Early development of the digestive tract (pharynx and gut) in the embryos and pre-larvae of the European sea bass Dicentrarchus labrax

The European sea bass Dicentrarchus labrax is a marine teleost important in Mediterranean aquaculture. The development of the entire digestive tract of D. labrax , including the pharynx, was investigated from early embryonic development to day 5 post hatching (dph), when the mouth opens. The digestive tract is initialized at stage 12 somites independently from two distinct infoldings of the endodermal sheet. In the pharyngeal region, the anterior infolding forms the pharynx and the first gill slits at stage 25 somites. The other three gill arches and slits are formed between 1 and 5 dph. Posteriorly, in the gut tube region, a posterior infolding forms the foregut, midgut and hindgut. The anus opens before hatching, at stage 28 somites. Associated organs (liver, pancreas and gall bladder) are all discernable from 3 dph. Some aspects of the development of the two independent initial infoldings seem original compared with data in the literature. These results are discussed and compared with embryonic and post-embryonic development patterns in other teleosts.     

Hematopoietic stem cells in mice during embryonic development preceding the establishment of liver hematopoiesis]

We studied the time course of appearance of CFUs (7-8 days old) in embryos of (C57B1/6 x CBA)F1 mice from the 8th day of embryonic development. Significant amounts of CFUs could be detected from the 10th day of development, initially in the body of the embryo from the stage of 30-33 pairs of somites, then in the yolk sac and still later, from the stage of about 40 pairs of somites, in liver anlage. CFUs could not be reliably detected until the 9th day of development either in the embryo itself or in the yolk sac. However, after incubation of nine day old embryos for four days in organ culture, such cultures contained CFUs. CFUs could be found in significant levels in embryos explanted from the embryos at the stage no earlier than 24 pairs of somites. When the yolk sac and the embryo were cultivated separately, CFUs could also be detected, however, the removal of liver primordium from the embryo did not influence the amount of CFUs in its body. CFUs were not found in cultures of liver primordium from nine day old embryos. Thus, we can detect pre-CFUs in 9 day old embryos at the stage 25-28 pairs of somites using the system of organ culture; at the same time CFUs cannot be found in intact embryos of the same age. These data provide evidence that before the establishment of liver hemopoiesis precursors of CFUs are located both in the yolk sac and in the embryo outside rudimentary liver. However, our results do not provide any data for the conclusion about the primary source of pre-CFUs in the mouse embryo.     

Histological and morphological evaluations of the digestive tract and associated organs of haddock throughout post-hatching ontogeny

At hatching, the oesophagus of haddock Melanogrammus aeglefinus lacks goblet cells, the intestine is a simple undifferentiated tube, the liver is present as a rounded mass caudal to the heart, and numerous zymogen granules are present in the pancreas. The first intestinal convolution appears at day 2, at the posterior end of the digestive tract. The oesophagus displays alcian blue and PAS positive mucus secreting cells on day 12, which become numerous by day 15. By day 18, epithelial cells of the posterior intestine show evidence of protein absorption in the form of supranuclear vacuoles. The swimbladder inflates in 50% of the larvae by day 22, although inflation rate is highly variable. By day 35, or 10 mm, a pyloric caecal ridge appears which separates the presumptive stomach, which is now showing evidence of gastric gland formation, from the intestine. This marks the beginning of digestive features characteristic of the juvenile stage.     

The major yolk protein is synthesized in the digestive tract and secreted into the body cavities in sea urchin larvae

Major yolk protein (MYP), a transferrin superfamily protein contained in yolk granules of sea urchin eggs, also occurs in the coelomic fluid of male and female adult sea urchins regardless of their reproductive cycle. MYP in the coelomic fluid (CFMYP; 180 kDa) has a zinc-binding capacity and has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). CFMYP is thought to be synthesized in the digestive tract and secreted into the coelomic fluid where it is involved in the transport of zinc derived from food. To clarify when and where MYP synthesis starts, we investigated the expression of MYP during larval development and growth in Pseudocentrotus depressus. MYP mRNA was detected using RT-PCR in the early 8-arm pluteus stage and its expression persisted until after metamorphosis. Real-time RT-PCR revealed that MYP mRNA increased exponentially from the early 8-arm stage to metamorphosis. Western blotting showed that maternal EGMYP disappeared by the 4-arm stage and that newly synthesized CFMYP was present at and after the mid 8-arm stage. In the late 8-arm larvae, MYP mRNA was detected in the digestive tract using in situ hybridization, and the protein was found in the somatocoel and the blastocoel-derived space between the somatocoel and epidermis using immunohistochemistry. These results suggest that CFMYP is synthesized in the digestive tract and secreted into the body cavities at and after the early 8-arm stage. We assume that in larvae, CFMYP transports zinc derived from food via the body cavities to various tissues, as suggested for adults.     

