Conotruncal defects represent a significant proportion of congenital cardiac malformations, and several animal models now exist that demonstrate reproducible abnormalities of this developmental pathway. In some cases, a single mutant gene is responsible for the abnormal phenotype. The Splotch mouse represents an animal model for persistent truncus arteriosus and related abnormalities, in which the specific genetic defect has been identified. The Splotch mouse is the result of a mutated Pax-3 gene. Pax-3 is a member of a developmentally critical class of transcription factors.
The aim of this proposal is to identify molecular pathways regulated by Pax-3 during cardiac development. The identification of genes regulated by specific transcription factors remains a major stumbling block in the field of developmental genetics, and no target genes regulated by Pax-3 have been identified. In order to identify target genes, I propose a dual approach that requires both the presence of a high affinity Pax-3 binding site within the genomic sequence, and putative target gene expression in normal but not Pax-3 deficient mice. Specific DNA sequences recognized by the Pax-3 paired domain will be selected from amongst a pool of genomic fragments. Genomic clones corresponding to these fragments will be identified. These genomic clones will then be further screened with probes derived from a completely different approach. Differential display PCR will be used to identify cDNA fragments specifically expressed in normal litter mates of Pax-3 deficient mice at the time of peak Pax-3 expression just prior to conotruncal septation. Genomic clones identified by this dual approach will represent excellent candidates for regulation by Pax-3. Further evidence for direct regulation will be provided by demonstrating: 1) specific, high affinity binding of Pax-3 protein to promoter sequences, 2) the ability of Pax-3 to trans activate a reporter construct containing the candidate promoter region, and 3) appropriate spatial and temporal expression patterns. A specific candidate gene, Nf1, will be examined in detail. This gene is responsible for type I neurofibromatosis. Mice engineered to lack a functional Nf1 allele uniformly display a specific abnormality of conotruncal development that is phenotypically different from the Splotch mouse. Pax-3 and Nf1 may contribute to parallel pathways required for normal cardiac development. However, available evidence suggests that Nf1 lies downstream of Pax-3. If so, NF1 expression should be altered in Splotch mice, and mice bred to be homozygous deficient for both genes should resemble the Splotch homozygote embryos. Thus, a general scheme for identifying genes regulated by a specific transcription factor during development is proposed. The sequence of molecular events leading to conotruncal development will be clarified by using two mouse models in which the particular mutant gene is known. Under the guidance of several renowned and devoted mentors who already know and support the candidate, this proposal will provide the foundation necessary, and serve as a catalyst, for the transition to independent research.
