Coordinate Galpha13 and Wnt6-beta-catenin signaling in F9 embryonal carcinoma cells is required for primitive endoderm differentiation.

The mouse F9 embryonal carcinoma cell line is ideally suited to study the epithelial-to-mesenchymal transition accompanying the differentiation of primitive to parietal extraembryonic endoderm. In F9 cells, the application of exogenous agents including retinoic acid or activation of signal transduction cascades downstream of G-proteins triggers widespread changes in gene expression and leads to the formation of primitive endoderm. The epithelial-to-mesenchymal transition is completed and parietal endoderm develops as of result of increasing PKA activity in primitive endoderm cells. Expression of a constitutively active form of Galpha13(Q226L) is sufficient to induce F9 cells into parietal endoderm and a model is emerging that a signaling axis linking G-protein signaling to RhoA and the ERM protein moesin is required for differentiation. In this study, we found that expression of either p115RhoGEF or a constitutively active, GTPase-deficient form of RhoA(L63) promoted primitive, but not parietal, endoderm formation. The overexpression of Galpha13(Q226L) or p115RhoGEF, but not Rho(L63), caused beta-catenin to translocate to the nucleus. Surprisingly, the stimulation of the Wnt-beta-catenin pathway was accompanied by nuclear beta-catenin and primitive endoderm formation, even when a dominant negative was used to block the signaling axis at the level of p115RhoGEF or when ROCK activity was inhibited using the pharmacological agent Y-27632. Together, results indicate that the coordinate signaling by two independent pathways, one involving canonical Wnt-beta-catenin activation of target genes and the other with Galpha13 signaling to ERM proteins to modulate cytoarchitectural changes, is required during the retinoic acid induced differentiation of F9 cells to primitive endoderm.