CSPG2 GENE AND CARDIAC OUTLET MORPHOGENESIS

描述（由申请人提供）：Cspg 2/多功能蛋白聚糖基因对早期心脏形成的功能重要性已由称为心脏缺陷（hdf）的Cspg 2缺失小鼠清楚地证明。Cspg 2基因的完全缺失字面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。这产生了一个问题，用于确定Cspg 2/多功能蛋白聚糖在涉及隔膜和瓣膜成熟的心脏发育后期的功能机制和意义，此时与活产中最常见的心脏缺陷相关性最高。为了解决这些问题，我们已经开始分析一种新的小鼠模型，即Cspg 2的mRNA剪接形式null。我们的分析表明，缺失的纯合子小鼠可以存活和繁殖，但也具有与至少1/3的活产缺陷高度相关的心脏缺陷。我们的中心工作假设是Cspg 2/多功能蛋白聚糖调节心脏垫中的正常EGFR信号传导，并调节形成心脏垫的生长和肌肉化所需的细胞基质信号传导。要解决的主要问题是1）多功能蛋白聚糖如何发挥作用，在调节EGF信号传导和下游目标所需的垫间充质的形成，生长和成熟; ii）什么是积极的或许可的作用，多功能蛋白聚糖在两个基本步骤的出口垫心肌化; iii）多功能蛋白聚糖在稳定心肌表型的垫间充质成熟的作用是什么？拟议研究的目的是确定多功能蛋白聚糖在直接影响活产心脏缺陷的后期阶段的功能。我们将结合体外生物测定、形态学、高通量三维共聚焦成像和高通量蛋白质组学来确定多功能蛋白聚糖的功能。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)确定多功能蛋白聚糖V2/V0剪接形式介导与心脏出口隔与心室出口对齐相关的心肌化过程的机制。3)确定当置于影响EGFR信号传导和心脏发育的遗传背景上时，V2/V0无效心脏中的缺陷表型是如何调节的。一些形态发生机制，包括垫间充质已被报道是必要的重塑U形心管成四个腔。然而，Cspg 2基因产物多功能蛋白聚糖在这些中枢机制中的活性或允许作用是公认的，但在很大程度上未被探索。V2/V0基因敲除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部分缺乏细胞外基质蛋白聚糖versican而导致的这些心脏出口整合的中枢机制的破坏。Cspg 2基因产物多功能蛋白聚糖的活性或允许作用已被认识，但大部分未被探索。我们已经在心脏缺陷小鼠（Cspg 2 null）中显示，未能形成缓冲导致早期胚胎死亡。该提议中的V2/V0无效小鼠提供了第一个哺乳动物模型，其中我们可以直接研究多功能蛋白聚糖在发育阶段心脏中的作用，其直接影响临床相关的活产心脏缺陷。

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.