeus 1766

Las células oxinticopépticas de la glándula proventricular profunda en el cardenal rojo (paroaria gularis gularis) fueron estudiadas por medio de la microscopía electrónica de transmisión. Estas células tienen la estructura fina típica de una cimogénica (proteína) y célula hidroclórica ácido/secretora (parecido a esos hallazgos se han encontrado en las células parietales y principales de mamíferos y en las células oxínticas de anfíbios): la presencia de numerosas mitocondrias, desarrollando retículo endoplásmico liso y granuloso y gránulos de secreción

Complex carbohydrate presence in the gut and Malpighian tubules of Drosophila auraria larvae (Insecta, Diptera): a cytochemical study.

Cytochemical techniques were used for the qualitative characterization of carbohydrates in the foregut, midgut, hindgut and Malpighian tubules of Drosophila auraria larvae. Periodate-reactive (neutral) polysaccharides were present in the foregut cuticle, peritrophic membrane, cells of the proventriculus which produce the peritrophic membrane, as well as the glycocalyx of the midgut and Malpighian tubule cells. Sulphated and carboxylated complex carbohydrates were detected in the cuticle of both foregut and hindgut, as well as the 'vacuoles with granules' in the Malpighian tubules. Periodate-reactive sulphated and carboxylated polysaccharides were detected in dense bodies appearing in all cells 10-20 h before pupation of larvae. The significance of the presence or absence of complex carbohydrates in D. auraria larval digestive system is discussed and compared with results found in the digestive system of other insects. In addition, the efficiency of the HID-TCH-SP and LID-TCH-SP techniques in staining lysosomes is discussed.

Immunocytochemical and ultrastructural characterization of endocrine cells in chicken proventriculus.

The endocrine cells of the chicken proventriculus were investigated immunocytochemically, using the peroxidase-antiperoxidase technique on paraffin and semithin sections for light microscopy, and immunogold staining in osmium-fixed material for electron microscopy. The fixation procedure also allowed a detailed ultrastructural investigation. Twenty-three antisera were tested and 7 immunoreactive cell-types were identified: D-cells containing somatostatin-like peptide; EG-cells immunoreactive to anti-glucagon, anti-GLP1 and anti-neurotensin; NT-cells labelled only with anti-neurotensin; BN-cells containing bombesin-like material; ENK-cells showing met-enkephalin immunoreactivity; EC-cells reactive to anti-serotonin; and APP-cells positive to anti-avian pancreatic polypeptide. In addition, enterochromaffin-like (ECL) cells, were also detected by electron microscopy. The presence of ENK-cells and the ultrastructure of these and NT-cells are described for the first time in chicken proventriculus, and glucagon. GLP1 and neurotensin are shown to be colocalized in the EG-cells.

Mast cells in the chick digestive tract. I. Development.

Mast cells in the digestive tract of the developing chick embryo were studied with histochemistry and electron microscopy (EM). The cells appeared in the esophagus, proventriculus and small intestine on about the 13th day of incubation, whereas mast cells in the tongue appeared earlier. The staining properties and ultrastructure of the mast cells varied with development. In 13- and 15-day embryos, mast cells showed a pale metachromasia with toluidine blue, and stained blue with Alcian Blue-Safranin O (AB-S). In the 18-day embryo, mast cells stained a deep purple with toluidine blue. Stained with AB-S, most of the mast cell granules stained blue, but some red granules were also seen in a few cells. In the newly hatched chick, the cells stained a strong reddish purple with toluidine blue. Stained with AB-S, a few cells contained only blue or red granules, but most contained both. Observations with the EM revealed that the internal structure of the granules varied with the stage of embryonic development. The basis for the changes in staining properties and ultrastructure of the mast cell in the developing chick embryo were discussed.

Gizzerosine raises the intracellular cyclic adenosine-3',5'-monophosphate level in isolated chicken proventriculus.

Gizzerosine, which was originally found in fish meal, is a compound that causes gizzard erosion and ulceration in chicks. The action of gizzerosine on the isolated cells of chicken proventriculus was studied in the present investigations. Gizzerosine increased intracellular cyclic adenosine-3',5'-monophosphate (cAMP) levels, reaching a plateau within 30 min. Similar maxima of cAMP level were observed in the presence of histamine or gizzerosine. However, the potency of gizzerosine was approximately 1,000-fold higher than that of histamine. The action of gizzerosine was depressed by cimetidine, a histamine H2-receptor antagonist, but not by pyrilamine, a histamine H1-receptor antagonist, indicating that gizzerosine is a very strong histamine H2-receptor agonist. The mucosal cells isolated from the proventriculus by the present procedure had both histamine H1 and H2-receptors. Gizzerosine showed a higher affinity to the cell surface histamine receptor than histamine. These results partly explain the potent activity of gizzerosine in inducing gastric acid secretion and causing gizzard erosion and ulceration in chickens.