Conotruncal heart malformations result from a failure of the aorta and pulmonary trunk to separate into two distinct vessels during embryonic development. Several lines of evidence suggest that these defects are the result of insufficient numbers of cardiac neural crest (NC) cells migrating to and colonizing the developing outflow tract, The Splotch (Sp2H) mutation provides a mouse model system in which to study the relationship between NC cell Migration and cardiovascular development. The applicant hypothesizes that (i) Sp2H mutant NC cells are inherently defective, (ii) insufficient Sp2H NC cells emigrate from the neural tube and/or (iii) the neural tube and the immediate surrounding matrix is non-permissive in Sp2H embryos due to a failure of the mutant Pax3 to negatively regulate the expression of molecules known to be involved in cell adhesion. The overall goal of this proposal is to identify which mechanism is responsible for the resultant conotruncal heart defects by determining when an how the reduction in Sp2H NC cell numbers occurs and, to alter the function of the candidate molecules in Sp2H embryos to correct the heart defect, utilizing a genetically-defined mammalian model, The applicant will achieve this by monitoring the progress of NC cell migration temporally and spatially, and by supplementing cultured whole embryos with additional NC cells that can either correct the functional defect in the tissue microenvironment through which NC cells migrate or increase the number of migratory NC cells