The Role of Eomesodermin in Cardiac Development

脱中胚层蛋白在心脏发育中的作用

• 批准号: 6970895
• 负责人: KENNETH RYAN
• 金额: $ 33.2万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6970895
• 关键词: Xenopus; Xenopus oocyte; activins; binding proteins; binding sites; cardiogenesis; cell differentiation; cell type; developmental genetics; embryogenesis; gel mobility shift assay; gene expression; genetic promoter element; growth factor receptors; heart; in situ hybridization; mesoderm; protein isoforms; protein protein interaction; protein structure function; transcription factor; transforming growth factors

DESCRIPTION (provided by applicant): The role of Eomesodermin (Eomes), a T-box transcription factor, in cardiac development is incompletely understood. Eomes was first described by the PI and colleagues, and was shown to be required both in Xenopus and in mouse development. Eomes responds to TGF-a signaling in an immediate-early fashion, through a 571 bp enhancer element (ARE) involving Smad/FAST proteins. Shown herein, Eomes is required in heart development. Our hypothesis consists of a four-step mechanism. 1) TGF-a signaling results in cooperation of Eomes with Smad proteins in trans-activating downstream target genes. 2) Eomes-Smad complexes bind to sites within the 571 bp Eomes ARE, maintaining Eomes expression. 3) Proper Eomes dosage is required for downstream gene activations. 4) Sustained Eomes expression is required in ventricular cardiomyocytes and bulbus cordis. The proposed research will further elucidate this essential cardiac Eomes pathway. AIM 1. A dominant-negative (DN) activin receptor prevents Eomes activation of mesodermal genes. Wild type and DN-Smads are used to determine if Eomes interacts with Smads in activating target genes. The Eomes promoter driving a human globin reporter is used to ask if Eomes interacts functionally with Smads. AIM 2. Eomes binding sites lie in close proximity to Smad sites in a functionally important region of the ARE. Promoter mutation, gel shift, and a promoter bioassay are used to characterize the regions within the Eomes ARE that regulate Eomes-Smad signaling, and identify their interacting proteins. AIM 3. A conditional Eomes isoform is used to examine if the dose of Eomes is important in normal cardiac development. AIM 4. In situ hybridization is used to determine the full range of cardiac cell types expressing Eomes; a conditional dominant-negative isoform of Eomes examines the requirement for Eomes in the heart.

描述（由申请人提供）：Eomesodermin (Eomes)（一种 T 盒转录因子）在心脏发育中的作用尚不完全清楚。 Eomes 首先由 PI 和同事描述，并被证明是爪蟾和小鼠发育所必需的。 Eomes 通过涉及 Smad/FAST 蛋白的 571 bp 增强子元件 (ARE) 以立即早期方式响应 TGF-a 信号传导。本文所示，Eomes 在心脏发育中是必需的。我们的假设由四步机制组成。 1) TGF-a 信号传导导致 Eomes 与 Smad 蛋白合作反式激活下游靶基因。 2) Eomes-Smad 复合物与 571 bp Eomes ARE 内的位点结合，维持 Eomes 表达。 3) 下游基因激活需要适当的Eomes剂量。 4) 心室心肌细胞和心球细胞需要持续的 Eomes 表达。拟议的研究将进一步阐明这一重要的心脏 Eomes 途径。 目的 1. 显性失活 (DN) 激活素受体阻止 Eomes 中胚层基因的激活。野生型和 DN-Smads 用于确定 Eomes 是否与 Smads 相互作用来激活靶基因。驱动人类珠蛋白报告基因的 Eomes 启动子用于询问 Eomes 是否与 Smads 进行功能性相互作用。目标 2.Eomes 结合位点位于 ARE 功能重要区域中的 Smad 位点附近。启动子突变、凝胶位移和启动子生物测定用于表征 Eomes ARE 内调节 Eomes-Smad 信号传导的区域，并鉴定它们的相互作用蛋白。目的 3. 使用条件 Eomes 同种型来检查 Eomes 的剂量对于正常心脏发育是否重要。 AIM 4. 原位杂交用于确定表达 Eomes 的全范围心肌细胞类型； Eomes 的条件显性失活亚型检查心脏对 Eomes 的需求。