The Roles of Semaphorin Signaling During Valvuloseptal Development

描述(申请人提供)：心脏瓣膜间隔结构畸形是先天性心脏病(CHDS)的常见原因，在高达1%的新生儿中发生，仍然是婴儿发病率和死亡率的主要原因。这项研究的长期目标是确定控制正常瓣膜间隔形态发生的分子、细胞和遗传机制，并揭示它们对CHD的贡献。在小鼠胚胎心脏中，房室管(AVC)区的瓣膜间隔发育是通过细胞外基质(ECM)在~E9.0的区域扩张而形成缓冲的。此后不久，AVC中的心内膜细胞亚群在心肌释放的刺激信号下转分化为间充质细胞，并迁移到ECM。细胞化的气垫作为瓣膜和隔膜的原基，通过复杂的成熟过程进一步重塑成最终的结构。骨形态发生蛋白(BMP)通路在房室垫的形成、细胞化和重塑过程中起着至关重要的作用；在动物模型和人类患者中，干扰BMP信号的突变会导致各种瓣膜间隔缺陷。然而，调节BMP信号复杂活动的下游基因仍然难以捉摸。为了便于应用细胞和分子方法研究房室垫形态发生，我们建立了温度敏感的永生化房室垫间充质细胞系tsA58-AVM。据我们所知，在文献中还没有其他永久缓冲细胞系的报道。利用这个独特的细胞培养系统，我们确定Sema6D是房室垫中BMP信号的一个新的调控靶点。虽然信号素最初被认为是系统发育保守的神经元指导信号，但现在它们的功能已经被涉及到在各种生物/病理过程中调节细胞的形态、增殖、黏附和迁移。文献中没有直接涉及信号素信号在房室间隔形态发生中的潜在作用的研究。我们利用体外培养的细胞和体外培养的AV组织进行的初步研究表明，Sema6D促进了缓冲间充质的形成和迁移。此外，我们建立了Sema6D的条件性基因敲除小鼠系，并利用该系进行的初步研究支持了Sema6D在体内对正常房室缓冲细胞化的重要作用。我们推测Sema6D是一个关键的BMP调控靶点，在瓣膜间隔发育过程中对促进房室间充质形成和迁移至关重要。这项提案概述了三个具体目标。在目标1中，我们将利用永生细胞系测定Sema6D在心内膜和间充质细胞中的细胞活性。在目标2中，我们将利用体外培养的房室组织来测试Sema6D和BMP信号在房室间充质形成和迁移过程中的功能相互作用。在目标3中，我们将通过条件基因失活的方法来确定Sema6D在房室缓冲形态发生过程中的体内功能。这项研究的成功完成将极大地提高我们对正常瓣膜间隔形成和CHDS背后的分子机制的了解。

DESCRIPTION (provided by applicant): Malformations of cardiac valvuloseptal structures are prevalent causes of congenital heart diseases (CHDs), which occur in as many as 1% of newborns and remain the leading cause of infant morbidity and mortality. The long term goal of this study is to identify the molecular, cellular, and genetic mechanisms governing normal valvuloseptal morphogenesis and to reveal their contributions to CHDs. Valvuloseptal development in the atrioventricular canal (AVC) region is initiated with cushion formation by regional expansion of extracellular matrix (ECM) at ~E9.0 in mouse embryonic hearts. Shortly thereafter, in response to stimulatory signals released from the myocardium, a subgroup of endocardial cells in the AVC transdifferentiate into mesenchymal cells and migrate into the ECM. The cellularized cushions serve as the primordia of valves and septa, and are further remodeled into final structures through complicated maturation processes. Bone Morphogenic Protein (BMP) pathways play essential roles during AV cushion formation, cellularization, and remodeling; mutations that disturb BMP signaling cause various valvuloseptal defects both in animal models and in human patients. Nevertheless, the downstream genes that mediate the complex activities of BMP signaling remain elusive. To facilitate the application of cellular and molecular approaches to study AV cushion morphogenesis, we developed a temperature sensitive immortal AV cushion mesenchymal cell line, tsA58-AVM. To the best of our knowledge, no other permanent cushion cell line has been reported in the literature. Using this unique cell culture system, we identified Sema6D as a novel regulatory target of BMP signaling in AV cushions. While Semaphorins were initially recognized as phylogenetically conserved neuronal guidance cues, their functions have now been implicated in regulating cell morphology, proliferation, adhesion and migration during various biological/pathological processes. No study in the literature has directly addressed the potential roles of Semaphorin signaling during AV valvuloseptal morphogenesis. Our preliminary studies using in vitro cultured cells and ex vivo cultured AV tissues suggested that Sema6D promotes cushion mesenchyme formation and migration. Moreover, we established a conditional knockout mouse line of Sema6D, and our initial studies using this line supported the essential in vivo role of Sema6D for normal AV cushion cellularization. We hypothesize that Sema6D is a key BMP regulatory target critical for promoting AV cushion mesenchyme formation and migration during valvuloseptal development. Three specific aims are outlined in this proposal. In Aim 1, we will determine the cellular activity of Sema6D in endocardial and mesenchymal cells using immortal cell lines. In Aim 2, we will test the functional interaction between Sema6D and BMP signaling during AV cushion mesenchyme formation and migration using ex vivo cultured AV tissues. In Aim 3, we will determine the in vivo function of Sema6D during AV cushion morphogenesis through a conditional gene inactivation approach. Successful accomplishment of this study will significantly advance our knowledge of the molecular mechanisms underlying normal valvuloseptal formation and CHDs.