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PubMed1986

Epithelial and basement membrane responses to chick embryo primitive streak grafts.

Chick embryo primitive streak grafts, placed beneath the epiblast of host embryos, tend to result in the formation of either a neural plate in response to anterior streak grafts, or in de-epithelialization in response to posterior grafts. Ultrastructural and immunocytochemical examination shows that both reactions are preceded by basement membrane disruption and early removal of fibronectin therefrom. This disruption does not occur in response to non-streak grafts. It is suggested that the disruption, evoked by primitive streak cells, is a prerequisite first step, allowing direct graft-epiblast cell contact. This contact elicits a specific cytoskeletal reaction determining the epiblast response.

Animals↗

PubMed1986

Altered synthesis of myosin light chains is associated with contractility in cultures of differentiating chick embryo breast muscle.

Cultured chick embryo skeletal muscle cells normally synthesize only the embryonic isoform of mysoin. We have found that aneural muscle cultures that become or are provoked into an extremely contractile state will begin to synthesize a pattern of myosin light chains typical of maturing muscle. Immunoblots with neonatal and adult specific monoclonal antibodies did not reveal a corresponding isozyme transition in myosin heavy chain. These results demonstrate a correlation between contractility and the regulation of myosin light chain maturation, and also suggest that the transitions of heavy and light chain synthesis during development do not appear to be under close coordinate regulation.

Animals↗

PubMed1983

Differentiation of bones and skeletal muscles in chick embryos cultured on albumen.

Chick embryos were cultured in hemispheric dishes containing 25 ml fresh albumen. The addition of albumen, known to the relatively poor in free calcium, increased the deficit of calcium regularly observed in embryos cultured with other methods. In spite of this, the embryos survived and differentiated adequately for prolonged periods of time. Most of the cultured embryos presented malformations of the long bones of the limbs consisting of bowing and exuberant growth of bone trabeculae in the concavity of the bend. Block staining with alizarin showed that bones were undermineralized at the time in which bending occurred. Leg muscles differentiated adequately as judged from their electron microscopical observation. It is concluded that the malformations result from the action of normal muscles on undermineralized bones. The growth of bone trabeculae at the concavity of the bend probably results from the piezoelectrical stimulus produced by the bending of the bones. The concentrations of both the ionized and bound fractions of blood calcium were significantly reduced in the cultures. The system appears to constitute an adequate model for further explorations on the effects of prolonged hypocalcemia on the differentiation of different tissues and organs.

Albumins↗

PubMed2005

Expression pattern of LINGO-1 in the developing nervous system of the chick embryo.

We isolated a chick homologue of LINGO-1 (cLINGO-1), a novel component of the Nogo-66 receptor (NgR)/p75 neurotrophin receptor (NTR) signaling complex, and examined the expression of cLINGO-1 in the developing brain and spinal cord of the chick embryo by in situ hybridization and immunohistochemistry. cLINGO-1 was expressed broadly in the spinal cord, including the ventral portion of the ventricular zone, and motor neurons. cLINGO-1 was also expressed in the dorsal root ganglion and boundary cap cells at dorsal and ventral roots. In the early embryonic brain, cLINGO-1 was first expressed in the prosencephalon and the ventral mesencephalon, and later in the telencephalon, the rostral part of the mesencephalon and some parts of the hindbrain. cLINGO-1 was also expressed in the ventral part of the neural retina and trigeminal and facial nerves. We also found that cLINGO-1, cNgR1 and p75NTR were expressed in overlapped patterns in the spinal cord and the dorsal root ganglion, but that these genes were expressed in distinct patterns in the early embryonic brain.

Amino Acid Sequence↗

PubMed1967

Structural variations during mitosis in the chick embryo.

Selected tissues from chick embryos were fixed in 2% glutaraldehyde and 1% OsO(4), both buffered at pH 7.6 with Veronal-acetate, and were embedded in Maraglas or Araldite. Two types of cell division have been noted. Generally, epithelial cells divide predominantly by a shortening of the chromosome-to-pole distance rather than by spindle elongation; mesenchymal cells undergo extensive spindle elongation. The presence of numerous continuous microtubules in cells that undergo extensive spindle elongation functionally implicates these tubules in the elongation process. In most embryonic epithelia, the cleavage furrow converges to a fixed site forming a mid-body near the anchoring desmosomes at the free surface; symmetrical furrow formation is typical of mesenchymal cells which lack desmosomes. The hypothesis of cleavage furrow formation and the fate of the mid-body that is formed during cytokinesis are discussed.

Animals↗

PubMed1975

Probable synchronous replication of mitochondrial DNA in cultures of chick embryo fibroblasts.