Ontogenetic development of the digestive tract in Cuban gar (Atractosteus tristoechus) larvae

Histological method was used to describe the development of the digestive tract in Atractosteus tristoechus larvae reared under culture conditions. The larvae were kept at 28 ± 1 °C in three 15 L circular tanks for 18 days and they were fed with Artemia. According to the structural changes in the digestive system, three significant stages were established: (1) lecithotrophic, (2) lecithoexotrophic and (3) exotrophic. The first stage spanned from hatching to 3 days after hatching (DAH), the digestive system started to differentiate and larvae depended entirely on the endogenous nutrition from the yolk sac. During second stage (4–10 DAH), considered critical since it is the transition period to exotrophic feeding, the digestive tract was fully differentiated into buccopharynx, esophagus, non-glandular and glandular stomach, anterior and posterior intestine. First periodic Schiff reagent-positive goblet cells also appeared, interdispersed within the epithelium of the digestive tract, increasing substantially in numbers and distribution as development continued. At this early stage, gastric glands were only observed in the fundic stomach and not in the cardiac and pyloric region. Pyloric caeca, spiral valve and rectum were also clearly distinguishable in the intestine. After the onset of the exogenous feeding (11–18 DAH), the organization and differentiation of the digestive tract did not undergo any noticeable modification, only the increase in size and complexity of the structures, and it attained the four tissue layer arrangement characteristic of adult vertebrates.     

Development of VIP in the peripheral nervous system of avian embryo

The distribution of the VIP containing structures was studied in the gut and in the paravertebral sympathetic ganglia of the quail and chick embryos by immunocytochemistry. In the gut, development of peptidergic nerves followed a craniocaudal gradient. Immunoreactive fibres were first visible in the oesophagus at day 9 in the quail and day 10 in the chick, at 12 days they extended over the whole length of the gut. Cell bodies were localized at day 9 in the foregut and observed in the mid- and hind-gut just before hatching. Transplantations on the chorioallantoic membrane of fragments of various parts of the digestive tract clearly demonstrated that VIP nerve cell bodies belonged to the intrinsic innervation of the gut. Besides the gut, sympathetic paravertebral ganglia contained cells with VIP immunoreactivity detected at day 9 and 10 in quail and chick respectively. In order to find out whether VIP containing neurons differentiated normally in chick embryos in which quail neural crest cells had been implanted at an early stage of development we looked for the appearance of peptidergic neurones in the following situations: when the quail neural primordium had been grafted orthotopically and isochronically into chick host (1) at the adrenomedullary (somites 18-24) and (2) at the vagal (somites 1-7) levels of the neural axis. In all conditions VIP immunoreactivity was observed in quail cells located either in the sympathetic paravertebral ganglia of the trunk at the level of the graft or in the enteric ganglia according to the graft was made at the adrenomedullary and vagal levels respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus

We have developed a whole-mount immunocytochemical method for Xenopus and used it to map the expression of the intermediate filament protein vimentin during early embryogenesis. We used two monoclonal antibodies, 14h7 and RV202. Both label vimentin filaments in Xenopus A6 cells, RV202 reacts specifically with vimentin (Mr, 55 x 10(3] on Western blots of A6 cells and embryos. 14h7 reacts with vimentin and a second, insoluble polypeptide of 57 x 10(3) Mr found in A6 cells. The 57 x 10(3) Mr polypeptide appears to be an intermediate filament protein immunochemically related to vimentin. In the whole-mount embryo, we first found vimentin at the time of neural tube closure (stage 19) in cells located at the lateral margins of the neural tube. By stage 26, these cells, which are presumably radial glia, are present along the entire length of the neural tube and in the tail bud. Cells in the optic vesicles express vimentin by stage 24. Vimentin-expressing mesenchymal cells appear on the surface of the somites at stage 22/23; these cells appear first on anterior somites and on progressively more posterior somites as development continues. Beginning at stage 24, vimentin appears in mesenchymal cells located ventral to the somites and associated with the pronephric ducts; these ventral cells first appear below the anterior somites and later appear below more posterior somites. The dorsal fin mesenchyme expresses vimentin at stage 26. In the head, both mesodermally-derived and neural-crest-derived mesenchymal tissues express vimentin by stage 26. These include the mesenchyme of the branchial arches, the mandibular arch, the corneal epithelium, the eye, the meninges and mesenchyme surrounding the otic vesicle. By stage 33, vimentin-expressing mesenchymal cells are present in the pericardial cavity and line the vitelline veins. Vimentin expression appears to be a marker for the differentiation of a subset of central nervous system cells and of head and body mesenchyme in the early Xenopus embryo.     

