PAX3 AND CARDIAC CONOTRUNCAL DEVELOPMENT

Children'S Hospital Of Philadelphia, Philadelphia PA

Investigators

Linked publications & trials

Abstract

(Adapted from the Applicant's Abstract) 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, the investigators 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. The investigators have successfully established this method in the laboratory. A second approach will involve the use of "gene trapping" in embryonic stem (ES) cells. Those cells in which the gene has inserted under the regulation of a constitutive promoter will be eliminated by growth in gancyclovir. 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 a Pax3 responsive promoter. The candidate genes will be isolated by inverse PCR. Interesting clones will be appropriate for the construction of "knock-out" mice, since the target genes will in most cases have been inactivated by the insertion of the fusion gene. Finally, transgenic mice will be engineered in which rescue of the Splotch cardiac defect will be achieved using heterologous and portions of the endogenous Pax3 promoter. This will allow for structure/function analysis of the Pax3 gene in vivo to determine structural motifs required for cardiac development. Together, these approaches should begin to unravel the molecular cascades involved in the neural crest contribution to cardiac development, and in collaboration with the clinical project and core facilities will provide candidate genes to examine as causes of human congenital heart disease.