The formation and maturation of the outer epithelial layer is essential for maximal alphafetoprotein (AFP) production during differentiation of F9 embryoid bodies in the presence of 5 X 10(-8) M retinoic acid (Grover et al., 1983. J. Cell Biol. 96:1690-1696). The critical phase is between the third and the fourth day when the components of the extracellular matrix organize into a basement membrane. The role of some of these components in the process of epithelium formation and maturation is analyzed in this paper. The role of laminin was investigated by testing the effect of exogenous laminin and antilaminin in cultures of differentiating F9 aggregates. Tests included growth rates, morphological changes, AFP production, determination of AFP mRNA levels, and fluorescent staining for basement membrane components and for epithelial markers. At concentrations greater than 5 micrograms/ml, exogenous laminin inhibited the production of AFP and prevented AFP gene transcription. On the basis of immunofluorescence tests, exogenous laminin appeared to act by preventing the accumulation of a basement membrane and by disrupting the organization of the outer layer into an epithelium. No such effects were produced by fibronectin or collagens type I or IV. Aggregates cultured in the presence of antilaminin also failed to organize an epithelium and did not produce AFP, whereas those in normal rabbit serum differentiated normally. Therefore, endogenous laminin plays a key role not only as a basement membrane structural component but also in organizing the epithelial layer of endoderm cells and hence (indirectly) in gene expression.
F9 embryonal carcinoma (EC) cells, cultured in suspension in medium containing 5 X 10(-8) M retinoic acid, aggregate and differentiate into embryoid bodies with an outer layer of visceral endoderm cells that synthesize and secrete alphafetoprotein (AFP) (Hogan, B. L. M., A. Taylor, and E. Adamson, 1981, Nature (Lond.). 291:235-237). Here we analyze the formation of the outer layer of cells as a model for epithelial differentiation. Three morphological phases are described, but analyses of cell numbers and the synthetic rates of some proteins, as well as the appearance of markers of visceral endoderm and basement membrane, show that the formation of the outer layer occurs as an orderly progression of multiple events. The markers used to follow the ontogeny of epithelial layer formation include SSEA-1, l, and i blood group antigens, laminin, fibronectin, type IV collagen, cytoskeletal intermediate filament proteins (vimentin, Endo A, and B), and AFP. The onset of epithelium formation occurs between the third and fourth day of culture, but its function is maximally expressed only when it is well organized. We found the rate of AFP secretion to be a measure of the proper alignment and maturity of the epithelium which occurs at the seventh or eighth day. This model of epithelium formation may help to explain how similar processes occur during embryogenesis.