Dissecting the Molecular Hierarchy for Mesendoderm Differentiation Through A Combination of ES Cell Culture and RNA interference

¹ Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, 2-2-3 Minatojimaminamimachi, Chuo-ku, Kobe, 650-0047, Japan; Science of In-Home Medicine, Health and Community Medicine, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, 466-8550, Japan
² Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, 2-2-3 Minatojimaminamimachi, Chuo-ku, Kobe, 650-0047, Japan
³ Research & Development, Cardio Incorporated, 1-5-4 Minatojimaminamimachi, Chuo-ku, Kobe, 650-0047, Japan

^* To whom correspondence should be addressed. E-mail: tera{at}cdb.riken.jp.

	Abstract

Although there is a criticism that 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 RNAi-medicated gene knock down (KD) method, we show that Eomesodermin (Eomes), Mixl1, Brachyury (T) and GATA6 are major molecular determinants in the differentiation of mesendodem, DE, VE and mesoderm. Eomes plays a pivotal role in an early stage of mesendoderm differentiation, while Mixl1 does in the later stage where mesendoderm differentiates into DE. Further analyses of quantitative-PCR and over-expression of Mixl1 demonstrate that Mixl1 is genetically a downstream molecule of Eomes. Additionally, both Eomes and Mixl1 act as negative regulators of T expression. This strategy also reveals that Eomes and T play cell-autonomous roles in PDGFR⁺VEGFR2⁺ and PDGFR⁺ mesoderm generations, respectively. Our results obtained from this study are fully consistent with previous knock-out studies of those genes. The present study, therefore, demonstrate 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.

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