IDENTIFYING BRAIN WIRING MECHANISMS BY GENE TRAPPING

哺乳动物大脑发育研究中的一个主要挑战是确定指导大脑复杂连接模式组装的细胞外配体和受体。最近对胚胎干细胞中的基因陷阱技术进行了改进，即“分泌陷阱”，允许系统地捕获编码配体和受体的基因，并产生在这些基因中存在突变的小鼠，为阐明这些基因的功能提供了重要的工具。然而，研究大脑连接的问题是，由于追踪神经元轴突投射的困难，可能极难识别实验扰动导致的连接变化。特别是，通常很难确定突变小鼠的线路缺陷，无论突变是自发发生的，还是通过基因打靶或插入突变产生的。我们已经设想了对基因陷阱技术的进一步修改，现在应该可以极大地方便识别和表征参与连接哺乳动物神经系统的受体。在我们的方法中，一个组织化学轴突标记物被定位于正常表达捕获基因的神经元。这使得能够直接看到这些神经元建立的连接，并通过简单的组织化学染色直接看到纯合子突变动物中的线路缺陷。通过这种方式，可以快速评估捕获的基因在大脑连接中的作用。同样重要的是，这种方法对整个神经科学界也有很大的好处，同时还将产生一组在不同轴突群体中表达组织化学标记的小鼠，这将为有兴趣阐明哺乳动物大脑中神经元连接的正常模式的研究人员提供一个重要的工具。这里提出了一项请求，以支持使用该修改的载体分离大量的分泌陷阱ES细胞系。据建议，每年将有80株这样的细胞系通过胚系进行表达和表型分析，以识别那些参与连接神经系统的细胞，并为神经科学界提供在不同神经元群体中表达轴突标记的小鼠资源。我们预计，这种方法将有助于大大加快发现指导哺乳动物神经系统连接的机制的步伐。

A major challenge in the study of mammalian brain development is to identify the extracellular ligands and receptors that direct the assembly of the complex wiring pattern of the brain. A recent modification of the gene trap technique in embryonic stem cells, the "secretory trap", allows the systematic trapping of genes encoding ligands and receptors, and the generation of mice harboring mutations in these genes, providing an important tool to elucidate the functions of these genes. However, the problem with studying brain wiring is that it can be extremely difficult to identify changes in wiring resulting from experimental perturbation because of the difficulty of tracing the axonal projections of neurons. In particular, it is usually extremely difficult to identify wiring defects in mutant mice, whether the mutation arose spontaneously or was generated by gene targeting or insertional mutagenesis. We have conceived a further modification of the gene trap technique that should now dramatically facilitate the identification and characterization of receptors involved in wiring the mammalian nervous system. In our method, a histochemical axonal marker is targeted to neurons that normally express the trapped gene. This enables direct visualization of the connections made by these neurons, and direct visualization of wiring defects in homozygous mutant animals, through a simple histochemical stain. In this way the role of the trapped gene in brain wiring can be rapidly assessed. Of equal importance, and of great benefit to the Neuroscience community at large, the method will also simultaneously result in the generation of a bank of mice expressing the histochemical marker in different populations of axons, which will provide an important tool for researchers interested in elucidating the normal pattern of neuronal connections in the mammalian brain. A request is made here to support the isolation of a large number of secretory trap ES cell lines using this modified vector. It is proposed that 80 of these lines per year will be put through the germ line for expression and phenotypic analysis to identify those involved in wiring the nervous system, and to provide a resource of mice expressing the axonal marker in different populations of neurons for use by the Neuroscience community. We expect that this method will help greatly accelerate the pace of discovery of mechanisms that direct the wiring of the mammalian nervous system.