Real-time visualization of neural stem cell transcriptome

描述（申请人提供）：神经干细胞（NSC）具有细胞状态从休眠到增殖再到分化的精确转变的迷人生物学，最近开始在细胞和分子水平上理解。尽管成功地鉴定了与这些细胞步骤相关的基因，但在NSC的整个分化阶段，这些基因是如何实时调节的，目前还缺乏一个全面和多维的图像。这一多学科的建议将首次结合使用两种强大的方法，在单细胞分辨率下直接评估参与NSC调节的关键基因的转录状态。第一种技术，多重原位杂交，由其中一个pi共同开发，可以在单细胞分辨率下直接可视化几个基因的转录活性。第二种技术被称为G-Trace，它将被用来产生带有荧光蛋白标记的细胞克隆，这种荧光蛋白根据它们的时间产生来区分细胞谱系。在Aim 1中，这两种方法在果蝇幼虫大脑中的结合将用于同时创建参与NSC调节的几个基因的正常野生型表达谱。进一步的计算图像分析工具将开发分割图像和减少噪声。我们将测试光谱条形码系统的应用，作为原理证明，以扩大一次分析数百个活性基因的检测。在Aim 2中，将通过敲除关键转录因子来诱导脑肿瘤，以跟踪标记克隆中基因表达的全局变化。期望这些组合方法将揭示NSC分化为几个特定神经谱系的关键特性，对更好地操纵一般和其他生物（包括人类）的干细胞具有深远的影响。

DESCRIPTION (provided by applicant): Neural stem cells (NSC) have a fascinating biology of precise transition of cellular states from dormant to proliferative to differentiating, which most recently begun to be understood at the cellular and molecular levels. Despite successful identification of genes linked to each of those cellular steps, a comprehensive and multi-dimensional picture on how those genes are regulated in real-time throughout the differentiation stages of NSC is sorely lacking. This multi- disciplinary proposal will combine for the first time the use of two powerful methods to directly assess the transcriptional state of key genes involved in NSC regulation in vivo, at a single cell resolution. The first technique, Multiplex in situ hybridization, co-developed by one of the PIs, allows direct visualization of transcriptional activity of several genes with single cell resolution. The second technique, known as G-Trace, will be used to generate cellular clones labeled with fluorescent proteins that distinctively mark cell lineages according to their temporal generation. In Aim 1, the combination of these two methods in the Drosophila larval brain will be used to create profiles of normal wild type expression of several genes involved in NSC regulation simultaneously. Further computational image analysis tools will be developed to segment images and reduce noise. We will test the application of a spectral bar coding system as a proof-of-principle to expand the detection of hundreds of active genes analyzed at a time. In Aim 2, brain tumors will be induced by knocking-out key transcription factors to track global alterations in gene expression within labeled clones. The expectation is that these combined methods will reveal key properties of the differentiation progression of NSC into several specific neuronal lineages, with far-reaching implications to a better manipulation of stem cells in general and in other organisms, including humans. PUBLIC HEALTH RELEVANCE: Our studies will increase knowledge on stem cell regulation and has the potential to impact the fields of regenerative medicine and stem cell cancer research. A better understanding of global gene regulation at the single-cell level is expected to enhance methods employed in a number of applications, such as in vitro neural stem cell differentiation to repair tissue after damage and development of probes for detecting early stages of cancer.