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PubMed1992

Transient outward currents in cochlear ganglion neurons of the chick embryo.

Cochlear ganglion neurons were isolated from chick embryos and membrane currents recorded using the patch-clamp technique. Depolarizing voltage steps elicited transient outward currents whose inactivation was best fitted by a double-exponential function with time constants < 30 ms and > 100 ms. The fast inactivating transient outward current (Ito,f) had a threshold for activation of -61 +/- 5.5 mV; steady-state inactivation was voltage-dependent between -90 and -60 mV, with half-inactivation near -75 mV. The slowly inactivating outward current (Ito,s) showed an activation threshold of 34 +/- 4 mV. Half-inactivation was at -67 +/- 3 mV. Ito,f was blocked by 4-aminopyridine which did not affect Ito,s. The effect was concentration- and voltage-dependent. Tetraethylammonium had no effect on either fast or slow transient currents but reduced the amplitude of the non-inactivating outward current in a dose-dependent manner. Ito,f was strongly inhibited by removing Ca2+ from the extracellular bathing solution. Cobalt ions inhibited Ito,f in a dose-dependent manner between 2 and 20 mM. The inhibitory effect of Co2+ was voltage-dependent, displaying a bell-shaped inhibition curve as a function of membrane voltage, maximal inhibition occurring between -20 and 0 mV. Ca2+ removal did not affect Ito,s and partially reduced the amplitude of the steady-state current. These results provide kinetic and pharmacological evidence for the presence of two distinct transient outward currents in cochlear neurons. These currents may play a role in the first synaptic relay of sound transmission.

4-Aminopyridine↗

PubMed1990

Scorbutic and fasted guinea pig sera contain an insulin-like growth factor I-reversible inhibitor of proteoglycan and collagen synthesis in chick embryo chondrocytes and adult human skin fibroblasts.

Chick embryo chondrocytes cultured in sera from scorbutic and fasted guinea pigs exhibited decreases in collagen and proteoglycan production to about 30-50% of control values (I. Oyamada et al., 1988, Biochem. Biophys. Res. Commun. 152, 1490-1496). Here we show by pulse-chase labeling experiments that in the chondrocyte system, as in the cartilage of scorbutic and fasted guinea pigs, decreased incorporation of precursor into collagen was due to decreased synthesis rather than to increased degradation. There was a concomitant decrease in type II procollagen mRNA to about 32% of the control level. As in scorbutic cartilage, proteoglycan synthesis by chondrocytes in scorbutic serum was blocked at the stage of glycosaminoglycan chain initiation. Scorbutic and fasted guinea pig sera also caused a 50-60% decrease in the rates of collagen and proteoglycan synthesis in adult human skin fibroblasts, which synthesize mainly type I collagen. Decreased matrix synthesis in both cell types resulted from the presence of an inhibitor in scorbutic and fasted sera. Elevated cortisol levels in these sera were not responsible for inhibition, as determined by the addition of dexamethasone to chondrocytes cultured in normal serum. Insulin-like growth factor I (IGF-I, 300-350 ng/ml) reversed the inhibition of extracellular matrix synthesis by scorbutic and fasted guinea pig sera in both cell types and prevented the decrease in type II procollagen mRNA in chondrocytes. Therefore, in addition to its established role in proteoglycan metabolism, IGF-I also regulates the synthesis of several collagen types. An increase in the circulating inhibitor of IGF-I action thus could lead to the negative regulation of collagen and cartilage proteoglycan synthesis that occurs in ascorbate-deficient and fasted guinea pigs.

Animals↗

PubMed1975

Polypeptide composition of cell membranes from chick embryo fibroblasts transformed by rous sarcoma virus.

