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Effects of polyamine limitation on nucleolar development and morphology in early chick embryos.

Inhibition of polyamine synthesis in early chick embryos by in ovo treatment with DL-alpha-difluoromethylornithine (DFMO), injected beneath the blastoderm after 5 h of incubation, permanently blocks post-gastrular development. After the first day of polyamine limitation, the embryos possess a thickened primitive streak. Further morphogenesis is blocked and the ectoderm and mesoderm are condensed around the streak. There is obvious suppression of nucleolar formation in 24 h DFMO-treated embryos. In the mesoderm obliquely in front of Hensen's node the frequency of nucleolus-possessing cells is only a few percent lower in DFMO-treated than in control embryos. However, in the same area the frequency of mesoderm cells possessing multiple nucleoli is about 50% lower in the polyamine-depleted embryos. At the ultrastructural level, mesoderm cells from 24 h DFMO-treated embryos show a reduction of the fibrillar component of the nucleolus with a resulting segregation of the nucleolar material. Our data indicate that stimulation of polyamine synthesis is an obligatory step in the differentiation of epiblast cells into mesoderm cells.

Animals↗

Localization of motor neuron pools supplying identified muscles in normal and supernumerary legs of chick embryo.

Neuromuscular specificity has been investigated in chick embryos with a grafted supernumerary leg. The nerves of the lumbar plexus are divided between the two legs so that rostral nerves innervate the grafted leg and the caudal nerves supply the host's original leg. The basic topographic organization of the histologically definable motor neuron clusters of the lateral motor columns remains unchanged by the addition of a supernumerary leg. Intramuscular injections of identified leg muscles have been used to map the intraspinal location of specific motor pools in stage 38 (12-day) embryos. In the normal embryo, the gastrocnemius muscle is innervated by neurons in a central dorsal cluster of motor neurons in segments 26-29. In six experimental cases, the motor neurons supplying the gastrocnemius muscle of a rostrally placed grafted leg were consistently located in a specific medial cluster of neurons in segments 23-25. Motor neurons in this location never normally innervate a gastrocnemius muscle, even in the very young embryos during the period of naturally occurring cell death. This observation of a systematic mismatch between a particular motor cluster and an abnormally innervated muscle indicates the operation of a selective developmental process. A hierarchy of selective chemoaffinities may best explain our experimental results.

Animals↗

Development of receptors for insulin and insulin-like growth factor-I in head and brain of chick embryos: autoradiographic localization.

In whole brain of chick embryos insulin receptors are highest at the end of embryonic development, while insulin-like growth factor-I (IGF-I) receptors dominate in the early stages. These studies provided evidence for developmental regulation of both types of receptors, but they did not provide information on possible differences between brain regions at each developmental stage or within one region at different embryonic ages. We have now localized the specific binding of [125I]insulin and [125I]IGF-I in sections of head and brain using autoradiography and computer-assisted densitometric analysis. Embryos have been studied from the latter part of organogenesis (days 6 and 12) through late development (day 18, i.e. 3 days before hatching), and the binding patterns have been compared with those in the adult brain. At all ages the binding of both ligands was to discrete anatomical regions. Interestingly, while in late embryos and adult brain the patterns of [125I]insulin and [125I] IGF-I binding were quite distinct, in young embryos both ligands showed very similar localization of binding. In young embryos the retina and lateral wall of the growing encephalic vesicles had the highest binding of both [125I]insulin and [125I]IGF-I. In older embryos, as in the adult brain, insulin binding was high in the paleostriatum augmentatum and molecular layer of the cerebellum, while IGF-I binding was prominent in the hippocampus and neostriatum. The mapping of receptors in a vertebrate embryo model from early prenatal development until adulthood predicts great overlap in any possible function of insulin and IGF-I in brain development, while it anticipates differential localized actions of the peptides in the mature brain.

Animals↗

Electron microscopy of Lednice virus in chick embryo cells.

Replication of Lednice virus in chick embryo cells was studied for 72 hr after inoculation by infectivity titration and the indirect immunofluorescence technique. At 24 and 48 hr after inoculation, electron microscopy revealed spherical virions of uniform morphology, 80-105 nm in diameter, which were localized mostly extracellularly.

Animals↗

Changes in protein glycosylation during chick embryo development.

