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C Vanroelen

Publications and source records attributed to C Vanroelen.

16 recordsLinked to original sources
PubMed1988

Morphological and immunocytochemical studies of fibronectin-coated, plasma membrane-limited vesicles in the early chicken embryo.

Vesticles with a mean outside diameter of 32.8 nm have been observed in the early chicken embryo after fixation with a mixture of glutaraldehyde and tannic acid. Densitometric tracing has revealed that the vesicles are limited by a unit membrane. The presence of complex carbohydrates is suggested by the increased electron density of the vesticles after addition of tannic acid to the fixative. Immunocytochemical staining with a monoclonal antibody directed against chicken cellular fibronectin demonstrated the presence of this glycoprotein along the surface of the vesicles. These results suggest a cellular origin of the vesicles, since their surface shares morphological and biochemical similarities with the cell surfaces of the embryonic tissue layers. Recycling of plasma-membrane vesicles may occur, as vesicles were found in the vicinity of coated vesicles. We postulate that extracellular materials of the cell surface, which may affect cell and tissue interactions, are shed in the environment together with plasma-membrane vesicles. The difficulties encountered in observing the vesicles stems from the facts that an adequate visualization method is necessary and that they are few in number. The latter reason suggests their transient nature. The vesicles probably rapidly disintegrate in the extracellular milieu or are recycled by the cell surface.

Animals↗
PubMed1986

On the presence of proteolytic activity in glycosaminoglycan-degrading enzyme preparations.

A solid-phase protease assay has been used to screen different commercial preparations of glycosaminoglycan-degrading enzymes for the presence of proteolytic activity. Proteases cannot be detected in preparations of testicular hyaluronidase and of chondroitinase at the concentration used for histochemical purposes. Commercial Streptomyces hyaluronidase contains proteolytic contaminants detectable at the concentration used for histochemistry. At higher concentrations, all preparations appear to be contaminated with proteases. The results obtained using this assay suggest that addition of a mixture of proteinase inhibitors containing N-ethylmaleimide, EDTA, pepstatin, and phenylmethanesulfonylfluoride or soybean trypsin inhibitor has little effect on the proteolytic activity of the glycosaminoglycan-degrading enzyme preparations, irrespective of the pH used. Moreover, the use of EDTA in this mixture is questionable. This study also describes two testicular hyaluronidase preparations that may be particularly useful in functional studies of the living organism, as they are only slightly contaminated.

Animals↗
PubMed1985

Fibronectin and its relation to the basal lamina and to the cell surface in the chicken blastoderm.

The ultrastructural distribution of fibronectin immunoreactivity was investigated in the chicken embryo during late gastrulation. Sites of binding of anti-fibronectin antibodies were ascribed to the basal lamina and associated structures, and to the cell surface. The fibronectin-rich basal lamina was resolved into a lamina densa, which appears as a continuous, dense sheet, a lamina lucida, consisting of anchoring cords between lamina densa and epithelial cells, and a lamina intima, closely juxtaposed to the cell surface. Cell-surface labelling was also observed in mesoblast cells, and along the dorsal side of the deep-layer cells. The ventral side of the latter cells was poorly stained in the endophyllic crescent, except in coated pits, and more regularly stained at the level of definitive endoblast. Some structures associated with the basal lamina reacted intensely with anti-fibronectin antibodies. These are the interstitial bodies, which are aggregates of extracellular material, and a kind of fibril or tubule, embedded in a fibronectin matrix and mainly found in the endophyllic crescent. Some intracellular labelling was found in most deep-layer cells, in few epiblast cells, never in mesoblast cells. These results extend previous studies on the localization of fibronectin, and correlate its presence and surface topology with its postulated role in migration of mesoblast cells on the basal lamina which, chemically, constitutes an appropriate substrate.

Animals↗
PubMed1985

Transfer of extracellular matrix components between germ layers in chimaeric chicken-quail blastoderms.

