Formation of the cardiac outflow tract is a complex morphogenetic process, whose failure results in a variety of clinically important congenital heart malformations. We have found that the homeodomain transcription factors encoded by the Pbx gene family are required for development of the cardiac outflow tract in mice. Mutations in Pbx genes lead to persistent truncus arteriosus, tetralogy of Fallot, overriding aorta and bicuspid aortic valves. These defects resemble cardiac anomalies observed in chick when neural crest cells are ablated. We hypothesize that Pbx genes are essential for neural crest cell function in mice, and propose to use those mouse models to investigate the mechanisms of how Pbx1 gene regulates neural crest cell function required for cardiac outflow tract development. These studies will provide insights to the developmental mechanisms of congenital heart defects. Specifically, we will study (1) whether Pbx1 acts cell intrinsically within the neural crest cells to regulate their function, (2) whether Pbx1 is essential to program the developmental fates of neural crest cells and (3) the molecular pathways downstream of Pbx1 in cardiac development.
Aim #1 : Determine the cellular sites of Pbx1 action required for cardiac development We will use the genetic Cre-lox methods and several mouse lines that express Ore recombinase in neural crest cells, myocardial cells and endocardial cells to study the cellular sites where Pbx1 may function to regulate the development of cardiac neural crest cells.
Aim #2 : Determine the role of Pbx1 in neural crest cell development. To study if Pbx1 is required for specifying the developmental identities of cardiac neural crest cells, we plan to employ RNA in situ hybridization and rhombomeric differentiation markers to study the rhombomeric identities in Pbx1-/- mouse embryos. As cardiac neural crest cells originate from rhombomeres, expression of rhombomeric markers in the PbxT1'embryos will provide insights to the fate specification of cardiac neural crest cells. Additional markers of neural crest cells will also be used to study the differentiation of neural crest cells as they migrate out of the neural tube.
Aim #3 : Define the molecular pathways affected by the absence of Pbx1. We will use standard molecular biology and embryology methods to study if Pax3 pathway is downstream of Pbx1 in cardiac development.
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