TRANSCRIPTIONAL REGULATION OF EMBRYONIC CARDIOGENESIS

该项目和该计划项目的统一主题是，心脏谱系和心脏限制性基因的表达的规范需要在心脏祖细胞出现期间发现富集的核心转录因子与这些基因的调控区域的模块化组织之间的组合相互作用，以指导心脏和 腔室特异性基因活性。这些核心因子之一是Nkx 2 -5，一种同源结构域基因，其对于正常心脏形态发生、肌发生和肌细胞分化是必需的。小鼠Nkx 2 -5基因座的发育调控是高度复杂和模块化的。目前，左心室和心房的增强剂仍然没有确定。我们建议使用Nkx 2 -5 BAC转基因建立一个有效的小鼠Nkx 2 -5基因增强子定位的体内系统。使用的策略，包括嵌套转座子10删除，鉴定保守的基因组序列比对，转基因和敲除的组合，我们建议检测和表征潜在的腔室特异性增强子/抑制子区域的Nkx 2 -5。另一个核心因子，血清反应因子（SRF）可能发挥主导作用的承诺的心脏祖细胞凭借其强制性要求的心脏中胚层的形成，并通过其相互作用的能力，以组合的方式与其他早期心脏富集的转录因子。SRF作为辅助因子（如Nkx 2 -5和加塔-4）结合的对接表面辅助，其可赋予特定基因程序的调节。我们发现SRF的限制性表达与Nkx 2 -5、GATA 4和CRPI/2（平滑肌Lim only因子）在早期脊椎动物胚胎中的表达密切重叠，与平滑肌靶基因和新生心肌细胞的最早出现相一致。该转录因子复合物是平滑肌特异性基因从头上调的中心。我们发现，dHAND与Nkx 2 -5、SRF、GATA 4和CRPt/2组合的出现激活了横纹β-肌动蛋白基因活性。我们建议研究心肌细胞分化的特化和维持的分子基础，以及Nkx 2 -5的模块化调节，以了解多腔心脏的早期发育，并为细胞替代治疗和心脏再生提供机会。以下目的是：确定模块化的遗传区域如何提供Nkx 2 -5基因位点的心源性调节活性？确定SRF如何将心前中胚层指定为定向的心源性细胞类型？确定CRP Lim家族成员如何指导早期心前基因活性以及dHAND的出现 CRP 3在定向心肌细胞中开启心源性基因活性？

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?