Department of Biology, Molecular Genetics Unit, and Child Health Research Institute, Western University, London, ON, Canada.
Cell Signal. 2012 Dec;24(12):2337-48. doi: 10.1016/j.cellsig.2012.07.024. Epub 2012 Aug 3.
Primitive endoderm formation from the inner cell mass is one of the earliest known cell fate decisions made in the mouse embryo. The mechanisms involved in orchestrating this process are not fully understood and are difficult to study in vivo. The F9 teratocarcinoma cell line is an in vitro model used to circumvent many technical problems surrounding the study of extraembryonic endoderm differentiation. F9 cells treated with retinoic acid differentiate to primitive endoderm and this is accompanied by the activation of canonical Wnt-β-catenin signalling. Reactive oxygen species can modulate this signalling pathway, but whether they are sufficient to induce extraembryonic endoderm in vitro is not known. In the present study, a sustained increase in ROS levels was found in retinoic acid-treated F9 cells. An increase in Tcf-Lef transcriptional activity, a read out of Wnt-β-catenin signalling, was also seen in response to exogenous H(2)O(2). Analysis from immunoblots, immunocytochemistry and real time PCR revealed the presence of markers of differentiation and a reduction in the expression of a marker of proliferation, confirming that H(2)O(2)-treated F9 cells developed into primitive endoderm. In contrast, exposing retinoic acid-treated cells to antioxidants impeded differentiation. Real time PCR was also used to identify candidates responsible for the observed elevation in ROS production. Results indicated that the NADPH oxidase 1, 2, 3 and 4 and Duox2 genes were RA responsive. Furthermore, the NADPH oxidase inhibitor, diphenyleneiodonium chloride was shown to attenuate primitive endoderm formation. Together, these results shed new light on how early mouse embryogenesis might be influenced by the crosstalk involving ROS and the Wnt-β-catenin signalling pathway.
原始内胚层的形成是小鼠胚胎中最早出现的已知细胞命运决定之一。目前还不完全了解协调这一过程的机制,并且很难在体内进行研究。F9 畸胎瘤细胞系是一种体外模型,用于规避围绕胚外内胚层分化研究的许多技术问题。用维甲酸处理的 F9 细胞分化为原始内胚层,这伴随着经典 Wnt-β-连环蛋白信号的激活。活性氧可以调节这个信号通路,但是它们是否足以在体外诱导胚外内胚层尚不清楚。在本研究中,发现维甲酸处理的 F9 细胞中 ROS 水平持续增加。外源 H2O2 也导致 Tcf-Lef 转录活性增加,这是 Wnt-β-连环蛋白信号的读出。免疫印迹、免疫细胞化学和实时 PCR 的分析显示分化标志物的存在和增殖标志物表达减少,证实 H2O2 处理的 F9 细胞分化为原始内胚层。相比之下,向维甲酸处理的细胞中添加抗氧化剂会阻碍分化。实时 PCR 还用于鉴定导致观察到的 ROS 产生升高的候选物。结果表明,NADPH 氧化酶 1、2、3 和 4 和 Duox2 基因对 RA 有反应。此外,NADPH 氧化酶抑制剂二苯并碘氯酸盐被证明可以减弱原始内胚层的形成。总之,这些结果为 ROS 和 Wnt-β-连环蛋白信号通路的相互作用如何影响早期小鼠胚胎发生提供了新的见解。