Regulatory logic driving stable levels of defective proventriculus expression during terminal photoreceptor specification in flies.

How differential levels of gene expression are controlled in post-mitotic neurons is poorly understood. In the Drosophila retina, expression of the transcription factor Defective Proventriculus (Dve) at distinct cell type-specific levels is required for terminal differentiation of color- and motion-detecting photoreceptors. Here, we find that the activities of two cis-regulatory enhancers are coordinated to drive dve expression in the fly eye. Three transcription factors act on these enhancers to determine cell-type specificity. Negative autoregulation by Dve maintains expression from each enhancer at distinct homeostatic levels. One enhancer acts as an inducible backup ('dark' shadow enhancer) that is normally repressed but becomes active in the absence of the other enhancer. Thus, two enhancers integrate combinatorial transcription factor input, feedback and redundancy to generate cell type-specific levels of dve expression and stable photoreceptor fate. This regulatory logic may represent a general paradigm for how precise levels of gene expression are established and maintained in post-mitotic neurons.

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.

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.

Distribution and ontogeny of gastrin- and serotonin-immunoreactive cells in the proventriculus of developing chick, Gallus gallus domestica.

The ontogeny and distribution of gastrin- and serotonin-immunoreactive (IR) cell in the proventriculus of chicks (Gallus gallus domestica, n = 60) in different growth periods was examined immunohistochemically using antisera specific to gastrin and serotonin. Gastrin and serotonin-IR cells were detected in chick proventriculus. Gastrin-IR cells were first evident after 12 days of incubation in lamina epithelialis and compound glands, while serotonin- IR cells were observed only in compound glands at that same time. Gastrin-IR and serotonin-IR cells increased in frequency on incubation day 14 and 16, respectively. Towards the end of incubation, gastrin- and serotonin-IR cell numbers decreased. In adult chicken, both IR cells were present but not lower numbers. The observations demonstrate the presence of gastrin- and serotonin-IR cells in the proventriculus of developing chicks in temporally changing frequencies.

Down-regulation of endodermal Shh is required for gland formation in chicken stomach.

During the development of the proventriculus (glandular stomach) of the chicken embryo, the endodermal epithelium invades into the surrounding mesenchyme and forms glands. The glandular epithelial cells produce pepsinogen, while the non-glandular (luminal) epithelial cells secrete mucus. Sonic hedgehog is expressed uniformly in the proventricular epithelium before gland formation, but its expression ceases in gland cells. Here we present evidence that down-regulation of Sonic hedgehog is necessary for gland formation in the epithelium using a specific inhibitor of Sonic hedgehog signaling and virus mediated overexpression of Sonic hedgehog. We also show that gland formation is not induced by down-regulation of Sonic hedgehog alone; a mesenchymal influence is also required.

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.

Development and birthdates of vasoactive intestinal peptide immunoreactive neurons in the chick proventriculus.

To gain insight into the mechanisms regulating expression of transmitter phenotypes in the enteric nervous system, we have studied the development and birthdate of vasoactive intestinal peptide immunoreactive (VIP-IR) myenteric neurons in the chicken proventriculus (secretory portion of the avian stomach) by a combination of immunocytochemistry and radioautography. The appearance and numbers of VIP-IR neurons in whole mounts of the myenteric plexus from chick embryos and chickens were examined. We found that VIP-IR neurons first appeared at embryonic day (E) 5.5-6.5 in the distal part of the proventriculus. At E7.5, VIP-IR neurons were found singly, in pairs, or in small groups, which together with unlabeled cells formed primitive myenteric ganglia. VIP-IR fibers were found within the developing fiber tracts which connected the ganglia. The number of VIP-IR neurons was found to be maximum in the E15.5 embryo and to decline to 68% of maximum in the 4 week old chicken. Birthdate studies were performed by application of either single pulses or cumulative doses of [3H]-thymidine to embryos between E3 and E14. Whole mounts of the myenteric plexus from the proventriculus of these embryos were immunostained for VIP at E10 or E17. The whole mounts were subsequently sectioned and processed for radioautography. We found that VIP-IR myenteric neurons were born between E3 and E10 with a peak at E7. Most cells underwent terminal division between E5 and E9. These data will be useful in determining the time and conditions when cells make decisions about transmitter phenotypes.

Developmental changes of DNA methylation pattern of embryonic chick pepsinogen gene.

