The Role of TGFbeta Signaling During Atrioventricular Canal Remodeling In Mice

描述（由申请人提供）：先天性心脏病（CHD）是婴儿发病率和死亡率的主要原因。由于房室通道（AVC）区域发育不良而导致的左心房和右心房单独连接到左心室的缺陷被称为双入口左心室（DILV），并且是研究最少的CHD之一。该项目的长期目标是揭示控制AVC区域正常形态发生的分子和遗传因素及其对DILV缺陷的贡献。细胞因子的TGF β家族在心血管发育中起关键作用。我未发表的工作表明，内皮特异性失活Tgfbr 2，它编码唯一已知的TGF β配体的II型受体，导致DILV缺陷，提供了一个独特的小鼠DILV遗传模型。为了促进AVC重塑的细胞和分子研究，我们目前正在开发一种条件永生化的AV垫间充质细胞系，称为tsA 58-AVM。据我所知，文献中还没有建立AV垫间充质细胞系的报道。该建议的主要重点是利用小鼠模型和tsA 58-AVM细胞系来探索AVC重构期间AV内皮细胞/间充质细胞上的TGF β信号传导的功能。提出了三个具体目标。具体目的1：检验Tgfbr 2介导的TGF β信号传导不是胚胎中上皮-间充质转化所需的假设。我们将使用Tgfbr 2无效胚胎来检查Tgfbr 2对EMT的要求。具体目标2：验证TGF β信号调节AV垫间充质细胞的增殖、存活和基因表达以促进适当的AVC重塑的假设。我们将对DILV小鼠模型进行详细的形态学和分子表征，主要关注的是AVC区域。具体目标3：为了检验TGF β配体可以剂量依赖性方式刺激tsA 58-AVM细胞增殖、存活和/或迁移的假设，我们将首先通过使用蛋白质印迹和免疫荧光分析检查其分子标记物的表达来确认tsA 58-AVM细胞的间充质身份。在第二部分中，我们将进一步研究在37 ℃的非允许温度下，tsA 58-AVM细胞对TGF β刺激的细胞反应，在该条件下，这些细胞表现得像原代培养的细胞。

DESCRIPTION (provided by applicant): Congenital heart diseases (CHDs) are the leading cause of infant morbidity and mortality. The defect in which both left and right atria are solely connected to the left ventricle as a result of maldevelopment of the atrioventricular canal (AVC) region is referred to as Double-inlet-left-ventricle (DILV), and is among the least studied CHDs. The long term goal of this project is to reveal the molecular and genetic factors governing normal morphogenesis of the AVC region and their contributions to the DILV defect. The TGFbeta family of cytokines plays critical roles in cardiovascular development. My unpublished work shows that endothelium specific inactivation of Tgfbr2, which encodes the only known type II receptor of TGFbeta ligands, causes the DILV defect, providing a unique mouse genetic model for DILV. To facilitate cellular and molecular studies on AVC remodeling, we are currently developing a conditionally immortalized AV cushion mesenchymal cell line called tsA58-AVM. To my best knowledge, there has not been any report of establishment of an AV cushion mesenchymal cell line in the literature. The primary focus of this proposal is to utilize mouse models and the tsA58-AVM cell line to explore functions of TGFbeta signaling on AV endocardial/mesenchymal cells during AVC remodeling. Three specific aims are proposed. Specific aim 1: To test the hypothesis that Tgfbr2 mediated TGFbeta signaling is not required for epithelial-mesenchymal-transformation in embryos. We will examine the requirement of Tgfbr2 on EMT using Tgfbr2 null embryos. Specific aim 2: To test the hypothesis that TGFbeta signaling regulates proliferation, survival, and gene expression of AV cushion mesenchymal cells to promote proper AVC remodeling. We will perform detailed morphological and molecular characterization of the DILV mouse model, with the primary focus on the AVC region. Specific aim 3: To test the hypothesis that TGFbeta ligands can stimulate proliferation, survival and/or migration of tsA58-AVM cells in a dose dependent manner, we will first confirm the mesenchyme identity of tsA58-AVM cells by examining their expression of molecular markers using Western blot and Immunofluorescence analysis. In the second part, we will further examine the cellular responses of tsA58-AVM cells upon TGFbeta stimulation at the non- permissive temperature of 37 C, the condition under which these cells behave like primary cultured cells.