Cultures of chick embryo fibroblasts were synchronized using a procedure previously described. The profile of incorporation of tritiated thymidine showed a main peak of nuclear DNA replication followed by a small peak between 18 and 24 hr after induction of the cell division, and representing 10 to 25% of the main peak. To identify this small peak, cells were treated with ethidium bromide(EB) chloramphenicol (CAP) or 9-B-D arabinofuranosyl adenine (Ara-A). When EB (1 mug ml-1) and CAP(25mug ml-1) were added at time of induction of mitosis (T0) or 14 hr later (T14) the small peak was suppressed whereas the main peak was not decreased. On the contrary, only the main peak was suppressed when Ara-A was added at T0 or T14. These results suggest that the peak might correspond to the synchronous replication of the mitochondrial DNA during the G2 and M phases of the cell division cycle.

Animals↗

PubMed1977

Magnesium and calcium effects on uptake of hexoses and uridine by chick embryo fibroblasts.

Cultures of chick embryo fibroblasts were incubated for varying periods in media containing different concentrations of Ca2+ and Mg2+-Mg2+ deprivation produced a gradual decrease in the Vmax of the glucose transport system for the D-glucose analogues 3-O-[3H]methyl-D-glucose and 2-deoxy-D-[3H]glucose and a parallel decrease in the rate of production of lactate from glucose in the medium. It greatly reduced the rates of [3H]uridine uptake and incorporation by decreasing the Vmax of the uridine transport system. Addition of Mg2+ to Mg2+-deprived cultures rapidly increased the rate of [3H]uridine uptake without requiring protein synthesis and increased the rate of 2-deoxy-D-[3H]glucose uptake without requiring RNA synthesis. These effects of changes in Mg2+ concentration qualitatively reproduce the effects of such variables as cell density and serum and insulin concentrations. Ca2+ deprivation resulted in similar, though much smaller, changes in the activities of the two transport systems, but also greatly increased the "leakiness" of the cells to the nontransported hexose L-[3H]glucose.

Animals↗

PubMed1983

Alterations of glycosaminoglycans synthesized by chick embryo cartilage treated with 6-amino-nicotinamide.

Treatment of day-4 chick embryos with 6-aminonicotinamide (6-AN) impairs limb chondrogenesis and produces micromelia. However, treatment of day-10 chick embryos with 6-AN does not produce micromelia. In the present study, the glycosaminoglycan (GAG) biosynthesis in the cartilage isolated from the day-10 chick embryos treated with 6-AN in vitro and in the cartilage from the day-11 chick embryos treated with 6-AN in ovo at day-10 was examined. In the epiphyseal cartilage treated in vitro, the radioactivity incorporated into the GAG fraction was significantly decreased and the 35S/3H ratio in the GAG fraction and the molecular size of GAG chain were also decreased. However, the percent distribution of delta Di-0S was almost unchanged. In the epiphyseal and diaphyseal cartilage treated in ovo at day-10, the alteration in GAG biosynthesis was not observed except the reduction of the molecular size of GAG in epiphyseal cartilage. Furthermore, the biosynthetic activity of GAG in the process of recovery/repair was also examined using the micromelial cartilage isolated from day 11-chick embryos given 6-AN in ovo at day-4. In the diaphyseal cartilage, the radioactivity incorporated into the GAG fraction and the 35S/3H ratio were increased, whereas the percent distribution of delta Di-0S was almost unchanged. These results indicate that treatment of day-4 chick embryos with 6-AN produces the rebound phenomena in the diaphyseal cartilage isolated at day-11.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Aminonicotinamide↗

PubMed1987

In vitro morphogenesis of chick embryo hypertrophic cartilage.

Dedifferentiated chick embryo chondrocytes (Castagnola, P., G. Moro, F. Descalzi-Cancedda, and R. Cancedda, 1986, J. Cell Biol., 102:2310-2317), when transferred to suspension culture on agarose-coated dishes in the presence of ascorbic acid, aggregate and remain clustered. With time in culture, clusters grow in size and adhere to each other, forming structures that may be several millimeters in dimension. These structures after 7 d of culture have the histologic appearance of mature hypertrophic cartilage partially surrounded by a layer of elongated cells resembling the perichondrium. Cells inside the aggregates have ultrastructural features of stage I (proliferating) or stage II (hypertrophic) chondrocytes depending on their location. Occurrence and distribution of type I, II, and X collagens in the in vitro-formed cartilage at different times of culture, show a temporal and spatial distribution of these antigens reminiscent of the maturation events occurring in the cartilage in vivo. A comparable histologic appearance is shown also by cell aggregates obtained starting with a population of cells derived from a single, cloned, dedifferentiated chondrocyte.

