Genetic and imaging tools to visualize neuronal subsets in developing zebrafish

描述(申请人提供)：动物的行为取决于大量重叠的神经回路的功能。为了完全理解一种特定行为背后的电路，人们必须识别涉及的神经元，确定它们彼此之间建立了什么突触连接，并在电路激活期间测量它们的电反应。斑马鱼幼体是研究回路的极好系统：它有良好的行为，可以通过遗传操作，最重要的是，它是透明的。通过基因表达荧光报告或光激活通道，一个人可以光学成像神经元的形态、连通性和活动，甚至光学控制它们的电活动，在一个完整的活动物(Scott，2009)。直到最近，在很大程度上缺少的是表达基因的方法，特别是感兴趣的神经元。斑马鱼中的Gal4“增强子陷阱”屏幕为这个问题提供了一个强有力的解决方案(Scott等人，2007年；Asakawa等人，2008年)。Gal4基因起到了遗传触发的作用，它被随机整合到斑马鱼的基因组中；根据它落在哪里，它将在另一组细胞(通常包括特定的神经元类型)中被打开，由附近基因的调控元件控制。通过筛选许多Gal4突变系，人们可以找到在自己最喜欢的神经元中表达的线，然后将这些线交叉到UAS的“响应线”，这样荧光报告器或其他基因就会在这些神经元中被打开。这项提案将进行第二代Gal4增强子陷阱筛选，并进行几项改进。(1)一种新的DNA捕获构建物，它不仅表达Gal4，而且可以转化为表达不同的遗传开关，Cre重组酶。这将允许通过将Gal4模式与Cre模式“相交”，在更特定的细胞集合中表达基因。(2)Gal4表达模式的在线数据库，包括三维视图。这将允许合作者，最终是整个斑马鱼群体，快速确定哪些线路可能在他们研究的组织或神经元中表达。(3)针对共聚焦显微镜数据进行了优化的新的公共领域3D可视化程序包--FluoRender。这将改进和加快表达式模式的文档编制。(4)UAS反应器线的“工具包”，用于统一表达水平的验证，以可视化神经元形状和连接性。间脑多巴胺能神经元，其特定的增强剂尚不清楚，将作为测试案例进行分析。综上所述，该项目将产生大量具有良好特性的Gal4增强子陷阱系和UAS响应系，这将使斑马鱼神经生物学家以及其他斑马鱼研究人员能够在许多不同的神经元类别和非神经组织中表达感兴趣的基因。为跨部门基因表达、UAS响应器的生成和3D可视化开发的技术也将在该领域得到广泛应用。该项目将显著增加Gal4-UAS方法在斑马鱼中的应用，帮助分析许多器官的发育和功能，以及神经元回路。 与公共卫生相关：从自闭症、脑瘫、多发性抽动症到帕金森氏症的各种神经系统疾病都是由于大脑中神经回路的故障造成的，但在许多情况下，对这些回路的了解还很初级。这项提议将开发基因工具来研究斑马鱼的大脑，斑马鱼的大脑具有许多组织甚至详细的人类大脑特征，这将使分析许多不同回路中神经细胞的形状、突触连接和电活动成为可能，长期目标是了解这些回路在人类疾病中可能发生的错误。

DESCRIPTION (provided by applicant): Animal behavior depends on the function of a large collection of overlapping neural circuits. To fully under- stand the circuit underlying a particular behavior, one must identify the neurons involved, determine what synaptic connections they make with each other, and measure their electrical responses during activation of the circuit. The zebrafish larva is an excellent system to study circuits: it has well-established behaviors, can be manipulated genetically, and most importantly, is transparent. By genetically expressing fluorescent reporters or light-activated channels, one can optically image neurons' morphology, connectivity, and activity, and even optically control their electrical activity, in an intact, living animal (Scott, 2009). What has been largely missing, until recently, are methods to express genes in particular neurons of interest. A powerful solution to this problem is provided by Gal4 "enhancer trap" screens in zebrafish (Scott et al., 2007; Asakawa et al., 2008). The Gal4 gene, which acts as a genetic trigger, is integrated randomly into the zebrafish genome; depending on where it lands, it will be turned on in a different set of cells (often including specific neuronal types), controlled by the regulatory elements of nearby genes. By screening through many Gal4 mutant lines, one can find lines that express in one's favorite neurons, then cross these to UAS "responder lines", so that fluorescent reporters or other genes are turned on in those neurons. This proposal will carry out a second-generation Gal4 enhancer trap screen with several improvements. (1) A new DNA trapping construct that not only expresses Gal4, but can be converted to instead express a different genetic switch, Cre recombinase. This will allow expression of genes in even more specific sets of cells by "intersecting" a Gal4 pattern with a Cre pattern. (2) An online database of Gal4 expression patterns, including three-dimensional views. This will allow collaborators, and eventually the zebrafish community at large, to quickly determine which lines may express in the tissues or neurons that they study. (3) A new public- domain 3D visualization package, FluoRender, that has been optimized for confocal microscopy data. This will improve and speed up documentation of expression patterns. (4) A "toolkit" of UAS responder lines, validated for uniform expression levels, to visualize neuronal shape and connectivity. Diencephalic dopaminergic neurons, for which no specific enhancer is yet known, will be analyzed as a test case. In summary, then, this project will generate a large number of well-characterized Gal4 enhancer trap lines and UAS responder lines, which will allow zebrafish neurobiologists as well as other zebrafish researchers to express genes of interest specifically in many different neuron classes and nonneural tissues. Techniques developed for intersectional gene expression, generation of UAS responders, and 3D visualization will also be widely applicable in the field. The project will significantly increase the utility of the Gal4-UAS method in zebrafish, aiding analysis of the development and function of many organs, in addition to neuronal circuits. PUBLIC HEALTH RELEVANCE: Neurological diseases ranging from autism, cerebral palsy, and Tourette's syndrome to Parkinson's disease are due to malfunction of neural circuits in the brain, yet in many cases the understanding of these circuits is only rudimentary. This proposal would develop genetic tools to study the zebrafish brain, which shares many organizational and even detailed features of the human brain, which will enable the analysis of the shape, synaptic connections, and electrical activity of nerve cells in many different circuits, with the long-term goal of understanding how these circuits may go wrong in human disease.