T. S. Okada

The calcium-dependent cell-cell adhesion system regulates inner cell mass formation and cell surface polarization in early mouse development.

The monoclonal antibody ECCD-1 inhibits Ca2+-dependent cell-cell adhesion in teratocarcinoma cells, recognizing a cell surface component of MW 124,000. When mouse embryos at various preimplantation stages were cultured in the presence of ECCD-1, the compacted morphology of the 8- to 16-cell-stage embryos was destroyed. In these embryos, cell proliferation normally occurred and development of blastocyst-like vesicles was attained. However, these embryos did not contain inner cell mass (ICM). We found that ECCD-1 affects the pattern of polarization of the cell surface in late 8- and 16-cell-stage blastomeres, as detected by staining with fluorescence-labeled concanavalin A. In normal blastomeres, the pole was always apart from the cell-cell contact plane. In those cultured in ECCD-1, formation of the pole tended to be inhibited, and if observed, the pole was close to the cell-cell contact plane. We discuss the possible mechanisms for inhibition of ICM formation caused by blocking Ca2+-dependent cell-cell adhesion between blastomeres.

Differentiation of lens in cultures of neural retinal cells of chick embryos.

Abstract When dissociated cells of neural retinae of 8-day-old chick embryos were cultured, monolayer sheets of epithelial cells were obtained. These cells proliferated actively. After about 30 days of culture, both lentoid bodies and pigment cells were differentiated in all plates. In the second and the third generation cultures, both differentiations were also observed. Lentoid bodies showed positive immunofluorescence for fluorescein-isothiocyanate-conjugated antiserum against δ-crystallin. Molecular constituents of lentoid bodies were very similar to those of lenses developing in situ , as revealed by immunodiffusion tests. Several lines of evidence for the “neural retinal” origin of lentoid bodies, as opposed to their being derived from lens cells inadvertently included in the original culture inocula are given. Some implications of the present results for the problem of “determination” are discussed.

Selective adhesion of embryonal carcinoma cells and differentiated cells by Ca2+-dependent sites☆

Abstract The specificity of adhesion between embryonal carcinoma cells and fibroblastic cells of various origins was studied. Embryonal carcinoma cells have intercellular adhesion sites requiring Ca2+ (CDS). These sites were found to be sensitive to proteases but resistant to them in the presence of Ca2+. CDS with a similar protease sensitivity is present in fibroblastic cells. When embryonal carcinoma cells of different lines were mixed, they adhered to each other nonselectively by CDS. Nonselective adhesion by CDS occurred also between fibroblastic cells of various lines. When embryonal carcinoma and fibroblastic cells were mixed, they preferentially adhered to homotypic cells. Fab fragments of antibodies raised against F9 cells (a nullipotent line of embryonal carcinoma) inhibited the adhesion between embryonal carcinoma cells but not between fibroblastic cells. This inhibitory activity of Fab was absorbed with embryonal carcinoma cells with CDS, but not with fibroblastic cells with CDS or embryonal carcinoma cells from which CDS was experimentally removed. SDS-polyacrylamide gel electrophoresis of radioiodinated cell surface proteins showed that the presence of a 140K-dalton component correlated with the presence of CDS in embryonal carcinoma cells, while the presence of a 150K-dalton component correlated with the presence of CDS in fibroblastic cells. These results suggest that CDS in embryonal carcinoma and fibroblastic cells comprise distinct molecules.

THE EXPRESSION OF DIFFERENTIATION BY CHICKEN LENS EPITHELIUM IN IN VITRO CELL CULTURE

Dissociated cells of the lens epithelium of newly hatched chickens were cultured in vitro to investigate whether cells actively grown in culture retain their own differentive entiative traits to form lens fibers. After an exponential growth phase of the flattened epithelial cells, a number of “islets” of smaller epithelial cells with polygonal shape appeared. Along the periphery of these islets, the characteristic morphological change which leads to the formation of spherical bodies was observed. Electron microscopic observation showed the differentiation of lens fibers in these spherical bodies comparable to those in the lens in situ. Accumulation of δ‐chrystallin was confirmed in such “lentoid” bodies. Outgrowth of the lens epithelial cells was maintained in in vitro culture up to about 50 days with several subculturings. The formation of lentoid bodies occurred in each subculture generation, which started from a homogeneous population of flattened epithelial cells. The present culture conditions permit the maintenance of such a population of cells that have a high growth potential and stably retains their differentiative trait to form lens fiber, even after repeated replication under in vitro conditions.

