Sara E. Zalik

On the possible role of endogenous lectins in early animal development

SummaryIn this review I have tried to summarize the information available on the lectins of developing embryos. The emerging evidence indicates that during fertilization carbohydrate-binding proteins play a role in sperm adhesion and in the reorganization of the extracellular matrix of the fertilized egg. Results also indicate that in adult tissues lectins participate in cell recognition and adhesion, and that several galactose-binding lectins function as receptors for laminin and, in principle could also interact with polylactosamine groups of other extracellular matrix glycoproteins. Since in developing embryos lectins are located at the cell surface, and colocalize with extracellular matrix glycoproteins, they could play a role in transitory adhesive interactions and in the segregation of organ primordia. On the basis of experiments in cultured cell lines, it has been suggested that lectins are involved in lysosomal and nuclear glycoprotein transport. These carbohydrate-binding proteins could also regulate development by modulating these processes in the embryo. Since galactose-binding lectins are mitogenic, and are present in high concentration in the chick yolk sac, these proteins could be released into the embryonic circulation, bind to cells expressing appropriate receptors, and act as growth regulators, by modulating cell division of specific cell lineages.

The blastomere periphery ofXenopus laevis, with special reference to intercellular relationships

SummaryThe peripheral region of theXenopus laevis blastomere is considered to be comprised of the extracellular material, the plasma membrane and the subsurface cytoplasm. These regions were examined with the electron microscope during cleavage and blastula stages (stages one to seven, Nieuwkoop and Faber, 1967). The cell contact relationships varied according to the location of the cell in the embryo. Superficially, a dense terminal junction occurred, possessing point contacts where the membranes approached to within 30 Å. More deeply, a variety of relationships appeared: wide intercellular spaces bridged by pseudopodia, long regions of unbridged parallel membrane or complex interdigitation. Tight junctions were found in limited numbers and developed at about stage seven. Extracellular material was examined using histochemical techniques on dissociated andin situ cells. The latter had appreciable amounts of such material, but dissociated cells reacted inconsistently to different techniques. The cytoplasm subjacent to the membrane possessed a filamentous network at all stages examined, but extensive microfilament tracts and microtubules appeared only at gastrulation.

Microelectrophoresis of early amphibian embryonic cells

Abstract Cells of different areas of amphibian embryos from blastula to early neurula stages were separated by density gradient centrifugation and their electrophoretic mobility (EPM) was measured. Differences in EPM were present between cells derived from different areas of the embryo. Cells of the marginal zone show the greatest increase in mobility between early and midgastrula. Vegetal pole cells had lower mobilities when compared with cells of other embryonic regions. Differences in EPM were found between stages. Early gastrula cells had the lowest mobility of any stage while those at midgastrula attained the highest values. Marginal zone cells showed the greatest increase in mobility between early and mid gastrula.

Distribution of two β-d-galactoside-binding lectins in the gastrulating chick embryo

We report that two lectin activities specific for β-d-galactoside groups can be isolated from gastrulating chick blastoderms. A soluble lectin LI(s) is associated with a protein that gives rise to a band of Mr 11 000 in SDS-PAGE electrophoresis. A second particle-associated lectin LII(p) is associated with a doublet band with Mr values of 70 000 and 72 000. The soluble lectin is present in tissue from the area pellucida and the area opaca. The particle-associated lectin activity is present mainly in the epiblast and endoderm of the area opaca, the highest specific activity being found in the endoderm cells.

Growth of newt iris epithelial cells in vitro: A study of the cell cycle☆

Abstract An in vitro system has been developed for the study of cellular metaplasia occurring during lens regeneration from the dorsal iris of the newt. Epithelial cells from the normal iris and the 10-day regenerating iris were used. The behavior and growth of these cells in culture are described. Depigmentation begins prior to mitosis. A small nondividing population of heavily pigmented cells remains in normal and 10-day iris cell cultures. The lag phase is considerably longer in normal than in 10-day cultures. A doubling time of 85 h was found in the 10-day cultures and 150 h in the normal cultures. Exponential growth is due to proliferation of fully depigmented cells. Repigmentation occurs during the stationary phase. The cell cycle was measured by autoradiography. S was 36 h and G 2 6 h in both cultures. In the 10-day cultures the total cell cycle was 69 h and G 1 25 h. It is concluded that at least in regard to cell cycle parameters the in vitro system does not differ greatly from that observed in vivo.