Embryonic chick pepsinogen (ECPg) is one of the pepsinogen isozymogens and its expression is restricted to epithelial cells of the embryonic chick proventriculus (glandular stomach). To examine whether DNA methylation is involved in the regulation of organ-specific and developmental stage-specific expression of ECPg gene, we analyzed the extent of methylation of ECPg gene in normal embryonic and hatched chick organs using methylation-sensitive restriction enzymes. In the proventriculus some CCGG sites underwent demethylation in the gene region after the onset of transcription of the ECPg gene. By contrast, these sites were kept methylated throughout the development in the other organs which do not express ECPg gene. GCGC sites in the gene region became methylated in organs which do not express the ECPg gene, after the initiation of transcription of the ECPg gene in the proventriculus. In the proventriculus, GCGC sites, which were methylated in other organs, were kept unmethylated throughout the development. The methylation state of CpG sites showed no change in the proventriculus of a chick 2 weeks after hatching when the expression of the ECPg gene had completely ceased. The data presented here demonstrate that the DNA methylation is involved in the regulation of organ-specific expression, but stage-specific expression might be brought about by some other mechanisms.

Versatile roles for sonic hedgehog in gut development.

Sonic hedgehog (Shh) is a gene encoding a protein that can be secreted and act as a morphogen. The protein exerts versatile and important effects on the surrounding cells by binding a specific receptor, named patched. So far Shh has been shown to be involved in the morphogenesis and cytodifferentiation of many organ systems, such as notochord, floor plate, limb, pancreas, and pituitary gland, to mention only a few examples. Shh is also involved in the determination of left-right asymmetry, at least in the chicken embryo. Here we present evidence that Shh is one of the key genes whose activity is pivotal for the normal morphogenesis and differentiation of digestive organs. Epithelial Shh regulates the formation of stomach glands and stratification of the mesenchyme into connective tissue and smooth muscle. It exerts its effect often through the induction of bone morphogenetic protein (BMP) genes in the mesenchyme. Thus, Shh is a key player in the epithelial-mesenchymal interactions in the development of the gut.

Can a non-gut mesenchyme support differentiation of gut endocrine cells?

This experiment was designed to find out if endoderm lacks an intrinsic ability to give rise to gut endocrine cells, and, if not, whether differentiation of endocrine cells can be supported by mesenchyme from a source outside the digestive tract. Heterospecific combinations of proventricular endoderm and flank mesenchyme from chick and quail embryos at 3.25-4 days of incubation were grown as chorio-allantoic grafts to a final incubation age of 21 days. Re-associated proventricular endoderm and mesenchyme served as controls. The proventricular endoderm induced some smooth muscle in the flank mesenchyme but the latter did not support as advanced glandular morphogenesis as did proventricular mesenchyme. Nevertheless, endocrine cells differentiated in experimental as in control grafts and at similar frequencies. The various types were distinguished immunocytochemically by their contained peptides; the range of types found was specific for the proventriculus. Hence it is concluded not only that the particular non-gut mesenchyme used does support differentiation of gut endocrine cells, but also that the determination of the progenitors of endocrine cells, and the selection of the range of types destined to differentiate in a particular part of the digestive tract under normal circumstances, occurs early in development--before 3.25 days of incubation in the case of the proventriculus.

The ontogeny and distribution of glucagon- and pancreatic polypeptide-immunoreactive cells in the gastrointestinal tract of the chicken.

The times of first appearance and the distribution of APP- and glucagon-immunoreactive cells have been established in the embryonic chick gut between 11 days of incubation and hatching. These immunoreactive cell types appeared for the first time at 13 days of incubation, APP-immunoreactive cells in the duodenum and upper ileum and glucagon-immunoreactive cells in the proventriculus and duodenum. At 14 days, APP-immunoreactive cells were detected in the proventriculus and lower ileum and glucagon-immunoreactive cells in the pyloric region, upper and lower ileum. Thereafter both APP- and glucagon-immunoreactive cells increased in frequency until the numbers at hatching were approximated, APP-immunoreactive cells at 19 days and glucagon immunoreactive cells at 17 1/2 days of incubation. No APP- or glucagon-immunoreactive cells were found in the gizzard, caecum or rectum at any of the selected stages examined. When these types of endocrine cells first appeared, the surface epithelium of the gastrointestinal tract was relatively undifferentiated. A few glands were present in the proventriculus only, at this stage. Thereafter immunoreactive cells of both types were found in the glandular epithelium of the proventriculus, pyloric region and small intestine soon after morphogenesis had begun.

Gastric gross and microscopic lesions caused by the UNAM-97 variant strain of infectious bronchitis virus after the eighth passage in specific pathogen-free chicken embryos.

