ABSTRACT Congenital heart disease (CHD) is the most common birth defect, yet most etiologies remain unknown. SHROOM3 is a novel CHD candidate gene that functions through interactions with F-ACTIN, Rho Associated Coiled-Coil Containing Protein Kinase (ROCK) and Disheveled2 (DVL2) and thus participates in the noncanonical WNT/Planar Cell Polarity (PCP) signaling pathway. Disruption of PCP signaling leads to cardiac defects, including left-right patterning defects, left ventricle noncompaction (LVNC), ventricular septal defects (VSDs) and outflow tract (OFT) defects. However, SHROOM3’s precise role in the PCP signaling cascade, and in cardiac development, is only partially understood. For example, the Ware laboratory recently identified that SHROOM3 variants are associated with the left-right patterning defect heterotaxy and CHD in patients. This finding prompted me to study the impact of the loss of SHROOM3 expression on cardiac development. Utilizing a gene trap mouse model (Shroom3gt/gt), I demonstrated that SHROOM3 loss-of-function results in CHDs in embryonic hearts, including VSDs, LVNC and OFT defects in homozygous mutant animals. We also showed evidence there may be genetic interaction between Shroom3 and Dvl2 during cardiac development. Moreover, molecular studies have established a protein-protein interaction (either direct or through a common binding partner) between SHROOM3 and DVL2, though the binding site is poorly defined. Interestingly, a recent study revealed that canonical WNT/PCP signaling is controlled in part through the deubiquination of DVL2 via USPX9, a highly conserved deubiquitylase. Utilizing immunoprecipitation (IP)/mass spectrometry (MS) of SHROOM3- transfected Cos7 cells, I have shown protein-protein interaction between SHROOM3 and USPX9. These observations have collectively led to the hypothesis that signaling between SHROOM3, DVL2 and USP9X is necessary for normal cardiac development and that disruption of this signaling can contribute to CHD. There is evidence SHROOM3 interacts with USP9x and DVL2 however the interactions are at best poorly defined. Genetic interactions between Shroom3 and Dvl2 during cardiac development have not been not fully delineated and genetic interaction between Shroom3 and Usp9x are unexplored. Aim 1 will assay genetic interaction between Shroom3, Dvl2 and Usp9x, analyzing cardiac defects and disrupted PCP endpoints. Although data demonstrate SHROOM3 binds DVL2, and USP9X, the binding site for DVL2 is only roughly localized to a 490 amino acid region within SHROOM3 and SHROOM3’s USP9X binding site is completely unknown. Aim 2 will assay protein-protein interactions between SHROOM3, USP9X and DVL2. Defining SHROOM3’s interaction partners will help determine its role in PCP, cardiac development and CHD pathogenesis. The plan will also build skills, under close mentorship as an Early Career Investigator, in molecular biology, mouse disease models and genomics analysis, providing a skillset to i...