Animals↗

PubMed1980

Effects of divalent cations on collagen biosynthesis in isolated chick embryo tendon cells.

Isolated chick embryo tendon cells were used in [14C]proline and [14C]lysine labelling experiments to investigate the effect of divalent cations on collagen biosynthesis with a special reference to prolyl hydroxylation and lysyl modifications. The following metals were studied by adding them to the incubation medium of the cells: Ca2+, Cd2+, Co2+, Hg2+, Mg2+, Mn2+, Ni2+, Pb2+ and Zn2+. Zn2+ caused a potent reductin in collagen prolyl hydroxylation with a concomitant increased cellular retention of collagenase-digestible material. These effects were detectable even at physiological concentrations. At the same concentrations of Zn2+, lysyl hydroxylation was considerably less inhibited than prolyl hydroxylation, and the extent of hydroxylysyl glycosylation was even increased. Co2+ was also an efficient inhibitor of collagen prolyl hydroxylation, but at concentrations ten times higher than those of Zn2+. In the presence of other metal ions, no or only up to 10% inhibition of prolyl hydroxylation was noted even at those concentrations at which [14C]proline incorporation into the protein was decreased. However, an increased cellular retention of collagen was detected in the presence of some metal ions. No reduction in lysyl hydroxylation was found in the presence of Ca2+ or Mg2+.

Animals↗

PubMed1977

Cardiac jelly arrangement during the formation of the tubular heart of the chick embryo.

The hearts of chick embryos from stages 9 to 11 according to Hamburger and Hamilton are studied by means of the electron microscopy, and the arrangement of the cardiac jelly (CJ) during the different steps of the fusion of the paired heart anlage is described. It is shown that two different areas can be distinguished in CJ: the CJ located between the endocardium and the myocardium (MECJ) and the CJ located between the endocardium and the ventral foregut endoderm (EECJ). MECJ is a zone poor on ultrastructural components most of which are unbanded filamentous material and low-density amorphous material, and its arrangement remains constant during the whole fusion process; EECJ, on the contrary, is very rich in ultrastructural components containing greater amounts of high-density amorphous material, collagen fibrils and cellular, detritus, and the arragement of this area of the CJ undergoes changes during the different fusion steps. Alcian blue staining does not show difference between both areas of CJ. It is suggested that the CJ can play a role during the fusion of the heart. In addition, some observations are reported which suggest that all the epithelial tissues surrounding the CJ can take part in the elaboration of that extracellular material.

Animals↗

PubMed2001

Development of endometriosis-like lesions after transplantation of human endometrial fragments onto the chick embryo chorioallantoic membrane.

The chick embryo chorioallantoic membrane (CAM) bioassay was used to investigate the early pathogenesis of endometriosis. Endometrial fragments were explanted onto the CAM. The grafts including the surrounding CAM were excised at 24, 48 or 72 h after explantation, fixed and embedded in paraffin. Immunohistochemical analysis was used to distinguish endometrial cells. To identify cells of human origin, in-situ hybridization was performed using a probe specific for human chromosome 1. After 24 h, direct contact between endometrial stromal as well as epithelial cells and the mesenchymal layer of the CAM was observed. Invasion of both stromal cells and intact endometrial glands into the mesenchymal layer was observed after 48 h. At 72 h, endometriosis-like lesions were observed in the mesenchymal layer. Positive staining with antibodies to vimentin and pan-cytokeratin was observed in the invading cells as well as in the lesions. In the lesions these positively stained cells showed in-situ hybridization signals for human chromosome 1, confirming their human origin. In conclusion, after 3 days of incubation, endometriosis-like lesions consisting of human endometrial glands and stromal cells were found in the mesenchymal layer of the CAM. These lesions apparently resulted from the invasion of intact human epithelial structures and stromal cells.

Allantois↗

PubMed1995

Pharmacology of nicotine-induced increase in cytosolic Ca2+ concentrations in chick embryo ciliary ganglion cells.

