Transgenic and knockout approaches to study protocadherin function

描述(申请人提供)：脊椎动物大脑的发育需要复杂的遗传网络来协调跨越时间和空间的无数过程，包括细胞增殖、分化和迁移，以及轴突引导、突触形成和消除。这些过程的协调导致微电路和分布式系统的组装，这些系统构成了一个功能神经系统。神经发育的中断可能会产生戏剧性的影响，导致复杂的大脑疾病，如自闭症谱系障碍、精神分裂症、精神发育迟滞和癫痫。越来越多的遗传数据表明，在这些复杂的大脑疾病中，参与轴突指导和突触发生的基因。此外，有证据表明这些疾病的常见病因，因为与一种疾病相关的突变也与其他疾病的发病率增加有关。与这些复杂的大脑疾病有关的分子中，有一种是原钙粘附素。特别是，pcdh19的突变会导致女性局限于婴儿发作的癫痫。尽管这些分子在神经发育和神经发育障碍的病因中扮演着明确的角色，但人们对它们在体内的功能知之甚少。在这里，我们建议使用锌指核酸酶(ZFN)来产生其中的pcdh7a、pcdh9、pcdh17和pcdh19已经失活的斑马鱼品系。此外，我们还建议在pcdh7a、pcdh9、pcdh17和pcdh19调控元件的控制下建立表达绿色荧光蛋白的BAC转基因株系。为了研究原钙粘附素突变对神经发育的影响，我们将GFP系交叉进入突变背景，并在体内使用双光子显微镜来确定突变对神经系统发育的影响。这项工作将极大地促进我们对原钙粘附素功能的理解，为更多的机械性研究提供基础，并有助于更好地理解原钙粘附素的破坏如何导致复杂的大脑疾病。

DESCRIPTION (provided by applicant): The development of the vertebrate brain requires complex genetic networks to coordinate myriad processes across time and space, which include cell proliferation, differentiation and migration, along with axon guidance, synapse formation and elimination. Orchestration of these processes results in the assembly of microcircuitry and distributed systems that constitute a functional nervous system. Disruptions in neural development can have a dramatic impact, resulting in complex brain disorders, such as autism spectrum disorders, schizophrenia, mental retardation and epilepsy. Increasingly, genetic data implicate genes involved in axon guidance and synaptogenesis in these complex brain disorders. In addition, evidence suggests a common etiology for these disorders, as mutations associated with one disorder are also associated with increased incidence of others. Among the molecules that have been implicated in these complex brain disorders are the protocadherins. In particular, mutations in pcdh19 result in a female-limited form of infant-onset epilepsy. Despite the clear role for these molecules in neural development and the etiology of neurodevelopmental dysfunction, relatively little is known about their function in vivo. Here, we propose to generate lines of zebrafish in which pcdh7a, pcdh9, pcdh17 and pcdh19 have been inactivated using zinc finger nucleases (ZFNs). In addition, we propose to generate BAC transgenic lines expressing GFP under the control of the pcdh7a, pcdh9, pcdh17 and pcdh19 regulatory elements. To study the effects of the protocadherin mutations on neural development, we will cross the GFP lines into the mutant backgrounds and use in vivo 2-photon microscopy to determine the impact of the mutations on nervous system development. This work will significantly advance our understanding of protocadherin function, providing a foundation for more mechanistic studies and facilitating a better understanding of how disruption of protocadherins can lead to complex brain disorders.