A chemical basis for the transmission of signals during gastrulation has been investigated by using chimaeric embryos resulting from the combination of 3H-glucosamine-labelled and unlabelled hypoblast with epiblast taken from chicken and quail embryos at stage 3 of Vakaet (1970). The ability to distinguish chicken from quail cells on the basis of their different nuclear distribution of heterochromatin after Feulgen staining made it possible to determine the origin of the cells in the chimaerae. Tritiated quail hypoblast (after incubation of the embryo in the presence of 3H-glucosamine) was transplanted onto unlabelled chicken blastoderm deprived of its hypoblast. After culture of the chimaera for 5 h, the autoradiographic pattern shows silver grains not only over the graft, but also at the ventral surface of the epiblast of the host. Transfer of label may occur to mesoblast cells, but not between chicken and quail hypoblast cells. Chase experiments exclude the possibility that unprocessed, tritiated glucosamine is transferred. Chemical fixation of the host before transplantation of a labelled quail hypoblast also allows visualization of a transfer of macromolecules from hypoblast to the basement membrane of the epiblast, suggesting that an intervention of the epiblast cells in this process is not necessary. The morphology of the chimaeric embryos, as studied by scanning electron microscopy, suggests a direct deposition of these macromolecules by filopodia of the dorsal surface of the hypoblast. The possibility of diffusion of free macromolecules has been considered and can reasonably be discarded on the basis of several observations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Masking of antigenic sites of fibronectin by glycosaminoglycans in ethanol-fixed embryonic tissue.

We studied the interaction between glycosaminoglycans (GAGs) and fibronectin in the basement membrane of the epiblast in the chicken blastoderm using testicular-hyaluronidase digestion of GAGs either on fixed tissue sections or in vivo after microinjection of the enzyme preparation prior to immunostaining for fibronectin. In the choice of fixatives, special attention was paid to their preservation of GAGs. The controls included alcian-blue staining of serial sections to test the efficiency of the digestion, and incubations in the presence of protease inhibitors to abolish contaminating proteolytic activity in the commercial hyaluronidase preparations. The results indicate that fixation in solutions which preserve GAGs, i.e. ethanolic solutions or aqueous solutions containing cetylpyridinium chloride, allows the immunocytochemical demonstration of fibronectin in the basement membrane of the epiblast at the level of the endophyllic crescent, but masks this glycoprotein at the epithelial-mesenchymal interface. As shown by both approaches, this masking of immunoreactivity is reversible. Moreover, the in vivo clearance of GAGs before fixation shows that the masking at the epithelial-mesenchymal interface is not an experimental peculiarity due to the use of a particular technique, but is the consequence of an interaction between GAGs and fibronectin in that particular area of the basement membrane that is used by mesoblast cells as a substrate for migration. The observation that fibronectin may be masked by GAGs in ethanol-fixed tissue--a commonly used fixation method--may require the re-evaluation of some negative results mentioned in the literature.

Animals↗
PubMed1985

Inhibition of cell spreading on the band of extracellular fibres in early chick and quail embryos.

The ventral surface of the upper layer shows a band of extracellular fibrils around the anterior and lateral border of the area pellucida during gastrulation of the chick embryo. Using scanning electron microscopy, we found that this disposition is correlated with the motility of the middle-layer cells of gastrulating chick and quail embryos. Outside the fibrous band, single middle-layer cells and a sheet of mesoblast cells were spread out and possessed lamellae. Single cells on the fibrous band did not form lamellae. The same cell behaviour was obtained with the explants of deep layer on the fibrous band. The fibrous band is assumed to operate as a barrier that inhibits cell motility during gastrulation.

Animals↗
PubMed1984

Expression of different regional patterns of fibronectin immunoreactivity during mesoblast formation in the chick blastoderm.