Chick embryo ciliary ganglion cells were acutely isolated, and the mechanism(s) underlying the increase in the cytosolic Ca2+ concentration ([Ca]in) induced by high concentrations of nicotine examined using fura-2 microfluorometry. The order of potencies of nicotinic receptor agonists in increasing [Ca]in was ACh > nicotine = dimethylphenylpiperazinium > cytisine. The nicotine-induced increase in [Ca]in was inhibited not only by nicotinic antagonists but also by muscarinic antagonists, while the muscarine-induced [Ca]in increase was little affected by nicotinic antagonists. The nicotine-induced [Ca]in increase was inhibited by both L- and N-type Ca2+ channel blockers and potentiated by an L-type Ca2+ channel agonist, Bay-K-8644. Nicotine also increased the cytosolic Na+ concentration ([Na]in) as measured by sodium binding benzofuranisophthalate microfluorometry, and this [Na]in increase was inhibited by various agents which reportedly affected nicotinic receptor channels in adrenal chromaffin cells. These results suggest that nicotine increased Na+ influx through nicotinic receptor channels resulting in membrane depolarization, which in turn increased Ca2+ influx through voltage-dependent Ca2+ channels. However, nicotine still increased influxes of Ca2+ and Mn2+ in the absence of external Na+, suggesting that nicotinic receptor channels in these cells are permeable not only to monovalent cations but also to Ca2+ and Mn2+.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1997

Epithelium is required for maintaining FGFR-2 expression levels in facial mesenchyme of the developing chick embryo.

In the developing chick embryo, fibroblast growth factor-2 (FGFR-2) expression patterns correlate with outgrowth of facial prominences. Frontonasal mass prominences that form the pre-nasal cartilage and upper beak express high levels of FGFR-2 receptor, whereas maxillary prominences that form the flattened corners of the beak and palatal shelves express low FGFR-2 transcript levels. Facial epithelium is an abundant source of FGFs and is required to support outgrowth of mesenchymal tissue, including cartilage rod formation. Because FGFR-2 is highly expressed in regions of facial outgrowth and because epithelium is required for outgrowth of facial prominences, epithelium could be required to maintain FGFR-2 transcripts in facial mesenchyme. To test this hypothesis, we removed epithelium to inhibit outgrowth of regions of the embryonic face, grafted frontonasal mass and maxillary prominences into a host limb bud, and then examined changes in FGFR-2 expression using in situ hybridization. We also hybridized adjacent sections with collagen II probe to identify regions undergoing chondrogenesis. Our results indicate that removal of epithelium from frontonasal mass led to a decrease in FGFR-2 and collagen II expression 24 hr after grafting to host and that neither FGFR-2 nor collagen II expression increased to expected levels at 48 hr. These results suggest that there are signals in the epithelium required for increasing FGFR-2 and collagen II gene transcription, and the expression of these genes are linked to outgrowth of facial prominences.

Animals↗

PubMed1996

Angiogenesis in the gonadal capillary network of the chick embryo.

Seventy-one chick embryos of both sexes at the 35 Hamburger and Hamilton (H-H) developmental stage were processed for scanning electron microscopy of vascular corrosion casts and of critical point dried specimens, as well as transmission electron- and light microscopy, in order to study the angiogenic structures. The gonadal subepithelial capillary network was located at the level of the tunica albuginea under the covering epithelium. The casts showed a densely-meshed capillary network and numerous sprouting (nodular protrusions or capillary sprouts) and non-sprouting (enlarged vessels and angiogenic holes) angiogenic structures that were randomly distributed and mixed. Four types of angiogenic holes were encountered in the casts: primary (diameter < 2.5 microns), secondary (diameter > 2.5 microns), tertiary (variable diameter and circular narrowings on one side), and open angiogenic holes. We suggest that the different morphologies reflect evolution of these holes. Furthermore, the open angiogenic hole would probably either form nodular protrusions at its open ends, which tend to join with other nodular protrusions and neighboring capillaries and form new vessels; or there would be fusion with two or more neighboring open holes. Correlative critical point dried sections showed fenestrations in the capillary walls and transcapillary pillars that corresponded to the angiogenic holes found in the casts. Ultrathin sections of the vessels presented typical characteristics of growing endothelium: large nuclei with loosely textured chromatin, abundant cytoplasm rich in cell organelles and intraluminal endothelial processes.

Animals↗

PubMed1983

Differences in the organization of actin in the growth cones compared with the neurites of cultured neurons from chick embryos.

Sensory neurons from chick embryos were cultured on substrata that support neurite growth, and were fixed and prepared for both cytochemical localization of actin and electron microscopic observation of actin filaments in whole-mounted specimens. Samples of cells were treated with the detergent Triton X-100 before, during, or after fixation with glutaraldehyde to determine the organization of actin in simpler preparations of extracted cytoskeletons. Antibodies to actin and a fluorescent derivative of phallacidin bound strongly to the leading margins of growth cones, but in neurites the binding of these markers for actin was very weak. This was true in all cases of Triton X-100 treatment, even when cells were extracted for 4 min before fixation. In whole-mounted cytoskeletons there were bundles and networks of 6-7-nm filaments in leading edges of growth cones but very few 6-7-n filaments were present among the microtubules and neurofilaments in the cytoskeletons of neurites. These filaments, which are prominent in growth cones, were identified as actin because they were stabilized against detergent extraction by the presence of phallacidin or the heavy meromyosin and S1 fragments of myosin. In addition, heavy meromyosin and S1 decorated these filaments as expected for binding to F-actin. Microtubules extended into growth cone margins and terminated within the network of actin filaments and bundles. Interactions between microtubule ends and these actin filaments may account for the frequently observed alignment of microtubules with filopodia at the growth cone margins.

