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PubMed1999

Does the subepicardial mesenchyme contribute myocardioblasts to the myocardium of the chick embryo heart? A quail-chick chimera study tracing the fate of the epicardial primordium.

Morris (J. Anat., 1976;121:47-64) proposed that the subepicardial mesenchyme might represent a continuing source of myocardioblasts during embryonic and fetal development. Recent studies have shown that the epicardium and subepicardial mesenchyme, and the coronary vasculature are all derived from a region of the pericardial wall, called the proepicardial serosa. In avian embryos, the cells from the proepicardial serosa colonize the heart via a secondary tissue bridge formed by attachment of proepicardial villi to the heart. In the present study, Morris's hypothesis was tested by tracing the fate of the proepicardial serosa. This was achieved by constructing quail-chick chimeras. The proepicardial serosa was transplanted from HH16/17 quail embryos to HH16/17 chick embryos (ED3). A new transplantation technique facilitated an orthotopic attachment of the quail proepicardial villi to the chicken heart, and prevented the attachment of the chicken proepicardial villi to the heart. The fate of the grafted quail cells was traced in chimeras from ED4 to ED18 with immunohistochemistry, using quail-specific antibodies (QCPN, QH-1). From ED4 onward, the transplant was connected to the dorsal heart wall via its proepicardial villi. Starting from the point of attachment of the quail proepicardial villi to the heart, the originally naked myocardium became almost completely covered by quail-derived epicardium, and quail mesenchymal cells populated the subepicardial, myocardial, and subendocardial layers including the av-endocardial cushions. Quail cells formed the endothelial and smooth muscles cells of the coronary vessels, and the perivascular and intramyocardial fibroblasts. Quail myocardial cells were never found in the subepicardial, myocardial, and subendocardial layers. This suggests that the subepicardial mesenchyme normally does not contribute a substantial number of myocardioblasts to the developing avian heart. The new transplantation technique presented facilitates the production of chimeric hearts in which the derivatives of the proepicardial serosa are almost completely of donor origin. This technique might be useful for future studies analyzing the role of certain genes in cardiac development by the creation of somatic transgenics.

Animals↗
PubMed1991

Gastrointestinal emptying in the final days of incubation of the chick embryo.

1. The gastrointestinal (GI) emptying of the chick embryo was studied in days 19 and 20 of incubation using radioactive (14C-PEG-4000) and coloured (Evans Blue dye) tracer markers. 2. Following administration by oral cannula, the percentages of tracer marker recovered in the yolk sac and the GI tract showed the existence of a GI motor pattern in both 19 d and 20 d chick embryos and suggested that the fluids drunk by the chick embryo during the final days of incubation could have reached the yolk sac from the GI tract via the vitelline diverticulum.

Animals↗
PubMed1989

Hemodynamic effects of acetylcholine in the chick embryo and differences from those in the rat embryo.

It has been reported that acetylcholine induces cardiac anomalies in the chick embryo. Thus, we studied hemodynamic effects of this drug in the chick embryo and also compared them with those in the rat embryo since we found that the effect of caffeine was different between the chick and rat embryos. Acetylcholine was given at doses of 5, 0.5, and 0.05 micrograms into the vitelline vein in chick embryos at Hamburger-Hamilton stage 21 and at a dose of 0.5 micrograms into the placenta in rat embryos at gestational day 12. In the chick embryo, heart rate was reduced to 91, 88, and 87% of control at the end of injection of 0.05, 0.5, and 5 micrograms, respectively, then returned to the baseline level. Vitelline arterial blood pressure was 110% of control with 0.05 micrograms, 134% with 0.5 micrograms, and 142% with 5 micrograms at 1 min after injection. The dorsal aortic blood flow decreased with time after injection, but it was increased only by a 5 micrograms dose at the end of injection. The vascular resistance increased in a dose-dependent manner. In the rat embryo, the change of heart rate was qualitatively similar to that of the chick embryo. The blood pressure did not change significantly. The blood flow velocity at the outflow tract decreased at the end of injection, which indicated the decrease in cardiac output, along with slowing of heart rate, then returned to the control level thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed2004

[Establishment of model human stem cell of transplantation into myocardium of chick embryos].

