verbatim): This proposal suggests methods for identifying molecular pathways regulated by the transcription factor Pax3 during cardiac development. Homozygous deficiency of Pax3 in the mouse leads to mid-gestation embryonic lethality. The mutant embryos display persistent truncus arteriosus and other abnormalities that resemble the effects of over or underexposure to retinoic acid. A similar constellation of findings is seen in chick embryos after neural crest ablation and in human patients with DiGeorge syndrome. Pax3 is one of nine members of a developmentally critical gene family. Nevertheless, few downstream genes regulated by this family of transcription factors have been implicated. Here, I propose two methods to screen for downstream targets in order to identify pathways disrupted in congenital heart disease. The first method involves the use of representational difference analysis. This procedure, a modification of subtractive hybridization, will allow for the identification of genes expressed in normal but not Pax3 deficient embryos at the time when Pax3 expression is peaking and when neural crest cells are migrating towards the outflow region of the heart. We have successfully verified this method in our laboratory and have identified candidate target genes. A second approach will involve the use of """"""""gene trapping."""""""" A promoterless gene encoding a fusion of hygromycin resistance and thymidine kinase will be transfected into cultured cells. Those cells in which the gene has inserted under the regulation of a constitutive promoter will be eliminated. Pax3 will then be expressed in the remaining cells, and those resistant to hygromycin will be isolated. These will represent cells in which the fusion gene has inserted under the regulation of Pax3 responsive promoter. The candidate genes will be isolated by 5' RACE and inverse PCR. Candidate Pax3 target genes emerging from both screens will be evaluated by expression analysis in wild type and Splotch embryos. Finally, transgenic mice will be engineered to functionally analyze the Pax3 promoter and to determine the cell autonomy of the neural crest defect. Together, these approaches should begin to unravel the molecular cascades involved in the neural crest contribution to cardiac development and will provide candidate genes to examine as causes of human congenital heart disease.
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