The appearance and distribution of the extracellular material glycoprotein, fibronectin, was investigated in gastrulating chick embryos using affinity-purified anti-human plasma fibronectin antibodies. Preservation of tissue structure and immunoreactivity was carried out by ethanol/acetic acid fixation or by formaldehyde/glutaraldehyde fixation. Using the former fixation method, fibronectin immunoreactivity was detected (1) at the ventral surface of the upper layer or epiblast, mainly anterior and lateral to Hensen's node, in regions where middle-layer or mesoblast cells are not yet present, and (2) sparsely in extracellular spaces of the deep layer. Using the latter fixation method, fibronectin immunoreactivity was, moreover, found at the entire ventral surface of the upper layer, i.e., also at the epithelial-mesenchymal interface, where a basement membrane was previously described. At the light microscope level, we could not detect significant immunoreactivity in the middle layer. Treatment of sections of ethanol-fixed blastoderms with testicular hyaluronidase before immunostaining for fibronectin partially demasked the antigenic sites of this glycoprotein at the epithelial-mesenchymal interface. The present report indicates that the different regional patterns of fibronectin immunoreactivity in the basement membrane of the upper layer are spatially and temporally correlated with migration and positioning of mesoblast cells. These regional patterns are probably due to differences in the composition of fibronectin-associated material such as chondroitin sulfate A and/or C proteoglycans, and/or hyaluronate, before and after mesoblast expansion, rather than to differences in the distribution of fibronectin itself. In this respect. In this respect, it is noteworthy that the chemical composition of the basement membrane of an epithelium changes as mesenchyme cells migrate over it. The results also favor the idea that fibronectin is a structural component of the whole basement membrane which is used as a substrate for migration of mesenchymal cells.

Animals↗
PubMed1983

The relationship between the presence of cysteine lyase in the yolk sac endoderm and the disposition of the area vasculosa in the chicken blastoderm.

Two series of experiments were carried out to test the possible relationship between the presence of cysteine lyase and area vasculosa in the chicken blastoderm. In the first series, the localization of cysteine lyase was compared with the disposition of the area vasculosa at the successive stages of gastrulation and neurulation. In definitive streak stage chicken blastoderms, the enzyme first appeared laterally at the area pellucida-area opaca border. At older stages, this positivity extended rostrally and only finally did it appear in the caudal area. Based on these in toto observations, the relationship with area vasculosa, the distribution of which has been studied by Settle ('54), seems only incidental. In the second series, intermediate streak stage blastoderms were transversely dissected so that the rostral part did not contain any mesodermal component. The presence of cysteine lyase in this rostral part after 15 hr of culture suggests that the appearance of cysteine lyase in the yolk sac endoderm is not dependent upon the presence of area vasculosa. Moreover, the results suggest that the yolk sac endoderm of the chicken blastoderm is determined to produce cysteine lyase from the intermediate streak stage on.

Animals↗
PubMed1983

The subgerminal yolk surface and its relationship with the inner germ wall edge of the stages X to XIV chick and quail embryo. A SEM study.

SEM reveals that the subgerminal yolk surface of the chick and quail embryo during stages X to XIV possesses microvilli and small pits. Threadlike extensions and globular structures are found on the subgerminal yolk surface mainly in the central area. The cellular nature of the subgerminal yolk was confirmed with TEM that showed the presence of a plasma membrane, mitochondria, micro-invaginations and microfilaments. The ventral cells of the germ wall are yolky and can be attached to the subgerminal yolk surface with filopodial extensions during stages X to XII. From stage XIII, the shape of these cells is usually more flattened and they protrude lamellae and filopodia.

Animals↗
PubMed1983

The dorsal surface of the animal pole of the just laid quail egg, studied with SEM.

The epiblast and the surface of the perigerminal yolk of the just laid quail blastoderm were examined by scanning electron microscopy (SEM). The dorsal surface structures are microvilli, mainly along the cell borders. The scarcity of the dimples does not support that ingression occurs at this stage. Flat or round cells on the epiblast are possibly deep layer cells that failed to incorporate after passing through the epiblast. The majority of the blastoderms have an irregular margin. The large edge cells possess microvilli at their borders only. A few blastoderms, probably the more developed, have a smooth edge with closely packed cells. The margin of these germs shows round cells and lamellae that could be protruded by deep cells. The process of cell rounding and extension of lamellae may be the onset of the formation of the margin of overgrowth. Concentric zones are present on the surface of the perigerminal yolk, on which microvilli and blebs are found near the germ. The presence of cell projections on the perigerminal surface suggests its living nature.

