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

在这个研究项目中，我们首先假设，通过整合素与细胞外基质的黏附以及通过钙粘附素的细胞与细胞之间的黏附所产生的细胞外信号可能与可溶性因子协同作用参与维持小鼠ES细胞的多能性。为了验证这一模型，我们绘制了通过过度表达整合素连接激酶(ILK)的显性-负性突变体来抑制整合信号的情况，并发现在分化抑制剂LIF存在的情况下，它可以部分诱导分化。我们还尝试在悬浮培养条件下检测E-钙粘附素介导的信号在诱导集合体表面原始内胚层分化中的作用。当我们将E-钙粘蛋白胞外区的可溶性形式放入这种培养物中时，原始内胚层的分化会受到干扰，这表明E-钙粘附素信号量的减少可能会触发位于聚集体表面的细胞的分化。这些细胞外信号通过调节转录因子的表达来决定细胞的命运。我们发现锌指转录因子GATA6的异位表达可以诱导ES细胞分化为与胚胎Xen细胞表型相似的原始内胚层细胞。作为转录因子控制细胞命运的模型系统，我们揭示了决定滋养外胚层分化的CDX2和多能性相关转录因子Oct3/4之间的相互抑制系统涉及多能细胞和滋养外胚层谱系之间的分离。此外，我们还阐明了另一种多潜能相关转录因子Sox2在ES细胞中的作用。这些发现将有助于分析原始内胚层分化的机制。

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.