Regulatory mechanisms to maintain epithelial barrier integrity in the placenta.

构成上皮屏障的细胞频繁翻转。上皮屏障的维持取决于祖细胞的行为，祖细胞要么繁殖以补充自己，要么分化为组织功能所需的特化上皮。胎盘上皮是一个很好的例子，在胎盘上，祖细胞滋养层细胞要么分裂以补充现有的祖细胞池，要么通过融合成覆盖的合胞体(合体滋养层细胞)而分化。合体滋养层细胞构成分隔母体和胎儿组织的主要屏障，但在整个妊娠过程中进入母体循环，需要底层细胞滋养层细胞的补充。从细胞滋养层细胞增殖到分化的调控机制还不是很清楚，但这一过程是理解基本的胎盘发育和功能的基础。*我们的主要目标是定义信号和转录网络，以调节细胞滋养层细胞增殖和分化之间的平衡。我们最近发现，OVO-like 1(OVOL1)是一种与果蝇Ovo基因同源的保守转录抑制因子，被认为是Wnt/-catenin信号的下游效应因子，对细胞滋养层细胞的分化至关重要。我们还发现，在人类滋养层细胞中，OVOL1结合在MYC、ID1、TP63和ASCL2基因的近端启动子区域，与包括滋养层细胞在内的各种上皮细胞的祖细胞性状保持相关。因此，我们假设OVOL1介导的抑制促进细胞滋养层祖细胞状态的基因对于促进细胞滋养层细胞分化至关重要。在这项应用中，提出了三个目标，将共同阐明OVOL1‘S在滋养层细胞中作用的上游和下游机制。每个目标都将纳入我们实验室常规使用的成熟的人类滋养层细胞分化模型。在目标1中，我们将揭示在滋养细胞分化过程中参与OVOL1转录激活的上游信号机制，重点是激活-catenin作为OVOL1转录的驱动力。在目标2中，我们将检测OVOL1与MYC、ID1、TP63、ASCL2的原位表达模式，并用生化方法评估OVOL1结合对每个基因抑制的重要性。在目标3中，我们将确定组蛋白脱乙酰酶(HDAC)1和HDAC2在介导OVOL1对基因转录抑制作用中的重要性。意义：我们的研究将为滋养细胞分化和胎盘发育的基本调控提供关键的见解，并将增进我们对上皮周转的分子调控的理解。**

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/-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 -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. **