In vivo functional analysis of ezrin during mouse blastocyst formation

During mouse blastocyst formation, a layer of outer cells differentiates in less than 48 h into a functional epithelium (the trophectoderm). Ezrin, an actin-binding structural component of microvilli in epithelial cells, is also involved in signal transduction and ionic pump control. In the mouse embryo, ezrin becomes restricted to the apical cortex of all blastomeres at compaction and of outer cells at later stages. Here we investigated the function of ezrin in living embryos during epithelial differentiation using mutant forms of ezrin tagged with green fluorescent protein (GFP). GFP-tagged wild-type ezrin (Ez/GFPc) behaved like endogenous ezrin and did not interfere with development. Deletion of the last 53 amino acids (Delta53/GFP) changed the localization of ezrin: after compaction, Delta53/GFP remained associated with the apical and basolateral cortex in all blastomeres, and its expression slightly disturbed the cavitation process. Finally, full-length ezrin with GFP inserted at position 234 (Ez/GFPi) was localized all around the cortex throughout development, although it was concentrated at the apical pole after compaction. In embryos expressing Ez/GFPi, the duration of the 16-cell stage was reduced, while the onset of cavitation was delayed. Moreover, cavitation was abnormal, and the blastocoele was small and retracted almost completely several times as if there were major leakages of blastocoelic fluid. Our results suggest that, in addition to its role in microvilli organization, ezrin is involved in the formation of a functional epithelium through a still unknown mechanism.