L. Vakaet

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 Hensens 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.

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

SummaryPrimitive 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 in 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.

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

SummaryThe 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.

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.

SummarySEM 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.

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.

The influence of tissue handling before fixation on the morphology of the chick blastoderm

The morphology of cells and tissues of chick blastoderms handled before fixation in different ways, was compared. Blastoderms were fixed in ovo or after mounting on a glass ring (New, 1955). Differences in the morphology were observed.

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

SummaryThe 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.