Deficiencies in cardiac neural crest cell (NC) development result defective remodeling of the aortic arch arteries and failure of outflow tract septation. By studying this heart defect in splotch (sp2H) mutant mice (mutation in pax3), we have shown that cardiac NC fail to undergo normal NC stem cell expansion. Insufficient NC cells migrate into the developing heart. Preliminary data indicate that sp2H aortic arch remodeling and outflow tract defects are secondary to myocardial dysfunction (poor excitation-contraction coupling). Surprisingly, it has been reported that the NC-associated heart defects can be genetically 'rescued' by crossing the sp mutants to the viable msx2 homeobox-containing null mice, demonstrating that pax3 is required for the repression of msx2 expression. Given that msx2 is a well-documented regulator of BMP signaling, we crossed the sp2H mutants with the Bmp4-1acZ knockout mice. Preliminary results indicate that Bmp4 is over-expressed in sp2H mutants and this is correlated with over-expression of several potential pax3 effector genes that may regulate mesenchymal morphogenesis (including the cell adhesion molecule periostin). Specifically, while the ubiquitous Yin Yang-1 (YY1) transcription factor is over-expressed, periostin is under-expressed in sp2H. Periostin may play a role in NC condensation and trans-differentiation into connective tissue within the pharyngeal arches and outflow tract. We hypothesize that modulation of msx2 expression via pax3/Bmp4/YY1 causes the sp2H cardiovascular defects. We also hypothesize that a lack of periostin-mediated epithelial-mesenchymal transformation of proepicardial cells causes the sp2H myocardial dysfunction. To determine whether these effector genes are primarily or secondarily affected and to separate the myocardial dysfunction from the NC-associated heart defects, we generated a pax3-1oxP mouse enabling us to re-create the sp2H mutation, within particular tissues at particular times during embryogenesis. We will determine the effects of the sp2H mutation within different regions of the cardiovascular system, if pax3 interacts directly or indirectly with YY and if pax3 regulates periostin. ? ?
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