Transcriptional regulation of fibroblast generation in the embryonic mouse heart.

在胚胎发育过程中，上皮细胞，如那些覆盖在生长中的心脏(心外膜)的细胞，经历了一个称为上皮向间充质转化(EMT)的过程，并转化为间充质前体细胞，从而产生了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内EMT程序的变化而产生，称为内皮细胞到间充质细胞的转变(EndMT)，这对心脏瓣膜的形态形成非常重要。细胞因子转化生长因子是EMT和EndMT的关键调节因子。间充质同源盒2(MEOX2)转录因子阻断转化生长因子介导的EMT。我们以前的工作证明，MEOX2抑制内皮细胞的增殖，促进内皮细胞的衰老。与MEOX2不同，锌指E盒结合同源框2蛋白(ZEB2)被转化生长因子激活，而ZEB2抑制Meox2和上皮性标志物如E-钙粘附素的表达。胚胎心脏成纤维细胞发育是否需要ZEB2/MEOX2转录开关尚不清楚。 我的研究计划的总体目标是确定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5年的目标是： 目的1.研究ZEB2/MEOX2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功能丧失的方法来测试这些转录开关在心脏细胞(心外膜和内皮细胞)向成纤维细胞间质转化过程中的作用。为了在我们对ZEB2/MEOX2开关的体外研究的基础上，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脏外膜中的这些基因。 目标2.确定控制ZEB2和Meox2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个调控基因表达的基因组调控元件尚不清楚。通过鉴定和验证调控Meox2和ZEB2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。 总而言之，我的HQP(2名博士生和1名硕士学生)进行的这些研究将描绘ZEB2/MEOX2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的重要途径的新机制。通过这些学习，我的HQP将获得实验室研究(实验设计、进行实验、分析结果)以及传播他们的发现(手稿写作和演讲)的技能，这将使他们在未来的广泛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.