OBJECTIVE: To establish an animal model of human stem cell transplantation into myocardium in chick embryos. METHODS: Hoechsst 33,258 labeled human primordial germ cells (hPGC) were microsurgically injected into the myocardium of 633 chick embryos of 3-4 days development. Ten days after, the hearts were isolated from the 94 surviving chick embryos, embedded, and sliced. In situ hybridization (ISH) with human specific DNA Alu probe was conducted on the sections with fluorescence to detect the existence of transplanted PGC. Immunohistochemistry with human-myocardium-specific antibody cTnT was conducted on the adjacent sections to observe the differentiation of human myocardial cells. RESULTS: ISH showed that PGC were detected in the myocardium of chick embryos 10 days post-operationally. Immunohistochemistry showed that the myocardium added with antibody in adjacent sections was cTnT-positive and the myocardium untreated with antibody was cTNT-negative. Successful cell transplantation occurred in 15.3% +/- 2.4% of chick embryos. CONCLUSION: Establishment of an animal model of cell transplantation of human stem cells into myocardium in chick embryos is feasible.

Animals↗
PubMed1997

[Inotropic effect of isoprenaline and noradrenaline on chick embryo heart].

Isolated ventricles of developing chick embryo heart, paced at 1 Hz, were used to assess the positive inotropic responses to isoprenaline and noradrenaline in order to characterize the adrenergic receptors involved in these effects. In 7 day-old-chick embryo heart ventricle, isoprenaline and noradrenaline exhibited similar potencies and efficacies. Moreover, propranolol (1 microM) inhibited the positive inotropic effect of isoprenaline and noradrenaline, while pentholamine (3 microM) failed to affect the latter response; in addition, phenylephrine (1 microM-1 mM) had no positive inotropic effect. It was therefore concluded that isoprenaline and noradrenaline induce their effect via stimulation of beta-adrenergic receptors. The efficacy of isoprenaline and noradrenaline and the potency of isoprenaline increased from the 7th to 10th day while the potency of noradrenaline decreased. The decrease in noradrenaline potency with age was attributed to its uptake, while the increase in isoprenaline potency was attributed to the increase in beta-adrenergic receptors. However, the increase in efficacy of both isoprenaline and noradrenaline with age might be due to the higher density and/or higher maturity of contractile proteins.

Age Factors↗
PubMed1988

Developmental hemodynamic changes in rat embryos at 11 to 15 days of gestation: normal data of blood pressure and the effect of caffeine compared to data from chick embryo.

We attempted to measure arterial blood pressure of the rat embryo. The embryo was excised within the uterus and immersed in Hanks' solution at 37 degrees C. The uterus wall and yolk sac were opened to expose the umbilical vessels. The umbilical artery was punctured with a glass micro-pipette, and blood pressure was measured by using a servo-null micro-pressure system. The mean blood pressure was 0.27 +/- 0.05 mm Hg in the embryo at the 11th day of gestation (n = 7), 0.48 +/- 0.03 mm Hg in the 12-day embryos (n = 19), 1.3 +/- 0.08 mm Hg in the 13-day (n = 11), and 2.6 +/- 0.1 mm Hg in the 15-day embryos (n = 10). Heart rate was 84 +/- 11 in 11-day, 122 +/- 3 in 12-day, 192 +/- 7 in 13-day, and 198 +/- 5 in 15-day embryos. These parameters were stable within 10 min after the excision. A comparison of the data with those of the chick embryo of comparable developmental stages revealed that the blood pressure was lower in 11- and 12-day rat embryos than in the chick embryo of Hamburger-Hamilton stages 18 and 21, but this was reversed in the later stages. In the stage 21 chick embryo, intravenous administration of caffeine (60 +/- 9 mg/kg embryo weight) induced an increase in blood pressure by 11 +/- 3% (n = 8), but did not result in a significant increase in dorsal aortic blood flow (6 +/- 6%, n = 9) or in heart rate. In contrast, caffeine (62 +/- 3 mg/kg) increased the heart rate by 8 +/- 2% (n = 10) without changing the blood pressure in the rat embryo of day 12. The velocity of blood flow in the truncus was measured by a pulsed Doppler flowmeter. Caffeine injection increased the mean velocity by 21 +/- 8%). Herein we indicate that measurement of blood pressure in the rat embryo is feasible, but with some limitations, and that there may be qualitative hemodynamic differences between the rat and chick embryos.

Animals↗
PubMed1991

Cultivation of helminths in chick embryos.

