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113 femurs and 111 tibias originating from chick embryos 8-21 days after incubation were injected with Indian ink and made transparent. The origin and distribution of cartilage canals are described. The existence of a pattern of segmental distribution, atypical canals and anastomosis between epiphysial canals and diaphysial marrow processes is reported.
In the present communication we describe a very simple procedure which allows measuring the specific activity of cholinergic enzymes (Acetylcholinesterase and cholinacetyltransferase) in microscopical areas of chick embryo spinal cord. Microhomogenates were prepared as follows: i) a number of fresh cryostat sections (20 mu thick) were stained with toluidine blue; ii) the adjacent unstained sections were microdissected under stereomicroscope and tissue fragments were collected from different areas (lateral, medial and posterior columns; anterior, lateral and posterior bundles). Tissue fragments were dissolved in 10 mM sodium-phosphate buffer pH 7.4 (20-40 sections). Protein assay was performed according to Bradford (1976). AChE and ChAc were measured by radiochemical methods of Fonnum (1969, 1970). In lumbar segments of 18-day chick embryo spinal cord AChE was found in all structure investigated, by contrast ChAc was localized almost exclusively in lateral somatomotor comumns. This result indicates that ChAc can be considered a specific marker for motoneurons. The ontogeny of the cholinergic enzymes in various areas of the chick embryo spinal cord has been investigated.
In 9-day-old chick embryos, decreased DNA synthesis and enhanced necrosis were observed in a defined area of the right female Müllerian ducts, supporting the idea of the existence of a regression process in this organ. In the male ducts, decreased DNA synthesis and a low level of necrosis were present all over the studied portion of the organ.
Routine methods of investigation were employed in parallel experiments for the isolation (in chick embryos), identification (serologically via HI), and typing (MDT, ICPI, IVPI, and via cloacal tests) of local isolates and standard strains of the Newcastle disease virus (NDV). The results were the same as those obtained by the single-phase complex investigation after the plaque technique method in a monolayer cell culture of chick embryo fibroblasts under agar cover. The cultures were inoculated with tissue suspension centrifugate in multiple dilutions of trachea and lung, spleen and liver, and brain of experimentally infected birds, using local isolates (13 in all) and standard strains of NDV (6 of the various types) as well as of birds that died of Newcastle disease (3 cases). The complex cell culture technique of investigation included plaque-forming under agar (and under agarose as well) following the direct inoculation with the infectious tissue suspensions in the presence of a normal (known negative), resp., specific hyperimmune serum against Newcastle disease. It also included a plaque-neutralization (plaque reduction) test and tests for hemadsorption and elution of erythrocytes--all made use of in a single planned programme of investigation. This technique, which is a modification of the one suggested by Beard et al. (1970) and known as single-phase complex plaque technique, provides the simultaneous isolation and identification of NDV isolates, which may well be coupled with some of the routinely practiced methods for definitive isolate typing (e. g., with the cloacal test for velogenic, and with MDT for lento- and mesogenic isolates). Thus, time can be saved, and labour and means are reduced, i. e., the laboratory diagnostics of ND may be perfected.
A nucleoside phosphotransferase purified about 40fold from chick embryos utilizes efficiently as phosphate donors deoxyribonucleoside and pyrimidine ribonucleoside monophosphates, whereas the pyrimidine deoxyribonucleoside appear to be the preferred acceptors of phosphate. The enzyme is very unstable to heat, dilution and dialysis. A marked enhancement in the stability is caused by nucleotides and it seems associated with the formation of an aggregated state of the protein.
We have previously shown that removal of the apical ectodermal ridge of the third interdigital space of the chick leg bud at stages 28 and 29 is followed by the appearance of ectopic cartilage, which in the course of development gives rise to extra digits. These in vivo studies suggest that the pattern of skeletal morphogenesis in the limb depends on the inhibitory effect of the ectoderm. In the present study we tested whether zone polarizing activity (ZPA) exerted an effect on the pattern of experimental chondrogenesis in the interdigital space of the leg bud in stage 29 HH chick embryos. A small fragment of tissue from the ZPA in chick embryos in which ZPA activity was most intense was grafted onto the interdigital space in which chondrogenesis had previously been experimentally induced. No significant changes were observed in the course of differentiation of the recipient interdigital spaces with ZPA grafts, leading us to conclude that the graft failed to modify the morphogenetic fate of interdigital tissue.
Chick embryo movements were picked up, by means of a vibration-recording technique, from the 5th day of incubation until the moment of hatching, and correlated with histological observations of the main joints. A close relationship could be demonstrated between the dates on which the first movements can be recorded and the commencement of joint formation.