Distribution and migration pathways of HNK-1-immunoreactive neural crest cells in teleost fish embryos.

Whole mounts and cross-sections of embryos from three species of teleost fish were immunostained with the HNK-1 monoclonal antibody, which recognizes an epitope on migrating neural crest cells. A similar distribution and migration was found in all three species. The crest cells in the head express the HNK-1 epitope after they have segregated from the neural keel. The truncal neural crest cells begin to express the epitope while they still reside in the dorsal region of the neural keel; this has not been observed in other vertebrates. The cephalic and anterior truncal neural crest cells migrate under the ectoderm; the cephalic cells then enter into the gill arches and the anterior truncal cells into the mesentery of the digestive tract where they cease migration. These cephalic and anterior trunk pathways are similar to those described in Xenopus and chick. The neural crest cells of the trunk, after segregation, accumulate in the dorsal wedges between the somites, however, unlike in chick and rat, they do not migrate in the anterior halves of the somites but predominantly between the neural tube and the somites, the major pathway observed in carp and amphibians; some cells migrate over the somites. The HNK-1 staining of whole-mount embryos revealed a structure resembling the Rohon-Beard and extramedullary cells, the primary sensory system in amphibians. Such a system has not been described in fish.     

Ethanol treatment induces a delayed segmentation anomaly in the chick embryo

A repeatable somite anomaly is described that results from the incubation of cultured chick embryos in the presence of ethanol. The anomaly comprises a misalignment of approximately five consecutive pairs of somites such that one of each pair is displaced cranially by up to one-half a somite length. The appearance of the malformation is delayed by approximately six somite pairs after the beginning of treatment. These characteristics were shared by embryos treated at the stage of gastrulation (no somites yet present) up to embryos possessing ten pairs of somites at treatment time. The deleterious effect did not appear to result from a disruption in the mechanics of the segmentation process itself, since isolated segmental plates were able to form normal intersomitic clefts in the presence of ethanol. Similarly, there were apparently no alterations in the compaction process that occurs at the cranial end of the segmental plate, since both the contractile and adhesive components were unaffected, as judged by the distributions of actin and fibronectin. The potential mechanisms of the anomaly are discussed with reference to similar segmental defects produced by heat shock. In view of earlier results indicating that cells in the primitive streak at gastrulation are sensitive to the presence of ethanol, it is proposed that this somite anomaly is due to a disruption in the contribution of these mesoderm cells to the segmental plate.     

Fate mapping study of the splanchnopleural mesoderm of the 1.5-day-old chick embryo

Various portions of the splanchnopleural mesoderm lateral to the somites of 1.5-day chick embryos were marked in ovo by local injection of Dil, and the distribution of the labelled cells in the digestive-tract mesoderm formed after 3 days' reincubation was analysed. The presumptive area of the digestive organs was confined to bands of splanchnic mesoderm lying lateral to the somites, on both sides, with a width two or three times that between the midline of the embryo and the lateral edge of the somite. Each band generally contributed cells to its own side of the digestive-tract mesoderm, except for the region around the bile duct. The anterior and posterior portion of the pre-gut area contributed cells to the anterior and posterior region of the digestive tract, respectively, but label originating from the portion furthest from the somite took the more ventral and posterior position. Thus, the presumptive areas of the respective digestive organs were located anteroposteriorly in the same order as in the digestive tract with their boundaries lying oblique to the embryonic axis.     

In vitro development of the postimplantation embryos of laboratory rodents in human blood serum

Rat and mouse embryos at the stage of the first somites formation (1-5 pairs) cultivated in human blood serum demonstrated its embryolethal and teratogenic effect. The embryos taken at a later stage (11-18 pairs of somites) developed normally and could be compared with the development of the rat embryos in homologous blood serum. There was no difference in the development when the embryos were cultivated either in male or female blood serum. The stage of embryogenesis 11-18 pairs of somites is recommended for in vitro experimental revealing in the human serum of embryotoxic factors induced by certain external influences.     

