CSPG2 GENE AND CARDIAC OUTLET MORPHOGENESIS

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基因的完全缺失意味着心脏发育在缓冲层形成和出口节段生长之前的阶段结束。这给确定Cspg2/versican在心脏发育后期的功能机制和意义带来了一个问题，包括间隔和瓣膜成熟，因为这与活产婴儿中最常见的心脏缺陷有最高的相关性。为了解决这些问题，我们已经开始分析一种新的小鼠模型，该模型是Cspg2的一个mRNA剪接形式。我们的分析表明，缺失纯合子的小鼠可以存活和繁殖，但也具有心脏缺陷，至少与1/3的活产出生缺陷高度相关。我们的主要工作假设是Cspg2/versican调节心脏缓冲中正常的EGFR信号，并调节形成心脏缓冲所需的生长和肌肉化的细胞基质信号。需要解决的主要问题是：1)versican如何在调节EGF信号和缓冲间质形成、生长和成熟过程中所需的下游目标中发挥作用；Ii)在出口缓冲心肌化的两个基本步骤中，versican的主动或纵容作用是什么；Iii)在缓冲间质成熟过程中，花青素在稳定心肌表型中的作用是什么？拟议研究的目的是确定versican在后期的功能，直接影响活产心脏缺陷。我们将结合体外生物测定、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。具体目的是：1)确定细胞和分子机制，通过剪接形成调节缓冲间质的形成和分化；2)确定version V2/V0剪接形式介导心出口间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中时，V2/V0 null心脏中的缺陷表型是如何被调节的。据报道，包括缓冲间质在内的几种形态发生机制是将u型心管重构为四个腔室所必需的。然而，Cspg2基因产物versican在这些中心机制中的积极或许可作用是公认的，但在很大程度上尚未探索。V2/V0空小鼠提供了第一个哺乳动物模型，我们可以直接研究细胞外基质蛋白聚糖的部分缺失导致的心脏出口整合的这些中心机制的破坏。虽然已经报道了一些涉及缓冲间质的形态发生机制是将u型心管重塑为四个腔室所必需的，但Cspg2基因产物versican的主动或允许作用是公认的，但在很大程度上尚未探索。我们已经在心脏缺陷小鼠（Cspg2 null）中证明，不能形成缓冲导致早期胚胎死亡。本研究中的V2/V0小鼠提供了第一个哺乳动物模型，我们可以直接研究versican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。

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.