Molecular mechanisms that specify the cardiac cell lineage

在这项研究中，我们通过分析心脏特异性转录因子Nkx2.5的增强子来研究指定心脏细胞谱系的分子机制。我们分离了覆盖Nkx2.5上游序列的60 kb的BAC克隆，并产生了约70个含有不同长度的上游序列的报告构建体。我们在大鼠新生心肌细胞的原代培养物中测量了每个构建体的荧光素酶活性，并鉴定了6个表现出显着高荧光素酶活性的区域。我们将这6个DNA片段亚克隆到LacZ报告载体中并产生转基因小鼠。在6个片段中，3个片段在体内显示增强子活性。特别是Nkx2.5上游5.2 ~ 6.4kb的DNA片段在从心新月形到直心管的心肌细胞中表现出强活性。该DNA片段含有串联的Smad结合位点，并且Smad结合位点的缺失几乎消除了培养的心肌细胞中的增强子活性。这一结果表明，Smad信号通路是Nkx2.5在心脏发育的早期阶段表达所必需的，并且可能在心脏细胞谱系的特化中起关键作用。我们现在正试图用转基因方法在体内证实这一结果。在该片段中，我们还发现了另一个在体外具有高报告活性的区域。由于该区域不含已知的顺式作用元件，我们正试图利用酵母单杂交系统分离结合蛋白。

In this study, we investigated molecular mechanisms that specify the cardiac cell lineage by analyzing enhancers of Nkx2.5, a cardiac specific transcription factor. We isolated BAC clones that cover 60kb of upstream sequence of Nkx2.5 and generated approximately 70 reporter constructs containing various length of the upstream sequence. We measured luciferase activity of each construct in primary culture of rat neonatal cardiac myoctyes and identified 6 regions exhibiting significantly high luciferase activities. We subcloned these 6 DNA fragments into a LacZ reporter vector and generated transgenic mice. Out of the 6 fragments, 3 fragments showed enhancer activities in vivo. Particularly, a DNA fragment between 5.2 and 6.4kb upstream of Nkx2.5 exhibited strong activity in cardiac myocytes from the cardiac crescent to the straight heart tube. This DNA fragment contained tandem Smad binding sites and deletion of the Smad binding sites almost abolished the enhancer activity in cultured cardiac myocytes. This result suggested that the Smad signaling pathway was required for expression of Nkx2.5 at early stages of cardiac development and may play a critical role in the specification of the cardiac cell lineage. We are now trying to confirm this result in vivo using transgenic approach. In this fragment, we also found another region having high reporter activity in vitro. Since this region did not contain known cis-acting elements, we are trying to isolate binding proteins using yeast one-hybrid system.