Dissecting the Molecular Hierarchy for Mesendoderm Differentiation Through a Combination of Embryonic Stem Cell Culture and RNA Interference
Article first published online: 19 APR 2007
Copyright © 2007 AlphaMed Press
Volume 25, Issue 7, pages 1664–1674, July 2007
How to Cite
Izumi, N., Era, T., Akimaru, H., Yasunaga, M. and Nishikawa, S.-I. (2007), Dissecting the Molecular Hierarchy for Mesendoderm Differentiation Through a Combination of Embryonic Stem Cell Culture and RNA Interference. STEM CELLS, 25: 1664–1674. doi: 10.1634/stemcells.2006-0681
- Issue published online: 2 JAN 2009
- Article first published online: 19 APR 2007
- Manuscript Accepted: 8 APR 2007
- Manuscript Received: 25 OCT 2006
- Embryonic stem cell;
- Short hairpin RNA;
- Definitive endoderm
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 interference-mediated gene knockdown (KD) method, we show that Eomesodermin (Eomes), Mixl1, Brachyury (T), and GATA6 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 Mixl1 does the same in the later stage where mesendoderm differentiates into DE. Further analyses of quantitative reverse transcription polymerase chain reaction and overexpression of Mixl1 demonstrated that Mixl1 is genetically a downstream molecule of Eomes. In addition, both Eomes and Mixl1 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 α (PDGFRα)+ vascular endothelial growth factor receptor 2 (VEGFR2)+ and PDGFRα+ 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 KD is an useful tool to elucidate the molecular hierarchy in in vitro ES cell differentiation.
Disclosure of potential conflicts of interest is found at the end of this article.