Chick embryo fibroblasts were transformed by the Bryan high-titer strain of Rous sarcoma virus (RSV-BH), or a mutant (RSV-BH-Ta) inducing temperature-dependent transformation. Surface membranes from normal and transformed cells were isolated as membrane vesicles by differential centrifugation, and as cell ghosts after ZnCl2 treatment and separation in an aqueous two-phase system. These preparations were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate or phenol/urea/acetic acid. In general a greater resolution of individual bands was found in gels containing phenol/urea/acetic acid, which separates polypeptides on the bases of size and charge. Electrophoresis of preparations from nontransformed cells showed that two polypeptides (molecular weights 200 000 and 250 000) found in cell ghosts were missing in membrane vesicles. In cell ghosts, transformation by RSV-BH resulted in a significant decrease of the 250 000 molecular weight complex. Also a polypeptide (molecular weight 73 000) prominent in membrane vesicles from nontransformed cells was decreased in transformed cells. Surfaces from cells transformed by RSV-BH-Ta at 37 degrees C presented patterns similar to those for RSV-BH infected cells. Shifting these cells to 41 degrees C resulted in an increase in the 250 000 molecular weight complex, although the amount of this protein(s) never reached that found in noninfected cells. Inhibitors of RNA and protein synthesis failed to block the morphological changes occurring in RSV-BH-Ta cells after temperature shifts from 41 degrees C to 37 degrees C or vice-versa. The same inhibitors caused a reduction in the levels of the 250 000 molecular weight complex at both temperatures. These data indicate that these large membrane-associated polypeptides play little or no role in the morphological changes associated with transformation and its reversal.

Animals↗

PubMed2004

Unveiling the establishment of left-right asymmetry in the chick embryo.

Vertebrates display striking left-right asymmetries in the placement of internal organs, which are concealed by a seemingly bilaterally symmetric body plan. The establishment of asymmetries about the left-right axis occurs early during embryo development and requires the concerted and sequential action of several epigenetic, genetic and cellular mechanisms. Experiments in the chick embryo model have contributed crucially to our current understanding of such mechanisms and are reviewed here. Particular emphasis is given to the elucidation of a genetic network that conveys left-right information from Hensen's node to the organ primordia, characterized to a significant degree of detail in the chick embryo. We also point out a number of early and late events in the determination of left-right asymmetries that are currently poorly understood and for whose study the chick embryo model presents several advantages. We anticipate that the availability of the chick genome sequence will be combined with multidisciplinary approaches from experimental embryology, biophysics, live-cell imaging, and mathematical modeling to boost up our knowledge of left-right organ asymmetry in the near future.

Animals↗

PubMed1983

The culture of chick embryo mesoderm cells in hydrated collagen gels.

Chick embryo mesoderm cells are various stages of differentiation were cultured in three-dimensional matrices of hydrated collagen. The tissues used were: stage 5 mesoderm from regions adjacent to the primitive streak; stage 12 mesoderm, comprising somitic, unsegmented (segmental plate) and lateral plate mesoderm; and stage 18 sclerotome. Explants were examined by phase contrast microscopy, including time-lapse, and scanning and transmission electron microscopy. The cells showed an increased ability to adhere to, and move in, the collagen gel with advancing stage. Of the stage 12 tissues, the unsegmented mesoderm was initially the slowest to grow out of the explant. Sclerotome cells showed by far the greatest ability to move within the gel. Where the collagen fibrils were randomly oriented, the cell morphology was polypodial and advancing lamellipodia showed clear undulations at their leading edges. A distinction was drawn between these undulations and the classical major ruffles which are seen in two-dimensional culture to uplift and pass back along the cell surface. The latter were not seen in the collagen matrix and were presumably suppressed by the three-dimensional culture configuration while the leading edge undulations were not. Ultrastructural examination showed that the cells possessed patches of amorphous material on their surface, which was sometimes interposed between the plasma membrane and collagen fibrils. Addition of hyaluronic acid (2 mg/ml) had an effect only the segmented mesoderm, where outgrowth was enhanced. Although the addition of plasma fibronectin (50 micrograms/ml) to the cultures did not affect any of the tissues, the removal of this substance, by antifibronectin antiserum or by the use of fibronectin depleted serum, inhibited outgrowth in most cases. The only tissue not reproducibly inhibited in this way was sclerotome. Alignment of the collagen fibres by the explants was observed, accompanied by an elongation of the outgrowing cells which, in bipolar form, preferentially moved up and down the aligned tracts. Scanning electron microscopy suggested that cell processes attached to, and presumably exerted tension on, bundles of fibrils thereby pulling them into line. Cell-to-cell contact was not accompanied by contact paralysis as judged by time-lapse micrography.

