Synaptic-GFP Tags for Zebrafish

描述(由申请人提供)：GFP标签在调节线虫等模式生物中突触形成的分子途径的遗传解剖中非常有用。同样，许多这样的融合蛋白现在被用来检测培养的哺乳动物神经元的突触发育。然而，斑马鱼的这种融合在很大程度上仍然没有得到利用，尽管斑马鱼独特地适合于突触发育的正向遗传解剖，因为它的快速发育，幼体发育期间的透明身体，以及易于遗传操作。随着斑马鱼基因组序列的接近完成，这些工具在斑马鱼身上开发的时机已经成熟。作为开发斑马鱼研究计划的第一步，旨在剖析调节突触靶标选择特异性的分子机制，我提议创建一组GFP标记的突触蛋白，以便于分析斑马鱼胚胎和幼虫体内突触的形成。利用现代重组DNA技术(GAL4/DAS系统、Gateway克隆技术、转座子载体和细菌人工染色体)，我建议创建斑马鱼突触蛋白-GFP融合表达载体和表达这些突触蛋白-GFP构建体的转基因斑马鱼动物。我们将专注于我们在线虫中展示的“移动”结构，以强健和准确地表达，并标记突触小泡群体或突触的活性区域。具体地说，我们最近创造了一种与线虫突触囊泡蛋白的新型融合，使活体蠕虫中突触的检测灵敏度提高了10倍。此外，这种线虫融合跨物种工作，因为当在果蝇中表达时，蠕虫蛋白融合牢固地定位于神经肌肉接头。初步研究表明，当在神经元中瞬时表达系统中表达时，等同的斑马鱼融合形成斑点，这表明它定位于突触。我们建议在斑马鱼中开发这种融合来标记突触前特化。同样，我们建议测试最近开发的几个线虫活动区标签是否允许当类似的斑马鱼蛋白融合在斑马鱼神经元中表达时，斑马鱼活动区的可视化。

DESCRIPTION (provided by applicant): GFP-tags have been extremely useful in the genetic dissection of molecular pathways that regulate the formation of synapses in model organisms such as C. elegans. Similarly, many such fusion proteins are now being used to examine synaptic development in cultured mammalian neurons. However, such fusions remain largely unutilized in Zebrafish even though the fish is uniquely suited for forward genetic dissection of synaptic development because of its fast development, transparent body during larval development, and the ease of genetic manipulation. With the genome sequence of Zebrafish nearing completion, such tools are ripe for development in the Zebrafish. As a first step in developing a research program in Zebrafish aimed at dissecting the molecular mechanisms that regulate the specificity of synapse target selection, I propose to create a set of GFP-tagged synaptic proteins to facilitate the analysis of synapse formation in vivo in Zebrafish embryos and larvae. Using a combination of modern recombinant DNA technologies (the GAL4/ DAS system, Gateway cloning technology, transposon vectors, and bacterial artificial chromosomes), I propose to create vectors for expression of Zebrafish synaptic protein-GFP fusions and transgenic Zebrafish animals expressing these synaptic protein-GFP constructs. We will concentrate on 'moving' constructs that we have shown in C. elegans to express robustly and precisely and label either synaptic vesicle populations or the active zone domain of the synapse. Specifically, we have recently created a novel fusion to a C. elegans synaptic vesicle protein that allows for 10-fold more sensitive detection of synapses in live worms. Furthermore, this C. elegans fusion works across species as the worm protein fusion localizes robustly to the neuromuscular junction when expressed in Drosophila. Preliminary studies indicate that the equivalent Zebrafish fusion forms puncta when expressed in neurons in a transient expression system suggesting it localizes to synapses. We propose to develop this fusion in Zebrafish to label presynaptic specializations. Similarly, we propose to test whether several recently developed C. elegans active zone tags will permit the visualization of Zebrafish active zones when the analogous Zebrafish protein fusions are expressed in Zebrafish neurons.