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The preimplantation development of the mouse embryo leads to the divergence of the first two cell lineages, the inner cell mass and the trophectoderm. The formation of a microvillus pole during compaction at the eight-cell stage and its asymmetric inheritance during mitosis are key events in the emergence of these two cell populations. Ezrin, a member of the ERM protein family, seems to be involved in the formation and stabilization of this apical microvillus pole. To further characterize its function in early development, we mutated the key residue T567, which was reported to be essential for regulation of ezrin function through phosphorylation. Here, we show that expression of ezrin mutants in which the COOH-terminal threonine T567 was replaced by an aspartate (to mimic a phosphorylated residue; T567D) or by an alanine (to avoid phosphorylation; T567A) interferes with E-cadherin function and disrupts the first morphogenetic events of development: compaction and cavitation. The active mutant ezrin-T567D induces the formation of numerous and abnormally long microvilli at the surface of blastomeres. Moreover, it localizes all around the cell cortex and inhibits cell-cell adhesion and cell polarization at the eight-cell stage. During the following stages, only half of the embryos are able to compact and finally to cavitate. In those embryos, the amount of ezrin-T567D decreases in the basolateral areas, while the proportion of adherens junctions increases. The reverse inactive mutant ezrin-T567A is mainly cytoplasmic and does not perturb compaction at the eight-cell stage. However, at the 16-cell stage, it relocalizes at the basolateral cortex, leading to a strong decrease in the surface of adherens junctions, and finally, embryos abort development. Our results show that ezrin is directly involved in the formation of microvilli in the early mouse embryo. Moreover, they indicate that maintenance of ezrin in basolateral areas prevents microvilli breakdown and inhibits the formation of normal cell-cell contacts mediated by E-cadherin, thereby impairing blastomeres polarization and morphogenesis of the blastocyst.

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., (Copyright 2001 Academic Press.)

The preimplantation development of the mouse embryo leads to the formation of two populations of cells: the trophectoderm, which is a perfect epithelium, and the inner cell mass. The divergence between these two lineages is the result of asymmetric divisions, which can occur after blastomere polarization at compaction. The apical pole of microvilli is the only asymmetric feature maintained during mitosis and polarity is reestablished only in daughter cells that inherit all or a sufficient part of this pole. To analyze the role of ezrin in the formation and stabilization of the pole of microvilli, we isolated and cultured inner cell masses (ICM). These undifferentiated cells can differentiate very quickly into epithelial cells. After isolation of the ICMs, ezrin relocalizes at the cell cortex before the formation of microvilli. This redistribution occurs in the absence of protein synthesis. The formation of microvilli at the apical surface of the outer cells of ICM correlates with a major posttranslational modification of ezrin. We show here that this posttranslational modification is not controlled by a serine/threonine kinase but an O-glycosylation may partially contribute to it. These data suggest that ezrin has at least two roles during development. First, ezrin may be involved in the formation of microvilli because it localizes at the cell cortex before microvilli appear in ICMs. Second, ezrin may stabilize the pole of microvilli because it is modified posttranslationally when microvilli form.