College of Veterinary Medicine, Shaanxi Center of Stem Cells Engineering and Technology, Northwest A&F University, 712100, Yangling, China.
State Key Laboratory of Genetically Engineered Veterinary Vaccines, Yebio Bioengineering Co.Ltd of Qingdao, Qingdao, China.
Mol Biol Rep. 2020 Dec;47(12):9259-9269. doi: 10.1007/s11033-020-05969-4. Epub 2020 Nov 6.
Previous studies have demonstrated that transcription factor Etv5 plays an important role in the segregation between epiblast and primitive endoderm at the second fate decision of early embryo. However, it remains elusive whether Etv5 functions in the segregation between inner cell mass and trophectoderm at the first cell fate decision. In this study, we firstly generated Etv5 knockout mouse embryonic stem cells (mESCs) by CRISPR/Cas9, then converted them into extended potential stem cells (EPSCs) by culturing the cells in small molecule cocktail medium LCDM (LIF, CHIR99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride), and finally investigated their differentiation efficiency of trophoblast stem cells (TSCs). The results showed that Etv5 knockout significantly decreased the efficiency of TSCs (CDX2) differentiated from EPSCs. In addition, Etv5 knockout resulted in higher incidence of the differentiated cells with tetraploid and octoploid than that from wild type. Mechanistically, Etv5 was activated by extracellular-signal-regulated kinase (ERK) signaling pathway; in turn, Etv5 had a positive feedback on the expression of fibroblast growth factor receptor 2 (FGFR2) which lies upstream of ERK. Etv5 knockout decreased the expression of FGFR2, whose binding with fibroblast growth factor 4 was essentially needed for TSCs differentiation. Collectively, the findings in this study suggest that Etv5 is required to safeguard the TSCs differentiation by regulating FGFR2 and provide new clues to understand the specification of trophectoderm in vivo.
先前的研究表明，转录因子 Etv5 在早期胚胎的第二次命运决定中，在滋养外胚层和原始内胚层的分离中发挥重要作用。然而，Etv5 是否在第一次细胞命运决定中，在内细胞团和滋养外胚层的分离中起作用，仍不得而知。在这项研究中，我们首先通过 CRISPR/Cas9 生成了 Etv5 敲除的小鼠胚胎干细胞（mESCs），然后通过在小分子鸡尾酒培养基 LCDM（LIF、CHIR99021、(S)-(+)-二甲茚二酮马来酸盐、盐酸米诺环素）中培养细胞，将其转化为扩展潜能干细胞（EPSCs），最后研究了它们向滋养层干细胞（TSCs）分化的效率。结果表明，Etv5 敲除显著降低了 EPSCs 分化为 TSCs（CDX2）的效率。此外，Etv5 敲除导致分化细胞中四倍体和八倍体的发生率高于野生型。从机制上讲，Etv5 被细胞外信号调节激酶（ERK）信号通路激活；反过来，Etv5 对 ERK 上游的成纤维细胞生长因子受体 2（FGFR2）的表达有正反馈作用。Etv5 敲除降低了 FGFR2 的表达，其与成纤维细胞生长因子 4 的结合对于 TSCs 分化是必不可少的。总之，这项研究的结果表明，Etv5 通过调节 FGFR2 来维持 TSCs 的分化，为理解体内滋养外胚层的特化提供了新的线索。
