New Transgenic Tools for Studying Neural Circuit Formation

描述(申请人提供)：最近，高分辨率共聚焦和多光子成像通过促进对活体模型生物体中神经元发育的近乎实时的解析，为神经电路的形成提供了独特的见解。作为一种模式系统，斑马鱼促进了这类研究，因为它们是在外部发育，在胚胎和幼体阶段高度透明。已经建立了一些在特定神经亚群中表达荧光报告蛋白的转基因斑马鱼品系，并已被证明对详细描述神经元形态的发展是有用的。然而，在神经回路形成方面，必须建立促进突触前和突触后伙伴群体同时成像的方法。使用互补的“颜色”的荧光记者促进了树突和轴突成分的成像，并已成功地应用于揭示视网膜分层亚回路形成的潜在机制。然而，实际问题阻碍了这一方法的广泛实施：1)现有转基因株系中的荧光报告表达水平对于详细和/或长期的成像研究往往是次优的。2)识别能够将转基因表达定向到不同神经元亚群的调控DNA序列，更不用说神经元伙伴亚群，是一个耗时的过程。本文建议实施新的转基因方法，旨在绕过目前的实用限制，创建在独特的神经亚群中表达细胞报告和/或神经元信号分子的斑马鱼系，包括神经元伙伴亚群。这些线将有助于从各种亚回路的特定角度研究正常和异常回路形成的分子和细胞机制。一系列稳定的转基因斑马鱼品系将由两种模块化转基因表达系统之一组成；一个基于LexA的系统将被开发出来，以补充现有的Gal4/UAS系统。拥有两个可用的二元表达系统将促进转基因在特定神经元亚群中表达的最大多样性。例如，两个不同的神经元亚群可以用荧光记者进行不同的标记。在两个亚群相互作用的情况下，神经元回路的形成可以可视化。此外，除了促进细胞标记，该系统还可以用于在特定神经元内表达任何感兴趣的基因(S)。旨在改变神经元活动、标记突触、追踪神经电路等的转基因有助于一系列分析，以研究电路形成和功能的潜在机制。由于拟议的项目代表了普遍适用的工具包，这些资源在未来许多年仍将是相关的。促进单个神经元亚型荧光标记的公共卫生相关工具有助于揭示大脑的单个组成部分是如何发育的。这里提出的新工具集允许用蓝色、绿色、黄色和红色荧光蛋白对不同的神经元亚群进行不同的标记。此外，这些工具箱旨在促进在活体动物模型系统中发生的神经元电路形成的直接可视化和分子操作，为了解脑细胞如何连接提供了一个独特的窗口。

DESCRIPTION (provided by applicant): High-resolution confocal and multiphoton imaging has recently provided unique insights into how neural circuits form by facilitating near real-time resolution of neuronal development in living model organisms. As a model system, zebrafish facilitate such studies due to that fact that they develop externally and are highly transparent during embryonic and larval stages. A number of transgenic zebrafish lines expressing fluorescent reporter proteins in specific neural subsets have been established and have proven useful for detailing how neuronal morphologies develop. However, in terms of neural circuit formation, methods must be established which promote simultaneous imaging of both pre- and postsynaptic partner populations. The use of complimentary "colors" of fluorescent reporters facilitates imaging of both dendritic and axonal elements and has been successfully applied to reveal mechanisms underlying the formation of stratified subcircuits in the retina. However, practical issues hamper widespread implementation of this approach: 1) Fluorescent reporter expression levels in available transgenic lines are often suboptimal for detailed and/or long-term imaging studies. 2) Identifying regulatory DNA sequences competent for directing transgene expression to distinct neuronal subpopulations, let alone subsets of neuronal partners, is a time consuming process. Proposed here is the implementation of new transgenic approaches designed to circumvent current practical limitations and create zebrafish lines expressing cellular reporters and/or neuronal signaling molecules in unique neural subsets, including neuronal partner subpopulations. These lines will be instrumental for investigating molecular and cellular mechanisms underlying proper and aberrant circuit formation from a variety of subcircuit-specific perspectives. A series of stable transgenic zebrafish lines will be derived comprised of one of two modular transgene expression systems; a LexA-based system will be developed to complement the existing Gal4/UAS system. Having two binary expression systems available will promote maximum versatility regarding the expression of transgenes in specific subsets of neurons. For instance, two different neuronal subpopulations can be differentially labeled with fluorescent reporters. In cases where the two subpopulations interact, neuronal circuit formation can be visualized. Moreover, in addition to promoting cell labeling, this system can be used to express any gene(s) of interest within specified neurons. Transgenes designed to alter neuronal activity, label synapses, trace neural circuitry, etc., facilitate an array of assays for investigating mechanisms underlying circuit formation and function. Because the proposed lines represent universally adaptable toolsets these resources will remain relevant for many years to come. PUBLIC HEALTH RELEVANCE Tools that promote fluorescent labeling of individual neuronal subtypes have been instrumental in revealing how single components of the brain develop. Proposed here are new toolsets that allow separate subpopulations of neurons to be differentially labeled with blue, green, yellow, and red fluorescent proteins. Moreover, these toolsets have been designed to facilitate direct visualization and molecular manipulation of neuronal circuit formation as it occurs in a living animal model system, providing a unique window into how brain cells get "wired up".