Animals↗

PubMed1992

Vertebral development of the chick embryo during days 3-19 of incubation.

Chick embryo vertebral development has been studied during the period from 3 to 19 days of incubation. Whole mount preparations stained with alcian blue for cartilage and alizarin red for bone show the vertebral bodies as cartilaginous at 5 days, with dorsal spinous processes at 6 1/2 days, the onset of bone formation at the ventral and dorsal surfaces of the bodies at 13 days, ossification extending dorsally within the neural arches at 13 1/2 days, and bone development occurring throughout the vertebrae at 16 days. Descriptions at each time period center on the thoracic vertebrae with occasional inclusion of adjacent lower cervical and upper lumbar vertebrae. Histologic development is correlated with the Hamburger-Hamilton stages, which are based on external characteristics. The notochord and neural tube are well developed by 3 days and surrounded by sclerotome, myotome, and dermatome cells. Cartilage formation in the perinotochordal region of the vertebral body is seen at 5 days and precedes development of the neural arches and spinous processes. Separate centers of chondrocyte hypertrophy occur in the body (9 days), the lateral neural arches and the dorsal spinous process. Bone formation is under way at 13 days, beginning in the vertebral bodies. Intramembranous periosteal bone formation is seen adjacent to internal regions of chondrocyte hypertrophy. Vascular invasion of hypertrophic chondrocyte regions occurs, but the mechanism of endochondral ossification differs from that of mammals. The cartilage is resorbed by multinucleated chondroclasts and marrow round cells. Clumps of growth plate cartilage cells and matrix are surrounded occasionally by newly synthesized bone, but invasion of individual hypertrophic chondrocyte lacunae by vessels with bone deposition by accompanying osteoblasts on single trabeculae of cartilage does not occur. 3H-thymidine autoradiography shows high uptake at 3 and 5 days in the germinal neuroepithelial cells of the neural tube (spinal cord) and notochord. By 7 days, notochordal uptake is markedly diminished, and no uptake of isotope occurs from 8 days onward. Spinal cord uptake is highest in the first 8 days but persists in lessened amounts to 19 days. From 5 days onward, both undifferentiated mesenchymal cells and differentiated chondrocytes show positive 3H-thymidine uptake, but labeling is never seen in hypertrophic chondrocytes.

Animals↗

PubMed2005

Adenohypophysis regulates cell proliferation in the gonads of the developing chick embryo.

The aim of the present study was to evaluate the effect of hypophysectomy on cell proliferation in the left ovary and the left testis of 8- to 14-day-old chick embryos. Hypophysectomy was performed by the partial decapitation technique. At 44-46 h of incubation, chick embryo heads were sectioned at the mesencephalic level and the prosencephalic region removed. Embryos were further incubated until 8-14 days of development. Cell division was evaluated by bromodeoxyuridine (BrdU) incorporation and by counting the total number of somatic and germ cells in the gonads. The ovary displayed an exponential increase in the number of somatic and germ cells and a higher rate of BrdU incorporation compared to the testis. BrdU incorporation was reduced in the ovary of hypophysectomized embryos at 9-14 days of incubation, while in the testis, the reduction was significant at 14 days of development. Changes in the total number of somatic and germ cells further suggest that the absence of hypophysis affects the growth of the ovary earlier than the growth of the testis. Reduction in the number of somatic and germ cells after hypophysectomy in the ovary was reversed by a hypophyseal graft on the chorioallantoic membrane. The adenohypophysis regulates, probably through gonadotropic hormones, proliferation of somatic and germ cells in the gonads during chick embryo development.

Animals↗

PubMed1984

Effects of alcohol on functional development of the auditory pathway in the brainstem of infants and chick embryos.

