Identification of enhancers whose activity defines cortical interneuron types

 描述（由申请人提供）：神经细胞类型的分子定义主要取决于通过原位杂交、RNA阵列和测序以及免疫组织化学评估的RNA或蛋白质的表达。然而，最近的研究表明，基因调控元件，如增强子，可以在发育中的大脑中具有高度特异性的空间和时间活动模式。因此，增强子活性可用于定义神经细胞类型，并且重要的是，还具有其他广泛的应用。首先，它们可以用作驱动特定细胞类型中的基因表达的工具，然后可以用于可视化和/或纯化细胞（GFP），修饰细胞中的基因表达（Cre），修饰电活性（通道视紫红质），以及可视化细胞中的电活性（GCaMP）。其次，增强子的性质和位置的知识使遗传学家能够识别在外显子外基因组空间中映射的疾病等位基因。在这里，我们将集中在确定增强子，活跃在皮层中间神经元，在发展和成熟状态。我们选择研究皮质GABA能中间神经元，因为它们在皮质功能和疾病中的中心作用。我们将集中在这些GABA能神经元来自内侧神经节隆起（MGE），产生了大部分的皮质中间神经元。我们的目的是使用三种测定来鉴定在皮质中间神经元中有活性的新型增强子：1）组蛋白（H3 K27 Ac）ChIP-seq（活性增强子的标记物）; 2）转录因子（TF）ChIP-seq，使用调节皮质中间神经元发育和功能的TF（Arx，Dlx 2和Lhx 6）; 3）基于未成熟皮质中间神经元中驱动Cre和GFP的增强子的病毒转导的增强子功能的新开发的体内测定。然后，我们将使用一系列方法来定义具有增强子活性的中间神经元亚型。总之，我们独特的方法应该定义interneuronal细胞类型（发育和成人），并同时产生一个强大的工具包，使新的方法来评估神经功能和疾病。

 DESCRIPTION (provided by applicant): Molecular definitions of neural cell types largely depend on the expression of RNAs or proteins as assessed by in situ hybridization, RNA array and sequencing, and immunohistochemistry. However, recent studies are demonstrating that gene regulatory elements, such as enhancers, can have highly specific spatial and temporal activity patterns in the developing brain. Thus, enhancer activity can be used to define neural cell types, and importantly, also have other broad applications. First, they can be used as tools to drive gene expression in specific cell types, which can then be used to visualize and/or purify the cells (GFP), modify gene expression in the cells (Cre), modify electrical activity (channel rhodopsin), and visualize electrical activity in the cells (GCaMP). Secondly, knowledge about the nature and position of enhancers enables geneticists to identify disease alleles that map in extra-exonic genomic space. Herein, we will focus on identifying enhancers that are active in cortical interneurons, during development and in the mature state. We choose to study cortical GABAergic interneurons because of their central role in cortical function and diseases. We will focus on these GABAergic neurons derived from the medial ganglionic eminence (MGE), which generates the majority of cortical interneurons. Our aim is to identify novel enhancers that are active in cortical interneurons using three assays: 1) histone (H3K27Ac) ChIP-seq (marker of active enhancers); 2) transcription factor (TF) ChIP- seq using TFs that regulate cortical interneurons development and function (Arx, Dlx2 and Lhx6); 3) a newly developed in vivo assay of enhancer function based on viral transduction of the enhancer driving Cre and GFP in immature cortical interneurons. We will then use a series of methods to define the interneuron subtypes that have enhancer activity. Together, our unique approach should define interneuronal cell types (developmental and adult), and simultaneously generate a powerful toolkit that will enable new ways to assess neural function and disease.