To investigate the molecular changes in cell-surface glycoproteins during chick embryo development, fibroblasts from 8- and 16-day embryos were extensively digested by pronase after (i) metabolic labeling with radioactive precursors and (ii) external labeling. Two main classes of glycopeptide pronase digestion product were distinguished by Sephadex G-50 column chromatography. The large material excluded was mostly composed of glycosaminoglycans. The small retarded glycopeptides underwent age-related modifications. Those in the 8-day cells were mainly N-linked, whereas 16-day cells contained both O- and N-linked glycopeptides. The evolution of high-mannose chains in younger cells to complex-type chains in the older cells is suggested by (i) the decrease in the mannose-to-galactose and mannose-to-N-acetylglucosamine ratio with embryo development, and (ii) the fact that endo-beta-N-acetylglucosaminidase H treatment released more oligomannosyls from younger than from older embryo cell glycopeptides. Small glycopeptides were also more highly sialylated in 16-day cells than in 8-day cells. The present results provide the first biochemical evidence that both quantitative and qualitative modifications occur in cell-surface glycoconjugates during the late stages of chick embryo development.

Animals↗

Activin and its receptors during gastrulation and the later phases of mesoderm development in the chick embryo.

We have cloned chick homologues of the type-II activin receptor, which we have designated cActR-IIA and -IIB. Binding assays show that the two receptors are indistinguishable in their ability to bind activin-A, with comparable kds. Injection of mRNAs encoding these receptors into Xenopus embryos causes axial duplications. Expression of both receptors can first be detected in the primitive streak by in situ hybridization. This suggests that these genes may be activated in response to mesoderm induction. In agreement with this, we find that treatment of preprimitive streak chick embryos with activin-A leads to rapid induction of the expression of cActR-IIB. At later stages, cActR-IIA transcripts become localized mainly in the notochord and myotome and cActR-IIB in the dorsal neural tube, proximal-anterior part of the limb bud, sensory placodes, and specific regions of the fore- and midbrain. To test the response of early chick embryonic tissues to activin, we designed a new in vitro assay for differentiation. We find that explants of area opaca epiblast or posterior primitive streak from various stages can respond to activin treatment by differentiating into a variety of mesodermal cell types in a dose-dependent manner. These results suggest that the importance of activin-related signaling pathways is not confined to pregastrulation stages and that these receptors may be involved in mediating the effects of inducing signals during later stages of development of the mesoderm, limbs, and nervous system.

Activin Receptors↗

Pituitary control of estrogen secretion in the chick embryo: true or spurious?

The aim of the present study was to examine whether the hypophysis played a role in ovarian 17 beta-estradiol secretion in the chick embryo. Hypophysectomy was fulfilled by partial decapitation, which has long been codified as a practical method of hypophysectomy in the chick embryo. Ovaries from decapitated, sham-operated, and intact embryos 9-19 days old were cultured in vitro during a 4-hr period, and 17 beta-estradiol released was determined by radioimmunoassay. A difference in estradiol secretion rate between decapitated and control embryos was noticed at 10-13 days, on both a per ovary and a per gram basis. At 15-19 days, there was no difference. These results suggest that the hypophysis influences the estrogen secretion of the chick embryo ovary during a limited space of time.

Analysis of Variance↗

Effect of antifungal compounds on aspergillosis in hatching chick embryos.

Aspergillosis was induced experimentally in hatching chick embryos by dipping them in water containing spores of Aspergillus fumigatus or A. flavus. The addition to the dip water of antifungal compounds prevented the development of this syndrome. Of the compounds studied, amphotericin B was most effective, followed by pimaricin and nystatin. Sorbic acid was without effect.

Acids↗

Activity of liver fructose diphosphatase from chick embryos treated with aminoguanidine sulfate.

Injection of chick embryos with aminoguanidine sulfate (AGS) on the fourth day of incubation resulted in a decreased specific activity of liver fructose diphosphatase (FDPase) prior to hatch time. This decreased FDPase specific activity was found to be the consequence of increased levels of an enzyme inhibitor (adenosin 5'-monophosphate) rather than a specific repression of enzyme synthesis.

Adenosine Monophosphate↗

Origin of the avian glycogen body: I. Effects of tail bud removal in the chick embryo.

The tail bud was removed from chick embryos at stage 16-17 in a first study directed to learn of the origin of the glycogen body in the lumbosacral spinal cord of birds. Results of tail bud removal and chorio-allantoic grafting of caudal portions of the embryo containing the tail bud or the neural tube suggest that the glycogen body does not arise from the tail bud, but from the preexisting neural tube craniad or anterior to the tail bud. The stem cells of the glycogen body are most likely among those components of the anterior portion of the lumbosacral neural tube derived from primary neurulation.

