Sox10 Alleles for Functional Analysis of Glial Lineages

描述（由申请人提供）：胶质细胞类型的胶质发生和维持对神经系统的发育和功能至关重要。Sox10是一种转录因子， 对多种神经胶质细胞谱系的发育至关重要，包括中枢神经系统中的少突胶质细胞以及神经嵴衍生的雪旺细胞、卫星神经胶质细胞和肠神经系统神经元以及外周中的神经胶质细胞。来自Sox10基因的调控区先前已被用于驱动单个荧光团转基因的表达，以跟踪神经胶质祖细胞的迁移和成熟神经胶质细胞类型的分布。这些第一代工具是有价值的，但不允许同时成像细胞核，形态，或单个细胞之间的信号。钙信号传导是一种基本的细胞机制，细胞通过该机制响应于外部刺激而传递细胞内信号或将信号传递到邻近细胞，并且对于神经胶质细胞发育和维持的许多方面是必不可少的。这些过程的研究主要是在细胞培养系统中进行的，这些系统适合于加载荧光染料或转染外源质粒。然而，最近在开发遗传编码钙指示剂（GECIs）方面的进展已经产生了荧光报告物，其允许监测活细胞和生物体中的钙瞬变。在R21机制的背景下，我们建议在小鼠中产生Sox10的多顺反子转基因等位基因作为成像迁移，细胞形态和神经胶质细胞之间的信号传导的新工具。在具体目标1中，我们将构建并测试多顺反子表达载体，以确定报告基因的最佳组合，以监测钙信号传导、核定位和细胞形态。在特定目标2中，我们将把多光谱表达盒纳入Sox10细菌人工染色体骨架，并建立在体内重现内源性Sox10表达的转基因系。同时跟踪神经胶质细胞群中的迁移、细胞形态和钙信号传导的能力将通过使得能够分析与祖细胞的定向分化相关的发育机制来显著影响该领域，并且将使得能够进行药理学分析以鉴定用于治疗中枢和外周神经病的潜在治疗剂。

DESCRIPTION (provided by applicant): Gliogenesis and maintenance of glial cell types are critical to development and function of the nervous system. Sox10 is a transcription factor that is essential for development of multiple glial lineages including oligodendrocytes in the central nervous system as well as neural crest- derived Schwann cells, satellite glia and enteric nervous system neurons and glia in the periphery. Regulatory regions from the Sox10 gene have previously been used to drive expression of single fluorophore transgenes for tracking the migration of glial progenitors and the distribution of mature glial cell types. These first generaton tools have been valuable but have not permitted concurrent imaging of cell nucleus, morphology, or signaling between individual cells. Calcium signaling is a fundamental cellular mechanism by which cells transmit intracellular signals in response to extrinsic stimuli or transmit signals to adjacent cells and is essential for many aspects of glial cell development and maintenance. Studies of these processes have been primarily investigated in cell culture systems that are amenable to loading with fluorescent dyes or transfection by exogenous plasmids. However recent progress in development of Genetically Encoded Calcium Indicators (GECIs) has produced fluorescent reporters that allow monitoring of calcium transients in living cells and organisms. In the context of the R21 mechanism we propose generation of a multi-cistronic transgenic allele of Sox10 in mice as a novel tool for imaging migration, cell morphology and signaling between glial cells. In Specific Aim 1 we will construct and test multi-cistronic expression vectors in vitro to identify the optimal combination of reporters to monitor calcium signaling, nuclear localization and cell morphology. In Specific Aim 2 we will incorporate a multi-spectral expression cassette into a Sox10 bacterial artificial chromosome backbone and establish transgenic lines that recapitulate endogenous Sox10 expression in vivo. The ability to concurrently track migration, cell morphology and calcium signaling among glial populations will significantly impact the field by enabling analysis of developmental mechanisms that are relevant for directed differentiation of progenitors cells and will enable pharmacologic analyses to identify potential therapeutic agents for treatment of central and peripheral neuropathies.