The Roles of Semaphorin Signaling During Mouse Valvuloseptal Development

描述（由申请方提供）：心脏瓣膜间隔结构畸形是先天性心脏病（CHD）的常见原因，其发生在多达1%的新生儿中，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）区域的瓣膜间隔发育始于细胞外基质（ECM）在~E9.0的区域扩张形成垫。此后不久，响应于从心肌释放的刺激信号，AVC中的内皮细胞亚群转分化成间充质细胞并迁移到ECM中。细胞化的垫作为阀和隔膜的原基，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（BMP）通路在AV垫形成、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信号传导的突变会导致各种瓣膜间隔缺损。尽管如此，介导BMP信号传导复杂活动的下游基因仍然难以捉摸。为了促进应用细胞和分子方法研究AV垫形态发生，我们建立了一个温度敏感的永生AV垫间充质细胞系，tsA 58-AVM。据我们所知，没有其他永久垫细胞系已在文献中报道。使用这种独特的细胞培养系统，我们确定Sema 6D作为一种新的调节目标的BMP信号在AV垫。虽然Semaphorins最初被认为是神经遗传学上保守的神经元引导信号，但它们的功能现在已经涉及在各种生物/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讨论脑信号蛋白信号传导在房室瓣间隔形态发生中的潜在作用。我们使用体外培养的细胞和离体培养的AV组织的初步研究表明，Sema 6D促进垫间充质形成和迁移。此外，我们建立了Sema 6D的条件性敲除小鼠系，我们使用该系的初步研究支持Sema 6D对正常AV垫细胞化的重要体内作用。我们假设Sema 6D是一个关键的BMP调节靶点，在瓣膜间隔发育过程中促进AV垫间充质形成和迁移至关重要。该提案概述了三个具体目标。在目的1中，我们将使用永生细胞系确定Sema 6D在内皮细胞和间充质细胞中的细胞活性。在目标2中，我们将使用离体培养的AV组织测试AV垫间充质形成和迁移期间Sema 6D和BMP信号传导之间的功能相互作用。在目标3中，我们将通过条件性基因失活方法确定Sema 6D在AV垫形态发生过程中的体内功能。这项研究的成功完成将大大提高我们对正常心瓣膜间隔形成和冠心病的分子机制的认识。 公共卫生相关性：心脏瓣膜间隔结构畸形占先天性心脏病的很大比例。BMP信号传导在房室（AV）瓣间隔发育中起关键作用。我们已经确定Sema 6D作为AV垫中BMP信号传导的下游调节靶点，并将应用多种互补方法来探索Sema 6D在支持AV垫正常发育中的潜在关键功能。

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.