Analysis of an ethanol-sensitive pathway regulating anterior craniofacial development

 描述（由申请人提供）：胎儿酒精谱系障碍（FASD）描述了一系列乙醇诱导的发育缺陷，包括颅面畸形，如下颌发育不全和腭裂。它影响每年在美国出生的100名儿童中的大约1名。虽然胎儿暴露于酒精会导致FASD，但这些酒精诱导的颅面缺陷背后的遗传学事实上还不清楚。颅面骨骼的大部分是由颅神经嵴细胞部分通过与上皮组织的复杂相互作用产生的。这些相互作用由骨形态发生蛋白（Bmp）信号通路等协调。我的初步数据表明，BMP信号，具体到内胚层，是下颌形态发生所需的，而BMP信号调节腭形态发生部分通过转录因子，gata 3。这些结果表明Bmp信号通过多种信号通路调节前部颅面形态发生的模型。越来越多的证据支持遗传易感性在这些乙醇诱导的缺陷中起作用，但对这些基因-乙醇相互作用的了解甚少。Eberhart实验室帮助开创了遗传筛查，以确定这些乙醇敏感的遗传位点。从这些屏幕上，我已经编制了一套突变体与乙醇诱导的缺陷表明，乙醇相互作用的遗传层次调节前颅面发育。首先，通过对斑马鱼突变体的筛选，我发现Bmp信号通路的几个成员表现出乙醇诱导的胚胎下颌和腭缺陷。其次，在正向遗传筛选中，我回收了乙醇敏感突变体，其表型复制Bmp表型的特定方面。这些研究结果支持这一假设，BMP信号调节一种新的乙醇敏感性途径调节颅面前骨骼的形态发生。在具体目标1中，我将1）使用4D共聚焦分析确定乙醇如何破坏BMP介导的内胚层-口腔外胚层-神经嵴途径，以及2）阐明乙醇作用的靶点BMP途径。在具体目标2中，我将1）克隆并表征au 15，au 24和au 25的乙醇敏感性，2）分析乙醇处理的au 15突变体中的神经嵴细胞行为，3）表征au 15突变体胚胎中乙醇作用的机制。在具体目标3中，我将a）克隆和表征au 26，B）可视化乙醇处理的BMP突变体和au 26胚胎中的腭形态发生，以及c）表征BMP途径成员au 26和gata 3之间的上位关系。总的来说，这项建议中概述的工作将提供深入了解乙醇敏感的途径调节前颅面发育和基因-乙醇相互作用，这些途径的基础。由于斑马鱼和人类之间基因功能的保守性，这项工作将直接转化为人类群体中候选基因的研究，并允许更好地诊断和治疗FASD。

 DESCRIPTION (provided by applicant): Fetal Alcohol Spectrum Disorders (FASD) describes a wide array of ethanol-induced developmental defects, including craniofacial dysmorphology, such as lower jaw hypoplasia and cleft palate. It affects approximately 1 in 100 children born in the United States each year. Although fetal exposure to alcohol causes FASD, virtually nothing of the genetics behind these ethanol-induced craniofacial defects is understood. The majority of the craniofacial skeleton is generated by cranial neural crest cells in part through complex interactions with epithelial tissues. These interactions are orchestrated by, among others, the Bone Morphogenetic Protein (Bmp) signaling pathway. My preliminary data demonstrates that Bmp signaling, specific to the endoderm, is required for lower jaw morphogenesis, while Bmp signaling regulates palate morphogenesis in part through the transcription factor, gata3. These results suggest a model where Bmp signaling regulates anterior craniofacial morphogenesis via multiple signaling pathways. Growing evidence supports that genetic predisposition plays a role in these ethanol-induced defects, yet little is understood about these gene-ethanol interactions. The Eberhart lab has helped pioneer genetic screens to identify these ethanol-sensitive genetic loci. From these screens, I have compiled a set of mutants with ethanol induced defects suggesting that ethanol interacts with a genetic hierarchy regulating anterior craniofacial development. First, through a screen of available zebrafish mutants, I found that several members of the Bmp signaling pathway exhibit ethanol-induced defects to the embryonic lower jaw and palate. Second, in a forward genetic screen I recovered ethanol-sensitive mutants that phenocopy specific aspects of the Bmp phenotype. These findings support the hypothesis that Bmp signaling regulates a novel ethanol-sensitive pathway regulating morphogenesis of the anterior craniofacial skeleton. In Specific Aim 1, I will 1) determine how ethanol disrupts a Bmp mediated endoderm-oral ectoderm- neural crest pathway using 4D confocal analyses and 2) elucidate the target of ethanol action the Bmp pathway. In Specific Aim 2, I will 1) clone and characterize the ethanol sensitivity of au15, au24 and au25, 2) analyze neural crest cell behaviors in ethanol-treated au15 mutants and 3) characterize the mechanism of ethanol action in au15 mutant embryos. In Specific Aim 3, I will a) clone and characterize au26, b) visualize palate morphogenesis in ethanol-treated Bmp mutants and au26 embryos and c) characterize the epistatic relationship between Bmp-pathway members, au26 and gata3. Overall, the work outlined in this proposal will provide insight into ethanol-sensitive pathways regulating anterior craniofacial development and the gene- ethanol interactions that underlie these pathways. Because of the conservation of gene function between zebrafish and humans, this work will directly translate to studies of candidate genes in human populations and allow for better diagnosis and treatment of FASD.