Genetic and imaging tools to visualize neuronal subsets in developing zebrafish

描述（由申请人提供）：动物行为依赖于大量重叠神经回路的功能。为了充分理解一个特定行为背后的电路，我们必须识别参与其中的神经元，确定它们彼此之间的突触连接，并测量它们在电路激活时的电反应。斑马鱼的幼虫是研究电路的一个极好的系统：它有完善的行为，可以从基因上操纵，最重要的是，它是透明的。通过基因表达荧光报告基因或光激活通道，人们可以在完整的活体动物中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（Scott, 2009）。直到最近，在特定的感兴趣的神经元中表达基因的方法在很大程度上是缺失的。在斑马鱼中使用Gal4“增强子陷阱”筛查提供了一个强有力的解决方案（Scott等人，2007；Asakawa等人，2008）。作为基因触发器的Gal4基因被随机整合到斑马鱼的基因组中；根据它的位置不同，它会在一组不同的细胞（通常包括特定的神经元类型）中被激活，由附近基因的调节元件控制。通过筛选许多Gal4突变系，人们可以找到在自己喜欢的神经元中表达的系，然后将这些系与UAS“响应系”交叉，这样荧光报告或其他基因就会在这些神经元中被激活。该提案将进行第二代Gal4增强子陷阱屏，并进行了几项改进。(1)一种新的DNA捕获结构，不仅表达Gal4，而且可以转化为表达另一种基因开关，Cre重组酶。这将允许通过将Gal4模式与Cre模式“交叉”，在更特定的细胞组中表达基因。(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，最终整个斑马鱼群落，快速确定哪些细胞系可能在他们研究的组织或神经元中表达。(3)一个新的公共领域三维可视化包，FluoRender，已经优化了共聚焦显微镜数据。这将改进和加快表达式模式的文档编制。(4)通过统一表达水平验证的UAS应答线“工具包”，以可视化神经元形状和连通性。间脑多巴胺能神经元，其具体的增强剂尚不清楚，将作为一个测试案例进行分析。总之，该项目将产生大量具有良好特征的Gal4增强子陷阱线和UAS反应线，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特异性表达感兴趣的基因。交叉基因表达、UAS应答器生成和3D可视化技术的发展也将广泛应用于该领域。该项目将显著提高Gal4-UAS方法在斑马鱼中的实用性，帮助分析许多器官的发育和功能，以及神经元回路。

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.