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回路，我们必须识别相关的神经元，确定它们之间的突触连接，并测量它们在回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，可以通过基因操纵，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、连接性和活性进行光学成像，甚至可以光学控制它们的电活动（Scott，2009）。直到最近，在很大程度上缺少的是在特定的感兴趣的神经元中表达基因的方法。 斑马鱼中的Gal 4“增强子陷阱”筛选提供了该问题的有力解决方案（Scott等人，2007; Asakawa等人，2008年）。Gal 4基因作为一种遗传触发因子，被随机整合到斑马鱼基因组中;根据它的位置，它将在不同的细胞（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Gal 4突变株系，人们可以找到在自己喜欢的神经元中表达的株系，然后将这些株系与UAS“响应株系”杂交，从而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Gal 4增强子陷阱筛选，并进行了几项改进。(1)一种新的DNA捕获构建体，不仅表达Gal 4，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Gal 4模式与Cre模式“交叉”而在甚至更特定的细胞组中表达基因。(2)Gal 4表达模式的在线数据库，包括三维视图。这将允许合作者，最终整个斑马鱼社区，快速确定哪些线可能在他们研究的组织或神经元中表达。(3)一个新的公共领域的三维可视化软件包，荧光渲染，已优化的共聚焦显微镜数据。这将改善和加快表达模式的文档。(4)UAS应答细胞系的“工具包”，经验证具有统一的表达水平，可可视化神经元形状和连接。间脑多巴胺能神经元，其中没有特定的增强子是已知的，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Gal 4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表达感兴趣的基因。为交叉基因表达、生成UAS响应者和3D可视化开发的技术也将在该领域广泛应用。该项目将显著提高Gal 4-UAS方法在斑马鱼中的实用性，有助于分析许多器官的发育和功能，以及神经元回路。 公共卫生相关性：从自闭症、脑瘫、图雷特综合征到帕金森病的神经系统疾病都是由于大脑中的神经回路故障造成的，但在许多情况下，对这些回路的理解只是初步的。这项提议将开发遗传工具来研究斑马鱼大脑，它与人类大脑有许多组织甚至详细的特征，这将使我们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可能出现的问题。