Paired box 3 (Pax3) is a transcription factor that is vital for cardiac neural crest (NC) specification and morphogenesis. CNC derivatives are essential for outflow tract (OFT) septation and remodeling of the pharyngeal arch arteries (AA) to give rise to the great vessels exiting the heart. Significantly, Pax3 mutations in humans and mice lead to persistent truncus arteriosus (PTA), interventricular septal defects (VSD), and abnormal AA remodeling, which phenocopies both surgical and genetic ablation of premigratory neural crest in chick and mouse embryos. Ap2aCre NC-restricted Pax3 deletion results in OFT alignment defects, and transgenic re-expression of Pax3 driven by a NC promoter (1.6kbPax3) rescues the Pax3 null phenotype. Data demonstrate that Msh homeobox protein 2 (Msx2) is transcriptionally regulated by Pax3 and failure of Msx2 transcriptional repression leads to a range of NC-related defects (including PTA/VSD). These data suggest that Pax3 mediates cardiac NC specification via an early and cell autonomous mechanism. Intriguingly, Pax3 nulls do not entirely prevent cardiac NC colonization of the OFT, indicating potential genetic compensation. Mouse Pax3 is expressed in the early neural crest and dorsally-restricted through neural tube closure, following which Pax3 expression is ventrally expanded. In contrast, mouse Pax7 (Pax3 paralogue) expression is first detected following neural tube closure (~E8.5) and is normally restricted from the dorsal-most region of the neural tube. Our preliminary data show that Pax7 is upregulated and dorsally expanded within the neural tube of a novel Pax3 hypomorphic mouse (~10% normal Pax3 protein;Pax3Hypo). Data demonstrate that Pax7 nulls are viable and do not exhibit any cardiac defects, however our preliminary data reveal that Pax7 systemic mutants on a Pax3Hypo background (Pax7-/-;Pax3Hypo) develop 100% penetrance of PTA and VSD. Both Pax3 and Pax7 contain nearly identical DNA-binding domains, and both contain differentially spliced transactivation domains vital for Pax3 and Pax7 transcriptional activity. While Pax3 splice variants are evolutionarily conserved, the role of Pax3 splice variatio in embryogenesis remains unclear. Preliminary data suggest that Pax3 splice variants may differentially regulate Pax7 in vitro. The goal of this proposal is to understand the tissue-specifc role of a conserved Pax3 transactivation domain that may provide insight into both distinct and redundant functions of Pax3 and Pax7 during embryogenesis and, more precisely, cardiac NC specification. Thus, the unifying hypothesis of this proposal is that a conserved Pax3 transactivation domain facilitates Pax7 exclusion from the NC domain and mediates CNC specification but that both Pax3 and Pax7 can specify cardiac NC lineage in abnormal situations. We will use tissue-specific compound transgenic mice combined with in vitro approaches to test this novel hypothesis.
Aim 1 will test the hypothesis that Pax3/Pax7 genetic compensation occurs via a conserved ability to repress Msx2 expression within the early cardiac NC lineage.
Aim 2 will test the hypothesis that Pax3 transactivation domain is essential for CNC specification.
Cardiac neural crest cells (CNC) are vital for heart development, Pax3 protein is necessary for proper CNC function and Pax3 mutations result in abnormal heart formation, and data suggest that Msx2, another protein within CNC, must be repressed by Pax3 or the great arteries exiting the heart will not be appropriately remodeled to give rise to a separate aorta and pulmonary trunk. Our preliminary data reveals that Pax7, a close relative of Pax3, may be able to compensate for Pax3 deficiency in the CNC. Thus, we will test the hypothesis that Pax7 compensates for Pax3 deficiency similarly by repressing Msx2, and that this results in normal heart development.