Actins↗

PubMed1991

Interactions between mesoderm cells and the extracellular matrix following gastrulation in the chick embryo.

In the gastrulating chick embryo, the mesoderm cells arise from the epiblast layer by ingression through the linear accumulation of cells called the primitive streak. The mesoderm cells emerge from the streak with a fibroblastic morphology and proceed to move away from the mid-line of the embryo using, as a substratum, the basement membrane of the overlying epiblast and the extracellular matrix. We have investigated the roles of fibronectin and laminin as putative substrata for mesoderm cells using complementary in vivo and in vitro methods. We have microinjected agents into the tissue space adjacent to the primitive streak of living embryos and, after further incubation, we have examined the embryos for perturbation of the mesoderm tissue. These agents were: cell-binding regions from fibronectin (RGDS) and laminin (YIGSR), antibodies to these glycoproteins, and a Fab' fragment of the antibody to fibronectin. We find that RGDS, antibody to fibronectin, and the Fab' fragment cause a decrease in the number of mesoderm cells spread on the basement membrane, and a perturbation of cell shape suggesting locomotory impairment. No such influence was seen with YIGSR or antibodies to laminin. These results were extended using in vitro methods in which mesoderm cells were cultured in fibronectin-free medium on fibronectin or laminin in the presence of various agents. These agents were: RGDS; YIGSR; antibodies to fibronectin, fibronectin receptor, laminin and vitronectin; and a Fab' fragment of the fibronectin antiserum. We find that cell attachment and spreading on fibronectin is impaired by RGDS, antiserum to fibronectin, the Fab' fragment of fibronectin antiserum, and antiserum to fibronectin receptor. The results suggest that although the RGDS site in fibronectin is important, it is probably not the only fibronectin cell-binding site involved in mediating the behaviour of the mesoderm cells. Cells growing on laminin were perturbed by YIGSR, RGDS and antibodies to laminin, suggesting that mesoderm cells are able to recognise at least two sites in the laminin molecule. We conclude that the in vivo dependence of mesoderm cells on fibronectin is confirmed, but that although these cells have the ability to recognise sites in laminin as mediators of attachment and spreading, the in vivo role of this molecule in mesoderm morphogenesis is not yet certain.

Analysis of Variance↗

PubMed1994

Ovotransferrin and ovotransferrin receptor expression during chondrogenesis and endochondral bone formation in developing chick embryo.

Ovotransferrin expression during chick embryo tibia development has been investigated in vivo by immunocytochemistry and in situ hybridization. Ovotransferrin was first observed in the 7 day cartilaginous rudiment. At later stages, the factor was localized in the articular zone of the bone epiphysis and in the bone diaphysis where it was concentrated in hypertrophic cartilage, in zones of cartilage erosion and in the osteoid at the chondro-bone junction. When the localization of the ovotransferrin receptors was investigated, it was observed that chondrocytes at all stages of differentiation express a low level of the oviduct (tissue) specific receptor. Interestingly, high levels of the receptor were detectable in the 13-d old tibia in the diaphysis collar of stacked-osteoprogenitor cells and in the layer of derived osteoblasts. High levels of oviduct receptor were also observed in the primordia of the menisci. Metabolic labeling of proteins secreted by cultured chondrocytes and osteoblasts and Northern blot analysis of RNA extracted from the same cells confirmed and completed the above information. Ovotransferrin was expressed by in vitro differentiating chondrocytes in the early phase of the culture and, at least when culture conditions allowed extracellular matrix assembly, also by hypertrophic chondrocytes and derived osteoblast-like cells. Osteoblasts directly obtained from bone chips produced ovotransferrin only at the time of culture mineralization. By Western blot analysis, oviduct receptor proteins were detected at a very low level in extract from differentiating and hypertrophic chondrocytes and at a higher level in extract from hypertrophic chondrocytes undergoing differentiation to osteoblast-like cells and from mineralizing osteoblasts. Based on these results, the existence of autocrine and paracrine loops involving ovotransferrin and its receptor during chondrogenesis and endochondral bone formation is discussed.

Animals↗