Allantois↗

[Glycogen metabolic enzymes in the skeletal muscles of the developing chick embryo].

In the skeletal muscles of the chick embryo from the 10th till the 15th day of embryogenesis, phosphorylase (EC. 2.4.1.1) is represented by two isozymes one of which corresponds, by electrophoretic mobility, to the liver phosphorylase and another to phosphorylase of the skeletal muscles of the adult rat. From the 17th day of embryogenesis on only one isozyme of phosphorylase is found in the skeletal muscles which is identical with that of the skeletal muscles of the adult bird. The isozyme spectrum of phosphorylase of the whole 4 days old embryo contains, besides phosphorylase L, a special "embryonic" isozyme which differs from that of the skeletal muscles by immunochemical characteristics and electrophoretic mobility. From the 10th day of embryogenesis till hatching, the activity of phosphorylase of the skeletal muscles increases more than 50 times and that of glycogen synthetase (EC. 2.4.1.11) only 4 times.

Animals↗

In situ hybridization analysis of globin mRNAs in the primitive erythroid cells of the chick embryo.

The possibility that the minor embryonic chick hemoglobins might be present in a particular subgroup of primitive erythroid cells has been investigated by in situ hybridization. Probe to detect the mRNA for the alpha A globin chain of the minor embryonic hemoglobin was used, and the results of the hybridization were compared with those obtained using as probes the cDNAs for total globin mRNAs. All erythroid cells circulating in a 4-day-old chick embryo gave positive signals with both probes at an approximately constant ratio. This shows that all cells contain a similar assortment of hemoglobin types, excluding the possibility that a subgroup might contain the minor primitive hemoglobins exclusively. However, the cells are not homogeneous, since about 10% of them show a distinctly higher concentration of mRNA of all globin types.

Animals↗

The two prolongations of spinal ganglia neuroblasts: an ultrastructural study during chick embryo development.

Peripheral and central prolongations of chick embryo spinal ganglia neuroblasts were studied and compared at an ultrastructural level during development. Important differences in the terminal tracts of the prolongations and their endings were demonstrated. The possibility of environmental influences being responsible for structural differences in the two prolongations is suggested. It was not possible, at any stage of development, to demonstrate significant polarization of any cytoplasmatic structure in the emerging cones.

Animals↗

Abnormal characteristics of the blood from chick embryos maintained in "shell-less" culture.

Chick embryos were maintained in shell-less culture up to a total age of 15 days. The composition of their blood was analyzed together with the blood from coontrol embryos of comparable degree of differentiation. The blood from cultured embryos had lower hematocrit values; their serum contained a reduced concentration of proteins, phospholipids and total calcium and an increased concentration of inorganic phosphorus. In view of the reduced concentration of proteins and phospholipids, the concomitant hypocalcemia must represent, at least in part, a reduction in the binding capacity of the serum and not only a decrease in the ionic fraction of serum calcium.

Animals↗

[Intra-encephalic injections of actinomycin D in the chick embryo. Autoradiographic study].

42 hour-old chick embryos receive intraencephalic injections of a radioactive Actinomycin D solution. The autoradiographic study of the embryonic head after treatment shows that the injected solution remains in the encephalon. In order to avoid some diffusion of the solution in the surrounding territories, it is necessary to take care of the encephalic inner pressure. These experiments allow to a better approach of studying the influence of Actinomycin D upon the embryonic head morphogenesis.

Animals↗

Laser-induced defects of retinal development in chick embryos.

Retinae of 8-day-old chick embryos were exposed to laser beam irradiation and examined after hatching. The most prominent feature in the irradiated area was the presence of rosettes consisting of photoreceptors surrounded by an orderly arrangement of outer plexiform layer, inner nuclear layer and inner plexiform layer. The histological character of these rosettes raises questions of regeneration of retinal cells that have apparently withdrawn from the mitotic cycle and also the possibility of the regeneration of ganglion cell axons.

Animals↗

Formation of coronary arteries sprouting from the primitive aortic sinus wall of the chick embryo.

The formation of coronary arteries in chick embryos was observed by scanning electron microscopy on injected casts as well as by transmission electron microscopy. Usually, 2-4 primitive coronary arteries appear from the right aortic sinus below the level of the cusp margin, and 1-3 from the left one. As development proceeds, the arteries are generally reduced in number to form a single definitive coronary artery on each side. Canalization of the arteries seems to take place by partially degenerative changes of the primordia.

Animals↗