Regulatory mechanisms to maintain epithelial barrier integrity in the placenta.

构成上皮屏障的细胞频繁更替。上皮屏障的维持取决于祖细胞的作用，祖细胞要么增殖以补充自身，要么分化成该组织功能所需的特化上皮。一个很好的例子是胎盘上皮，其中祖细胞滋养层细胞要么分裂以补充现有的祖细胞库，要么通过融合成覆盖的合胞体（合胞体滋养层）而分化。合胞体滋养层形成分离母体和胎儿组织的主要屏障，但在整个妊娠期间脱落到母体循环中，并且需要由下面的细胞滋养层细胞补充。从细胞滋养层增殖到分化的调控机制尚不清楚，但这一过程是了解胎盘基本发育和功能的基础。 我们的主要目标是确定信号和转录网络，调节细胞滋养层繁殖和分化之间的平衡。我们最近发现，OVO-like 1（OVOL 1），一个保守的转录抑制同源的果蝇Ovo基因，是作为Wnt/β-连环蛋白信号的下游效应牵连，是至关重要的细胞滋养层分化。我们还发现，在人类滋养层细胞中，OVOL 1结合在MYC、ID 1、TP 63和ASCL 2基因的近端启动子区域内，这些基因与维持各种上皮细胞（包括滋养层细胞）中的祖细胞性状有关。因此，我们假设OVOL 1介导的抑制基因，促进细胞滋养层祖细胞的状态是至关重要的促进细胞滋养层分化。在本申请中，提出了三个目标，它们将共同阐明OVOL 1在滋养层细胞中作用的上游和下游机制。每个目标将纳入我们实验室常规使用的人类滋养层分化的成熟模型。在目标1中，我们将揭示在滋养层分化过程中参与OVOL 1转录激活的上游信号传导机制，重点是激活β-连环蛋白作为OVOL 1转录的驱动力。在目标2中，我们将研究OVOL 1与MYC，ID 1，TP 63，ASCL 2的原位表达模式，并以生物化学方法评估OVOL 1结合对抑制每个基因的重要性。在目的3中，我们将确定组蛋白去乙酰化酶（HDAC）1和HDAC 2在介导OVOL 1对基因转录的抑制作用中的重要性。重要性：我们的研究将为滋养层细胞分化和胎盘发育的基本调控提供关键的见解，并将提高我们对上皮细胞更新的分子调控的理解。

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/ß-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 ß-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.