Analysis of Clustered Protocadherin Function in Mammalian Neurodevelopment

描述(申请人提供)：为了成熟神经系统的正常功能，神经元在发育过程中发出过程，必须从大量细胞中选择正确的突触伙伴。越来越多的人认识到，许多神经发育障碍，如自闭症和癫痫，以及成人障碍，如双相情感障碍和精神分裂症，都可能是神经元回路形成不当的结果，这突显了这一过程在哺乳动物大脑中的重要性。因此，充分了解调节突触伴侣选择的分子对于提高我们对这些疾病的理解和治疗是至关重要的。虽然已经确定了许多指导线索，帮助超过生长的轴突到达大脑的某个区域，但轴突如何选择其确切的突触伙伴还不太清楚。聚集的原钙粘附素基因家族(Pcdhs)在哺乳动物脑发育中显示出巨大的潜力来填补这一角色。Pcdh基因家族由三个基因簇(1、2和3)中的58个基因组成，每个基因簇编码一种独特的粘附性跨膜蛋白。已有研究表明，单个神经元表达不同的PcdH亚型，研究表明它们在某些类型的神经元中对突触形成和轴突靶向具有重要作用。此外，对精神分裂症和双相情感障碍患者的连锁分析发现，在人类基因组中靠近聚集的原钙粘素基因家族的这两种疾病的易感基因座。然而，对Pcdhs功能的理解一直受到Pcdh-3基因敲除动物致死性的限制，而对其他簇的研究很少(对于Pcdh-1)或根本不存在(对于Pcdh-2)。然而，通过使用带有双标记的嵌合体分析(MADM)，应该有可能对Pcdhs促进神经发育的机制有新的见解。简而言之，MADM允许在单个细胞中同时进行标记和基因敲除。通过在18号染色体上插入适当的标记盒，并将这些小鼠(称为MADM18小鼠)与各种Pcdh杂合突变小鼠(1、2和3个突变小鼠，或一个所有三个簇都已缺失的突变小鼠)杂交，将有可能分析Pcdh缺失神经元的轴突投射、树突分枝和突触形成，旁边是已知表达这些蛋白的各种大脑区域中野生型标记神经元。与以前的Pcdh基因敲除研究相比，这种策略有几个好处：它避免了与完全丧失Pcdh-3相关的致命性，并且应该通过单细胞敲除基因来提供更准确的Pcdh功能评估。表征成簇的原钙粘附素在神经元发育中的作用也可能对理解几种神经疾病的分子机制有价值，如自闭症和精神分裂症，人们认为这些疾病可能涉及不正确的突触形成。最后，为这项研究生产的MADM小鼠未来可以用于分析18号染色体上MADM盒远端的任何基因，这可能是各种科学领域的研究人员的有用工具。

DESCRIPTION (provided by applicant): For proper function of the mature nervous system, neurons send processes out during development that must choose their correct synaptic partners from an enormous population of cells. The importance of this process in the mammalian brain is underscored by the growing knowledge that many neurodevelopmental disorders, such as autism and epilepsy, as well as adult disorders like bipolar disorder and schizophrenia, may result from improper formation of neuronal circuits. Therefore, a full understanding of the molecules mediating synaptic partner choice is crucial for make advancements in our comprehension and treatment of these diseases. While many guidance cues have been identified that assist outgrowing axons to reach an area of the brain, it's less clear how an axon chooses its exact synaptic partner. The clustered protocadherin family of genes (Pcdhs) show great potential to fill this role in mammalian brain development. The Pcdh gene family consists of 58 genes in three gene clusters (1, 2, and 3), each encoding for a unique adhesive transmembrane protein. It has been shown that individual neurons express a wide diversity of Pcdh isoforms, and studies have suggested their importance for synapse formation and axon targeting in certain classes of neurons. Furthermore, linkage analyses of schizophrenia and bipolar disorder patients have uncovered susceptibility loci for both disorders in the human genome near the clustered protocadherin gene family. Yet progress in understanding the function of Pcdhs has been limited by lethality in Pcdh-3 knockout animals, while studies of the other clusters are few (for Pcdh-1) or non-existent (for Pcdh-2). However, by using Mosaic Analysis with Double Markers (MADM), it should be possible to gain novel insight into the mechanisms by which Pcdhs contribute to neural development. Briefly, MADM allows for simultaneous labeling and gene knockout in individual cells. By inserting the appropriate labeling cassettes into chromosome 18 and breeding these mice (called MADM18 mice) with various Pcdh heterozygous mutant mice (1, 2, and 3 mutants, or a mutant mouse where all three clusters have been deleted), it will be possible to analyze axonal projections, dendritic arborizations, and synapse formation of Pcdh-lacking neurons next to wildtype-labeled neurons in a variety of brain regions where these proteins are known to be expressed. Compared to previous Pcdh-knockout studies, this strategy has several benefits: it avoids the lethality associated with complete loss of Pcdh-3, and should provide a more accurate assessment of Pcdh function through single cell knockout of the genes. Characterizing the role of the clustered protocadherins in neuronal development may also have value for understanding the molecular mechanisms of several neurological diseases, such as autism and schizophrenia, where it's thought that improper synapse formation may be involved. Finally, the MADM mouse produced for this study could be used in the future to analyze any gene distal to the MADM cassettes on the chromosome 18, which may be a useful tool for researchers in a variety of scientific fields.