Role of RNA-binding protein DDX3X in the endocardium during development and disease - PROJECT SUMMARY Cardiac development is a complex process that occurs early during embryonic development and requires precise regulation. Early cardiac development, especially of the endocardial and myocardial lineages, is implicated in the development of congenital heart diseases (CHDs). Unfortunately, despite CHDs being the most frequent birth defect affecting approximately one in 100 live births, very few mechanisms are understood about many different forms of CHDs. Proper cardiac development requires precise post-transcriptional regulation, such as alternative splicing, translation initiation, and mRNA modification. These functions are mediated by RNA-binding proteins (RBPs), and unsurprisingly, mutations to many RBPs are implicated in cardiac development and formation of CHDs. One such RBP is DDX3X, which is known from clinical evidence to have implications in CHDs: patients with mutations in DDX3X exhibit DDX3X syndrome, marked by neurodevelopmental disorders and increased risk of CHDs. However, DDX3X has not been well-studied to date in the context of heart development. My proposed research will study the dosage-sensitive effects and mechanisms of DDX3X in the endocardial lineage during cardiac development. In Aim 1 of this proposal, I will determine the effects of reduced DDX3X levels on endocardium formation and function. Using transgenic mice, I will conditionally delete DDX3X in male and female mouse endocardium. Expression level of DDX3X in the endocardium will be quantified using immunofluorescent imaging and flow cytometry. I will then characterize resulting phenotypes by examining phenotypic onset, structural and functional consequences, and cellular and molecular consequences in the heart using a combination of brightfield and immunofluorescent imaging, weight measurements, and echocardiograms. In Aim 2 of this proposal, I will identify the molecular mechanisms of DDX3X in the endocardium. Using immunofluorescent imaging, I will characterize the subcellular localization of DDX3X in the endocardium. I will then identify the direct regulatory network of DDX3X in the endocardial lineage using RNA-seq and Ribo-seq. Targets will be validated against pre-existing eCLIP data as well as in an in vitro system of human pluripotent stem cell-derived endocardial cells. The results of this research will contribute to our understanding about RNA biology, gene dosage, development and disease.
