Pax genes in cardiac development

Pax基因在心脏发育中的作用

• 批准号: 8315116
• 负责人: Zachary Paul Neeb
• 金额: $ 2.29万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-02 至 2012-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8315116
• 关键词: Ablation; Aorta; Arteries; Binding; Biological Assay; Boxing; Branchial arch structure; Cardiac; Cell Separation; Cell physiology; Cells; Co-Immunoprecipitations; Competitive Binding; Congenital Heart Defects; DNA Binding Domain; Data; Defect; Development; Dorsal; EMSA; Embryo; Embryonic Development; Energy Transfer; Exclusion; Exhibits; Failure; Financial compensation; Fluorescence; Genes; Genetic; Goals; Heart; Heart Septal Defects; Heterodimerization; Homeodomain Proteins; Human; Image; In Situ Hybridization; In Vitro; Lead; Luciferases; Lung; Mediating; Morphogenesis; Mus; Mutation; Neural Crest; Neural Crest Cell; Neural Tube Closure; Neural tube; Operative Surgical Procedures; Penetrance; Persistent Truncus Arteriosus; Phenocopy; Phenotype; Play; Prevention; Proteins; RNA Splicing; Regulation; Regulatory Element; Relative (related person); Repression; Role; Specific qualifier value; Staging; Testing; Tissues; Transactivation; Transcriptional Regulation; Transgenic Mice; Transgenic Organisms; Up-Regulation; Variant; cardiogenesis; gene repression; in vivo; insight; mutant; novel; prevent; promoter; spatiotemporal; transcription factor

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.

描述（由申请人提供）：配对框3 （Pax3）是一种转录因子，对心脏神经嵴（NC）规范和形态发生至关重要。CNC衍生物对于流出道（OFT）的分离和咽弓动脉（AA）的重塑产生出心脏的大血管至关重要。值得注意的是，人类和小鼠的Pax3突变会导致持续性动脉干（PTA）、室间隔缺损（VSD）和异常AA重塑，这反映了鸡和小鼠胚胎中迁移前神经嵴的手术和遗传消融。Ap2aCre NC限制性Pax3缺失导致OFT比对缺陷，由NC启动子（1.6kbPax3）驱动的Pax3转基因重新表达挽救了Pax3的空表型。数据表明，Msh同源盒蛋白2 （Msx2）受Pax3的转录调控，Msx2转录抑制失败导致一系列nc相关缺陷（包括PTA/VSD）。这些数据表明Pax3通过早期和细胞自主机制介导心脏NC规范。有趣的是，Pax3无基因并不能完全阻止OFT在心脏NC的定植，这表明可能存在遗传补偿。小鼠Pax3在早期神经嵴表达，并通过神经管闭合向背侧受限表达，随后Pax3在腹侧扩增表达。相比之下，小鼠Pax7 （Pax3旁对话）的表达在神经管关闭后首次被检测到（~E8.5），并且通常被限制在神经管的背侧大部分区域。我们的初步数据显示，Pax7在一种新型Pax3半胚小鼠（~10%正常Pax3蛋白；Pax3Hypo）的神经管中上调并背侧扩增。数据表明Pax7无基因是可行的，不会表现出任何心脏缺陷，然而我们的初步数据显示Pax7系统突变体在Pax3Hypo背景下（Pax7-/-;Pax3Hypo）会产生100%的PTA和VSD外显率。Pax3和Pax7都含有几乎相同的dna结合域，并且都含有对Pax3和Pax7转录活性至关重要的不同剪接的转激活域。虽然Pax3剪接变异在进化上是保守的，但Pax3剪接变异在胚胎发生中的作用尚不清楚。初步数据表明，Pax3剪接变异体可能在体外对Pax7有差异调节。本研究的目的是了解保守的Pax3转激活结构域的组织特异性作用，这可能有助于了解Pax3和Pax7在胚胎发生过程中的不同和冗余功能，更准确地说，是心脏NC规范。因此，该建议的统一假设是，保守的Pax3转激活域促进Pax7从NC域排除并介导CNC规范，但Pax3和Pax7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化合物转基因小鼠结合体外方法来验证这一新的假设。目的1将验证Pax3/Pax7遗传补偿通过抑制早期心脏NC谱系中Msx2表达的保守能力发生的假设。目标2将测试Pax3交互激活域对CNC规范至关重要的假设。