Placental cytotrophoblast microvillar stabilization is required for cell-cell fusion

The placenta is an essential organ of pregnancy required for maternal-fetal transport and communication. The surface of the placenta facing the maternal blood is formed by a single giant multinucleate cell: the syncytiotrophoblast. The syncytiotrophoblast is formed and maintained via fusion of progenitor cytotrophoblasts. Cell-cell fusion is a tightly regulated process, and in non-trophoblastic cells is accompanied by stereotypical alterations in cell shape by cells that have attained fusion-competence. The most prominent feature is the formation of actin-based membrane protrusions, but whether stereotypic morphological changes occur in fusion-competent cytotrophoblasts has not been characterized. Using a human placental explant model and trophoblast organoids, we identify apical microvilliation as an obligate morphological feature of fusion-competent cytotrophoblasts. Disruption of microvilli using an inhibitor of the actin-membrane cross linker protein ezrin prevented cytotrophoblast fusion and differentiation. We provide evidence that these polarized apical microvillar domains function to regulate polarized endocytosis and to spatially localize and accumulate a key glycoprotein for fusion, CD98. Thus, we propose that the polarized assembly of microvillar domains is critical for mediating efficient syncytiotrophoblast development. Overall design: To understand the role of microvili in cytotrophoblast fusion we utilized an ezrin inhibitor to block the stabilization of microvili in both explants and organoids. We performed RNA-seq analysis at 2 different time points and two different conditions for the explants (Ezrin inhibitor vs control). For the organoids, there were 3 different time points across the two conditions. Model characterization was also performed for both the explants and the organoids. Three replicates were performed for the explants, and two replicates were performed for the organoids.