TRANSCRIPTIONAL REGULATION OF ENDOCARDIAL DEVELOPMENT

描述（由申请方提供）：本提案的长期目标是了解特定内皮细胞亚群（前瓣膜内皮细胞（PVEC））的遗传控制，并确定其在瓣膜形成中的作用。在胚胎发生期间，NFATd表达限于内皮细胞，并且其表达在PVEC（在心脏瓣膜形成期间不经历间充质转化的内皮细胞）中增强。这些观察结果表明，专注于NFATd调节可能提供独特的见解的转录程序编排前阀内皮细胞的发展和阀门的形成。最近鉴定了NFATd的第一内含子内的243 bp序列，该序列用作增强NFATd基因表达所需的PVEC特异性转录增强子，并确定了该增强子区域内的保守58 bp序列用作关键的顺式调节元件，控制PVEC群体中NFATd表达的强度和特异性，我们假设该NFATd增强子区域定义了一个增强体，该增强体在正常PVEC发育和瓣膜形成所必需的NFATd表达的调节中起关键作用。因此，我们提出1）使用新的心内膜细胞系进行凝胶位移、DNA亲和纯化和质谱测定，确定PVEC特异性NFATd转录增强复合物活化所需的分子组分和相互作用，2）利用基于cre-loxp的双标记酶，确定PVEC增强体在PVEC发育和瓣膜形成中的体内作用。用于NFATd表达的增强体抑制和激活的顺序分析的遗传小鼠模型，和3）通过使用双基因cre-/loxp，puAtk条件遗传消融小鼠系时空消融这些细胞来描述PVEC内皮细胞亚群的体内作用。这项工作将定义心脏瓣膜发育所需的关键调控机制，并为瓣膜退化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.