Jane Overton

Desmosome development in normal and reassociating cells in the early chick blastoderm.

Abstract Desmosomes have been studied in the 1–2-day chick blastoderm in a strip just lateral to the embryonic axis in normal development and in reaggregating cells. A correlation was found between the time at which desmosomes appear and the degree of cohesion of developing epithelial sheets. Desmosomes in ectoderm, mesoderm, and endoderm are similar in structure. Development of fine structure appeared to be as follows: first, an electron-dense region in and between cell surfaces; second, condensation of desmosomal plaques; and third, development of an extensive fibrillar system. In reassociating cells, stages appearing to be parallel to those in normal development could be found. In addition, in newly dispersed cells intracellular desmosomal components appeared very frequently in channels or vacuoles within the cell, and after reassociation misaligned desmosomes occasionally occurred at adjacent cell surfaces where aligned desmosomes were also present.

Formation of junctions and cell sorting in aggregates of chick and mouse cells

Abstract The frequency of desmosome formation was examined in aggregates of old cells, which form many junctions, combined with young cells, which form few. Cells of chick corneal epithelium and mouse epidermis, which can be distinguished morphologically, were combined. Desmosomes between these cell types are stable. Further, young cells make more desmosomes than they otherwise would on those surfaces adjoining old cells. Desmosomes increase in number in aggregates while cell sorting is occurring. Cells consistently sort, with those which form most desmosomes lying internally. Gap junctions and intermediate junctions are also present, but are uncommon. A carbohydrate cell-surface coat has regenerated by the time desmosome formation starts. The possible relation of desmosome formation to cell sorting is discussed.

Response of epithelial and mesenchymal cells to culture on basement lamella observed by scanning microscopy

The basement lamella of Xenopus tadpole skin has been viewed in situ by scanning microscopy, then isolated by trypsin treatment and used as a substrate for cell culture. The basal lamina may also be viewed after EDTA treatment. Responses of epithelial and mesenchymal cells to the lamella have been compared. Mesenchymal cells from chick skin and heart ventricle flatten and attach between the plies of the lamella, then infiltrate it. Myoblasts appear to move less readily within the lamella. Embryonic Xenopus skin epithelium spreads over the surface. Isolated chick skin epithelial cells first begin to spread, then round up and eventually attach to each other in clusters which form a flat basal surface above the lamella. Thus epithelial and mesenchymal cells cultured on this isolated extracellular material mimic aspects of normal tissue organization.

Scanning electron microscopy of chick epiblast expansion on the vitelline membrane. Cell-substrate interactions.

Abstract Cell-substrate interactions have been studied by examining migrating edge cells of the expanding chick extraembryonic epiblast on their normal substrate and in culture. Scanning electron microscopy shows that the outer face of the vitelline membrane is a random meshwork of fibrils (80 nm diam). The inner face, which is the normal substrate of epiblast expansion, is composed of a random branched system of fibers (400 nm diam) overlain by a network of fibrils (40 nm diam). The epiblast edge in situ has radially oriented filopodia (20 μm long, 200 nm diam.), frequently extending from broad lamellipodia. Blastoderms cultured on the inner face of unincubated vitelline membrane expand at a normal rate but display ruffles as well as filopodia and lamellipodia. When the blastoderm is cultured on the outer membrane face there is no expansion, but cells leave the edge and migrate across the membrane. In these cultures, ruffles are observed on the ventral epiblast face. Absence of the mass of yolk in culture appears to permit or provoke the observed ruffling. Comparison of dissociated epiblast edge cells and skin epithelial cells, cultured on glass and on the vitelline membrane inner face, indicates that epiblast cells remain flattened and display characteristic filopodia on both substrates, whereas skin cells display ruffles on the vitelline membrane but are flattened on glass. The mode of migration of epiblast edge cells seems to be more dependent on intrinsic factors than that of skin cells.

Chapter 1 Experiments with Junctions of the Adhaerens Type1

Publisher Summary This chapter describes some recent findings concerning the structure of adhaerens junctions, followed by the consideration of what is known of their development, their fate upon cell isolation, and after recombination of the cells of like and of unlike types. The desmosome or macula adhaerens exists in its mature form typically as a symmetrical structure spanning two cells and, therefore, requires coordinated activity in the adjacent cells for its formation. In addition to the relatively precise and symmetrical structure of the desmosome itself, this adhesive junction as well as the junctions of the intermediate type termed zonulae or fasciae adhaerentes have the advantage that the cytoplasm of the cell may be more or less extensively organized in close relation to these sites in an obvious way. Groups of the junctions on the cell surface are present in characteristic complexes and may be intimately related. There are a few particularly striking examples of the positioning of the junctions in relation to the polarity of the tissue. The corneal epithelium of the chick is suitable material for work on desmosome development, because at a given stage, there is a marked increase in junction formation over a relatively short period.

