F9 embryonal carcinoma (EC) cells were used as a model system to study endoderm 
formation during mammalian embryogenesis. F9 cells treated with retinoic acid 
(RA) or RA plus dibutyryl cyclic AMP (cAMP) were examined for the expression of 
stage-specific embryonic antigen-3 (SSEA-3), a cell surface marker of primitive 
and visceral endoderm. SSEA-3 was not detected by indirect immunofluorescence on 
the surface of undifferentiated stem cells; however, a subset of SSEA-3-positive 
cells appeared with time in culture, amounting to 20% of cells 10 days after 
plating. When cultured in the presence of RA, the percentage of SSEA-3-positive 
cells increased to 70% of cells 10 days after plating. In contrast, treatment of 
cells with RA plus cAMP yielded differentiated cells that were SSEA-3-negative. 
These SSEA-3-negative cells exhibited ultrastructural features of parietal yolk 
sac endoderm. In contrast, SSEA-3-positive cells appearing in cultures treated 
with RA alone exhibited ultrastructural features of primitive endoderm on day 3, 
switching to ultrastructural features of parietal endoderm on day 10. Cells with 
hybrid features, resembling both visceral and parietal yolk sac, were also seen. 
We suggest that differentiation of F9 EC cells into parietal yolk sac-like cells 
can occur along two distinct pathways: 1) direct under the combined influence of 
RA and cAMP; and 2) indirect, under the influence of RA alone, in which cells 
first differentiate into primitive endoderm. Parietal yolk sac-like cells 
induced through the latter pathway continue to express SSEA-3, a cell surface 
marker of primitive endoderm that is not normally found on parietal endodermal 
cells in vivo.