The effects of alcohol on the development of neural function in the auditory pathway of the brainstem have been examined in human neonates and chick embryos. Auditory brainstem evoked responses were recorded in six infants of mothers for whom there was evidence of alcohol abuse during pregnancy. In four of the infants, evoked responses were abnormal, with poorly defined, inconsistent peaks. Neurophysiological and histological studies were done on chick embryos which received a single dose of alcohol early incubation. In most surviving embryos the neuroanatomical arrangement of the auditory nuclei in the brainstem was normal. However, Golgi impregnation revealed abnormalities in the length and structure of the dendrites in nucleus laminaris. Electrically evoked neuronal activity revealed abnormalities of synaptic function and the distribution of nerve terminals. The results indicate that in infants and chick embryos the functional development of the auditory pathway in the brainstem is disrupted by exposure to alcohol. Such early changes in this sensory pathway may contribute to the behavioural and intellectual handicaps associated with the fetal alcohol syndrome.

Action Potentials↗

PubMed1986

Chick embryo nerve growth factor. Fractionation and biological activity.

A nerve growth factor (NGF)-like factor initiating nerve fibre outgrowth from sympathetic ganglia in culture was partially purified from chick embryo extract by cation-exchange chromatography followed by hydrophobic interaction chromatography on octylsulfide agarose. The NGF-like factor was markedly activated upon gel filtration in the presence of 6 M urea. Further analysis of the activated chick NGF by immunoblotting following SDS-PAGE, and by inhibition of bioassay response using antibodies to mouse beta NGF demonstrated a distinct antigenic cross-reactivity. The size of the chick embryo NGF was also indistinguishable from that of the mouse beta NGF with a molecular weight (MW) of about 14,000. The findings demonstrate directly the presence of biologically active NGF protein in the developing 18-day chick embryo.

Animals↗

PubMed1980

Chick embryo DNA polymerase beta. Purified enzyme consists of a single Mr = 40,000 polypeptide.

DNA polymerase (EC 2.7.7.7) beta was purified to near homogeneity from chick embryos. The final preparation has a specific enzyme activity of 1,100,000 units (nanomoles of dTMP incorporation/h) per mg of protein with (rA)n X (dT)12-18 as a template-primer. The molecular weight of DNA polymerase beta is about 40,000 as judged by gel filtration on Sephadex G-150 column. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the polypeptide of Mr = 40,000 accounted for 95% of total protein in the final preparation. The fingerprint analysis of tryptic peptides of polypeptide shows that 14 out of 24 125I-peptides from the Mr = 40,000 polypeptide are the same as those of the Mr = 40,000 polypeptide of DNA polymerase beta purified from rat ascites hepatoma cells. A Mr = 27,000 polypeptide, which was present in the less pure preparation, did not show any structure similar to Mr = 40,000 polypeptides from rat and chick cells. Thus, it is concluded that chick embryo DNA polymerase beta consists of a single polypeptide of Mr = 40,000. The minimal number of DNA polymerase beta molecules per chick embryo cell was estimated as about 5,000.

Animals↗

PubMed1992

Monoclonal antibodies as probes for the analysis of the secretory ependymal differentiation in the subcommissural organ of the chick embryo.

Monoclonal antibodies directed against components of the subcommissural organ (SCO) of the chick embryo were produced by immunizing mice with SCO homogenate. In three series of production, 788 hybridomas were screened by immunofluorescence microscopy. Four hybridoma cell lines producing antibodies that specifically recognize both SCO cells and Reissner's fiber (RF) were selected and cloned. Using these immunological probes, the ontogenetic development of the SCO and RF was investigated in the chick embryo. Immunoreactive material could be detected in the SCO anlage from stage 17 on and RF was first observed in the central canal of the thoracal part of the spinal cord in 10-day-old embryos. Monoclonal antibodies can be useful as markers for analyzing molecular mechanisms involved in the specific function of these ependymal cells.

Animals↗

PubMedDate not supplied

Sulphonated phthalocyanine induced caudal malformative syndrome in the chick embryo.