Differential response of embryonic cells to culture on tissue matrices

Cell responses to different natural substrates have been followed by scanning microscopy in order to evaluate the role of these substrates in morphogenesis. Matrix has been isolated then repopulated with suspensions of embryonic cells from chick skin, spinal ganglia, duodenal epithelium and heart. In some cases outgrowth from amphibian embryonic tissue was used. Basal lamina of the Xenopus tail may be exposed by freezing and thawing the tissue, or by EDTA treatment. The underlying lamella of orthogonally oriented collagen fibers may be exposed by use of trypsin or hyaluronidase. Trypsin causes more clumping of collagen fibers and a coarser texture of the matrix. On trypsin isolated basement lamella, nerve cell processes grow out on the surface and show no strong tendency to penetrate the lamella while skin mesenchymal cells commonly burrow among the collagen plies. Epithelial cells remain on the surface. On the basal lamina mesenchymal cells ruffle in early stages of culture, then flatten. Epithelial cells flatten rapidly on the lamina. These differences in cell response are in some cases closely related to cell behavior in vivo and suggest that cells show a selective response to the chemical composition of the substrate as well as to its physical conformation.

The development and fine structure of centrifuged eggs of Chironomus thummi

Abstract A total of 630 eggs of Chironomus thummi were centrifuged at 8000 g for 10 minutes, and their subsequent development was observed. Of these eggs 509 survived. Eggs were centrifuged with either the anterior or the posterior end in the centrifugal position. The majority of the eggs developed normally, but about 20% of the time, double abdomen or double cephalon was produced, confirming the observations of Yajima (1960) . The two types of duplications were produced irrespective of the orientation of the egg. In eggs centrifuged with the posterior end directed centrifugally, the number of duplications was ten times as great as with the opposite orientation. The fine structure of the cortical region of normal and centrifuged eggs was examined. All the components of this region exclusive of the plasma membrane appeared to be shifted by centrifugation. These results are discussed in connection with conflicting observations and it is concluded that cytoplasmic regions controlling development of body axes are shiftable with centrifugation.

Organization of the migrating chick epiblast edge: Attachment sites, cytoskeleton, and early developmental changes

Abstract The radial expansion of the chick extraembryonic epiblast on the inner side of the vitelline membrane in yolk sac formation provides a useful system for study of adhesion and migration of an epithelial cell sheet. A band of specialized cells at the epiblast edge adheres by its dorsal side to the overlying vitelline membrane. The attached edge was examined by scanning electron microscopy. The attachment region (av 0.06 mm wide) extends from the advancing edge to a transitional ridge. The ridge appears to be an area of adhesion and de-adhesion. The attached surface is smooth with small surface projections and filopodia. These become more numerous and prominent with cold treatment. Epiblast cells display a filopodial/lamellipodial mode of migration in vivo and in vitro. The distribution of 4- to 7-nm microfilaments in edge cells is examined using transmission electron microscopy of whole cells. Decoration with heavy meromyosin shows that these components of the cytoskeleton contain actin. Treatment of intact blastoderms and dissociated edge cells with cytochalasin B and cold suggests that microfilaments rather than microtubules are primarily responsible for edge cell morphology. Early blastoderm cells which have not initiated migration respond to cytochalasin B, cold, and colcemid in the same way as migrating edge cells. This suggests that the differentiative change that produces the rapidly migrating edge cells does not involve a shift in the relative contribution of microtubules and microfilaments to the cytoskeleton.

Scanning electron microscopic visualization of collagen fibers in embryonic chick skin

Abstract The disposition of collagen fibers in embryonic chick skin can be visualized by use of scanning microscopy (SEM). Chick back skin was removed from 8-day embryos, the epithelial and mesenchymal components were separated, and the mesenchyme was subjected to 10% trypsin treatment (Stuart, E. S., and Moscona, A. A. (1967) Science 157, 947–948), after which it was prepared for SEM by critical point drying and coating. Such preparations were largely free of cellular material. Cavities which presumably had contained the cells were present in a network of fibers. Skin of the scaleless mutant was also studied. In this mutant the collagen network was more irregular and collagen fiber diameter was more variable. These findings are discussed in connection with the formation of feather germ pattern.

Inhibition of desmosome formation with tunicamycin and with lectin in corneal cell aggregates.

Abstract Desmosome formation in chick corneal epithelium can be used as a tool to study cell interaction. In the present work, the possible role of carbohydrates in initiating junction formation is investigated. When corneal cells of 15-day chicks are dispersed, then aggregated in rotating medium, desmosomes form on a regular schedule and can be quantitated as desmosomes per micrometer of cell membrane cross section. Tunicamycin, at 0.05 μg/ml has little effect on incorporation of leucine into TCA-precipitable cell fractions, but lowers mannose incorporation, and inhibits desmosome formation. The inhibition is counteracted by the presence of leupeptin, a protease inhibitor. This finding can be interpreted as favoring a role of stabilization, rather than recognition for the carbohydrate moiety. Cytochalasin B inhibits cell surface turnover in isolated cells and enhances desmosome formation. Enhancement occurs even in the presence of tunicamycin. Ferritin-conjugated succinyl-Con A will label surfaces of freshly dispersed cells and when cells are aggregated in its presence, the label is internalized in cytoplasmic vacuoles and desmosomes do not form. To test further the possibility that a lectin binding component of the surface may be a specific recognition factor, cells were aggregated in the presence of a number of sugars. None inhibited junction formation. Thus, evidence favors a stabilizing role for carbohydrates, acting at some point in the process of junction formation and leaves open the possibility that a “recognition” function may also be involved.