Regulatory mechanisms to maintain epithelial barrier integrity in the placenta.

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Cells that comprise epithelial barriers turnover frequently. Maintenance of epithelial barriers is contingent on the actions of progenitor cells, which either propagate to replenish themselves, or differentiate into the specialized epithelium required for that tissue's function. An excellent example of this is the placental epithelium, where progenitor cytotrophoblast cells either divide to restock the existing pool of progenitor cells, or differentiate by fusing into an overlying syncytium (syncytiotrophoblast). Syncytiotrophoblast forms the primary barrier separating maternal and fetal tissue, but is shed into maternal circulation throughout pregnancy and needs to be replenished by underlying cytotrophoblast cells. The regulatory mechanisms that govern the transition from cytotrophoblast propagation to differentiation is not well known, but this process is fundamental to understanding basic placental development and function. *** Our primary objective is to define signalling and transcriptional networks that regulate the balance between cytotrophoblast propagation and differentiation. We recently discovered that OVO-like 1 (OVOL1), a conserved transcriptional repressor homologous to the Drosophila Ovo gene that is implicated as a downstream effector of Wnt/β-catenin signalling, is vital for cytotrophoblast differentiation. We also identified that, in human trophoblast cells, OVOL1 binds within the proximal promoter region of MYC, ID1, TP63, and ASCL2 – genes linked with maintaining progenitor traits in various epithelial cells, including trophoblast cells. Thus, we hypothesize that OVOL1-mediated repression of genes that promote the cytotrophoblast progenitor state is critical for promotion of cytotrophoblast differentiation. In this application, three Aims are proposed that will collectively shed light on the upstream and downstream mechanisms of OVOL1's actions in trophoblast cells. Each Aim will incorporate well-established models of human trophoblast differentiation that are routinely used in our lab. In Aim 1, we will reveal upstream signalling mechanisms involved in transcriptional activation of OVOL1 during trophoblast differentiation, focusing on activation of β-catenin as a driving force for OVOL1 transcription. In Aim 2, we will examine in situ expression patterns of OVOL1 with MYC, ID1, TP63, ASCL2, and biochemically assess the importance of OVOL1 binding for repression of each gene. In Aim 3, we will determine the importance of histone deacetylase (HDAC) 1 and HDAC2 in mediating the repressive actions of OVOL1 on gene transcription. Significance: Our studies will provide key insights on the fundamental regulation of trophoblast differentiation and placental development, and will improve our understanding of the molecular regulation of epithelial turnover. **