CSPG2 GENE AND CARDIAC OUTLET MORPHOGENESIS

CSPG2 基因与心脏出口形态发生

• 批准号: 7368114
• 负责人: COREY H MJAATVEDT
• 金额: $ 36.5万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7368114
• 关键词: Address; Adult; Affect; Alternative Splicing; Be++ element; Beryllium; Breeding; Cardiac; Cell Density; Cells; Childhood; Congenital Abnormality; Congenital Heart Defects; Defect; Development; Disruption; EGF gene; Embryo; Epidermal Growth Factor Receptor; Event; Extracellular Matrix; Failure; Funding; Gene Proteins; Genes; Genetic; Growth; Heart; Hyaluronan; Image; In Vitro; Knockout Mice; Length; Live Birth; Mediating; Mesenchymal; Mesenchyme; Messenger RNA; Modeling; Molecular; Morphogenesis; Morphology; Mus; Mutation; Myocardial; Phenotype; Play; Process; Proteins; Proteoglycan; Proteomics; Pulmonary artery structure; Purpose; RNA Splicing; Reagent; Reporting; Role; Shapes; Signal Pathway; Signal Transduction; Staging; Techniques; Testing; Tissues; Tube; Ventricular; Work; cardiogenesis; clinically relevant; design; in vitro Bioassay; in vivo; insight; mouse model; mutant; nestin protein; novel; promoter; protein expression; receptor; research study; stathmin; three dimensional structure; vector; versican

DESCRIPTION (provided by applicant): The functional importance of the Cspg2/versican gene to early heart formation has been clearly demonstrated by the Cspg2 null mice called heart defect (hdf). Total absence of the Cspg2 gene literally means the end of heart development at a stage prior to cushion formation and outlet segment growth. This creates a problem for determining the functional mechanism and significance of Cspg2/versican at later stages of heart development involving septa and valve maturation when there is the highest relevance to the cardiac defects most often seen in live births. To approach these questions, we have begun to analyze to a new mouse model that is a mRNA splice form null of Cspg2. Our analysis shows that mice homozygous for the deletion can survive and breed, but also possess cardiac defects highly relevant to at least 1/3 of all live birth defects. Our central working hypothesis is that Cspg2/versican modulates normal EGFR signaling in the heart cushions and regulates cell-matrix signaling needed for growth and muscularization of the forming heart cushions. The major questions to be addressed are 1) how does versican play a role in modulating EGF signaling and downstream targets needed during cushion mesenchyme formation, growth and maturation; ii) what is the active or permissive role of versican in the two fundamental steps of outlet cushion myocardialization; iii) what is the role of versican in stabilizing the myocardial phenotype in maturing cushion mesenchyme? The purpose of the proposed studies are to determine versican's function at later stages that impact directly on live birth heart defects. We will use a combination of in vitro bioassays, morphology, high throughput 3D confocal imaging and high throughput proteomics to determine versican's function. The Specific Aims are: 1) Determine the cellular and molecular mechanisms through which versican splice forms modulate formation and differentiation of the cushion mesenchyme.; 2) Determine the mechanism through which the versican V2/V0 splice form mediates the process of myocardialization that is associated with alignment of the cardiac outlet septa to the ventricular outlets.; 3) Determine how the defective phenotype in the V2/V0 null hearts is modulated when placed on genetic backgrounds affecting EGFR signaling and heart development. Several morphogenetic mechanisms involving cushion mesenchyme have been reported to be necessary for remodeling the U-shaped heart tube into four chambers. However, the active or permissive role of the Cspg2 gene product, versican, on these central mechanisms is recognized but largely unexplored. The V2/V0 null mouse provides the first mammalian model in which we can directly investigate the disruption of these central mechanisms of cardiac outlet integration that result from a partial absence of the extracellular matrix proteoglycan versican.Although several morphogenetic mechanisms involving cushion mesenchyme have been reported to be necessary for remodeling the U-shaped heart tube into four chambers, the active or permissive role of the Cspg2 gene product, versican, is recognized but largely unexplored. We have shown in the heart defect mouse (Cspg2 null), the failure to form cushions results in early embryonic lethality. The V2/V0 null mouse in this proposal provides the first mammalian model in which we can directly investigate the role of versican in the heart at developmental stages that directly impact clinically relevant live birth heart defects.

描述（由申请人提供）：Cspg2/versican 基因对早期心脏形成的功能重要性已被称为心脏缺陷（hdf）的 Cspg2 缺失小鼠清楚地证明。 Cspg2 基因的完全缺失实际上意味着心脏发育在垫形成和出口段生长之前的阶段结束。这就产生了确定 Cspg2/versican 在涉及隔膜和瓣膜成熟的心脏发育后期的功能机制和重要性的问题，此时与活产中最常见的心脏缺陷具有最高的相关性。为了解决这些问题，我们开始分析一种新的小鼠模型，该模型是 Cspg2 的 mRNA 剪接形式无效的。我们的分析表明，缺失纯合子的小鼠可以存活和繁殖，但也具有与所有活产缺陷中至少 1/3 高度相关的心脏缺陷。我们的中心工作假设是 Cspg2/versican 调节心垫中的正常 EGFR 信号传导，并调节形成的心垫生长和肌肉化所需的细胞基质信号传导。需要解决的主要问题是1）多功能蛋白聚糖如何在调节缓冲间充质形成、生长和成熟过程中所需的EGF信号和下游靶标中发挥作用； ii) 多功能蛋白聚糖在出口垫心肌化的两个基本步骤中的主动或许可作用是什么？ iii) 多功能蛋白聚糖在稳定成熟缓冲间充质心肌表型方面的作用是什么？拟议研究的目的是确定多功能蛋白在后期对活产心脏缺陷有直接影响的功能。我们将结合使用体外生物测定、形态学、高通量 3D 共聚焦成像和高通量蛋白质组学来确定 versican 的功能。具体目标是： 1) 确定多功能蛋白聚糖剪接形式调节垫间充质形成和分化的细胞和分子机制。 2) 确定多功能蛋白 V2/V0 剪接形式介导心肌化过程的机制，该过程与心脏出口隔膜与心室出口的对齐相关。 3) 确定当置于影响 EGFR 信号传导和心脏发育的遗传背景下时，V2/V0 无效心脏中的缺陷表型如何受到调节。据报道，涉及垫间充质的几种形态发生机制对于将 U 形心管重塑为四个腔室是必要的。然而，Cspg2 基因产物 versican 对这些中心机制的主动或许可作用已得到认可，但很大程度上尚未被探索。 V2/V0 缺失小鼠提供了第一个哺乳动物模型，在该模型中我们可以直接研究由于细胞外基质蛋白多糖多功能蛋白部分缺失而导致的心脏出口整合的这些中央机制的破坏。尽管已经报道了几种涉及垫间充质的形态发生机制将 U 形心管改造成四个心室所必需的，Cspg2 基因产物 versican 的主动或许可作用已被认识，但很大程度上尚未被探索。我们已经在心脏缺陷小鼠（Cspg2 null）中证明，未能形成垫会导致早期胚胎死亡。本提案中的 V2/V0 缺失小鼠提供了第一个哺乳动物模型，我们可以在其中直接研究多功能蛋白聚糖在心脏发育阶段的作用，直接影响临床相关的活产心脏缺陷。