Site-finding and pairing of Echinostoma revolutum (Trematoda) on the chick chorioallantois

Site-finding of 14-day-old Echinostoma revolutum from the domestic chick was studied by inoculating single worms into various sites on the chorioallantoic membrane (CAM) of 13-day-old chick embryos. Regardless of the site of inoculation, single worms were attracted significantly to the area of the CAM above the embryo. More worms were found in this site at 24 than at 1 hr postinoculation. Worm-pairing was studied in chick embryos by inoculating 2 worms in separate windows, 2 cm apart. Worm-pairing, i.e., worms in contact or within 5 mm of each other, was very evident at 24 hr. The percentage of paired worms on the CAM above the embryo was considerably less than single worms.     

DIFFERENTIATION OF THE DIGESTIVE TRACT EPITHELIUM UNDER THE INFLUENCE OF THE HETEROLOGOUS MESENCHYME OF THE DIGESTIVE TRACT IN THE BIRD EMBRYOS

The endoderm of the oesophagus, proventriculus, gizzard or small intestine of the 5-day-old chick or quail embryo was cultivated in combination with homologous or heterologous mesenchyme on a WxxxOLFFyyy and HxxxAFFHNyyy medium for 7 to 21 days or on the chorio-allantoic membrane (CAM) for 8 days. With homologous mesenchyme the epithelium always differentiated homotypically. In association with heterologous mesenchyme, the differentiation of the epithelium was both homotypical and heterotypical depending on the region of the digestive tract. The oesophagus and small intestine differentiate mainly homotypically both in culture and on CAM, but the gizzard and proventriculus show heterotypic differentiation particularly on CAM. Thus, the endoderm of the digestive tract of the 5-day-old chick or quail embryo, though rather "determined", still reacts to the heterologous stimuli of the mesenchyme to some degree.     

Cultivation of excysted metacercariae of Echinostoma revolutum (Trematoda) in chick embryos

Three techniques were used to study post-metacercarial growth and development of chemically excysted metacercariae of Echinostoma revolutum on the chorioallantoic membrane of domestic chick embryos. The in ovo technique of Zwilling (1959) and the in vitro technique of Auerbach et al. (1974) provided for better worm recovery in chick embryos than the in ovo technique of Woodruff &; Goodpasture (1931). Regardless of the technique used, postmetacercarial development was obtained for this species, and 6- to 8-day-old chorioallantoic-wornis achieved sexual development to the coiled uterus stage comparable to worms grown in domestic chicks for 7 days. However, somatic growth of worms from the chorioallantois was stunted when compared to worms grown in chicks. Worms grown on the chorioallantois voided their excretory concretions and showed histochemical lipid staining identical to that seen in worms grown in chicks.     

Immunohistochemical studies on the development of avian embryo pituitary corticotrophs under normal and experimental conditions

Summary Immunofluorescent ACTH cells are present in the developing chick pituitary gland from the 9th day of incubation.Rathke pouch grafts from 4–5 day or 5.5 and 6.5 day-old chicks, grafted into chick chorioallantoic membrane and grown for 12 days, gave rise to tinctorially normal pituitary glands in both cases.The early grafts were of pouch epithelium alone, separated from mesenchyme by trypsinization. The later grafts were surrounded by their attached mesenchyme, from which they are virtually inseparable.In 17 out of 18 of the 4.5 day grafts no immunofluorescent ACTH cells developed. (In the 18th case a few feebly stained single cells). In 16 out of 30 of the 5.5 and 6.5 day grafts ACTH cells were present in normal numbers.Of the 3 hypotheses put forward to explain these findings only one appears valid. This is that the ACTH cells are contributed directly by the mesectoderm (neural crest) surrounding the 5.5 and 6.5 day pituitary primordia.     

Proteases are mitogenic to mesenchyme in vivo : A study using the chick embryo chorioallantoic membrane

The chorioallantoic membrane (CAM) of the 9 and 10 day chick embryo has been used as a system to investigate the control of growth of mesenchymal tissue in vivo. The mesenchyme (but not the chorionic epithelium or allantoic epithelium) responded to the mitogenic stimuli of trypsin, activated macrophages and the secreted products of activated macrophages. The response to trypsin mimics the response of chick embryo fibroblasts in vitro. Under the conditions used, the increased labeling index was observed in CAM mesenchyme by 6 h (the shortest time assayed) and remained at this level for at least 24 h. This system could be used to evaluate the mitogenicity in vivo of other substances known to be mitogenic to fibroblasts in vitro.     

