Reactive oxygen species and Wnt signalling crosstalk patterns mouse extraembryonic endoderm

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 [1], 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 H2O2. 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 H2O2-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. ► RA activation of Wnt–β-catenin signalling induces F9 cells into primitive endoderm. ► A burst of ROS precedes and is necessary for this differentiation event. ► H2O2 affects β-catenin-dependent Tcf-Lef transcription and induces differentiation. ► The Nox1–4 and Duox 2 genes encoding NADPH oxidases are RA-responsive. ► RA-induced differentiation is affected by Nox activity inhibitors.