OVOL2 Reinforces Epithelial Identity and Promotes SynT-I Differentiation in the Mouse Placenta

The placenta establishes the maternal-fetal interface during pregnancy, enabling the selective exchange of nutrients and gases between maternal and fetal circulations. In humans and mice, this exchange surface is lined by multinucleated epithelial layers called the syncytiotrophoblast (SynT), which in the mouse placenta is organized into two distinct layers, SynT-I and SynT-II. SynT formation from trophoblast precursors requires epithelial plasticity to permit cell differentiation, fusion, and morphogenesis, while preserving epithelial integrity to maintain the maternal-fetal exchange barrier. However, the mechanisms that safeguard epithelial features during SynT development remain unclear. OVO-like 2 (OVOL2), a transcriptional repressor of mesenchymal-associated programs and key regulator of epithelial identity, is highly expressed in the mouse placenta and essential for its development. We hypothesized that OVOL2 promotes SynT lineage formation by restraining mesenchymal-associated transcriptional programs during trophoblast differentiation. To test this, placental development was examined following Ovol2+/- matings, and wild-type and Ovol2-deficient trophoblast stem cells were analyzed under stem conditions or differentiated with CHIR99021 to enrich for SynT lineages. SynT-I lineage development was disrupted in both Ovol2-deficient placentas and differentiating trophoblast stem cells, whereas SynT-II-associated differentiation appeared less severely affected. Chromatin profiling identified OVOL2 binding near genes associated with epithelial-to-mesenchymal transition, including Id1, Zeb1, and Vim, which were upregulated in Ovol2-deficient trophoblasts. Consistent with these observations, Ovol2-deficient cells showed elevated levels of mesenchymal markers such as ZEB1 and Vimentin and reduced levels of epithelial markers including E-cadherin. These findings identify OVOL2 as a critical regulator of SynT-I lineage formation and epithelial identity in the mouse placenta.