Experimental manipulation of cell surface to affect cellular recognition mechanisms

Abstract The mechanism of selective cell adhesion was studied using Chinese hamster V79 and chick embryonic neural retinal cells. Both of these cell types have been shown to have two experimentally separable mechanisms of adhesion; Ca 2+ -dependent and Ca 2+ -independent. Cells can be dispersed so that either or both of the mechanisms remain intact by use of different treatments. A method of labeling cells with FITC was devised to identify one of the two types of cells in a binary cell population. When cells with one of the two adhesion mechanisms were mixed with cells with the other mechanism, they segregated completely, forming independent aggregates, not only in the heterotypic combination of these cell types but also in the homotypic combination of each cell type. In contrast, when cells were mixed with others with the same adhesion mechanism, either Ca 2+ -dependent or -independent, they formed chimeric aggregates, even in the heterotypic cell combination. These results suggest that the specificity in each of those two mechanisms of cell adhesion plays an important role in cellular recognition processes.

The retention of differentiated properties by lens epithelial cells in clonal cell culture.

Abstract A small number of cells of lens epithelium from newly hatched chickens were cultured at clonal density to investigate the retention of differentiated properties during cellular growth in vitro . Singly plated cells proliferated to produce colonies, at least some of which were considered to be true clones of single cell origin. The differentiation of lens fibers occurring in many colonies was identified through observations by electron microscopy as well as immunofluorescence utilizing specific antiserum against lens fibers. Primary or secondary mass cultures of cells of lens epithelium contained cells which produce differentiated colonies when cultured at clonal density. Colony-producing cells can be differentially dissociated from monolayers by EDTA treatment without using tyrpsin. For successful culture of cells of lens epithelium at clonal density, the use of conditioned medium is necessary.

Recent progress in studies of the transdifferentiation of eye tissue in vitro.

A major switch in the overt differentiation phenotypes, which we call transdifferentiation, occurs very often in cultures of embryonic eye tissues. The systems provide irrevocable evidence for instability in the results of cell differentiation, and hence, studies on this topic are expected to contribute much to the understanding of the basic mechanisms of cell differentiation. In this article, recent work on transdifferentiation, mostly with chick embryos, was reviewed. Several new systems of lens transdifferentiation starting from brains, adenohypophyses, and other tissues have recently been discovered. Regarding the widely known cases of transdifferentiation starting from neural and pigmented retina cells, there has been considerable progress in elucidating the factors controlling such major switches in differentiation. In particular, efforts are under way to attempt to understand the mechanisms of transdifferentiation in relation to the transcriptional control of genes coding lens-specific proteins.

Cleavage stage mouse embryos share a common cell adhesion system with teratocarcinoma cells.

Abstract We examined similarities in adhesive properties of mouse cleaving embryos at one- to eight-cell stages and of teratocarcinoma cells by aggregation studies. Teratocarcinoma cells and fibroblastic cells have a Ca 2+ -dependent cell-cell adhesion site (CDS), which is resistant to trypsin in the presence of Ca 2+ but sensitive in the absence of Ca 2+ . When several embryos treated with trypsin in the presence of Ca 2+ (TC) were kept in contact with each other, they fused into a single aggregate in the medium with Ca 2+ but not without Ca 2+ . Embryos treated with trypsin in the absence of Ca 2+ (TE) did not show such Ca 2+ -dependent aggregation. Aggregation of TC-treated embryos was inhibited by Fab fragments of antibody raised against TC-treated teratocarcinoma F9 cells. The aggregation-inhibitory effect of the Fab was removed by absorption with TC-treated teratocarcinoma cells, but not with TE-treated teratocarcinoma cells. This effect was not removed by absorption with fibroblasts and some other tissue cells. TC-treated embryos adhered to TC-treated teratocarcinoma cells, but not to TC-treated fibroblastic cells. These results suggest that early mouse embryos share a common CDS molecule with teratocarcinoma cells but not with fibroblastic cells.

Gene coding for a lens-specific protein, δ-crystallin, is transcribed in nonlens tissues of chicken embryos☆

The question of whether or not delta-crystallin gene (delta-gene) is transcribed in tissues of chicken embryos other than lens was examined by Northern blot analysis, using cloned delta-crystallin cDNA as a probe. delta-Crystallin mRNA was detected in neural retinas, brains, and limb buds of 3.5-day-old embryos. However, the distribution of delta-gene transcripts is limited to lens only in posthatched chickens. It was indicated that the ratio of large delta-crystallin RNA, probably precursor RNA, to mature delta-crystallin mRNA is always much higher in nonlens tissues.

THE DIFFERENTIATION OF PIGMENT CELLS IN CULTURES OF CHICK EMBRYONIC NEURAL RETINAE

Neural retinal cells of 8–9 day‐old chick embryos were differentiated into pigment cells in the conditions of cell culture for about 25 days. The increase of pigment cells in vitro was semi‐quantitatively shown, by counting the number of black foci of pigmented cells per plate throughout the culture period. The increase paralleled the increase in the activity of tyrosinase. The addition of a small number of pigment cells freshly dissociated from tapeta to the cultures of neural retinae did not increase the number of black foci in vitro. Electron microscopic observations revealed the morphological differences of melanin granules between those in pigment cells of the neural retinal cultures and those in cultured tapetum cells. It was discussed that pigment cells appearing in the neural retinal cultures were derived from neural retinal cells, but not from contaminated cells of the tapetum.