TRANSCRIPTIONAL REGULATION OF ENDOCARDIAL DEVELOPMENT

描述(由申请人提供)：这项建议的长期目标是了解特定的心内膜亚群--瓣膜前心内膜细胞(PVECs)的遗传控制，并确定它们在瓣膜形成中的作用。在胚胎发育过程中，NFATd的表达仅限于心内膜，在PVECs(在心脏瓣膜形成过程中不经历间充质转化的心内膜细胞)中表达增强。这些观察表明，对NFATd调节的关注可能为协调瓣膜前心内膜发育和瓣膜形成的转录程序提供独特的见解。最近在NFATd的第一内含子中发现了一个243 bp的序列，它是PVEC特异的转录增强子，是增强NFATd基因表达所必需的，并确定了这个增强子区域中一个保守的58bp序列是一个关键的顺式调控元件，控制着PVEC群体中NFATd表达的强度和特异性，我们假设这个NFATd增强子区域定义了一个增强体，它在调节NFATd表达的过程中发挥关键作用，这对正常的PVEC发育和瓣膜形成是至关重要的。因此，我们建议1)确定激活PVEC特异性NFATd转录增强复合体所需的分子组成和相互作用，用于凝胶漂移、DNA亲和纯化和质谱分析；2)利用基于cre-loxP的双基因小鼠模型连续分析增强体抑制和激活NFATd表达的序列；2)确定PVEC增强体在PVEC发育和瓣膜形成中的体内作用；3)通过使用Biggencre/loxP，puatk条件遗传启动子小鼠系在时空上禁用这些细胞来描述PVEC心内膜亚群在体内的作用。这项工作将确定心脏瓣膜发育所需的关键调节机制，并将为瓣膜退变提供新的组织再生疗法。

DESCRIPTION (provided by applicant): The long-term objectives of this proposal are to understand the genetic control of a specific subpopulation of endocardium, the pro-valve endocardial cells (PVECs), and determine their role in valve formation. During embryogenesis, NFATd expression is restricted to the endocardium and its expression is accentuated in the PVECs (endocardial cells that do NOT undergo mesenchymal transformation during the formation of cardiac valves). These observations suggest that a focus on NFATd regulation may provide unique insights into the transcriptional program orchestrating pro-valve endocardial development and valve formation. Having recently identified a 243 bp sequence within the first intron of NFATd that serves as a PVEC specific transcriptional enhancer required for accentuation of NFATd gene expression and having determined that a conserved 58 bp sequence within this enhancer region functions as a critical cis-regulatory element, controlling both the intensity and specificity of NFATd expression in the PVEC population, we hypothesized that this NFATd enhancer region defines an enhanceosome that plays a critical role in the regulation of NFATd expression essential for normal PVEC development and valve formation. Therefore, we propose to 1) Determine the molecular components and interactions required for activation of the PVEC-specific NFATd transcriptional enhancing complex using a novel enodardial cell line for gel shift, DNA affinity purification and mass psectrometry, 2) Define the in vivo role of the PVEC enhanceosome in PVEC development and valve formation utilizing a cre-loxp based dual-genetic mouse model for sequential analysis of enhanceosome suppression and activation of NFATd expression, and 3) Delineate the in vivo role of the PVEC endocardial subpopulation by spatiotemporal ablation of these cells using bigenic cre-/loxp, puAtk conditional genetic ablator mouse line. This work will define critical regulatory mechanisms required for heart valve development and will inform novel tissue-regeneration remedies for valve degeneration.