Animals↗
PubMed1982

The effects of partial hypoblast removal on the cell morphology of the epiblast in the chick blastoderm.

The hypoblast of early-primitive-streak-stage chick blastoderms was partially removed. This experiment provokes a reaction in the epiblast which curls up and becomes even at its ventral surface. The basal lamina underlying the epiblast is also dependent upon the presence of hypoblast. During culture after partial hypoblast removal, active hypoblast wound healing is observed. Where the hypoblast underlies the epiblast again, the effects of the removal disappear and normal development proceeds. The results suggest that the normal epiblast morphology is dependent upon the presence of hypoblast. This influence of hypoblast on epiblast is thought to be concerned with the morphology of the epiblast and not directly with its morphogenesis.

Animals↗
PubMed1981

Resolution in light microscope autoradiography using a carbon-14 labelled line source.

Light microscope autoradiographs were prepared from a 14C line source Two factors that affect resolution were studied: emulsion thickness and section thickness. The distribution around the line source was determined using the half-distance (HD) value to quantify the resolution. An increase in HD value was observed with thicker sections or emulsion layers. The shape of the curve reflecting the grain density distributions around these line sources was very similar. After normalization in HD and relative grain density units, an average distribution was calculated and compared with the shape of normalized density distributions obtained from electron microscope autoradiographs. Other than a discrepancy near the source, an acceptable correlation was observed.

Autoradiography↗
PubMed1980

Alcian blue staining during the formation of mesoblast in the primitive streak stage chick blastoderm.

The distribution of alcian blue (AB) positivity, and its sensitivity to streptococcal and testicular hyaluronidase, were studied in primitive streak stage chick blastoderms. Accumulation of hyaluronate was observed in deep layer (DL) cells and on laterally migrating middle layer (ML) cells. During the formation of the middle layer, a first stage, namely de-epithelialization of the upper layer cells, is recognized and correlated with the absence of hyaluronate. A second stage, namely migration of the de-epithelialized upper layer cells laterally to the edge of the area pellucida, is correlated with the accumulation of AB-positivity. The AB-staining also demonstrated the accumulation of both sulphated and not-sulphated mucopolysaccharides, where a basal lamina is present.

Alcian Blue↗
PubMed1980

Distribution and turnover of testicular hyaluronidase sensitive macromolecules in the primitive streak stage chick blastoderm as revealed by autoradiography.

Primitive streak stage chick blastoderms were cultured for 30 min on a medium containing tritiated glucosamine. Light microscope autoradiography revealed extracellular labeling, and pretreatment of the sections with testicular hyaluronidase suggested the glycosaminoglycan nature of the labeled products. After incorporation of the tritiated precursor, some blastoderms were transferred to a chase medium, and cultured for 30, 90, 210 min. The changes is distribution of the labeled testicular hyaluronidase-sensitive macromolecules during the chase experiment illustrated the ingression of cells in the primitive streak stage chick blastoderm. Grain density differences, resulting from the various chase periods, suggested the renewal of the testicular hyaluronidase-sensitive fraction.

Animals↗
PubMed1980

Localisation and characterization of acid mucopolysaccharides in the early chick blastoderm.

Acid mucopolysaccharides in the extracellular compartment of early chick blastoderms (16 h of incubation) were labelled with tritiated glucosamine and/or [35S]sulphate. The incorporation pattern was studied autoradiographically. Treatment with testicular hyaluronidase revealed a testicular hyaluronidase-sensitive fraction, mainly at the periphery of Middle Layer and Deep Layer cells, and a testicular hyaluronidase-resistant fraction, mainly at the ventral side of the Upper Layer. A biochemical analysis, utilizing chondroitinase ABC and nitrous acid, followed by cellulose acetate electrophoresis, demonstrated the synthesis of a non-sulphated fraction, i.e. hyaluronic acid and/or chondroitin, and a sulphated fraction, comprising two undersulphated components, i.e. chondroitin sulphate, and heparan sulphate or heparin. The appearance of different AMPS in specific areas of the early chick blastoderm is regarded as an early specialization of the extracellular compartment.

Animals↗