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Biomedical subjects

L Vakaet

Publications and source records attributed to L Vakaet.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1984

Microinjection in the chick blastoderm. An improved method to study the extracellular matrix in the living organism.

A microinjection technique for the chick blastoderm is described. With a micropipette attached to a de Fonbrune micromanipulator, 25-45 nl of a reagent was injected into the entophyllic crescent of a chick blastoderm explanted in vitro according to New [7]. This procedure offers the advantage of eliminating the concentration variability which was observed after subblastodisc injection, and in contrast to the in ovo techniques, it allows one to stage the blastoderms properly. To check its applicability, testicular hyaluronidase was injected. On the basis of morphological and histochemical observations we ascertained that the experimental procedure itself did not interfere with the results. This method may provide a reliable experimental procedure with which to study the interactions between several macromolecules and the tissues during morphogenesis.

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 relationship between the folliculo-stellate network and the thyrotropic cells of the avian adenohypophysis.

The folliculo-stellate network of the avian adenohypophysis consists of stellate cells surrounding colloid-containing follicular cavities into which cilia and microvilli project. Other identifying criteria are agranularity, junctional complexes at the apical pole, presence of cytoplasmic processes ramifying between adjacent secretory cells, and close appositions of plasma membranes linking folliculo-stellate cells and presumptive thyrotropic cells. Transmission electron microscopy reveals that TRH and L-DOPA induce simultaneous ultrastructural changes in the folliculo-stellate network and in the thyrotropic cells. TRH transforms at cell of the cephalic lobe into a highly hypertrophic cell in which enlargement of cisterns of rough endoplasmic reticulum containing secretory granules, development of a large Golgi complex, presence of newly synthesized secretory granules, and granulation of the cytoplasm are the main features. In the meantime, the follicular cavities become dilated by large amounts of homogeneous colloid. The administration of L-DOPA also leads to the development of dilated cisterns in presumptive thyrotropic cells of the cephalic lobe. Intracisternal granules, immature secretory granules, and large Golgi complexes, however, are not observed. Degranulation of the cytoplasm is obvious. The follicular cavities of both cephalic and caudal lobes are enlarged and filled with colloid in which granular elements are noted. The ultrastructural changes observed in thyrotropic cells and in the folliculo-stellate network reflect functional changes induced by the experimental manipulation. These changes may be related, directly or indirectly, or completely independent.

Animals↗
PubMed1981

Peripheral avian yolk assemblage and its persistence in the blastoderm, studied by trypan blue-induced fluorescence.

Shortly after subcutaneous or intraperitoneal injection of nontoxic quantities of trypan blue into laying Japanese quails, red fluorescent yolk granules appear in the peripheral ooplasm of their oocytes at the end of the lampbrush stage or subsequently. Later a red fluorescence can be observed in the apical cytoplasm of the granulosa cells. The results obtained by this method confirm our previous results (Callebaut 1979) obtained by autoradiography after 3H-leucine administration and furnish interesting additional data. The trypan blue-induced fluorescence method gives a good indication of the permeability of the oocytal cortex and its derivative the germinal disc. The avian yolk which is, or has been peripherally assembled (primordial, true white and yellow yolk) can be characteristically labelled by the administration of trypan blue. The injection of higher, still nontoxic quantities of trypan blue has a prolonged "retarding" effect and permits the marking of a broader part of the germinal disc or eventually of the blastoderm which develops from it.

Animals↗
PubMed1981

Microspectrographic analysis of trypan blue-induced fluorescence in oocytes of the Japanese quail.

It was shown that the vital dye trypan blue injected subcutaneously is adsorbed on exogenous yolk and stored in oocytes of Japanese quails. The binding sites of the dye could be visualized by fluorescence microscopy. The spectral distribution of the trypan blue-induced fluorescence emitted by yolk granules was analyzed microspectrographically. The analysis revealed that yolk granules exhibit a deep red fluorescence radiation with a maximum intensity at 670 nm, when blue or green excitation light is used. This fluorescence was exclusively induce by the presence of trypan blue, and not by contaminants of the dye. The fluorescence intensity did not decrease during processing of the tissue throughout the different solvents routinely used in light microscopy, especially after fixation in Heidenhain's fluid, nor did it suffer from pronounced fading during irradiation of the tissue. Model experiments showed that the value of the fluorescence emission maximum was concentration-dependent, and that amounts as little as 5 x 10(-3) mg trypan blue per ml solution containing an excess of yolk as a substrate for the dye, could clearly be detected and measured. It is suggested that a highly diluted solution of trypan blue can be used without teratogenic effects, as a tracer for exogenous yolk uptake and migration into oocytes, and that fluorescence microscopy is a reliable method for its further localization. A detailed account of the procedure is reported.

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

Microspectrograpic analysis of formaldehyde-induced fluorescence in the quail adenohypophysis after injection of L-dopa and 5-hydroxytryptophan.

The spectral distribution of the formaldehyde-induced fluorescence emitted by model solutions and by adenohypophpyses after intraperitoneal injection of L-dopa or 5-hydroxytryptophan was analyzed microspectrographically. Based on previously reported studies and on present findings, it seems that dopamine is stored in the strongly fluorescent cells after injection of L-dopa, and that a compound closely related to 5-hydroxytryptophan. A non-specific, granular fluorescence appeared after 5-hydroxytryptophan and, to a lesser extend, L-dopa treatment. It probably represents autofluorescence of lysosomes, which are numerous in these circumstances.

5-Hydroxytryptophan↗
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↗