Scanning electron microscopy of Echinostoma revolutum (Trematoda) during development in the chick embryo and the domestic chick

Fried B. and Fujino T. 1984. Scanning electron microscopy of Echinostoma revolutum (Trematoda) during development in the chick embryo and the domestic chick. International Journal for Parasitology14: 75–81. Scanning electron microscopy (SEM) was used to study the development of chemically excysted metacercariae of Echinostoma revolutum on the chick chorioallantois. SEM studies were also made on preovigerous adults of E. revolutum grown in the domestic chick. During worm development on the chorioallantois the tegument changed from smooth to granular and sensory papillae on the suckers became well-defined. As worms developed on the chorioallantois the cephalic collar spines became thicker and more curved and the tegumentary spines showed marked changes in shape, size and distribution on both ventral and dorsal aspects of the body. Changes in the surface ultrastructure of worms grown on the chorioallantois were essentially similar to those observed in preovigerous worms from chicks.     

Cultivation of excysted metacercariae of Echinostoma caproni (Trematoda) to ovigerous adults on the chick chorioallantois

Excysted metacercariae of Echinostoma caproni were cultivated on the chick chorioallantoic membrane (CAM) maintained at 38.5 +/- 1 C and a relative humidity of 60-65%. Of 59 6-day-old embryos, each inoculated with 25 metacercariae, 29 (49.2%) were infected 2-12 days postinoculation. The total number of worms recovered from the infected eggs was 163 or 22.5% of the 725 inoculated metacercariae. Eggs contained from 1 to 12 (average 5.6) worms per CAM. Worm length increased rapidly from an average of 0.5 mm at 2 days to about 3.0 mm at 6 days postinoculation. Ovigerous worms first were seen on day 8 PI, but fluke eggs did not develop embryos. Worm development in ovo lagged about 1 day behind that of in vivo worms. One worm maintained for 17 days on 2 successive CAMs reached 6 mm in length, contained about 100 eggs in its uterus, and laid an additional 100 eggs on the CAM surface.     

Regulation of parental investment in the Antarctic petrel Thalassoica antarctica: an exchange experiment

An experiment was conducted on the Antarctic petrel to test whether the parents were able to respond to changes in food demand of their offspring. Two experimental groups were formed by replacing eight 20-day-old chicks with 10-day-old chicks, and vice versa. The growth rate of chicks in the experimental groups was compared with that in two control groups with chicks of known age. The growth rate of 10-day-old chicks in the nests of parents which initially had 20-day-old chicks did not differ significantly from that in their respective control groups. This indicates that those parents were able to raise a new young nestling, despite having already raised another chick from hatching to 20 days. However, the 20-day-old chicks placed in nests with 10-day-old chicks had a significantly lower growth rate than their control group. Feeding rate per day and nest did not differ significantly among any of the groups. This suggests that the observed difference in growth rate between 20-day-old chicks is related to a lower amount of food delivered per visit to experimental chicks. Thus, in the Antarctic petrel, the feeding rate apparently is not regulated by the status of the chick, but by the parents' ability to gather food or willingness to provide food for the chicks.Publication no. 137 from the Norwegian Antarctic Research Expedition (NARE) 1989/90     

Excystation and development in the chick and on the chick chorioallantois of the metacercaria of Sphaeridiotrema globulus (Trematoda)

Fried B. and Huffman J. E. 1982. Excystation and development in the chick and on the chick chorioallantois of the metacercaria of Sphaeridiotrema globulus (Trematoda). International Journal for Parasitology12: 427–431. Encysted metacercariae of Sphaeridiotrema globulus were obtained from naturally infected Goniobasis virginica snails in Morris County, New Jersey, U.S.A. These metacercariae were successfully excysted within l h in an alkaline bile salts trypsin medium maintained at 41°C in the absence of acid pepsin pretreatment. Encysted metacercariae treated in 3% sodium bicarbonate produced successful infections in day-old domestic chicks and worms became ovigerous in the lower ileum by day 4. Excysted metacercariae transferred to the chorioallantois of 6–10-day-old chick embryos maintained at 41°C developed into ovigerous adults by day 4.     