Lednice (Yaba 1) virus was found to multiply in primary cultures of goose, duck and chick embryo cells. The size of inoculum and age of embryos used for preparation of primary cultures was decisive for the amount of multiplied virus. One strain (110) of Lednice virus showed reduced ability to induce a cytopathic effect in the first passage in chick embryo cells. Virus multiplication in all three kinds of cells was confirmed by immunofluorescence.
There is considerable interest in the discovery of compounds which inhibit angiogenesis dependent (neovascular) diseases. The chick embryo, due to the rapid development of an extensive vascular capillary network in the chorioallantoic membrane (CAM), has been used extensively as a model for studying angiogenesis. Angiostatic steroids are a new class of compounds which inhibit the growth of new capillaries in the chick CAM and in other models of neovascularization. Despite the potential therapeutic importance of these compounds, little is known about the ability of the CAM to metabolize these steroids. We have evaluated the ability of the chick CAM to metabolize cortisol which is both an angiostatic steroid as well as a glucocorticoid. When CAM homogenate was incubated with [3H]cortisol and NADPH at 37 degrees C and pH 7.4, and the reaction products analyzed by reverse phase HPLC, [3H]cortisol was converted exclusively to 20 beta-dihydrocortisol (4-pregnen-11 beta,17 alpha,20 beta,21-tetrol-3-one). The cortisol metabolite, 20 beta-dihydrocortisol, has very little glucocorticoid activity, but shows significant angiostatic activity in the CAM comparable to cortisol. The apparent Km determined for cortisol metabolism was 12 microM and the observed Vmax was 1.4 mumol cortisol/mg protein/min. The majority of the 20 beta-reductase activity was found in the soluble (242,000 g) fraction of CAM homogenate. 20 beta-Reductase activity in chick embryo CAM has not been previously reported.
The immunological properties of the surface of chick embryo cells infected with strain MC29 avian leukosis virus were investigated by immune electron microscopy in conjunction with antiviral and anticellular immune sera. When samples were taken at sequential times after infection, it was found that cells stained strongly for viral antigen very early after infection. Staining reached a minimum 6 to 12 hr after infection and then increased to maximum at 15 hr after infection, remaining at this level at later times. Anticellular immune sera showed the presence of a distinct antigen or antigens which appeared concurrently with morphological alteration of the infected chick cells. This antigen was cross-reactive or identical to an antigen appearing on the surface of chick embryo cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus.
Pulse and chase BrdU labeling of early chick embryos represents a serious technical problem due to the hindrance of removing unincorporated BrdU after the pulse. We have developed a simple method that allows BrdU washout and control of pulse/chase duration. In this method, BrdU pulses are carried out in ovo. Afterwards, embryos are removed from the yolk, BrdU is washed out, and the embryos are maintained in a wholemount culture. Under these conditions, HH8-12 embryos continue with their normal development for at least 30 h. Morphological development of the nervous system and cell cycle kinetics of precursor cells seem to be normally maintained in cultured embryos. To prove the feasibility of the method, it has been applied to determine the onset of TUJ1 expression. TUJ1 is frequently considered an early neuronal marker, yet some reports have shown its expression in dividing progenitor cells and differentiating neurons. The application of this new method demonstrates that TUJ1 is expressed in newborn neurons as early as 1 h after cell cycle exit.
The posterior marginal zone (PMZ) of the chick embryo has Nieuwkoop centre-like properties: when transplanted to another part of the marginal zone, it induces a complete embryonic axis, without making a cellular contribution to the induced structures. However, when the PMZ is removed, the embryo can initiate axis formation from another part of the remaining marginal zone. Chick Vg1 can mimic the axis-inducing ability of the PMZ, but only when misexpressed somewhere within the marginal zone. We have investigated the properties that define the marginal zone as a distinct region. We show that the competence of the marginal zone to initiate ectopic primitive streak formation in response to cVg1 is dependent on Wnt activity. First, within the Wnt family, only Wnt8C is expressed in the marginal zone, in a gradient decreasing from posterior to anterior. Second, misexpression of Wnt1 in the area pellucida enables this region to form a primitive streak in response to cVg1. Third, the Wnt antagonists Crescent and Dkk-1 block the primitive streak-inducing ability of cVg1 in the marginal zone. These findings suggest that Wnt activity defines the marginal zone and allows cVg1 to induce an axis. We also present data suggesting some additional complexity: first, the Vg1 and Wnt pathways appear to regulate the expression of downstream components of each other's pathway; and second, misexpression of different Wnt antagonists suggests that different classes of Wnts may cooperate with each other to regulate axis formation in the normal embryo.
After infection of primary chick embryo cells with an influenza A virus (FPV) the synthesis of polar lipids was specifically inhibited, while mono-, di- and triacylglycerols and fatty acids accumulated. Influx of Ca++ accelerated and Ca++ accumulated in the infected cells. Since enzymes like choline phosphotransferase are sensitive to high concentrations of Ca++, specific inhibition of the synthesis of polar lipids is presumably due to an increased influx of Ca++ by the infection.
