Dual Expression Control for Studying Drosophila Neural Circuits

描述(由申请人提供)：具有不同亚型特定驱动因素的双元转基因诱导允许标记和/或操纵完整生物体中的不同脑细胞亚组。然而，仅用一个二元转基因诱导系统不能对同一生物体中不同的脑细胞(如特定的神经元及其突触伙伴)进行差异标记或独立操作。在这里，我们建议用两个非干扰的二元转录系统(GAL4/UAS和LexA/LexAop)来建立研究果蝇神经回路发育的工具。我们将分离不同的LexA驱动程序，以独立于GAL4/UAS靶向多种不同的脑细胞。我们还将产生各种依赖于LexA的转基因，以选择性地标记或操纵LexA阳性细胞。这些试剂将构成大多数果蝇神经生物学家立即将两个独立的二元转基因诱导系统的力量应用于他们的神经回路发育研究所需的东西。此外，我们将在LexA/lexAop的基础上构建新的遗传马赛克工具包，而不使用GAL4抑制因子GAL80。这些独立于GAL4/UAS/GAL80的遗传镶嵌系统保证了在多种遗传/转基因工具的共同应用中最大的通用性。两个独立的二元转基因诱导系统的同时应用有望进一步革新现代神经生物学研究，因为它支持同时涉及不同脑细胞的不同分析或差异处理的复杂遗传学研究。这将允许对神经回路发育进行更彻底的阐明和更精细的时空操作，并可能导致识别新的治疗靶点，以纠正大脑发育异常或在各种神经退行性疾病中恢复神经回路。与公共健康相关的神经回路布线涉及神经元之间以及神经元和神经胶质细胞之间复杂的细胞-细胞相互作用，这些相互作用有助于控制神经突起的单个生长锥体，以导航、阐述并最终与特定目标进行突触接触。许多先天性大脑功能障碍是由神经回路的异常连接引起的。要了解在这种神经疾病中电路的连接是如何出错的，需要阐明细胞与细胞相互作用的各种复杂过程的细胞和分子基础。此外，有关神经回路发育的知识可能为退行性疾病期间或损伤后的干预提供强大的临床方法。这个项目的目标是建立工具，更好地研究高度复杂的中枢神经系统中的细胞与细胞之间的相互作用。我们建议在果蝇身上开发这样的工具，果蝇是一种了解大脑发育和功能的强大模式生物。具有不同亚型特异性驱动因素的二元转基因诱导允许标记和/或操作完整生物体中的不同脑细胞亚群。然而，人们不能用一个二元转基因诱导系统同时对不同的脑细胞进行不同的标记或独立操作。为了独立靶向多种神经元类型或突触前细胞与突触后细胞或神经元与神经胶质细胞，我们建议在果蝇中建立第二个广泛适用的二元转基因诱导系统。这涉及到产生亚型特异的驱动程序和一组依赖于驱动程序的转基因基因，用于标记或操纵各种特定的果蝇脑细胞，而不依赖于现有的二进制转录系统及其所有派生的遗传/转基因工具。此外，我们将在新的二元转基因诱导系统的基础上建立更多功能的遗传镶嵌技术。两个独立的二元转基因诱导系统的同时应用有望进一步革新现代神经生物学研究，因为它支持复杂的遗传学研究，涉及对同一生物体中不同脑细胞的不同分析或差异处理。这些研究将对我们的神经回路的详细图谱、神经回路发育的阐明和操作以及对大脑发育和功能的最终理解产生根本影响。

DESCRIPTION (provided by applicant): Binary transgene induction with distinct subtype-specific drivers permits marking and/or manipulation of different subsets of brain cells in intact organisms. However, one cannot differentially mark or independently manipulate distinct brain cells (e.g. specific neurons and their synaptic partners) in the same organism with only one binary transgene induction system. Here we propose to build tools for studying Drosophila neural circuit development with two noninterfering binary transcriptional systems (GAL4/UAS and LexA/lexAop). We will isolate diverse LexA drivers for targeting a large variety of different brain cells independently of GAL4/UAS. We will also generate various LexA-dependent transgenes for selectively marking or manipulating LexA-positive cells. These reagents would constitute what most Drosophila neurobiologists need to immediately apply the power of two independent binary transgene induction systems to their studies of neural circuit development. In addition, we will build new genetic mosaic toolkits on top of LexA/lexAop and without the GAL4 repressor GAL80. These GAL4/UAS/GAL80-independent genetic mosaic systems guarantee maximal versatility in the co-application of multiple genetic/transgenic tools. The simultaneous application of two independent binary transgene induction systems promises to further revolutionize modern neurobiological research by supporting complex genetic studies involving respective analysis or differential manipulation of distinct brain cells at the same time. This will allow more thorough elucidation and finer spatiotemporal manipulation of neural circuit development, and potentially lead to the identification of new therapeutic targets for remedying abnormal brain development or restoring neural circuitry in various neurodegenerative conditions. PUBLIC HEALTH RELEVANCE Wiring of neural circuits involves intricate cell-cell interactions among neurons and between neurons and glial cells that help govern individual growth cones of neurites to navigate, elaborate, and finally make synaptic contacts with specific targets. Many congenital malfunctions of the brain result from aberrant wiring of neural circuits. To understand how wiring of circuitry goes awry in such neurological disorders requires elucidation of the cellular and molecular bases of the diverse complex processes of cell-cell interactions. Furthermore, knowledge about neural circuit development may provide powerful clinical approaches for interventions during degenerative disorders or after injury. The goal of this project is to build tools for better studying cell-cell interactions in the highly convoluted central nervous system. We propose to develop such tools in the fruit fly Drosophila, a powerful model organism for understanding brain development and function. Binary transgene induction with distinct subtype-specific drivers permits marking and/or manipulation of different subsets of brain cells in intact organisms. However, one cannot differentially mark or independently manipulate distinct brain cells at the same time with only one binary transgene induction system. To independently target multiple neuron types or presynaptic versus postsynaptic cells or neurons versus glia, we propose to establish a second widely applicable binary transgene induction system in Drosophila. This involves generation of both subtype-specific drivers and a set of driver-dependent transgenes for marking or manipulating various specific Drosophila brain cells independently of the existing binary transcriptional system and all its derived genetic/transgenic tools. In addition, we will build more versatile genetic mosaic techniques on top of the new binary transgene induction system. The simultaneous application of two independent binary transgene induction systems promises to further revolutionize modern neurobiological research by supporting complex genetic studies involving respective analysis or differential manipulation of distinct brain cells in the same organism. Such studies will have fundamental impacts on our detailed mapping of neural circuitry, the elucidation and manipulation of neural circuit development, and the ultimate understanding of brain development and function.