Analysis of the apoptotic threshold at the onset of embryonic differentiation

Abstract

The first step of embryonic differentiation involves a transition from a naïve to a primed pluripotency state that occurs between the pre- and post-implantation stages of development. Mouse embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) are used as in vitro models of each of these pluripotent states. Although significant insight has been gained into the regulation of the self-renewal and differentiation capacity of pluripotent cells, little is known regarding how their survival is controlled. It has been demonstrated that cells in the primed pluripotent state are hypersensitive to cell death stimuli. Here we show the importance of miRNAs ensuring cell survival of EpiSCs and post-implantation embryos preventing apoptosis by inhibiting the expression of the pro-apoptotic protein BIM. In this way, miRNAs maintain primed pluripotent cells in a state that is also primed for death. We next compared if ESCs and EpiSCs have different apoptotic requirements. We observed that while EpiSCs are very sensitive to apoptotic stimuli and require the expression of the anti-apoptotic factors Bcl2, Bcl-xL, Bcl-w and Mcl1 for their survival, ESCs are relatively resistant to apoptosis and mainly rely on Mcl1 to block apoptosis. To unravel the possible reasons for these different apoptotic requirements we studied mitochondrial morphology and dynamics and found that as cells exit the naïve pluripotent state they change from having fragmented to having fused mitochondria. Furthermore, we demonstrated that inhibiting mitochondrial fusion by inhibiting DRP1 or inhibiting mitochondrial activity by blocking the activity of the ATP-synthase, increased the sensitivity of ESCs to cell death. Therefore, these results highlight how miRNA expression, pluripotency status, metabolism and mitochondrial dynamics determine the apoptotic threshold during the early stages of embryonic development.