Department of Genetics and Animal Breeding, Poznan University of Life Sciences, Wolynska 33, 60-637, Poznan, Poland.
Dev Biol. 2020 Jul 1;463(1):63-76. doi: 10.1016/j.ydbio.2020.04.004. Epub 2020 Apr 28.
Capturing stable embryonic stem cell (ESC) lines from domesticated animals still remains one of the challenges of non-rodent embryology. The stake is high, as stable ESCs derived from species such as cattle present high economic and scientific value. Understanding of the processes leading to the embryonic lineage segregation is crucial to provide species-orientated molecular environment capable of supporting self-renewal and pluripotency. Therefore, the aim of this study was to validate the action of the two core regulatory pathways (WNT and MEK/ERK) during bovine embryo development. In vitro produced bovine embryos were obtained in the presence of inhibitors (i), which enable activation of the WNT pathway (via GSK3i, CHIR99021) and suppression of MEK signalling by PD0325901 in the 2i system and PD184325 and SU5402 in the 3i system. We have followed the changes in the distribution of the key lineage specific markers both at the transcript and protein level. Our results showed that WNT signalling promotes the expression of key inner cell mass (ICM) specific markers in bovine embryos, regardless of the MEK/ERK inhibitor cocktail used. MEK/ERK downregulation is crucial to maintain OCT4 and NANOG expression within the ICM and to prevent their exclusion from the trophectoderm (TE). At the same time, the classical TE marker (CDX2) was downregulated at the mRNA and protein level. As a follow up for the observed pluripotency stimulating effect of the inhibitors, we have tested the potential of the 2i and the 3i culture conditions (supported by LIF) to derive primary bovine ESC lines. As a result, we propose a model in which all of the primary signalling pathways determining embryonic cell fate are active in bovine embryos, yet the requirement for pluripotency maintenance in cattle may differ from the described standards. WNT activation leads to the formation (and stabilisation of the ICM) and MEK/ERK signalling is maintained at low levels. Unlike in the mouse, GATA6 is expressed in both ICM and TE. MEK/ERK signalling affects HP formation in cattle, but this process is activated at the post-blastocyst stage. With regard to self-renewal, 2i is preferable, as 3i also blocks the FGF receptor, what may prevent PI3K signalling, important for pluripotency and self-renewal.
从驯化动物中获得稳定的胚胎干细胞 (ESC) 系仍然是非啮齿动物胚胎学的挑战之一。 stakes 是高的，因为从牛等物种中获得的稳定 ESC 具有很高的经济和科学价值。了解导致胚胎谱系分离的过程对于提供具有物种定向分子环境以支持自我更新和多能性至关重要。因此，本研究的目的是验证两条核心调控途径 (WNT 和 MEK/ERK) 在牛胚胎发育过程中的作用。在存在抑制剂 (i) 的情况下获得体外产生的牛胚胎，这些抑制剂能够激活 WNT 途径（通过 GSK3i、CHIR99021）并通过 PD0325901 在 2i 系统和 PD184325 和 SU5402 在 3i 系统中抑制 MEK 信号。我们已经在转录和蛋白质水平上跟踪了关键谱系特异性标记物的分布变化。我们的结果表明，WNT 信号通路促进了牛胚胎中关键内细胞团 (ICM) 特异性标记物的表达，而与使用的 MEK/ERK 抑制剂鸡尾酒无关。MEK/ERK 的下调对于维持 OCT4 和 NANOG 在 ICM 内的表达以及防止它们从滋养外胚层 (TE) 中排除至关重要。同时，经典的 TE 标记物 (CDX2) 在 mRNA 和蛋白质水平上均下调。作为对抑制剂观察到的多能性刺激作用的后续研究，我们已经测试了 2i 和 3i 培养条件（由 LIF 支持）在衍生主要牛 ESC 系中的潜力。结果，我们提出了一个模型，其中决定胚胎细胞命运的所有主要信号通路都在牛胚胎中活跃，但牛维持多能性的要求可能与描述的标准不同。WNT 激活导致 ICM 的形成（和稳定），并且 MEK/ERK 信号保持在低水平。与小鼠不同，GATA6 在 ICM 和 TE 中均表达。MEK/ERK 信号影响牛中 HP 的形成，但该过程在胚泡后阶段被激活。就自我更新而言，2i 是优选的，因为 3i 还会阻断 FGF 受体，这可能会阻止对多能性和自我更新很重要的 PI3K 信号。
