BACKGROUND The segregation of the hypoblast and the emergence of the pluripotent epiblast mark the final stages of blastocyst formation in mammalian embryos. In bovine embryos the formation of the hypoblast has been partially studied, and evidence shows that MEK signalling plays a limited role in the segregation of this lineage. Here we explored the role of different signalling pathways during lineage segregation in the bovine embryo using immunofluorescence analysis of NANOG and SOX17 as readouts of epiblast and hypoblast, respectively. RESULTS We show that SOX17 starts to be expressed in 16-32-cell stage embryos, whereas NANOG is first detected from 8-cell stage. SOX17 is first co-expressed with NANOG, but these markers become mutually exclusive by the late blastocyst stage. By assessing the expression kinetics of NANOG/SOX17 we show that inhibition of MEK signalling can eliminate SOX17 expression in bovine blastocysts, without altering NANOG expression. Modulation of WNT, PKC and LIF did not affect NANOG expression in the epiblast when used in combination with the ERK inhibitor. CONCLUSIONS This study shows that SOX17 can be used as a reliable early marker of hypoblast in the bovine, and based on its expression profile we show that the hypoblast segregates in day 7 blastocysts. Furthermore, SOX17 expression is abolished using 1 μM of PD0325901, without affecting the NANOG population in the epiblast. Modulation of WNT, PKC and LIF are not sufficient to support enhanced NANOG expression in the epiblast when combined with ERK inhibitor, indicating that additional signalling pathways should be examined to determine their potential roles in epiblast expansion.

中文翻译：

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对 MEK 抑制的剂量依赖性反应决定了牛胚胎中的下胚层命运。

背景下胚层的分离和多能外胚层的出现标志着哺乳动物胚胎囊胚形成的最后阶段。在牛胚胎中，已经部分研究了下胚层的形成，证据表明 MEK 信号在该谱系的分离中发挥的作用有限。在这里，我们使用 NANOG 和 SOX17 的免疫荧光分析分别作为外胚层和下胚层的读数，探讨了不同信号通路在牛胚胎谱系分离过程中的作用。结果 我们表明 SOX17 开始在 16-32 细胞阶段的胚胎中表达，而 NANOG 首次从 8 细胞阶段检测到。SOX17 首先与 NANOG 共表达，但这些标记在胚泡后期变得相互排斥。通过评估 NANOG/SOX17 的表达动力学，我们表明抑制 MEK 信号可以消除牛胚泡中 SOX17 的表达，而不会改变 NANOG 的表达。当与 ERK 抑制剂联合使用时，WNT、PKC 和 LIF 的调节不影响外胚层中 NANOG 的表达。结论 本研究表明，SOX17 可用作牛下胚层的可靠早期标志物，基于其表达谱，我们表明下胚层在第 7 天的胚泡中分离。此外，使用 1 μM 的 PD0325901 消除了 SOX17 表达，而不会影响外胚层中的 NANOG 群体。当与 ERK 抑制剂结合时，WNT、PKC 和 LIF 的调节不足以支持外胚层中 NANOG 表达的增强，