Analysis of Clustered Protocadherin Function in Mammalian Neurodevelopment

描述（由申请人提供）：为了成熟神经系统的正常功能，神经元在发育过程中发出必须从大量细胞中选择正确的突触伙伴的过程。人们越来越认识到，许多神经发育障碍（例如自闭症和癫痫症）以及成人疾病（例如双向情感障碍和精神分裂症）可能是由于神经元回路形成不当造成的，这一过程在哺乳动物大脑中的重要性得到了强调。因此，充分了解介导突触伙伴选择的分子对于我们对这些疾病的理解和治疗取得进展至关重要。虽然已经确定了许多引导线索可以帮助轴突生长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。簇状原钙粘蛋白基因家族（Pcdhs）显示出在哺乳动物大脑发育中发挥这一作用的巨大潜力。 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跨膜蛋白。研究表明，单个神经元表达多种 Pcdh 亚型，研究表明它们对于某些神经元类别的突触形成和轴突靶向的重要性。此外，精神分裂症和双相情感障碍患者的连锁分析发现，人类基因组中靠近聚集的原钙粘蛋白基因家族的这两种疾病的易感位点。然而，对 Pcdhs 功能的理解进展受到 Pcdh-3 敲除动物的致死率的限制，而对其他簇的研究很少（对于 Pcdh-1）或不存在（对于 Pcdh-2）。然而，通过使用双标记马赛克分析 (MADM)，应该可以对 Pcdhs 促进神经发育的机制获得新的见解。简而言之，MADM 允许在单个细胞中同时进行标记和基因敲除。通过将适当的标记盒插入 18 号染色体，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 个突变体，或所有三个簇都被删除的突变小鼠）进行繁殖，将有可能分析已知这些蛋白质存在的各种大脑区域中缺乏 Pcdh 的神经元在野生型标记神经元旁边的轴突投影、树突分枝和突触形成。表示。与之前的 Pcdh 敲除研究相比，该策略有几个好处：它避免了与 Pcdh-3 完全丢失相关的致死性，并且应该通过单细胞基因敲除来提供对 Pcdh 功能的更准确的评估。表征簇状原钙粘蛋白在神经元发育中的作用对于理解几种神经系统疾病的分子机制也可能具有价值，例如自闭症和精神分裂症，人们认为这些疾病可能与不正确的突触形成有关。最后，为本研究生产的 MADM 小鼠将来可用于分析 18 号染色体上 MADM 盒远端的任何基因，这对于各个科学领域的研究人员来说可能是一个有用的工具。

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.