Herein we report a description of gross and microscopic lesions found in specific pathogen-free chicken embryos caused by UNAM-97 infectious bronchitis virus (IBV) variant strain after the eighth passage. Embryos were divided into three groups and were inoculated in the chorioallantoic sac with 0.2 mL of UNAM-97, Mass 41 IBV (positive control), or sterile PBS (negative control). Forty-eight hours later the allatoic fluid was taken and used to start a cycle of eight passages through 9-d-old embryos. Seven days after the last passage, embryos were harvested and macroscopic lesions in all organs were recorded. Proventriculus and gizzard samples were obtained from all embryos and routinely processed for microscopic and ultrastructural examinations. The UNAM-97 IBV variant strain caused two macroscopic lesions uncommon for Mexican strains: thin-walled proventriculus and gizzard, as well as urate accumulation within an extra-embryonic peritoneal sac, leaving the body through the umbilical duct and accompanied by the yolk sac. At microscopic level, two relevant findings were observed to be produced by this variant. In the proventriculus, there was a decrease in the gland papillary branching, while the gizzard showed a significant reduction in mucosa thickness and tubular-to-proliferative-cell ratio, as well as an absence of hyaline secretion in the lumen. Electrodense material scattered in proventricular and gizzard cells was observed, with a structure consistent with that of coronaviruses. These pathological chicken embryo findings have not been reported as being caused by other IBV strains in Mexico.

Developmental patterns of selected characteristics of the gastrointestinal tract of young turkeys.

Developing embryos and hatchling poults were sampled (n = 4) at Days 22, 24, 26, and 28 of incubation and at 1, 2, 4, 6, and 8 days after hatching, and selected characteristics of the gastrointestinal tract (GIT) were measured. Body weight increased linearly up to day of hatching and also from 2 to 8 days posthatching. Residual yolk weight decreased rapidly starting on Day 26 of incubation and was nearly depleted by 4 days posthatching. Changes in weight of segments of the GIT nearly paralleled the increase in body weight until day of hatching. Thereafter, weights of the proventriculus, small intestine, and pancreas increased more rapidly than body weight until 6 days after hatching. At this time, change in weight of small intestine and pancreas seemed to parallel that of body weight, whereas proventriculus weight continued to increase more rapidly. Gizzard weight, as a percentage of body weight, increased until Day 4 posthatching and then remained relatively constant through 8 days. Specific activities (SA) of pancreatic amylase, lipase, and trypsin were low until after hatching. Subsequently, amylase SA increased nearly threefold by Day 6. Lipase SA remained nearly constant between Days 1 and 8, and trypsin SA increased only slightly. Total activities of pancreatic enzymes, however, increased substantially after hatching, mainly because of increased pancreas weight. Jejunal maltase SA was high at hatching but decreased markedly by Day 4. This decrease in SA resulted in a notable reduction in total maltase activity of the jejunum despite an increase in jejunum weight.

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.

Expression and function of Wnt5a in the development of the glandular stomach in the chicken embryo.

The epithelium of the chicken embryonic glandular stomach (proventriculus) differentiates into both a glandular and a luminal epithelium, the cells of which express specific marker genes. The subsequent formation and differentiation of the glands then proceed under the influence of the mesenchyme. To search for possible candidates for the mesenchymal factors involved, we have now investigated the expression and function of Wnt5a in this process. Our current results show that Wnt5a is expressed in the mesenchyme during active gland formation and that overexpression of this gene in ovo results in the increased and ectopic expression of some of the marker genes of the luminal and glandular epithelia. In particular, the overexpression of Wnt5a markedly enhances the expression of the embryonic chicken pepsinogen gene, a marker of the glandular epithelium, indicating its role as a mesenchymal factor that regulates the differentiation of the proventricular epithelium.

Exogenous administration of octanoic acid accelerates octanoylated ghrelin production in the proventriculus of neonatal chicks.

Ghrelin is modified by fatty acid at the third serine residue. In this study, derivation of fatty acid for acylation of ghrelin was investigated using a hatchling chicken model. We first studied ghrelin gene expression and production in the neonatal chick proventriculus and then investigated the effect of exogenous octanoic acid (OA) administration on acylated ghrelin production. In a free-feeding condition on day 2.5 after hatching, the density of ghrelin mRNA-expressing (ghrelin-ex) cells was greater than that of ghrelin-immunopositive (ghrelin-ip) cells, but no difference was found between those densities in adult chickens. Intraperitoneal or oral administration of OA for a few days significantly increased the density of ghrelin-ip cells without any changes in ghrelin-ex cells and elevated only octanoylated ghrelin levels in the proventriculus. The results indicate that fatty acid absorbed from food is directly utilized in acylated ghrelin production in the chicken.

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.