Dual Expression Control for Studying Drosophila Neural Circuits

描述（由申请人提供）：具有不同亚型特异性驱动的二元转基因诱导允许在完整生物体中标记和/或操作不同的脑细胞亚群。然而，一个人不能区别标记或独立操纵不同的脑细胞（例如特定的神经元和它们的突触伙伴）在同一生物体中只有一个二元转基因诱导系统。在此，我们提出利用GAL4/UAS和LexA/lexAop这两个互不干扰的二元转录系统构建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，用于独立于GAL4/UAS靶向多种不同的脑细胞。我们还将生成各种依赖lexa的转基因，用于选择性标记或操纵lexa阳性细胞。这些试剂将构成大多数果蝇神经生物学家需要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。此外，我们将在LexA/lexAop之上构建新的基因镶嵌工具包，不含GAL4抑制因子GAL80。这些独立于GAL4/UAS/ gal80的遗传镶嵌系统保证了多种遗传/转基因工具协同应用的最大通用性。同时应用两个独立的二元转基因诱导系统，通过支持涉及不同脑细胞的各自分析或差异操作的复杂遗传研究，有望进一步革新现代神经生物学研究。这将允许更彻底的阐明和更精细的神经回路发育的时空操纵，并有可能导致新的治疗靶点的鉴定，以纠正异常的大脑发育或恢复神经回路在各种神经退行性疾病。神经回路的连接涉及神经元之间以及神经元与神经胶质细胞之间复杂的细胞间相互作用，这些相互作用有助于控制单个神经突生长锥的导航、精心设计并最终与特定目标建立突触接触。许多先天性脑功能障碍是由神经回路的异常连接引起的。要理解在这种神经系统疾病中，电路的布线是如何出错的，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，关于神经回路发育的知识可以为退行性疾病或损伤后的干预提供有力的临床方法。该项目的目标是建立工具，以便更好地研究高度复杂的中枢神经系统中的细胞-细胞相互作用。我们建议在果蝇中开发这样的工具，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的二元转基因诱导允许在完整生物体中标记和/或操纵不同亚型的脑细胞。然而，仅用一种二元转基因诱导系统无法同时对不同的脑细胞进行差异标记或独立操作。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胶质细胞，我们提出在果蝇中建立第二个广泛适用的二元转基因诱导系统。这包括产生亚型特异性驱动因子和一组驱动因子依赖的转基因，用于标记或操纵各种特异性果蝇脑细胞，独立于现有的二元转录系统及其衍生的遗传/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构建更多功能的遗传镶嵌技术。同时应用两个独立的二元转基因诱导系统，通过支持复杂的遗传研究，包括对同一生物体中不同脑细胞的各自分析或差异操作，有望进一步革新现代神经生物学研究。这些研究将对我们详细绘制神经回路、阐明和操纵神经回路的发育以及最终理解大脑的发育和功能产生根本性的影响。

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.