TRANSCRIPTIONAL REGULATION OF EMBRYONIC CARDIOGENESIS

这个项目和计划项目的一个统一主题是，心脏谱系的规范和心脏限制性基因的表达需要在心脏前体细胞出现期间被发现丰富的核心转录因子和这些基因的调节区的模块化组织之间的组合相互作用，以指导心脏和 小室特异性基因活性。Nkx2-5是这些核心因子之一，它是一个同源结构域基因，对正常的心脏形态发生、肌肉发生和心肌细胞分化是必不可少的。小鼠Nkx2-5基因座的发育调控是高度复杂和模块化的。目前，左心室和心房增强剂仍未确定。我们建议利用Nkx2-5 BAC转基因技术建立一个有效的体内系统，用于小鼠Nkx2-5基因的增强子定位。我们建议使用包括嵌套转座子10缺失、保守基因组序列比对、转基因和敲除在内的组合策略来检测和表征Nkx2-5潜在的小室特异性增强子/抑制子区域。另一个核心因素是血清反应因子(SRF)，由于它对心脏中胚层的形成是必需的，并且能够与其他早期心脏丰富的转录因子以组合的方式相互作用，因此可能在心脏前体细胞的承诺中发挥主导作用。SRF作为辅助因子结合的对接表面，如Nkx2-5和GATA-4，可能对特定的基因程序进行调节。我们发现在脊椎动物早期胚胎中，SRF的限制性表达与Nkx2-5、GATA4和CRPI/2(仅限平滑肌LIM因子)等密切重叠，与最早出现的平滑肌靶基因和新生心肌细胞相一致。这个转录因子复合体是平滑肌特异基因从头开始上调的中心。我们发现，DHand的出现与Nkx2-5、SRF、GATA4和CRPT/2一起激活了条纹β-肌动蛋白基因的活性。我们建议研究Nkx2-5在心肌细胞分化的规范、维持和模块化调控中的分子基础，以了解多室心脏的早期发育，并为细胞替代治疗和心脏再生提供机会。以下目的是：确定模块化遗传区如何提供Nkx2-5基因座的生心调节活性？为了确定SRF如何将心前中胚层指定为特定的心源性细胞类型？确定C反应蛋白LIM唯一家族成员如何引导早期促心绞痛基因活性以及DHAND如何出现 CRP3在承诺的心肌细胞中启动心脏基因活性吗？

A unifying theme of this project and the program project is that specification of cardiac lineages and expression of cardiac restricted genes requires combinatorial interactions between core transcription factors found to be enriched during the emergence of cardiac progenitor cells and the modular organization of regulatory regions of these genes to direct cardiac and chamber specific gene activity. One of these core factors is Nkx2-5, a homeodomain gene, which is essential for normal heart morphogenesis, myogenesis and myocyte differentiation. Developmental regulation of the murine Nkx2-5 genetic locus is highly complex and modular. Currently, the left ventricular and atrium enhancers are still not identified. We propose to establish an efficient in vivo system for enhancer mapping of the mouse Nkx2-5 gene using Nkx2-5 BAC transgenics. Using a combination of strategies, that include nested transposon 10 deletions, identification of conserved genomic sequence alignments, transgenics and knockouts, we propose to detect and characterize potential chamber specific enhancer/repressor regions of Nkx2-5. Another core factor, serum response factor (SRF) may play a leading role in the commitment of cardiac progenitors by virtue of its obligatory requirement for cardiac mesoderm formation and by its ability for interacting in a combinatorial manner with other early cardiac enriched transcription factors. SRF assists as a docking surface for the binding of cofactors, such as Nkx2-5 and GATA-4 that may confer the regulation of specific gene programs. We have found that restricted expression of SRF closely overlapped with such as Nkx2-5, GATA4 and CRPI/2 (smooth muscle Lim only factors), in early vertebrate embryos, coincident with the earliest appearance of smooth muscle target genes and nascent myocardial cells. This transcription factor complex,was central for the de novo upregulation of smooth muscle specified genes. We discovered that the appearance of dHAND in combination with Nkx2-5, SRF, GATA4, and CRPt/2 activated striated beta-actin gene activity. We propose to investigate the molecular basis underlying the specification, and maintenance of cardiac muscle cell differentiation, and the modular regulation of Nkx2-5 to understand early development of multichambered heart and provide opportunities for cell replacement therapy and heart regeneration. The following aims are: To determine how modular genetic regions provide for cardiogenic regulatory activity of the Nkx2-5 gene locus? To determine how SRF specifies procardiac mesoderm to committed cardiogenic cell types? To determine how CRP Lim only family members direct early procardiogenic gene activity and how the appearance of dHAND and CRP3 switches on cardiogenic gene activity in committed cardiomyoctyes?