Comparative investigations were carried out with local velogenic strains (2 viscerotropic--Haskovo and Bregovo, and neurotropic--Rosa and Toutrakan) and 6 standard strains of the Newcastle disease virus, belonging to various pathogenic groups, in tissue cultures of chick embryo fibroblasts. The four local strains as well as the velogenic one (The. GB) and the mesogenic standard strains (Komarov, Roakin, and Hertfordshire) were reproduced in such cultures, producing a clear cytopathic effect without preliminary adaptation passages. The two strains studied parallel to the others, that were of velogenic pathogenic type (La Sota and Hitchner B1) did not produce cytopathogenic effect, and the cultural liquids did not poses agglutination activity with regard to chicken erythrocytes. The average infection titers in tissue cultures of chick embryo fibroblasts (TCID50) for the particular strains in an experiment varied in a wide range and proved higher with the velogenic (regardless of the fact that they belonged to the viscero- and neurotropic pathogenic type) that with the mesogenic strains. The TCID50/cm3 titer ratio of the strains with regard to their corresponding infection titers in chick embryos (EID50/cm3) also varied in a wide range, but invariably in favour of EID50. The latter was more strongly expressed for the mesogenic strain than for the velogenic pathotype strain of the Newcastle disease in experimental conditions.
The growth of heart during the chick embryo development is accompanied by the increase in total protein content per weight unit of the organ and in the content of the actomyosin complex proteins calculated to the total heart protein. Changes in the rate of heart respiration during development are determined, mainly, by the mass of mitochondria. The growing heart at the early developmental stages is characterized by a very high rate of doubling of the mitochondrial protein mass.
We have tried to expose some characteristics of the embryonic structure of the ceco-appendicular region in the chick embryo during its development. And so, to establish a relation between this region in the chick embryo and the same region in the human embryo.
DNA polymerase alpha was purified from a chick embryo extract by ammonium sulfate fractionation and successive column chromatographies. The final preparation had a specific enzyme activity of 39,000 units/mg of protein with activated calf thymus DNA as a template.primer. Electrophoresis in nondenaturing polyacrylamide gel showed that the preparation contained two proteins, one of which was associated with DNA polymerase activity. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the electrophoretically pure enzyme gave two clusters of polypeptide bands. One was composed of 3-4 polypeptides with similar electrophoretic mobilities corresponding to Mr = 130,000-155,000, and the other consisted of four distinct polypeptide bands corresponding to Mr = 59,000, 56,000, 54,000, and 51,000. Two-dimensional tryptic peptide mapping analysis of these polypeptides after 125I-iodination indicated that the structures of all four Mr = 51,000-59,000 polypeptides were very similar. In addition, polypeptides in the Mr = 145,000-155,000 region had almost identical structure with those in the Mr = 130,000-140,000 region. No significant structural homology was observed between the high molecular weight polypeptides and low molecular weight polypeptides. These findings indicate that the four polypeptides of Mr = 51,000-59,000 are generated by minor modification(s) of a single polypeptide. Similarly, the heterogeneity of the high molecular weight polypeptides is brought about by minor modification(s). Thus, chick embryo DNA polymerase alpha is basically composed of two kinds of subunit polypeptides.
In all higher vertebrate embryos the sensory ganglia of the trunk develop adjacent to the neural tube, in the cranial halves of the somite-derived sclerotomes. It has been known for many years that ganglia do not develop in the most cranial (occipital) sclerotomes, caudal to the first somite. Here we have investigated whether this is due to craniocaudal variation in the neural tube or crest, or to an unusual property of the sclerotomes at occipital levels. Using the monoclonal antibody HNK-1 as a marker for neural crest cells in the chick embryo, we find that the crest does enter the cranial halves of the occipital sclerotomes. Furthermore, staining with zinc iodide/osmium tetroxide shows that some of these crest-derived cells sprout axons within these sclerotomes. By stage 23, however, no dorsal root ganglia are present within the five occipital sclerotomes, as assessed both by haematoxylin/eosin and zinc iodide/osmium tetroxide staining. Moreover, despite this loss of sensory cells, motor axons grow out in these segments, many of them later fasciculating to form the hypoglossal nerve. The sclerotomes remain visible until stages 27/28, when they dissociate to form the base of the skull and the atlas and axis vertebrae. After grafting occipital neural tube from quail donor embryos in place of trunk neural tube in host chick embryos, quail-derived ganglia do develop in the trunk sclerotomes. This shows that the failure of occipital ganglion development is not the result of some fixed local property of the neural crest or neural tube at occipital levels. We therefore suggest that in the chick embryo the cranial halves of the five occipital sclerotomes lack factors essential for normal sensory ganglion development, and that these factors are correspondingly present in all the more caudal sclerotomes.