Sulphonated phthalocyanine (Pht.) has been tested for its possible noxious effect on the developing chick embryo. When injected into the subembryonic cavity of 40-45 hours incubated chick embryos (mainly 10-20 somite pairs), Pht. induces a highly reproducible caudal malformative syndrome (trunk and taillessness, various anomalies of the limbs). The main effect is--in about 15% of the malformed specimens--associated with unilateral microphthalmy and, less frequently, with coelosomy. Microscopically developmental disturbances of the caudal axial organs, of the mesonephros and of the limbs are observed. The initial pathological changes, at microscopic level, are necrosis and hemorrhages in the caudal axial and paraxial area. The allantois is poorly developed or even absent. Skeletal changes involve anomalies of the ribs and of the vertebral column and total or partial absence of the pelvic girdle bones. The high mortality, mainly during the first week, is due--first of all--to the developmental disturbances including the poor development or absence of the allantois. Control experiments with CuCl2 suggest the ethiological role of Cu. Pathogenetic aspects are discussed.

Abnormalities, Drug-Induced↗

PubMed1988

Teratogenic effects of benzo[a]pyrene in developing chick embryo.

The effect of benzo[a]pyrene (BP), an established carcinogen, on developing chick embryos was investigated. The embryos were exposed in different stages of development to various doses of BP via the yolk sac route. This resulted in retarded growth, as reflected by lower embryonic body weight, reduced crown-rump length and bill length. Abnormal survivors showed remarkably twisted legs with shortening of the bones, abdominal oedema, haematomas, blisters and a short neck. These findings suggest that BP is a teratogen when injected via this route, and the 'oedema syndrome' is a possible mechanism causing teratogenic effects in developing chick embryos when treated with BP.

Abnormalities, Drug-Induced↗

PubMed1977

[A contribution to infection with yellow fever virus 17D in chick embryos (author's transl)].

During the production of yellow fever virus 17 D vaccine from chick embryos, few embryos die in the time between injection of the eggs and harvest of the embryos. In order to answer the question whether the death of the embryos is due to the infection or to the injury caused by the injection, it is necessary to examine the embryos macro- and microscopically for pathological changes. 8 and 9 days old chick embryos were inoculated into the amniotic cavity with different concentrations of yellow fever virus 17 D (table 1). The embryos were removed from the eggs between the 2nd and 7th day after inoculation. Heart, lung, kidney, brain, liver, and spleen were removed. Macroscopically observable pathological symptoms of the embryos and their organs were recorded. Sections of the organs were histologically investigated. Some embryos died soon after the infection, others on the 5th and 6th day of incubation (table 2). Their death was dependent on the dosage of the inoculum. Macroscopically, some embryos showed oedemas, petechiae on head and trunk, haemorrhages of liver and kidney, enlargement of liver and spleen, and a yellow discolouration of the liver (table 3). Microscopically, liver and brain showed the greatest pathological changes; heart and kidney were also affected, whereas lungs and spleen seemed to be unaffected. The following pathological changes were observed: slight fatty degeneration, oedemas, vascular inflammation, perivascular infiltrates, diffuse infiltrations, infiltrations in form of small nodules and necroses (table 3 and figures 1-12). the severity of the symptoms was evaluated using arbitrary units. These units are summarized on table 3 according to time of occurrence p.i., to virus dilution, to organ, and to type of symptom. An index of pathological changes was allived by dividing the sum of units by the number of organs. The maximum value of the index was demonstrable in liver and brain (table 4). Figure 13 adn table 5 show the development of the lesions during the infection. The maximum value of the index was reached on the 5th day p.i. The most significant pathological changes, as necrosis and perivascular infiltrate, were mainly observed from the 5th day p.i. on (table 6). The perivascular infiltrates were found in heart and brain, the necroses in brain and liver (table 7).

Animals↗

PubMed2006

Microscopic study of cell death in the adrenal glands of mouse and chick embryos.

Dying cells of both chromaffin and cortical cell types were found scattered throughout the adrenal gland of 14-18 day mouse embryos and 17-19 day chick embryos. The ultrastructural appearance of these dying cells was unlike that of cells undergoing apoptosis and there was no evidence of macrophages or other phagocytes removing these cells from the adrenal. Possible morphogenetic functions of cell death in the developing adrenal are discussed.

Adrenal Cortex↗

PubMed1987

Ontogenesis of immunocytochemically demonstrable somatotrophs in the adenohypophyseal pars distalis of the developing chick embryo.

