IDENTIFYING BRAIN WIRING MECHANISMS BY GENE TRAPPING

哺乳动物大脑发育研究的一个主要挑战是确定指导大脑复杂线路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”，可以系统地捕获编码配体和受体的基因，并产生携带这些基因突变的小鼠，这为阐明这些基因的功能提供了重要工具。然而，研究大脑线路的问题在于，由于难以追踪神经元的轴突投影，因此很难识别由实验扰动引起的线路变化。特别是，在突变小鼠中识别线路缺陷通常是极其困难的，无论突变是自发产生的还是由基因靶向或插入突变产生的。我们已经设想了一种基因陷阱技术的进一步改进，这种技术现在将极大地促进与哺乳动物神经系统连接有关的受体的识别和表征。在我们的方法中，组织化学轴突标记针对通常表达捕获基因的神经元。通过简单的组织化学染色，可以直接看到这些神经元之间的连接，也可以直接看到纯合突变动物的线路缺陷。通过这种方式，可以快速评估被困基因在大脑线路中的作用。同样重要的是，对于整个神经科学界来说，该方法也将同时产生在不同轴突群体中表达组织化学标记的小鼠库，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重要工具。在此提出请求，支持使用这种改良的载体分离大量分泌诱捕胚胎干细胞系。据建议，每年将有80个这样的细胞系通过生殖细胞系进行表达和表型分析，以确定那些与神经系统连接有关的细胞系，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。我们期望这种方法将有助于大大加快发现哺乳动物神经系统连接机制的步伐。

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.