Dissecting the molecular hierarchy for mesendoderm differentiation through a combination of embryonic stem cell culture and RNA interference

Although there is a criticism that embryonic stem (ES) cell differentiation does not always reflect the differentiation process involved in mouse development, it is a suitable model system to dissect the specific differentiation pathway. We established the culture conditions that selectively differentiated mouse ES cells into three germ layers containing mesendoderm, definitive endoderm (DE), visceral endoderm (VE), mesoderm, and neuroectoderm. However, the molecular mechanisms of differentiation under each specific condition still remain unclear. Here, in combination with the RNA interferencemediated gene knockdown (KD) method, we show that Eomesodermin (Eomes), Mix11, Brachyury (7), and GA TA 6 are major molecular determinants in the differentiation of mesendoderm, DE, VE, and mesoderm. Eomes plays a pivotal role in an early stage of mesendoderm differentiation, whereas Mix11 does the same in the later stage where mesendoderm differentiates into DE. Further analyses of quantitative reverse transcription polymerase chain reaction and overexpression of Mix11 demonstrated that Mix11 is genetically a downstream molecule of Eomes. In addition, both Eomes and Mix11 act as negative regulators of T expression. This strategy also reveals that Eomes and T play cell-autonomous roles in platelet-derived growth factor receptor a (PDGFRce)(+) vascular endothelial growth factor receptor 2 (VEGFR2)(+) and PDGFRa(+) mesoderm generations, respectively. Our results obtained from this study are fully consistent with previous knockout studies of those genes. The present study, therefore, demonstrates that the major molecular mechanism underlying in vitro ES cell differentiation largely recapitulates that in actual embryogenesis, and the combination of our culture system and RNAi-mediated gene KI) is an useful tool to elucidate the molecular hierarchy in in vitro ES cell differentiation.