How are the cells on the surface of embryoid body derived from ES cells induced to differentiate into primitive endoderm?

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In this research project, we first hypothesized that the extracellular signals derived from the adhesion to the extracellular matrix via integrin and the cell-to-cell adhesion via cadherin would involve in the maintenance of pluripotency in mouse ES cells in cooperation to the soluble factors. To test this model, we plotted the inhibition of the integrain signal by overexpressing the dominant-negative mutant of integrin-linked kinase (ILK) and found that it partially induce differentiation in the presence of differentiation inhibitor LIF. We also tried to test the function of the E-cadherin-mediated signal on induction of differentiation toward primitive endoderm on the surface of aggregates in suspension culture condition. When we put the soluble form of the extracellular domain of E-cadherin in this culture, differentiation of primitive endoderm is interfered, suggesting that the reduced amount of E-cadherin signal might trigger differentiation in the cells locating the surface of the aggregates.These extracellular signals determine the cell fate by modulating the expression of the transcription factors. We found that the ectopic expression of zinc-finger transcription factor Gata6 induces differentiation of ES cells into primitive endoderm cells that mimic the phenotype of embryo-derived XEN cells. As the model system to control cell fate by transcription factors, we revealed that the reciprocal inhibition system between Cdx2, which determines differentiation to trophectoderm, and the pluripotency-associated transcription factor Oct3/4 involves in segregation between pluripotent cell and trophectoderm lineages. In addition, we also made it clear how the other pluripotency-associated transcription factor Sox2 works in ES cells. These findings will contribute to analyze the mechanism to direct primitive endoderm differentiation.