Analysis of normal somite development

We describe how the first 6 somite pairs form, using the third somites as examples. This history is based upon time-lapse movies of carbon-marked embryos and histological studies by light and electron microscopy of embryos fixed in situ with glutaraldehyde and osmium tetroxide. At head-process stage a continuous sheet of mesoblast occupies the regions of the future third somites. Mesoblast cells attach either to hypoblast or to overlying neural plate which is already a simple pseudostratified columnar epithelium. Prospective somite cells are those attached to the neuroepithelium, and they extend laterally exactly as far as the neural plate does. By head-fold stage, regression of the node down the midline is shearing the sheet of mesoblast into right and left halves. Somite cells hang from the bottom of the neural plate. As the neural plate condenses toward the midline, attached somite cells are compacted. When the somite segments, somite cells are tightly apposed to one another, and, in addition to junctions binding their basal ends, new junctions appear between their apical ends. This leads to reorganization into the typical somite rosette configuration. Spaces filled with extracellular materials form around the whole somite.     

花背蟾蜍消化道5-羟色胺细胞的分布及形态学特征

采用卵白素-生物素-过氧化物酶复合物（avidin-biotin-peroxidase complex technique,ABC）免疫组织化学方法对花背蟾蜍（Bufo raddei）消化道5-羟色胺（5-HT）细胞的分布密度及形态学特征进行了观察。结果显示,5-HT细胞在花背蟾蜍整个消化道中均有分布,食管、贲门、胃体和幽门的分布密度都显著高于肠道各段,胃幽门部密度最高,胃体部其次,直肠部最低。消化道各个部位5-HT开放型和闭合型细胞的比值变化范围为2.48~4.71。消化道各段均以开放型细胞为主,大多呈锥体形、梭形或不规则形,少数为闭合型细胞,呈圆形或椭圆形。花背蟾蜍5-HT细胞的形态学特征与其他两栖动物相似,但分布密度有自身特征,可能与其食性和善于摄取活动性小的食物的生活习性有关。     

东亚腹链蛇消化道嗜银细胞的分布及形态学观察

应用Grimelius银染法研究了东亚腹链蛇（Amphiesma vibakari）消化道嗜银细胞的分布密度及形态。结果表明：在东亚腹链蛇的消化道中嗜银细胞分布广泛,见于全消化道。其分布密度曲线大致呈波浪形,其中胃部是嗜银细胞分布密度的高峰,胃幽门次之,回肠分布密度最低。嗜银细胞形态多样,主要以锥体形为主,其次还有圆形、椭圆形。广泛分布于上皮细胞基部、上皮细胞之间、腺泡上皮细胞之间。结论：东亚腹链蛇消化道嗜银细胞分布型的形成与各部位消化功能有关;根据其形态,我们认为东亚腹链蛇消化道内嗜银细胞具有内分泌、外分泌、旁分泌3种功能。     

Ingested Blood Contributes to the Specificity of the Symbiosis of Aeromonas veronii Biovar Sobria and Hirudo medicinalis, the Medicinal Leech

Hirudo medicinalis, the medicinal leech, usually carries in its digestive tract a pure culture of Aeromonas veronii bv. sobria. Such specificity is unusual for digestive tracts that are normally colonized by a complex microbial consortium. Important questions for the symbiotic interaction and for the medical application after microvascular surgery are whether other bacteria can proliferate or at least persist in the digestive tract of H. medicinalis and what factors contribute to the reported specificity. Using a colonization assay, we were able to compare experimentally the ability of clinical isolates and of a symbiotic strain to colonize H. medicinalis. The symbiotic A. veronii bv. sobria strain proliferated well and persisted for at least 7 days inside the digestive tract. In contrast, the proliferation of Pseudomonas aeruginosa and Staphylococcus aureus was inhibited inside the animal compared to growth in the in vitro control, indicating that the ingested blood was modified within the digestive tract. However, both strains were able to persist in the digestive tract for at least 7 days. For an Escherichia coli strain, the viable counts decreased approximately 1,000-fold within 42 h. The decrease of viable E. coli could be prevented by interfering with the activation of the membrane-attack complex of the complement system that is present in blood. This suggests that the membrane-attack complex remained active inside H. medicinalis and prevented the proliferation of sensitive bacteria. Thus, antimicrobial properties of the ingested vertebrate blood contribute to the specificity of the A. veronii-H. medicinalis symbiosis, in addition to modifications of the blood inside the digestive tract of H. medicinalis.     

Variation in development of rat embryos at the presomite period.

