New Transgenic Tools for Studying Neural Circuit Formation

描述（由申请人提供）：高分辨率共聚焦和多光子成像最近提供了独特的见解，神经回路是如何形成的，通过促进近实时分辨率的神经元发育在活的模式生物。斑马鱼作为一种模型系统，由于它们在胚胎和幼虫阶段的外部发育和高度透明，因此有助于此类研究。许多在特定神经亚群中表达荧光报告蛋白的转基因斑马鱼系已经建立，并已被证明对详细描述神经元形态如何发展有用。然而，就神经回路的形成而言，必须建立促进突触前和突触后伴侣群体同时成像的方法。荧光报告的互补“颜色”的使用促进了树突和轴突元件的成像，并已成功地应用于揭示视网膜分层亚回路形成的机制。然而，实际问题阻碍了这种方法的广泛实施：1)在现有的转基因系中，荧光报告基因的表达水平通常不适合进行详细和/或长期的成像研究。2)鉴定能够引导转基因表达到不同神经元亚群的调控DNA序列是一个耗时的过程，更不用说神经元伙伴亚群了。这里提出的是实施新的转基因方法，旨在绕过当前的实际限制，并创建斑马鱼系表达细胞报告和/或神经元信号分子在独特的神经亚群，包括神经元伴侣亚群。这些细胞系将有助于从各种亚电路特定的角度研究正常和异常电路形成的分子和细胞机制。由两种模块化转基因表达系统之一组成的一系列稳定的转基因斑马鱼系将得到；将开发一个基于lexa的系统，以补充现有的Gal4/UAS系统。拥有两种可用的二进制表达系统将促进转基因在特定神经元亚群中的表达的最大通用性。例如，两个不同的神经元亚群可以用荧光报告基因进行不同的标记。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，除了促进细胞标记外，该系统还可用于表达特定神经元中感兴趣的任何基因。转基因旨在改变神经元活动，标记突触，追踪神经回路等，促进了一系列研究回路形成和功能机制的分析。因为建议的线路代表了普遍适用的工具集，这些资源将在未来许多年保持相关性。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。这里提出了新的工具集，允许单独的神经元亚群用蓝色、绿色、黄色和红色荧光蛋白进行不同的标记。此外，这些工具集被设计用来促进在活体动物模型系统中神经元回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".