Nadine C. Milos

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.

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.

The effects of various nutritional supplements on the growth, migration and differentiation ofxenopus laevis neural crest cells in vitro

SummaryThis study investigates the nutritional requirements ofXenopus laevis neural crest cells and melanophores developing in vitro. A comparison is made between the growth and differentiation of cells in serum-containing medium and a chemically defined, serum-free medium that we have designed. Our chemically defined medium is more efficient than serum-supplemented medium in promoting proliferation of these cells. Several supplements are required to enhance culture development. These include insulin, α-melanocyte stimulating hormone, somatotropin, luteotrophic hormone, linoleic acid, uridine, and putrescine. In addition, collagen and fibronectin provide the most conductive environment tested for cell migration and 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.

Involvement of endogenous galactoside-binding lectin of Xenopus laevis in pattern formation of Xenopus neurites in vitro.

Galactoside-binding lectin has been purified from Xenopus laevis embryos at the stage of neural crest migration. Addition of this lectin to neurite cultures correlates with the appearance of fascicles of greater diameter and shorter length compared with controls. Lectin-treated neurites are also more spread out on the substratum than their controls. The potent hapten inhibitor of the endogenous lectin, thiodigalactoside (TDG), was also added to these cultures. TDG-treated neurites are less well spread out than the controls; fascicle diameters and lengths are not altered. These results suggest that galactoside-bearing receptors and endogenous galactoside-binding lectin are present in these neurites and can participate in controlling neuronal morphogenesis in vitro, although to differing extents.

The different effects on cranial and trunk neural crest cell behaviour following exposure to a low concentration of alcohol in vitro

Embryonic neural crest cells give rise to large regions of the face and peripheral nervous system. Exposure of these cells to high alcohol concentrations leads to cell death in the craniofacial region resulting in facial defects. However, the effects of low concentrations of alcohol on neural crest cells are not clear. In this study, cranial neural crest cells from Xenopus laevis were cultured in an ethanol concentration approximately equivalent to one drink. Techniques were developed to study various aspects of neural crest cell behaviour and a number of cellular parameters were quantified. In the presence of alcohol, a significant number of cranial neural crest cells emigrated from the explant on fibronectin but the liberation of individual cells was delayed. The cells also remained close to the explant and their morphology changed. Cranial neural crest cells did not grow on Type 1 collagen. For the purposes of comparison, the behaviour of trunk neural crest cells was also studied. The presence of alcohol correlated with increased retention of single cells on fibronectin but left other parameters unchanged. The behaviour of trunk neural crest cells growing on Type 1 collagen in the presence of alcohol did not differ from controls. Low concentrations of alcohol therefore significantly affected both cranial and trunk neural crest cells, with a wider variety of effects on cells from the cranial as opposed to the trunk region. The results suggest that low concentrations of alcohol may be more detrimental to early events in organ formation than currently suspected.

Localization of endogenous galactoside-binding lectin during morphogenesis of Xenopus laevis.

SummaryA monoclonal antibody has been produced againstXenopus laevis galactoside-binding neural-creststage lectin. This antibody inhibits lectin-mediated hemagglutination. Using this antibody in conjunction with immunohistochemical techniques, lectin deposition has been studied in embryos and tadpoles at different stages of morphogenesis, from initial neural crest migration, up to the formation of a swimming tadpole. Lectin levels change during development in different regions of the embryo and tadpole, decreasing in migratory cells, and increasing in sites where cells become more adhesive to one another. The results suggest that galactoside-binding lectins may be an important class of cellular adhesion molecules during these stages of development.

Release of β-d-galactoside-binding lectins into the cavities of aggregates of chick extraembryonic endoderm cells

Dissociated cells from the extraembryonic endoderm of gastrulating chick embryos form aggregates when cultured in rotating flasks. The large cellular aggregates are initially solid but subsequently cavitate to form hollow, thin-walled vesicles. These cells also contain an endogenous beta-D-galactoside-binding lectin. Previous work has shown that high extracellular concentrations of this lectin are associated with decreased cell-cell adhesion [Milos, N. and S.E. Zalik: Differentiation 21, 175-182 (1982)]. We have removed the fluid contents from aggregates cultured for 24 and 48 h and tested them for the presence of lectin activity. The results demonstrate that lectin activity is detectable in a higher number of aggregates cultured for 24 as opposed to 48 h (75% vs. 28%, respectively). The lectin activity per aggregate is also higher in aggregates cultured for 24 h (180 vs. 67 hemagglutinating units, respectively, for 24- and 48-h aggregates). Thus, at the time when cells are moving apart from one another during aggregate cavitation, detectable lectin activity is released into the vesicular contents of the aggregate.