A total of 23 species from 14 families of the Digenea have been studied in chick embryos, mainly on the chick chorioallantoic membrane (CAM). Most species for which cultivation has produced ovigerous adults in chick embryos have been avian digeneans with progenetic metacercariae. Less success has been obtained with the hermaphroditic, non-progenetic, economically important trematodes such as fasciolids and echinostomatids, although post-metacercarial development has been achieved for Fasciola hepatica and Echinostoma trivolvis (synonym of E. revolutum) (see Fried and Butler, 1979; Fried and Pentz, 1983). Success with human or animal blood flukes has been minimal, although adults of Schistosoma mansoni and Spirorchis spp. (turtle blood flukes) have at least been maintained on the CAM (see Fried et al., 1982; Fried and Tornwall, 1969). Schistosome cercariae and in vitro transformed cercariae (schistosomules) should be tested in chick embryos. Marine avian schistosomes in the genera Austrobilharzia and Ornithobilharzia, along with the freshwater avian schistosome Trichobilharzia, would provide useful material for avian embryo studies on non-human schistosomes. Studies on trematodes in chick embryos have been done mainly to gain basic biological information on these parasites. That is, to identify species for which definitive hosts are not available; for studies on worm-intraspecific variation, growth and development; for studies on worm feeding and digestion; and for studies on worm-mediated chemoattraction and worm site location on the CAM (Fried, 1989). Sites other than the upper surface of the CAM have not been well-explored for digeneans, although Irwin and Saville (1988a,b) have examined the subchorioallantois and allantois as habitats for stigeids and microphallids. They have also studied the effects of serum supplements to the embryo on the enhancement of worm growth and development. Irwin and Saville's work should be extended to other helminths. The albumen in the hen's egg is a good site for the development of Clinostomum marginatum and Amblosoma sawaense (see Larson and Uglem, 1990; Fried et al., 1981), but the reasons for the better growth of these parasites in the albumen than on the CAM are not known. The inoculation of trematode larvae into CAM blood vessels, the yolk sac, the amnion and the embryo proper have not been explored and may provide useful avenues of research. Only a single study has been done with a monogenean trematode, Polystomoides sp. (see Fried, 1965), in which worms were grown on the CAM at 30 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)

Allantois↗
PubMedDate not supplied

[Immunohistochemical study of the appearance of somatotropic hormone in the adenohypophysis of chick embryos].

The differentiation of somatotropocytes was studied in the Leghorn chick embryos during 11 to 18 days of incubation and in chickens during the first week of life using the immunohistochemical method of nonlabelled antibodies with PAP complex and antiserum against human somatotropic hormone (STH). Unlike in humans, the fixation of pituitaries in the Carnoy mixture is optimal for STH to be immunohistochemically estimated in the chickens and chick embryos. STH was found in adenohypophysis from 12-13 days of development. Besides predominant localization of somatotropocytes in the adenohypophysis caudal lobe, individual STH-positive cells are also present in the cephalic lobe of chickens and chick embryos. The results obtained suggest a relatively late appearance of STH during histotypical development of adenohypophysis in chick embryos.

Animals↗
PubMed1994

Insulin synthesis in chick embryo retinas during development.

Retinas of chick embryos contain insulin and further, are capable of synthesizing it, as demonstrated by incubating retinas at different ages (7th-18th day) with [3H]leucine. The synthesized radioactive insulin was isolated and assayed by means of a HPLC procedure. The synthesis of insulin was found to be highest in the youngest retinas studied (day 7), afterwards it declined with age except for an increment found at 14-15 day. Explants of chick embryo retinas, cultured in vitro, rapidly degraded insulin. Nevertheless, the content of immunoreactive insulin in retinal explants diminished slowly with the age of culture, so that, after 8 days of incubation, it was about 60% of the content found in the retinas at the beginning of incubation. This was proof that cultured explants are capable of efficiently synthesizing insulin. The synthesized [3H]insulin was released from explants into the medium. This was evident also after 6-8 days in culture.

Animals↗
PubMed1988

Origin of the iris sphincter muscle in chick embryo.

The origin of the iridial sphincter muscle in chick embryo was investigated by means of immunohistochemistry. Desmin immunoreactive cells are shown in the mesenchymal stroma overlying the anterior epithelial layer of the iris in 4 1/2-day chick embryos. In 9-11-day chick embryos also some cells of the posterior epithelium near the pupillary margin, and of the iridial lamella show a slighter desmin-immunoreactivity. This finding agrees with a double origin of the iridial sphincter muscle: an early mesenchymal one and a later epithelial other.

Animals↗
PubMed1983

Purification of 5-aminolaevulinate synthase from liver mitochondria of chick embryo.

