Genetic and imaging tools to visualize neuronal subsets in developing zebrafish

遗传和成像工具可可视化发育​​中的斑马鱼的神经元亚群

• 批准号: 7937648
• 负责人: Chi-Bin Chien
• 金额: $ 37.63万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-09 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7937648
• 关键词: Animal Behavior; Animals; Area; Autistic Disorder; Axon; Behavior; Biological Assay; Brain; Cell Count; Cell Nucleus; Cells; Cerebral Palsy; Collaborations; Collection; Communities; Computers; Confocal Microscopy; DNA; Data; Databases; Dendrites; Development; Documentation; Enhancers; Exposure to; Fertilization; Fishes; Future; Gene Expression; Gene Transfer Techniques; Generations; Genes; Genetic; Genomics; Gilles de la Tourette syndrome; Goals; Human; Image; Imagery; Imaging Device; Ions; Label; Larva; Life; Maps; Measures; Membrane; Methods; Morphology; Nervous system structure; Neurons; Organ; Output; Parkinson Disease; Pathway interactions; Pattern; Peptides; Property; Public Domains; Publications; Publishing; Regulatory Element; Reporter; Research Personnel; Resolution; Sampling; Scientist; Screening procedure; Shapes; Site; Solutions; Specificity; Speed; Staging; Structure; Synapses; System; Techniques; Technology; Testing; Tissue-Specific Gene Expression; Tissues; Transgenic Organisms; Universities; Utah; Vertebrates; Viral; Zebrafish; design and construction; dopaminergic neuron; human disease; imaging modality; improved; in vivo; insight; interest; movie; mutant; nervous system disorder; neural circuit; neuronal patterning; postsynaptic; presynaptic; public health relevance; recombinase; relating to nervous system; research study; response; shape analysis; tool; two-dimensional; zebrafish genome

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.

描述（由申请人提供）：动物行为取决于大量重叠神经回路的功能。为了充分理解特定行为背后的电路，我们必须识别所涉及的神经元，确定它们之间的突触连接，并测量它们在电路激活期间的电反应。斑马鱼幼虫是研究电路的绝佳系统：它具有完善的行为，可以进行基因操纵，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对神经元的形态、连接性和活动进行光学成像，甚至光学控制其电活动（Scott，2009）。直到最近，在特定感兴趣的神经元中表达基因的方法一直在很大程度上缺失。 斑马鱼中的 Gal4“增强子陷阱”筛选为这个问题提供了强有力的解决方案（Scott 等人，2007 年；Asakawa 等人，2008 年）。 Gal4 基因作为遗传触发因素，随机整合到斑马鱼基因组中；根据它着陆的位置，它将在一组不同的细胞（通常包括特定的神经元类型）中被打开，并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最喜欢的神经元中表达的系，然后将它们与 UAS“响应系”交叉，以便在这些神经元中打开荧光报告基因或其他基因。 该提案将进行第二代 Gal4 增强剂陷阱筛选，并进行多项改进。 (1) 一种新的 DNA 捕获结构，不仅表达 Gal4，而且可以转化为表达不同的基因开关 Cre 重组酶。这将允许通过将 Gal4 模式与 Cre 模式“交叉”来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。这将使合作者以及最终整个斑马鱼群体能够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) 一种新的公共领域 3D 可视化软件包 FluoRender，已针对共焦显微镜数据进行了优化。这将改进并加速表达模式的记录。 (4) UAS 响应系“工具包”，经过验证一致的表达水平，以可视化神经元形状和连接性。间脑多巴胺能神经元尚无特定增强子，将作为测试案例进行分析。 总之，该项目将产生大量特征良好的 Gal4 增强子捕获系和 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特异性表达感兴趣的基因。为交叉基因表达、UAS 响应器生成和 3D 可视化而开发的技术也将广泛应用于该领域。该项目将显着提高 Gal4-UAS 方法在斑马鱼中的实用性，帮助分析除神经元回路之外的许多器官的发育和功能。 公共健康相关性：从自闭症、脑瘫、图雷特氏综合症到帕金森氏病等神经系统疾病都是由于大脑神经回路功能障碍造成的，但在许多情况下，对这些回路的了解还很初级。该提案将开发遗传工具来研究斑马鱼大脑，斑马鱼大脑具有人类大脑的许多组织甚至细节特征，这将能够分析许多不同回路中神经细胞的形状、突触连接和电活动，长期目标是了解这些回路在人类疾病中如何出错。