Cell type-specific and developmentally regulated expression of the AE1 anion exchanger in the chicken chorioallantoic membrane

Antibodies specific for the chicken AE1 anion exchanger have been used to determine the cell-type specific pattern of expression of this electroneutral transporter in the chick chorioallantoic membrane (CAM) during embryonic development. Immunolocalisation analyses demonstrated that the AE1 anion exchanger accumulated in the basolateral membrane of a subset of cells in both the chorionic and allantoic epithelial layers. Double immunostaining indicated that the AE1-positive cells in the chorionic and allantoic epithelia were also positive for the carbonic anhydrase isoform, CAII, which serves as a marker for the villus cavity (VC) cells of the chorionic epithelium and the mitochondria-rich cells of the allantoic epithelium. Immunoelectron microscopy revealed that AE1 accumulated in extensive projections that extended from the lateral membrane of VC cells towards the adjacent capillary covering cells. These results represent the first demonstration of anion exchanger expression in the chick CAM, and they suggest a role for basolateral AE1 in bicarbonate reabsorption that is required in the embryo for maintaining acid-base balance during development.     

Mesenchymal control of branching pattern in the fetal mouse lung

The effect of mesenchyme on specialization of respiratory epithelium in the fetal mouse was tested in organ cultures. Heterologous combinations were made between respiratory and non-respiratory lung epithelia and the corresponding mesenchymes. Isolated terminal respiratory buds of fetal mouse lungs were recombined with mesenchyme from chick lung parabronchi, mouse trachea or from the avascular, non-respiratory air sacs of chick lungs. Isolated non-branching chick air sacs were combined with mouse terminal bud mesenchyme or mesenchyme from the respiratory branches of chick lungs. Air sac epithelia branched in a pattern characteristic of the chick lung when combined with chick respiratory mesenchyme and in a pattern characteristic of mouse lung when combined with mouse terminal bud mesenchyme. Mouse terminal bud epithelia did not branch with either mouse tracheal mesenchyme or chick air sac mesenchyme but branched in a chick pattern with chick parabronchial mesenchyme. Electron microscopic examination of the cultures showed that all chick air sac epithelial cultures failed to produce surfactant (lamellar bodies) even when they branched. Control cultures of mouse terminal buds contained large numbers of lamellar bodies; mesenchyme which suppressed branching reduced the number of lamellar bodies to only a few in a small proportion of the cells. Culture medium supplemented with growth factors and hormones increased the number of lamellar bodies in heterologous mouse combinations but did not bring the number to control levels. Supplemented medium had no effect on lamellar body production by chick air sac epithelium. The results indicate that branching pattern is determined by the mesenchyme surrounding the epithelial primordium. However, the capacity to synthesize surfactant is determined by the source of the epithelium; mesenchyme may control the degree of expression but not the absolute presence or absence of the differentiated condition.     

Phosphorylation of Synaptic Proteins in Chick Forebrain: Changes with Development and Passive Avoidance Training

We have used synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) to study protein phosphorylation at the synapse in the developing chick forebrain and in 1-day-old chick forebrain following training on a passive avoidance task. Endogenous phosphorylation patterns in SPMs and PSDs prepared by extraction with n-octylglucoside isolated from chick forebrain were investigated by labelling with [32P]ATP. The phosphoprotein components of the SPM and PSD fractions were separated using sodium dodecyl sulphate gradient polyacrylamide gel electrophoresis. Autoradiography and densitometry of the Coomassie Blue protein staining pattern revealed phosphate incorporation into several SPM components including those of molecular mass 52, 37, and 29 kilodaltons (kDa). Bands of similar molecular mass were not phosphorylated in PSD fractions. This difference in phosphorylation between SPMs and PSDs was not due to the detergent n-octylglucoside. In a developmental study in which SPM and PSD fractions were prepared from 1-day-old, 14-day-old, and 21-day-old chickens, the phosphorylation patterns of SPMs were similar throughout, but striking differences occurred in PSDs, both in the level of phosphorylation and in the components phosphorylated. A time-course study was carried out in which phosphorylation of SPMs and PSDs from 1-day-old chicks trained on a passive avoidance task was compared with patterns from control chicks trained on a water-coated bead and untrained chicks. In SPMs prepared from forebrains removed 10 mins following training, a consistent but nonsignificant decrease (-21%) in phosphorylation of a 52 kDa band occurred in chicks with passive avoidance training compared with water-trained and untrained chicks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of epithelial-mesenchymal interactions in the ontogenesis of intestinal brush-border enzymes