5-Aminolaevulinate synthase from chick-embryo liver mitochondria has, for the first time, been purified to homogeneity in its native non-degraded form by molecular sieve chromatography, chromatofocusing and affinity chromatography. The enzyme has a minimum molecular weight of 68000 as determined by sodium dodecylsulphate/polyacrylamide gel electrophoresis and a specific activity of 35000 units/mg of protein. This result conflicts with the previous report of Whiting, M.J. and Granick, G. [(1976) J. Biol. Chem. 251, 1340-1346] that the chick embryo enzyme has a molecular weight of 49000. We show here that the purified form can be degraded proteolytically to a smaller form of molecular weight around 50000 while retaining full enzymatic activity. It seem evident, therefore, that the enzyme isolated by Whiting & Granick (1976) was degraded. We have further established by pulse-labelling studies and immunoprecipitation that the enzyme isolated by our new and rapid procedure has the same minimum molecular weight as that which exists in vivo.

5-Aminolevulinate Synthetase↗
PubMed1986

Type X collagen synthesis during in vitro development of chick embryo tibial chondrocytes.

In the developing chick embryo tibia type X collagen is synthesized by chondrocytes from regions of hypertrophy and not by chondrocytes from other regions (Capasso, O., G. Tajana, and R. Cancedda, 1984, Mol. Cell. Biol. 4:1163-1168; Schmid, T. M., and T. F. Linsenmayer, 1985, Dev. Biol. 107:375-381). To investigate further the relationship between differentiation of endochondral chondrocytes and type X collagen synthesis we have developed a novel culture system for chondrocytes from 29-31-stage chick embryo tibiae. At the beginning of the culture these chondrocytes are small and synthesize type II and not type X collagen, but when grown on agarose-coated dishes they further differentiate into hypertrophic chondrocytes that synthesize type X collagen. The synthesis of type X collagen has been monitored in cultured cells by analysis of labeled collagens and in vitro translation of mRNAs. When the freshly dissociated chondrocytes are plated in anchorage-permissive dishes, most of the cells attach and dedifferentiate, as revealed by their fibroblastic morphology. Dedifferentiated chondrocytes, after several passages, can still reexpress the differentiated phenotype and continue their development to hypertrophic, type X collagen-synthesizing chondrocytes. Hypertrophic chondrocytes, when plated in anchorage permissive dishes, attach, maintaining the differentiated phenotype, and continue the synthesis of type X collagen.

Animals↗
PubMed1993

Protein synthesis measured in the whole body of chick embryos cultured in vitro.

1. Whole body protein synthesis was measured in chick embryos cultured in vitro. On day 7 of incubation chick embryos were cultured for 60 min in synthetic serum-free medium containing 4-[3H]phenylalanine. Specific radioactivities in free and protein-bound phenylalanine in the whole embryo were measured, starting 2 min after commencement of the culture process. 2. The values for fractional synthesis rate (FSR) estimated in vitro at 20, 30, 45 and 60 min during the embryo culture agreed well, ranging from 35 to 40%/d, suggesting that the method would serve as a useful model for studying the effect of growth promoters in chick embryos. 3. Bovine insulin in the synthetic medium did not affect FSR of protein in chick embryos cultured in vitro.

Animals↗
PubMed1986

A large chondroitin sulfate proteoglycan (PG-M) synthesized before chondrogenesis in the limb bud of chick embryo.

Extraction of stage 22-23 chick embryo limb buds that had been metabolically labeled with [35S]sulfate yielded heparan sulfate proteoglycan, small chondroitin sulfate proteoglycan, and large chondroitin sulfate proteoglycan (designated PG-M). PG-M constituted over 60% of the total macromolecular [35S]sulfates. It was larger in hydrodynamic size, richer in protein, and contained fewer chondroitin sulfate chains as compared to the predominant proteoglycan (PG-H, Mr congruent to 1.5 X 10(6)) of chick embryo cartilage. The chondroitin sulfate chains were notable for their large size (Mr greater than or equal to 60,000) and high content of nonsulfated chondroitin units (about 20% of the total hexosamine). Hexosamine-containing chains corresponding in size to N-linked and O-linked oligosaccharides were also present. The core protein was rich in serine, glutamic acid (glutamine), and glycine which together comprised about 38% of the total amino acids. Following chondroitinase AC II (or ABC) digestion, core molecules were obtained which migrated on sodium dodecyl sulfate gel electrophoresis as a doublet of bands with approximately Mr = 550,000 (major) and 500,000, respectively. The Mr = 550,000 core glycoprotein was structurally different from the core glycoprotein (Mr congruent to 400,000) of PG-H, as ascertained by tryptic peptide mapping and immunochemical criteria. Immunofluorescent localization of PG-M showed that the intensity of PG-M staining progressively became higher in the core mesenchyme region than in the peripheral loose mesenchyme, closely following the condensation of mesenchymal cells. Since the cell condensation process has been shown to begin with the increase of fibronectin and type I collagen concentration, the similar change in PG-M distribution suggests that PG-M plays an important role in the cell condensation process by means of its interaction with fibronectin and type I collagen.

