Experimental Study On Wdpcp Gene For Heart And Coronary Vessel Development

Congenital heart abnormalities is a life threatening disease for the children. The genetic mechanism of them is still not clear. In this subject, we use genetic tools to study the aberrant heart phenotype of Wdpcp mutant mouse, including atrial septal defect, ventricular septal defect, pulmonary atresia, outflow tract defect and abnormal coronary vessel. We want to reveal the relationship between Wdpcp gene and heart development.This study consists 4 parts. In the first part, we cultured the IMDC3 and do immunofluorescence staining to the Wdpcp protein and microtubules. The result showed that the Wdpcp protein is a key component of cilia.The second part, with the Gli1 Lac Z / + mouse, we trace Hh signaling pathway marker in Wdpcp mutant mice. The results showed that the wild type mouse embryo of E11.5d has the DMP structure in the heart and expressed the Gli1, but in Wdpcp mutant mice, Gli1 expression was significantly lower and DMP structure was dysplasia. Since DMP is the key component of primary atrial septum mediate by the Hh signaling, it is possible to conclude that atrial septal malformations in mice is due to Hh signaling pathway defect caused by Wdpcp mutation. In addition, The sixth pharyngeal arch artery of E11.5d mutant mouse embryos is completely absent. With the Gli1 Lac Z / + mouse to trace outflow tract cells, we found that the outflow tract of wild type mice express a large number of Gli1 positive cells, and mutant mice in the same position without any Gli1 expression. We conclude that due to absence of pharyngeal arch arteries, Hh signaling pathway is blocked and cardiac neural crest cells can not migrate to the outflow tract for growth.The third part, we observed the anterior descending coronary artery of Wdpcp mutant mice E18.5d embryo was short, rarefaction or even disappear. We used the endothelial cell specific antibodies(CD31) to stain the endothelial cells of coronary vessel in E12.5d, E13.5d, E14.5d embryo,but did not find obvious defect. Then, we extracted the RNA of E13.5d embryonic heart, for quantitative testing Gli1, Gli2, Gli3, Shh, Patch1 and other relevant factors. It was no statistically significant difference. But EMT markers exhibited difference. Snail1, Snail3, Twist1 of Wdpcp mutant mice expression were significantly reduced. Snail1 is the most important marker of EMT which is downstream regulated by Wt1. To confirm this finding, we use Wt1 Cre ERT2 / +, Rosa26 m Tm G as a tool to trace Wt1 positive epicardial cells and epicardium derived cells at E15.5d embryonic heart. The results showed that the subepicardium of wild type mice embryonic Heart hadl significantly more Wt1 positive cells than that of mutant mice. This result provides the evidence that there are EMT abnormalities in Wdpcp mutant mice, which will cause decreased production of smooth muscle cells. Coronary vessels remodeling process will be blocked.In the last part of this study, we selected 25 human cases of membranous ventricular septal defect with pulmonary hypertension, and to take blood samples for Wdpcp gene testing. The results were negative.This is the first time to study Wdpcp gene on the mouse heart and coronary vessel development. We reveal the mechanisms of heart defect caused by Wdpcp gene mutation.