The respective roles of embryonic intestinal mesenchyme and endoderm in the biochemical differentiation of brushborder enzymes have been investigated. As a first step of this study, the prenatal developmental pattern of several enzymes (maltase, sucrase, lactase, alkaline phosphatase), measured in brush-border membranes purified from chick and rat intestine, has been established. Xenoplastic recombinations between the intestinal tissue components of $\text{5-}\text{1}\text{2}\text{-}\text{day-old}$ chick embryos and 14-day-old fetal rats have been performed. After 11 days of intracoelomic graft in 3-day-old chick embryos, the combinations composed of chick mesenchyme and rat endoderm (Cm/Re) showed enzyme activities characteristic of the fetal rat intestine: high lactase activity and traces of sucrase activity. The inverse combinations composed of rat mesenchyme and chick endoderm (Rm/Ce) exhibited a chicken-like pattern: high sucrase activity and traces of lactase activity. In the latter combinations, the specific enzyme activities were similar to those present in the intestine of 15- to 16-day-old chick embryos (theoretical level reached after the grafting period). Conversely, the levels of enzyme activities of the Cm/Re combinations remained lower than those present in the normally developed rat intestine. These results show that the endodermal tissue carries the specific characteristics of its future biochemical differentiation. They also suggest that the important maturation events, which occur shortly before birth in the rat, are dependent upon other factors, presumably hormones.     

Recognition of extracellular matrix components by neonatal and adult cardiac myocytes

The histogenesis of renal basement membranes was studied in grafts of avascular, 11-day-old mouse embryonic kidney rudiments grown on chick chorioallantoic membrane (CAM). Vessels of the chick CAM invade the mouse tissue during an incubation period of 7-10 days and eventually hybrid glomeruli composed of mouse epithelium and chick endothelium form. Formation of basement membranes during this development was followed by immunofluorescence and immunoperoxidase stainings using polyclonal and monoclonal antibodies against mouse and chick collagen type IV and against mouse laminin. These antibodies were species-specific as shown in immunochemical and immunohistologic analyses. The glomerular basement membrane contained both mouse and chick collagen type IV, demonstrating its dual cellular origin. All other basement membranes were either exclusively of chick origin (mesangium, vessels) or of mouse origin (tubuli, Bowman's capsule).     

A gelatin film procedure for the localization of proteolytic activity in Leucochloridiomorpha constantiae (Trematoda) adults

A gelatin film procedure was used to localize proteolytic activity in the lumen of the intestinal ceca of Leucochloridiomorpha constantiae (Trematoda) adults. Slides were coated with a 7·5% gelatin solution and then fixed in 10% neutral buffered formalin (NBF). Cryostat sections of isolated worms or those attached to the bursa of Fabricius of the domestic chick were affixed to the gelatin film. Experimental and control slides were incubated in a humid chamber for 30 min at 37·5 and 4°C, respectively. Slides were again fixed in NBF, and then stained for protein with mercuric bromphenol blue (MBB). In experimental slides, the lumen of the intestinal ceca was lysed and did not stain, whereas worm and host tissue and the gelatin were protein-positive. Control sections stained uniformly positive for protein. In this procedure tissue is retained on the slide and proteolytic activity can be correlated with a tissue site on the same slide.     

The gut supports neurogenic differentiation of periocular mesenchyme, a chondrogenic neural crest-derived cell population

Periocular mesenchyme (PM) is a mesencephalic neural crest derived cell population which as a result of an interaction with the retinal pigment epithelium forms the scleral cartilage of the avian eye. Enteric neurons are derived from vagal crest cells which invade the gut. To study factors which regulate neuronal differentiation, we investigated whether the gut could direct neurogenesis in PM, a cell population that does not produce neurons in vivo. We report here that PM cultured in the presence of aneural chick hindgut on the chorioallantoic membrane (CAM), invaded the gut and formed large numbers of neurons. These were localized in enteric ganglia and contained neurofilament immunoreactivity, vasoactive intestinal peptide immunoreactivity, and somatostatin immunoreactivity. In the control PM cultured alone on the CAM, a small number of cells contained neurofilament immunoreactivity but lacked the appearance of mature neurons.     

