The Role of TGFbeta Signaling During Atrioventricular Canal Remodeling In Mice

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DESCRIPTION (provided by applicant): Congenital heart diseases (CHDs) are the leading cause of infant morbidity and mortality. The defect in which both left and right atria are solely connected to the left ventricle as a result of maldevelopment of the atrioventricular canal (AVC) region is referred to as Double-inlet-left-ventricle (DILV), and is among the least studied CHDs. The long term goal of this project is to reveal the molecular and genetic factors governing normal morphogenesis of the AVC region and their contributions to the DILV defect. The TGFbeta family of cytokines plays critical roles in cardiovascular development. My unpublished work shows that endothelium specific inactivation of Tgfbr2, which encodes the only known type II receptor of TGFbeta ligands, causes the DILV defect, providing a unique mouse genetic model for DILV. To facilitate cellular and molecular studies on AVC remodeling, we are currently developing a conditionally immortalized AV cushion mesenchymal cell line called tsA58-AVM. To my best knowledge, there has not been any report of establishment of an AV cushion mesenchymal cell line in the literature. The primary focus of this proposal is to utilize mouse models and the tsA58-AVM cell line to explore functions of TGFbeta signaling on AV endocardial/mesenchymal cells during AVC remodeling. Three specific aims are proposed. Specific aim 1: To test the hypothesis that Tgfbr2 mediated TGFbeta signaling is not required for epithelial-mesenchymal-transformation in embryos. We will examine the requirement of Tgfbr2 on EMT using Tgfbr2 null embryos. Specific aim 2: To test the hypothesis that TGFbeta signaling regulates proliferation, survival, and gene expression of AV cushion mesenchymal cells to promote proper AVC remodeling. We will perform detailed morphological and molecular characterization of the DILV mouse model, with the primary focus on the AVC region. Specific aim 3: To test the hypothesis that TGFbeta ligands can stimulate proliferation, survival and/or migration of tsA58-AVM cells in a dose dependent manner, we will first confirm the mesenchyme identity of tsA58-AVM cells by examining their expression of molecular markers using Western blot and Immunofluorescence analysis. In the second part, we will further examine the cellular responses of tsA58-AVM cells upon TGFbeta stimulation at the non- permissive temperature of 37 C, the condition under which these cells behave like primary cultured cells.