CSPG2 GENE AND CARDIAC OUTLET MORPHOGENESIS

描述(由申请人提供)：Cspg2/verscan基因对早期心脏形成的功能重要性已经被称为心脏缺陷(HDF)的Cspg2缺失小鼠清楚地证明了。Cspg2基因的完全缺失从字面上意味着心脏发育在缓冲形成和出口段生长之前的阶段结束。这为确定Cspg2/versican在心脏发育的后期阶段的作用机制和意义创造了一个问题，该阶段涉及间隔和瓣膜成熟，此时与最常见于活产的心脏缺陷的相关性最高。为了探讨这些问题，我们已经开始分析一种新的小鼠模型，即Cspg2的mRNA剪接形式为空。我们的分析表明，缺失纯合的小鼠可以存活和繁殖，但也有心脏缺陷，与至少三分之一的活体出生缺陷高度相关。我们的中心工作假设是，Cspg2/verscan调节心脏垫上正常的EGFR信号，并调节形成心脏垫生长和肌化所需的细胞基质信号。需要解决的主要问题是：1)Verscan如何在调节缓冲间充质形成、生长和成熟所需的EGF信号和下游靶点中发挥作用；ii)Verscan在出口垫状心肌化的两个基本步骤中起什么积极或允许的作用；iii)Verscan在稳定成熟的缓冲间充质中的心肌表型方面起什么作用？这项拟议的研究的目的是确定Verscan在后期直接影响活体出生心脏缺陷的功能。我们将结合体外生物分析、形态学、高通量3D共聚焦成像和高通量蛋白质组学来确定Verscan的功能。具体目的是：1)确定多种剪接形式调节缓冲间充质形成和分化的细胞和分子机制。2)确定多种剪接形式调节心肌化过程的机制，该机制与心脏出口隔膜与室口对齐有关。3)确定当V2/V0缺失心脏置于影响EGFR信号转导和心脏发育的遗传背景时，缺陷表型是如何调节的。据报道，几种涉及垫状间充质的形态发生机制是将U形心管重塑为四个心腔所必需的。然而，Cspg2基因产物versican在这些中枢机制中的积极或允许作用已被认识到，但在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部分缺乏细胞外基质蛋白多糖Versican而导致的心脏出口整合的这些中枢机制的破坏。尽管有报道称涉及缓冲间充质的几种形态发生机制是将U形心脏管重塑为四个腔室所必需的，但Cspg2基因产物versicA的活跃或允许作用已被认识到，但在很大程度上尚未被探索。我们已经在心脏缺陷小鼠(Cspg2缺失)中表明，未能形成垫子会导致早期胚胎死亡。该方案中的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Verscan在心脏发育阶段中的作用，这些阶段直接影响临床相关的活体出生心脏缺陷。

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.