Transcriptional regulation of fibroblast generation in the embryonic mouse heart.

在胚胎发育过程中，上皮细胞，如覆盖生长中的心脏（心外膜）的上皮细胞，经历称为上皮细胞向间充质细胞转化（EMT）的过程，并转化为间充质祖细胞，其产生发育中的心脏的成纤维细胞、内皮细胞和血管平滑肌细胞群。心脏成纤维细胞也可以通过内皮细胞中EMT程序的变化产生，称为内皮细胞向间充质转化（EndMT），这对心脏瓣膜形态发生很重要。EMT和EndMT的关键调节因子是细胞因子转化生长因子-（TGF-β）。间质同源框2（MEOX 2）转录因子阻断TGF-介导的EMT。我们先前的工作已经证明MEOX 2抑制内皮细胞增殖并促进内皮衰老。与MEOX 2相反，锌指E盒结合同源盒2蛋白（ZEB 2）通过TGF-处理活化，ZEB 2抑制Meox 2和上皮标志物（如E-钙粘蛋白）的表达。 ZEB 2/MEOX 2转录开关是否是胚胎心脏成纤维细胞发育所必需的尚不清楚。 我的研究计划的总体目标是确定在胚胎发育过程中心脏成纤维细胞分化所需的转录因子网络。 我未来五年的目标是： 目标1.检查ZEB 2/MEOX 2通路在体外和体内胚胎心脏成纤维细胞生成中的作用。 我们将使用功能获得和丧失方法来测试这些转录开关在心脏细胞（心外膜和内皮细胞）向成纤维细胞的间充质转化过程中的作用。 为了建立我们对ZEB 2/MEOX 2开关的体外研究，我们将使用转基因小鼠方法来特异性地删除心脏内皮细胞和心外膜中的这些基因。 目标2.鉴定控制Zeb 2和Meox 2的细胞类型特异性表达的增强子元件。在胚胎发育过程中控制这两种调控基因表达的基因组调控元件尚不清楚。通过鉴定和验证调节Meox 2和Zeb 2表达所需的增强子元件，我们将能够建立控制心脏成纤维细胞发育的转录因子网络。 总之，由我的HQP（2名博士生和1名硕士生）进行的这些研究将描述ZEB 2/MEOX 2如何控制胚胎成纤维细胞的生成。这项研究计划将描述一种控制哺乳动物心血管系统胚胎发育重要途径的新机制。通过这些学习，我的HQP将获得实验室研究技能（实验设计，进行实验，分析结果）以及传播他们的发现的技能（手稿写作和演讲），这将使他们能够在未来的职业生涯中取得成功。

During embryonic development, epithelial cells, such as those that cover the growing heart (epicardium), undergo a process termed Epithelial to Mesenchymal Transition (EMT) and convert into mesenchymal progenitors, which give rise to the fibroblast, endothelial and vascular smooth muscle cell populations of the developing heart. Cardiac fibroblasts can also be generated by a variation of the EMT program in endothelial cells termed Endothelial to Mesenchymal Transition (EndMT), which is important for cardiac valve morphogenesis. A key regulator of EMT and EndMT is the cytokine Transforming Growth Factor- (TGF-). The Mesenchyme Homeobox 2 (MEOX2) transcription factor blocks TGF- mediated EMT. Our previous work has demonstrated that MEOX2 inhibits endothelial cell proliferation and promotes endothelial senescence. In contrast to MEOX2, the Zinc Finger E Box-Binding Homeobox 2 protein (ZEB2) is activated by TGF- treatment and ZEB2 represses the expression of Meox2 and epithelial markers such as E-Cadherin. Whether the ZEB2/MEOX2 transcriptional switch is required for embryonic cardiac fibroblast development is not known. The overall goal of my research program is to identify the transcription factor networks that are required for cardiac fibroblast differentiation during embryonic development. My goals for the next 5 years are to: Goal 1. Examine the role of the ZEB2/MEOX2 pathway in the generation of embryonic cardiac fibroblasts both in vitro and in vivo. We will use both gain and loss of function approaches to test the roles of these transcriptional switches during mesenchymal transition of cardiac cells (epicardial and endothelial) into fibroblasts. To build on our in vitro studies of the ZEB2/MEOX2 switch, we will use transgenic mouse approaches to specifically delete these genes in cardiac endothelial cells and the cardiac epicardium. Goal 2. Identify enhancer elements that control cell type specific expression of Zeb2 and Meox2. The genomic regulatory elements that control expression of these two regulatory genes during embryonic development are not known. By identifying and validating the enhancer elements required to regulate the expression of Meox2 and Zeb2, we will be able to establish the transcription factor network that controls the development of cardiac fibroblasts. Together, these studies conducted by my HQP (2 PhD students and 1 MSc student) will delineate how ZEB2/MEOX2 control embryonic fibroblast generation. This research program will characterize a novel mechanism that controls an important pathway in embryonic development of the mammalian cardiovascular system. Through these studies, my HQP will acquire skills in laboratory research (experimental design, conducting experiments, analyzing results) as well as skills in disseminating their findings (manuscript writing and presenting talks) that will enable them to succeed in in a wide range of future careers.