Targeted inactivation of the clustered protocadherin genes in zebrafish

描述(申请人提供)：中枢神经系统(CNS)的功能依赖于发育过程中突触连接的建立和神经元连接的适当模式来产生功能电路。未能建立适当的突触连接可能会导致严重的神经系统疾病，包括智力低下、自闭症、精神分裂症和抑郁症。因此，了解负责组装突触连接和指导选择功能上合适的突触伙伴的分子机制是至关重要的。我们的长期目标是了解脊椎动物中枢神经系统中负责突触组装和选择的细胞和分子机制。为了解决这些问题，我们正在使用体内多光子成像的突触发生在发育中的斑马鱼中枢神经系统，重点是3-原钙粘附素所扮演的角色。胚胎斑马鱼是解决这些问题的理想系统，因为胚胎是透明的，外部发育迅速，具有相对简单和高度定型的中枢神经系统。此外，最近通过使用工程锌指核酸酶(ZFN)对基因组进行定点修饰的发展将能够有效地产生含有目标基因突变的斑马鱼品系。在这个探索性的R03提案中，我们将使用新兴的ZFN技术来产生斑马鱼的线条，这些斑马鱼携带着斑马鱼pcdh13和pcdh23中存在的每一个3-原钙粘附素基因的损伤。在生成并验证了这些品系之后，我们还将执行初始的表型分析。由于在小鼠中定向缺失pcdh3会导致整个中枢神经系统广泛的神经元死亡，我们将首先评估发育中的神经系统中发生的程序性细胞死亡的水平。我们还将描述对神经系统组织的任何影响，使用全细胞免疫细胞化学标记轴突束的早期支架。最后，我们将使用免疫细胞化学和实时成像来识别突触囊泡团的形成或维持中的任何缺陷。这项工作产生的pcdh3突变系将是研究Pcdh3细胞和发育功能的宝贵资源，使我们能够充分利用斑马鱼模型系统。 公共卫生相关性：突触连接的形成及其形成适当连接的神经元群体的回路对于中枢神经系统的正常发育至关重要。这些事件中的缺陷可能会对人类的行为产生深远的影响，并可能导致许多神经系统疾病，包括自闭症、智力低下和精神分裂症。我们的研究结果将极大地提高我们对3-原钙粘附素在神经回路组装中的作用的认识，并将为开发解决这些疾病的新的治疗策略提供坚实的基础。这些研究还将有助于坚定地理解负责突触发生的发育机制，这可能对于创造有效的方法来促进脑或脊髓损伤后的修复至关重要。

DESCRIPTION (provided by applicant): The function of the central nervous system (CNS) is dependent upon the establishment of synaptic junctions during development and the proper patterning of neuronal connectivity to generate functional circuits. Failure to establish the appropriate synaptic connections can result in severe disorders of the nervous system, including mental retardation, autism, schizophrenia and depression. Thus, it is essential to understand the molecular mechanisms responsible both for assembling the synaptic junction, and for directing the selection of functionally appropriate synaptic partners. Our long-term goal is to understand the cellular and molecular mechanisms responsible for synaptic assembly and selection in the vertebrate CNS. To address these questions, we are using in vivo multiphoton imaging of synaptogenesis in the developing zebrafish CNS, focusing on the roles played by 3-protocadherins. The embryonic zebrafish is an ideal system in which to address these questions, as embryos are transparent, develop externally and rapidly, and have a relatively simple and highly stereotyped CNS. In addition, the recent development of site-specific modification of the genome through the use of engineered zinc-finger nucleases (ZFNs) will enable the efficient production of zebrafish lines that harbor mutations in genes of interest. In this exploratory R03 proposal, we will use the emerging ZFN technology to generate lines of zebrafish carrying lesions in each of the 3-protocadherin genes present in zebrafish, pcdh13 and pcdh23. Having generated and validated these lines, we will also perform an initial phenotypic analysis. As targeted deletion of pcdh3 in mice results in extensive neuronal death throughout the CNS, we will first assess the levels of programmed cell death occurring in the developing nervous system. We will also characterize any effects on the organization of the nervous system, using wholemount immunocytochemistry to label the early scaffold of axon tracts. Finally, we will use both immunocytochemistry and live imaging to identify any defects in the formation or maintenance of synaptic vesicle clusters. The pcdh3 mutant lines produced from this work will be invaluable resources for investigating both the cellular and developmental function of Pcdh3, allowing us to take full advantage of the zebrafish model system. PUBLIC HEALTH RELEVANCE: The formation of synaptic junctions and their patterning into circuits of appropriately connected neuronal populations is essential for the normal development of the central nervous system. Defects in these events can have a profound influence on human behavior, and may underlie numerous disorders of the nervous system, including autism, mental retardation and schizophrenia. The results of our studies will greatly enhance our knowledge of 3-protocadherin function in neural circuit assembly, and will provide a strong foundation for developing novel therapeutic strategies for addressing these disorders. These investigations will also contribute to a firm understanding of the developmental mechanisms responsible for synaptogenesis, which could be essential for creating effective approaches to promote repair after brain or spinal cord injuries.