Animals↗
PubMed1986

Ultrastructural differentiation of glandular stomach (proventriculus) in chick embryo.

Cytochemical characterization of mucosubstances of chick glanular stomach (proventriculus) changes from 15 days of development to postnatal and adult stages was studied. To corroborate these data cytochemical, ultrastructural and ultracytochemical study of chick embryo proventriculus from 7 to 20 days of development was performed. At the 7th day several layers of undifferentiated cells formed an epithelium which covered the walls of the glandular stomach. Mocosubstances were not found. Between the 9th and 5th day a single layer of cylindrical cells was encountered forming invaginations which originated deep glands. Three types of cells were separated from the above mentioned layer, dark, clear and undifferentiated. The dark cells had organelles which are involved in protein synthesis and the clear ones were rich in mitochondria. Argentaffine cells appeared at 15th day instead mucosubstances formed a thin coat on the epithelium at 9th day which increased at the end of development in the apical cytoplasm and gland cells. These observations demonstrate that proventriculus of chick embryo has ultrastructurally differentiated cells involved with enzymatic and hydrochloric acid secretion after the 9th day. These progressive events are correlated with the digestion process of yolk during embryogenesis. At the end of development the proventriculus has completely organized the glandular layer.

Animals↗
PubMed1992

The lack of effect of basic and acidic fibroblast growth factors on the naturally occurring death of neurons in the chick embryo.

In vivo treatment of developing chick embryos with acidic and basic fibroblast growth factors (aFGF and bFGF) failed to affect the differentiation and survival of several populations of developing neurons in the CNS and PNS. All of the neuronal populations examined are known to undergo naturally occurring cell death, and they include spinal and cranial motoneurons, dorsal root ganglia, sympathetic ganglia, nodose ganglia, ciliary ganglia, and sympathetic preganglionic neurons in the PNS, as well as the accessory oculomotor nucleus, the isthmo-optic nucleus, and the brainstem auditory nuclei laminaris and magnocellularis in the CNS. Despite the lack of effect of bFGF on neuronal survival and differentiation, in vivo treatment increased the serum levels of bFGF and stimulated the proliferation of non-neuronal cells in the spinal cord. Therefore, although the administration of exogenous FGF to the developing chick embryo in vivo clearly has some biological activity in the CNS, it was nonetheless ineffective in promoting neuronal survival or differentiation. These data do not support the idea that FGF is a physiologically relevant neurotrophic agent in the developing avian nervous system.

Animals↗
PubMed1979

Use of recombinant plasmids to characterize collagen RNAs in normal and transformed chick embryo fibroblasts.

Two recombinant plasmids containing chick collagen DNA sequences have been used to characterize messenger RNAs for pro-alpha1 (type I) and pro-alpha2 collagen. Poly(A)-containing RNA from chick embryo calvaria and long bones, tissues which are very active in collagen synthesis, were electrophoresed on agarose gels containing methylmercuric hydroxide and transferred to diazobenzyloxymethyl paper; these covalently bound RNAs were hybridized to 32P-labeled pro-alpha1 or pro-alpha2 collagen DNA sequences derived from the recombinant plasmids. The pro-alpha1 collagen probe identified two RNAs, a major species of 5000 bases and a minor species of 7100 bases; the pro-alpha2 collagen probe hybridized to a major species very similar in size to the pro-alpha1 mRNA, about 5200 bases, and a minor species of 5700 bases. It is possible that the 7100 and 5700 base RNAs represent precursors of pro-alpha1 and pro-alpha2 collagen mRNA, respectively. When similar hybridization experiments were performed with RNA from chick embryo fibroblasts, both the pro-alpha1 and pro-alpha2 collagen mRNAs were observed, as well as their corresponding larger species. With RNAs from fibroblasts transformed by Rous sarcoma virus, however, the levels of all RNA species which hybridized with the pro-alpha1 and pro-alpha2 collagen DNA probes were significantly reduced.

Animals↗