Pax genes in cardiac development

描述（由申请人提供）：配对框3（Pax 3）是一种对心脏神经嵴（NC）特化和形态发生至关重要的转录因子。CNC衍生物对于流出道（OFT）分隔和咽弓动脉（AA）重塑以产生离开心脏的大血管是必不可少的。值得注意的是，人类和小鼠中的Pax 3突变导致持续性动脉干（PTA）、室间隔缺损（VSD）和异常AA重塑，这与鸡和小鼠胚胎中迁移前神经嵴的手术和遗传消融相似。Ap 2aCre NC限制性Pax 3缺失导致OFT比对缺陷，并且由NC启动子（1.6kbPax3）驱动的Pax 3的转基因再表达挽救了Pax 3无效表型。数据表明，Msh同源框蛋白2（Msx 2）的转录调控Pax 3和Msx 2转录抑制的失败导致一系列NC相关的缺陷（包括PTA/VSD）。这些数据表明，Pax 3介导的心脏NC规范通过早期和细胞自主机制。有趣的是，Pax 3无效并不能完全阻止心脏NC在OFT的定植，这表明潜在的遗传补偿。小鼠Pax 3在早期神经嵴中表达，并通过神经管闭合进行背侧限制，随后Pax 3表达腹侧扩展。相比之下，小鼠Pax 7（Pax 3 paraxin）表达首先在神经管闭合（~E8.5）后检测到，并且通常限于神经管的最背侧区域。我们的初步数据表明，Pax 7是上调和背侧扩展内的神经管的一种新的Pax 3 hypomorphic小鼠（~10%正常Pax 3蛋白; Pax 3 Hypo）。数据表明Pax 7空值是有活力的，并且不表现出任何心脏缺陷，然而我们的初步数据显示，Pax 3 Hypo背景上的Pax 7系统性突变体（Pax 7-/-; Pax 3 Hypo）100%地发生PTA和VSD。Pax 3和Pax 7都含有几乎相同的DNA结合结构域，并且都含有对Pax 3和Pax 7转录活性至关重要的差异剪接的反式激活结构域。虽然Pax 3剪接变异体在进化上是保守的，但Pax 3剪接变异体在胚胎发生中的作用仍不清楚。初步数据表明，Pax 3剪接变异体可能在体外差异调节Pax 7。该建议的目标是了解保守的Pax 3反式激活结构域的组织特异性作用，该结构域可以提供对胚胎发生期间Pax 3和Pax 7的不同和冗余功能的了解，更确切地说，心脏NC规格。因此，该建议的统一假设是保守的Pax 3反式激活结构域促进Pax 7从NC结构域排除并介导CNC规范，但Pax 3和Pax 7都可以在异常情况下指定心脏NC谱系。我们将使用组织特异性复合转基因小鼠结合体外方法来验证这一新的假设。目的1将测试的假设，Pax 3/Pax 7的遗传补偿发生通过保守的能力，抑制Msx 2的表达在早期心脏NC谱系。目的2将检验Pax 3反式激活结构域是CNC特化所必需的假设。 公共卫生相关性：心脏神经嵴细胞（CNC）对心脏发育至关重要，Pax 3蛋白是正常CNC功能所必需的，Pax 3突变导致异常心脏形成，数据表明CNC中的另一种蛋白Msx 2必须被Pax 3抑制，否则离开心脏的大动脉将无法适当重塑以产生单独的主动脉和肺动脉干。我们的初步数据表明，Pax 7，Pax 3的近亲，可能能够弥补CNC中的Pax 3缺陷。因此，我们将测试Pax 7通过抑制Msx 2类似地补偿Pax 3缺陷的假设，并且这导致正常的心脏发育。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.