Hyaluronic acid production and hyaluronidase activity in the newt iris during lens regeneration

The process of lens regeneration in newts involves the dedifferentiation of pigmented iris epithelial cells and their subsequent conversion into lens fibers. In vivo this cell-type conversion is restricted to the dorsal region of the iris. We have examined the patterns of hyaluronate accumulation and endogenous hyaluronidase activity in the newt iris during the course of lens regeneration in vivo. Accumulation of newly synthesized hyaluronate was estimated from the uptake of [3H]glucosamine into cetylpyridinium chloride-precipitable material that was sensitive to Streptomyces hyaluronidase. Endogenous hyaluronidase activity was determined from the quantity of reducing N-acetylhexosamine released upon incubation of iris tissue extract with exogenous hyaluronate substrate. We found that incorporation of label into hyaluronate was consistently higher in the regeneration-activated irises of lentectomized eyes than in control irises from sham-operated eyes. Hyaluronate labeling was higher in the dorsal (lens-forming) region of the iris than in ventral (non-lens-forming) iris tissue during the regeneration process. Label accumulation into hyaluronate was maximum between 10 and 15 days after lentectomy, the period of most pronounced dedifferentiation in the dorsal iris epithelium. Both normal and regenerating irises demonstrated a high level of endogenous hyaluronidase activity with a pH optimum of 3.5-4.0. Hyaluronidase activity was 1.7 to 2 times higher in dorsal iris tissue than in ventral irises both prior to lentectomy and throughout the regeneration process. We suggest that enhanced hyaluronate accumulation may facilitate the dedifferentiation of iris epithelial cells in the dorsal iris and prevent precocious withdrawal from the cell cycle. The high level of hyaluronidase activity in the dorsal iris may promote the turnover and remodeling of extracellular matrix components required for cell-type conversion.

Effect of theβ-d-galactoside-binding lectin on cell to substratum and cell to cell adhesion of cells from the extraembryonic endoderm of the early chick blastoderm

SummaryCells from the extraembryonic endoderm of the gastrulating chick embryo contain a β-d-galactoside-binding lectin inhibited by thiodigalactoside (TDG). When cell suspensions are cultured in stationary culture in the presence of exogenously added purified blastoderm lectin or TDG, their attachment to the substratum is delayed and decreased compared to controls. The cells take on a fibroblastic-like morphology and cell to cell contact becomes limited to localized areas of the cell surface. Many lectin or TDG-treated cells appear to be migrating over the substratum. This is in contrast to control cultures where the cells appear epithelial in morphology and tend to maximize their areas of apposition. These data suggest that the endogenous lectin may have a role to play in cell to substratum and cell to cell adhesion.

Distribution of a galactose-specific lectin in endoderm cells from early chick embryos.

SummaryCells from the endoderm of the area opaca of gastrulating chick embryos were maintained in stationary cultures, stained with antibodies against the endogenous β-D-galactoside-binding lectin and examined by immuno-fluorescence. In the majority of cells fluorescence was present as an irregular circular web in the central cytoplasm. In cells that appeared to be migrating increased fluorescence was observed in the peripheral cytoplasm and retraction fibers. In regions where a portion of the cell was detaching from the substratum high fluorescence was observed in the extracellular “footprints” deposited by the cell.

Calcium-independent adhesion of extra-embryonic endoderm cells from the early chick blastoderm is inhibited by the blastoderm β-d-galactoside-binding lectin and by β-galactosidase

Extra-embryonic endoderm cells from gastrulating chick embryos possess Ca2+-dependent and Ca2+-independent adhesive mechanisms. These cells also contain an endogenous beta-D-galactoside-binding lectin and cell surface receptors bearing galactose groups. The endogenous lectin inhibits cellular adhesion. To test whether the adhesive interactions involving lectin and galactose molecules are part of the Ca2+-independent or Ca2+-dependent adhesive mechanism, dissociated cells which were preincubated in beta-galactosidase were allowed to aggregate in the presence and absence of Ca2+ ions. Significant decreases in adhesion were observed in both cases. Cells were also allowed to aggregate in the presence and absence of Ca2+ ions when blastoderm lectin was present in the medium. Adhesion was decreased in both cases. The results suggest that cell surface galactose groups and the beta-D-galactoside-binding lectin are involved in Ca2+-independent adhesion.

Endogenous Lectins and Cell Adhesion in Embryonic Cells

In his classic studies on the amphibian blastula and gastrula, Holtfreter (1939) showed that the segregation of the embryonic germ layers taking place during the morphogenetic movements of gastrulation is accompanied by a change in the mutual affinities of the cells that constitute the presumptive germ layers. That these affinities were the result of discriminatory properties of the cells themselves was shown in later studies, by Townes and Holtfreter (1955). It was found, in recombination experiments, that dissociated cells from different regions of the amphibian gastrula would first adhere to each other in an indiscriminate fashion and then sort out into cell groups, each consisting of a similar cell type. These workers hypothesized that changes in the selectivity of cellular adhesion are due to alterations at the cell surface (see Fig. 27 in Townes and Holtfreter, 1955). Since that time, investigators have devoted considerable time and effort to studying the factors involved in cellular recognition.