Neural Crest and Cardiac Outflow Tract Formation

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(Adapted from applicant's abstract) The candidate is an MD with extensive medical training in the fields of pediatrics, neonatal-perinatal medicine and pediatric cardiology. He has acquired research experience in molecular and developmental biology and he is currently seeking intensive basic research training to transition to an independent investigator with the ultimate goal to become an academic physician scientist. At the time of the award the candidate will be a faculty member of the Children's Hospital of Philadelphia and Instructor in Pediatrics at the University of Pennsylvania. This proposal focuses on a critical time during embryonic cardiogenesis in which septation and formation of the outflow tract take place, a process involving neural crest contribution. Developmental defects occurring during this period account for a large number of congenital heart defects. Elucidation of the mechanisms involved in cardiac outflow tract formation will enhance our understanding of these diseases. Preliminary experiments performed by the applicant were aimed towards the identification of known and novel genes activated in the heart at the time when outflow tract septation begins (E11.5). One of them (Clone 35) encodes a protein of unknown function that is conserved through evolution. It is expressed specifically in the outflow tract and in neural crest derived mesenchyme at the time of aortopulmonary septation. Here, it is proposed an in depth analysis of this novel gene with particular attention to its potential role in neural crest mediated cardiac morphogenesis. In addition, a novel transgenic mouse created in the sponsor's laboratory will be used to analyze the fate of neural crest cells in several mutant mouse lines with congenital heart disease. Specific aims are: 1) In depth analysis of Clone 35. This will include isolation of full length cDNA, extensive expression analysis, preparation of antibodies to allow subcellular localization, genomic chromosomal mapping, and generation of transgenic mice with targeted deficiency of this gene. 2) Cell fate determination of neural crest cells in mouse mutant lines with congenital heart disease (Splotch, Patch, Nfl and collapsing 3 mutant embryos) by using transgenic mice expressing Cre-recombinase from the proximal Pax 3 promoter, that directs expression in neural crest cells, and appropriate Cre-reporter mice.