Jielin Liu

Date of Award

1-1-2016

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biomedical Sciences

First Advisor

Ke Zhang

Abstract

Congenital heart diseases (CHDs) are among the most common type of developmental anomaly affecting 8 in 1000 live births. Approximately 30% of CHDs involve cardiac outflow tract (OFT) malformation which leads to significant morbidity and mortality in both children and adults. Development of OFT is regulated by a complex genetic network including Sonic-Hedgehog signaling pathway, TGF-β and BMP signaling pathway. Mutation of a transcription factor, Gata4, has been known to cause OFT defects including double outlet right ventricle (DORV) in both human and mice for decades. Several transcription targets of Gata4 have been identified such as Mef2c, Ccnd2 and Cdk4, however none has been shown to be functionally involved in OFT development. Thus, the important role of Gata4 during OFT development remains unclear.

Here we analyzed the requirement of Gata4 in several cell progenitors which contribute to the development of OFT and found that knocking down Gata4 in myocardium, second heart field and cardiac neural crest cells was able to maintain normal OFT development. However, Gata4 haploinsufficiency in Hedgehog (Hh) receiving cells caused a high penetrance of DORV in embryos at embryonic stage 14.5. Elongation as well as rotation defect were also observed in these mutant embryos compared to wildtype. Through TUNEL apoptosis assay, we found that there was no significant increase in cell apoptosis within the outflow tract region in these mutant embryos compared to wildtype. However, BrdU proliferation assay showed a significant decrease of cell proliferation in

the conal portion of outflow tract in these mutant embryos. By performing real-time PCR, luciferase assay and ChIP-qPCR, we found Gata4 directly binds to Hh signaling effector Gli1 and regulates its expression. Also, by further blocking Hh signaling pathway in Gata4 mutant embryos or reducing such blockage, we found the occurrence and severity of OFT defect increased or decreased respectively. Together these results suggest that Gata4 interacts with Hedgehog signaling pathway in regulating outflow tract development.

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