Growth hormone (GH)-containing cells in the hypophyseal pars distalis of the chick embryo were identified immunohistochemically using anti-chicken GH sera. GH cells are first demonstrable in Rathke's Pouch as early as Day 4.5 of incubation. By Day 9.5, when the two lobes (rostral and caudal) of the pars distalis are easily recognized, GH cells are confined exclusively to the caudal lobe. Although the number of GH cells increases gradually during embryonic development, there is a statistically significant difference between Days 10.5 and 12.5 in both the cellular density and the percentage of somatotrophs. GH cells, which contain coarse granules evenly distributed throughout the cytoplasm, varied in diameter from 9.3 micron on Day 4.5 to 11.8 micron on Day 20.5, while the nuclear diameter of these cells increased from 2.8 micron on Day 4.5 to 4.9 micron on Day 20.5. There was a statistically significant difference in the nuclear/cytoplasmic ratio between Days 4.5 and 13.5 and between Days 13.5 and 20.5 of incubation. Aspects of the regulation of growth hormone synthesis and secretion in the chick embryo are discussed.

Animals↗

PubMed1980

Ultrastructural and biochemical alterations in the livers from chick embryos maintained in shell-less culture.

Ultrastructural and biochemical studies were conducted on the livers from chick embryos maintained in shell-less culture up to stage 39 (Hamburger-Hamilton) and from control embryos developed in ovo up to the same stage. The ultrastructural characteristics of hepatic cells from the cultured embryos were similar to those found in the controls except that they contained many large lipid droplets and were almost devoid of lipoprotein granules normally associated with the Golgi complex and the smooth endoplasmic reticulum. These changes suggest the existence of alterations in the lipid metabolism. The livers from cultured embryos showed also a decreased incorporation of tritiated leucine into proteins, which indicates a reduced rate of protein synthesis. These results are consistent with previous reports showing that cultured embryos possess hypoproteinemia. Lactic dehydrogenase activity was similar and pyruvic kinase higher in the livers from cultured with respect to control embryos. This appears to indicate that both aerobic and anaerobic glycolysis were not depressed and that the changes observed in the rate of protein synthesis should not be attributed to hypoxia. "Fat-storing cells" similar to those described in mammals were found both in control and cultured embryos. They had not been previously described in the livers from chick embryos.

Animals↗

PubMed1988

Onset and development of intersegmental projections in the chick embryo spinal cord.

The ontogeny of intersegmental (propriospinal) projections was studied in the chick embryo spinal cord between embryonic day 2.5 and day 6. Our goals were 1) to determine the earliest projections of intersegmental interneurons between specific spinal regions and to establish the cell types involved; and 2) to follow the ontogeny of these projections during the early formative stages of spinal cord development. Studies were carried out in vitro by using an isolated spinal cord/brainstem preparation. Horseradish peroxidase injections were made either uni- or bilaterally at various levels of the spinal cord along the rostrocaudal axis of the embryo. HRP histochemistry was done on Vibratome sections with diaminobenzidine as the chromogen. Following unilateral injections at day 2.5, labelled commissural interneurons were found contralaterally and were confined to the injected segment. Subsequently, labelled cells were found progressively further away from the injected segment. By day 4.5 reciprocal projections extended between lumbar and brachial regions. Interneurons with intersegmental axonal projections were often undifferentiated, consisting of primitive unipolar or bipolar cells with little, if any, dendritic development. In some cases migrating interneurons could be retrogradely labelled from two or three segments away from the location of their translocating cell body. Anterograde Golgi-like labelling of early undifferentiated cells revealed growing axons, axonal terminals, and growth cones. Five or six reasonably distinct classes of intersegmental interneurons were identified based on their location, axonal projections, and morphology of dendritic arbors. These appeared to be segmentally and bilaterally arranged along the rostrocaudal axis of the spinal cord. The axons of some of these types of interneurons exhibited preferences in their longitudinal projections within the ventral and ventrolateral marginal zone at the very onset of pathway formation. From the present observations it can be concluded that intersegmental connectivity precedes the development of ascending and descending supraspinal, as well as primary afferent connections in the chick embryo spinal cord.

Animals↗