Rat embryos at a single gestational time in the presomite period were studied for their variation in development and their fate after culture. They were explanted at 8 A.M. on day 9 of gestation from timed-pregnant Sprague-Dawley rats which were obtained by mating between 8 and 10 A.M. (plug day = day 0). In the first experiment, a total of 203 embryos from 20 litters were examined for their variation in development. Several dimensions of embryo/egg cylinder were measured and development of various embryonic/extraembryonic structures were assessed using a scoring system that we developed for the present study. Embryos were then divided into different stages of development based on their scores using the staging system that we developed previously. A large variation in developmental stage was demonstrated; the youngest embryo was at the early primitive streak stage with no signs of amniotic folds and the oldest one was at the late neural plate stage with a foregut pocket but without visible somites. No strong correlation was demonstrated between developmental stage and size of embryo/egg cylinder, nor between developmental stage and development of the proamniotic tube, ectoplacental cavity, or allantois. In the second experiment, embryos were explanted at the same time and those at different stages were cultured separately in rotating bottles and their outcomes were compared after 49 hours. The difference in mean somites number of embryos cultured from the mid primitive streak and late neural plate stages was 6.1. This difference corresponds to approximately 10 hours based on the known linear increase of somites number on day 11 of approximately 0.6 somites per hour. These results indicate a large variation in development of presomite period embryos supposedly of the same gestational age and suggest the importance of careful staging at the time of explantation if precision is needed for whole embryo culture experiments.     

An examination of the role of the digestive gland of two loliginid squids,with respect to lipid: storage or excretion?

The role of the digestive gland, with respect to non-structural lipid, was examined using proximal analysis, histochemistry and quantitative histological techniques in the tropical loliginid squids Sepioteuthis lessoniana (Lesson) and Photololigo sp. The digestive gland of both species was characterized by large and numerous lipid droplets in the apical portion of the digestive cells and very few in the basal portion. The apical lipid droplets were released into the lumen of the gland and subsequently rapidly removed. Despite the numerous large apical lipid droplets, the lipid concentration in the digestive glands of S. lessoniana and Photololigo sp. was lower than that reported for most squid species. There was no relationship between lipid concentration and stage of digestion, suggesting that lipid is not stored in the gland after a meal. There was also no relationship between lipid concentration and the sex of an individual or stage of reproductive maturity, suggesting that these squids are not storing lipid in the digestive gland for use in fuelling reproductive maturation or providing an energy source for oocytes. I believe this study is the first to combine proximal analysis and quantitative histological techniques to examine the role of the squid digestive gland with respect to non-structural lipids. The results indicate that the digestive gland of these tropical loliginid squids is excreting, not storing, excess dietary lipid.     

Fasciola gigantica: Histology of the digestive tract and the expression of cathepsin L

The digestive tract of Fasciola gigantica is composed of the oral sucker, buccal tube, pharynx, esophagus, and caecum. The tegumental-type epithelium lines the first four parts of the digestive tract while the caecal-type epithelium lines the remaining parts from the caecal bifurcation. The caecal-epithelial cells are classified into 3 types according to their staining properties and ultrastructural characteristics, as related to the amount of food contents in the caecal lumen. All caecal-type epithelial cells synthesize and secrete cathepsin L, a major group of enzymes in the digestive tract, as detected by in situ hybridization and immunolocalization. Moreover, the secreted cathepsin L is also adsorbed on the outer surface of the tegument and the glycocalyx coating of the surface of the tegument, whereas the tegumental cells and tegumental syncytium covering the parasite’s body and lining the proximal part of the digestive tract exhibit no in situ hybridization signal and immunostaining for cathepsin L.     

Survival, Physiology, and Lysis of Lactococcus lactis in the Digestive Tract

The survival and the physiology of lactococcal cells in the different compartments of the digestive tracts of rats were studied in order to know better the fate of ingested lactic acid bacteria after oral administration. For this purpose, we used strains marked with reporter genes, the luxA-luxB gene of Vibrio harveyi and the gfp gene of Aequora victoria, that allowed us to differentiate the inoculated bacteria from food and the other intestinal bacteria. Luciferase was chosen to measure the metabolic activity of Lactococcus lactis in the digestive tract because it requires NADH, which is available only in metabolically active cells. The green fluorescent protein was used to assess the bacterial lysis independently of death. We report not only that specific factors affect the cell viability and integrity in some digestive tract compartments but also that the way bacteria are administrated has a dramatic impact. Lactococci which transit with the diet are quite resistant to gastric acidity (90 to 98% survival). In contrast, only 10 to 30% of bacteria survive in the duodenum. Viable cells are metabolically active in each compartment of the digestive tract, whereas most dead cells appear to be subject to rapid lysis. This property suggests that lactococci could be used as a vector to deliver specifically into the duodenum the proteins produced in the cytoplasm. This type of delivery vector would be particularly appropriate for targeting digestive enzymes such as lipase to treat pancreatic deficiencies.