Pepsinogen Induction in Chick Stomach Epithelia by Reaggregated Proventricular Mesenchymal Cells In Vitro

In vitro organ culture system which permits embryonic chick proventriculus (glandular stomach) to synthesize pepsinogen de novo was developed. Explants of the proventricular rudiment were cultured on Millipore filters in Medium 199 with Earle's salts supplemented with 50% 12-day embryo extract at 38°C in 95% air and 5% CO₂.
In these culture conditions, pepsinogen, a functional marker protein of proventriculus, was first detected after 3 days of cultivation of 6-day chick proventricular rudiment. When recombined and cultured with 6-day proventricular mesenchyme, 6-day oesophageal, proventricular or gizzard (muscular stomach) epithelium expressed pepsinogen while small intestinal epithelium did not. These results were consistent with the previous results obtained by chorioallantoic membrane (CAM) grafting, and showed that the culture conditions are permissive for pepsinogen expression.
When recombined and cultured with reaggregated mesenchymal cells isolated from 6-day proventricular mesenchymal fragments, both 6-day proventricular and gizzard epithelia formed glandular structure and expressed pepsinogen. This indicates that the proventricular mesenchymal cells retain the ability to induce morphogenesis and cytodifferentiation of the proventricular epithelium even if the normal organization of proventricular mesenchyme is once destroyed.     

Ultrastructural and biochemical studies of the swelling of developing chick telencephalic slices

Summary An ultrastructural and biochemical study of the importance and localization of tissue swelling was performed on telencephalic slices of 1- and 30-day-old chicks incubated in an oxygenated or a non-oxygenated physiological medium. The swelling of slices is greater for 30-day-old chick material than for that from 1-day-old chicks. It also reaches higher values in the non-oxygenated than in the oxygenated medium. When the 30-day-old chick telencephalic slices are incubated in an oxygenated medium, swelling mainly affects astrocytes, and especially the astrocytic endfeet. When they are incubated in a non-oxygenated medium, the astrocytes and astrocytic endfeet are very swollen and in addition the swelling also affects the neurons and their organelles. Extracellular space is increased. When 1-day-old chick telencephalic slices are incubated in a non-oxygenated medium, the tissue structures are well preserved. Swelling predominantly affects astrocytes and astrocytic endfeet. Neurons are not affected and the extracellular space is reduced. However, when they are incubated in an oxygenated medium, tissue structures are greatly affected showing a high degree of disorganization. Extracellular space is greatly increased. This study thus indicates that the best incubation conditions are an oxygenated medium for 30-day-old chick telencephalic slices which are characterized by an aerobic metabolism, and a non-oxygenated medium for 1-day-old chick telencephalic slices which have a predominantly anaerobic metabolism.     

Intestinal mesenchyme provokes differentiation of intestinal endocrine cells in gizzard endoderm

The gizzard (muscular stomach) of chicks is deficient in endocrine cells at hatching. It has previously been shown that proventricular types and proportions of endocrine cells can be induced in gizzard endoderm under the influence of proventricular (glandular stomach) mesenchyme. In order to test its capacity to form nongastric endocrine cell types, gizzard endoderm of 3.75- to 5-day chick embryos was combined with mesenchyme from the small intestine of 3.5- to 4-day quail embryos. The combinations were grown as chorio-allantoic grafts until they attained an incubation age comparable to that of hatching chicks. Controls comprised reassociated endoderm and mesenchyme of chick gizzard and of quail intestine. In the experimental grafts, morphogenesis was predominantly intestinal but some grafts showed gizzard-like features, particularly if the endoderm had been provided by older donors. All intestinal endocrine cell types, including those also found in the normal proventriculus (serotonin-, glucagon-, pancreatic polypeptide-, neurotensin- and somatostatin-immunoreactive cells) differentiated in experimental grafts, some even where morphogenesis was gizzard-like. Hence progenitors of not only gastric, but also intestinal, endocrine cells are indeed present in gizzard endoderm. The possibility that gizzard mesenchyme is inhibitory to endocrine cell differentiation is mooted. Motilin- and secretin-immunoreactive cells, which are characteristic of the intestine but not of the proventriculus of chicks at hatching, were respectively sparse or absent when the endoderm was derived from older donors. Thus the ability of gizzard endoderm to differentiate into nongastric endocrine cell types declines before its capacity to form gastric types. The unexpected appearance of gastrin-releasing peptide (GRP)-immunoreactive cells, a proventricular type not found in normal chick intestine, suggests that the intestinal mesenchyme, at least in this instance, was exercising a permissive role.