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 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 alpha (PDGFRalpha)+ vascular endothelial growth factor receptor 2 (VEGFR2)+ and PDGFRalpha+ 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.
Izumi
Naoki
N
Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.
Era
Takumi
T
Akimaru
Hiroshi
H
Yasunaga
Masahiro
M
Nishikawa
Shin-Ichi
S
D028341:Activins / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology*; D004707:Endoderm / Q000166:cytology*; D050993:GATA6 Transcription Factor / Q000378:metabolism; D015870:Gene Expression; D018398:Homeodomain Proteins / Q000378:metabolism; D008648:Mesoderm / Q000166:cytology*; D051379:Mice; D008954:Models, Biological; D034622:RNA Interference; D020825:T-Box Domain Proteins / Q000378:metabolism
C491198:Eomes protein, mouse; D050993:GATA6 Transcription Factor; C494760:Gata6 protein, mouse; D018398:Homeodomain Proteins; C464781:Mixl1 protein, mouse; D020825:T-Box Domain Proteins; D028341:Activins