The Roles of Semaphorin Signaling During Mouse Valvuloseptal Development

描述（由申请人提供）：心脏瓣膜间隔结构畸形是先天性心脏病（CHDs）的常见原因，在新生儿中发病率高达1%，并且仍然是婴儿发病率和死亡率的主要原因。本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，并揭示它们对冠心病的影响。小鼠胚胎心脏在E9.0时，房室管（AVC）区域的瓣膜间隔发育是由细胞外基质（ECM）区域扩张形成的缓冲开始的。此后不久，响应心肌释放的刺激信号，AVC中的心内膜细胞亚群转分化为间充质细胞并迁移到ECM中。细胞化的软垫作为瓣膜和间隔的原基，并通过复杂的成熟过程进一步重塑为最终结构。骨形态发生蛋白（Bone Morphogenic Protein， BMP）通路在AV缓冲层形成、细胞化和重塑过程中发挥重要作用；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，介导BMP信号复杂活动的下游基因仍然难以捉摸。为了方便应用细胞和分子方法研究AV缓冲层的形态发生，我们建立了一个温敏的AV缓冲层间充质细胞系tsA58-AVM。据我们所知，文献中没有其他永久性缓冲细胞系的报道。利用这种独特的细胞培养系统，我们发现Sema6D是AV缓冲细胞中BMP信号的一个新的调控靶点。虽然信号蛋白最初被认为是系统发育上保守的神经元引导线索，但它们的功能现在已涉及在各种生物/病理过程中调节细胞形态、增殖、粘附和迁移。文献中没有研究直接解决信号素信号在房室瓣隔形态发生中的潜在作用。我们对体外培养细胞和离体培养AV组织的初步研究表明，Sema6D促进缓冲间质形成和迁移。此外，我们建立了一个条件敲除Sema6D的小鼠系，我们使用该系的初步研究支持Sema6D在正常AV缓冲细胞化中的重要体内作用。我们假设Sema6D是一个关键的BMP调控靶点，在瓣膜间隔发育过程中促进房室缓冲间质形成和迁移。这项建议概述了三个具体目标。在Aim 1中，我们将使用永生细胞系确定Sema6D在心内膜细胞和间充质细胞中的细胞活性。在Aim 2中，我们将使用体外培养的AV组织测试Sema6D和BMP信号在AV缓冲间质形成和迁移过程中的功能相互作用。在Aim 3中，我们将通过条件基因失活方法确定Sema6D在AV缓冲层形态发生中的体内功能。这项研究的成功完成将大大提高我们对正常瓣膜间隔形成和冠心病的分子机制的认识。

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. PUBLIC HEALTH RELEVANCE: Malformations of cardiac valvuloseptal structures account for a large proportion of congenital heart diseases. BMP signaling plays a critical role in atrioventricular (AV) valvuloseptal development. We have identified Sema6D as a downstream regulatory target of BMP signaling in AV cushions, and will apply multiple complementary approaches to explore the potentially critical function of Sema6D in supporting normal development of AV cushions.