The Role of GSK-3beta in Palate Development and Fusion

描述（由申请人提供）：颅面出生缺陷代表了毁灭性的社会心理并发症以及重大的社会经济负担。其中，2004年，仅直接住院费用一项，腭裂就造成了每年超过1亿美元的“国家账单”，并且非常普遍，发病率约为0.1%（千分之一），使其成为第二大常见的出生缺陷。以往的研究表明，腭裂的发病是多因素的，可能既有遗传因素，也有环境因素。我们对颅面裂的大部分知识来自患者的病例研究，尽管有限，但选择了动物模型。许多基因已被确定与腭裂表型相关，但大多数病例的病因仍然难以捉摸。受影响的儿童不仅需要进行多次手术来解决上颚关闭问题，还需要解决相关的语言、喂养、牙齿和其他颅面生长缺陷问题。GSK-32的功能先前已被证明与正常颅面发育密切相关并且是必要的。本应用的中心假设是依赖于GSK-32的信号转导通路控制着次级上颚的发育。本研究将利用遗传分析和新型蛋白调控技术研究GSK-32在腭形成中的作用。我们提出了两个具体的目的来探讨GSK-32在腭发生和融合中的作用。第一个目的是解决腭发育中GSK-32信号的机制要求，第二个目的是为腭发育研究提供一种控制GSK-32蛋白水平的新技术。在第一个目标中，将探讨GSK-32突变小鼠中继发腭裂的机制。该突变体的腭架在胚胎发生过程中似乎发育正常，但未能融合。这种缺陷可能是由于腭架生长缺陷、细胞凋亡和/或转分化造成的，本研究的第一部分将在体内研究突变型腭发育过程中与野生型腭发育过程的标志物。在第二部分中，体外器官培养将用于确定突变腭架在器官培养中放置时是否能够融合。在第二个目标中，使用药物依赖性GSK-32等位基因在不同阶段的腭发育的研究将进行。由于GSK-32在不同区域的不同时间需要，因此其活性的完全早期丧失可能会妨碍对其在下游时间和位置的作用的研究，例如，颌骨发育缺陷后上颚的生长和融合事件。该系统是新颖的，需要进一步的研究和适应体外方案。综上所述，这两个目标将明确GSK-32信号的时空要求，并极大地推进我们对人类唇腭裂机制原因的认识。此外，我们开发的新的蛋白质调控技术将为未来的发育研究提供模板。

DESCRIPTION (provided by applicant): Craniofacial birth defects represent devastating psychosocial complications as well as a significant socioeconomic burden. Among them, cleft palate posed an annual "national bill" exceeding $100 million in 2004 in direct hospital costs alone and is highly prevalent as the incidence is roughly 0.1% (one in 1000 children), making it the second most common birth defect. Previous studies indicate that the pathogenesis of cleft palate is multifactorial and likely has both genetic and environmental factors. Much of our knowledge of craniofacial clefting arises from case studies of patients and although limited, selected animal models. A number of genes have been identified to be associated with the cleft palate phenotype, but the etiology of the majority of cases remains elusive. Affected children require multiple operations to address not only palate closure, but also associated problems with speech, feeding, dentition, and other craniofacial growth deficiencies. The function of GSK-32 has previously been shown to be intimately related to and necessary for normal craniofacial development. The central hypothesis of this application is that signal transduction pathways dependent on GSK-32 control the development of the secondary palate. This proposal will use genetic analysis and novel protein regulation techniques to study the roles of GSK-32 in palate formation. Two Specific Aims are proposed to explore the roles of GSK-32 in palatogenesis and fusion. The first aim will address the mechanistic requirements of GSK-32 signaling in palatogenesis while the second aim brings a new technique to control levels of GSK-32 protein to the study of palate development. In the first aim, the mechanism(s) underlying the cleft of the secondary palate in GSK-32 mutant mice will be explored. Palatal shelves in this mutant have previously been shown to develop seemingly appropriately during embryogenesis, but fail to fuse. This deficiency may be the result of defective palatal shelf growth, cell apoptosis and/or transdifferentiation, and the first part of this aim will look in vivo to investigate markers of these processes during palatogenesis in mutant compared to the wild-type palates. In the second part, in vitro organ culture will be used to determine if the mutant palateal shelves are capable of fusion when placed in apposition in organ culture. In the second aim, the study of palatogenesis using a drug-dependent GSK-32 allele during distinct stages will be undertaken. Because GSK-32 is required at different times in different regions, complete early loss of its activity may preclude study of its action at a downstream time and location for example, growth and fusion events in the palate after defective maxillogenesis. This system is novel and requires further investigation and adaptation to in vitro protocols. Taken together, these two aims will pinpoint the spatial and temporal requirements of GSK-32 signaling and greatly advance our knowledge of the mechanistic causes of human orofacial clefting. Furthermore, the new protein regulation techniques that we develop will serve as a template for future developmental studies. PUBLIC HEALTH RELEVANCE: Children and families affected by cleft palate must not only endure multiple, physiologically challenging surgeries to address palate closure, but also associated problems with speech, feeding, dentition, and other facial growth deficiencies. In addition, cleft palate brings with it devastating psychosocial implications for many children as well as significant socioeconomic burden exceeding $100 million in direct hospital costs alone. These facts are significant and while certain genes and environmental factors have been associated with the development of cleft palate, the cause in the majority of cases remains unknown and further study is extremely important.