Nowotschin Sonja, Xenopoulos Panagiotis, Schrode Nadine, Hadjantonakis Anna-Katerina
Developmental Biology Program, Sloan-Kettering Institute, New York, NY, USA.
BMC Dev Biol. 2013 Apr 25;13:15. doi: 10.1186/1471-213X-13-15.
Live imaging provides an essential methodology for understanding complex and dynamic cell behaviors and their underlying molecular mechanisms. Genetically-encoded reporter expressing mouse strains are an important tool for use in live imaging experiments. Such reporter strains can be engineered by placing cis-regulatory elements of interest to direct the expression of desired reporter genes. If these cis-regulatory elements are downstream targets, and thus activated as a consequence of signaling pathway activation, such reporters can provide read-outs of the signaling status of a cell. The Notch signaling pathway is an evolutionary conserved pathway operating in multiple developmental processes as well as being the basis for several congenital diseases. The transcription factor CBF1 is a central evolutionarily conserved component of the Notch signaling pathway. It binds the active form of the Notch receptor (NICD) and subsequently binds to cis-regulatory regions (CBF1 binding sites) in the promoters of Notch responsive genes. In this way, CBF1 binding sites represent a good target for the design of a Notch signaling reporter.
To generate a single-cell resolution Notch signaling reporter, we used a CBF responsive element to direct the expression of a nuclear-localized fluorescent protein. To do this, we linked 4 copies of a consensus CBF1 binding site to the basal simian virus 40 (SV40) promoter, placed this cassette in front of a fluorescent protein fusion comprising human histone H2B linked to the yellow fluorescent protein (YFP) Venus, one of the brightest available YFPs. We used the CBF:H2B-Venus construct to generate both transgenic embryonic mouse stem (ES) cell lines and a strain of transgenic mice that would report Notch signaling activity.
By using multiple CBF1 binding sites together with a subcellular-localized, genetically-encoded fluorescent protein, H2B-Venus, we have generated a transgenic strain of mice that faithfully recapitulates Notch signaling at single-cell resolution. This is the first mouse reporter strain in which individual cells transducing a Notch signal can be visualized. The improved resolution of this reporter makes it ideal for live imaging developmental processes regulated by the Notch signaling pathway as well as a short-term lineage tracer of Notch expressing cells due to the perdurance of the fluorescent reporter. Taken together, the CBF:H2B-Venus mouse strain is a unique tool to study and understand the morphogenetic events regulated by the Notch signaling pathway.
实时成像为理解复杂且动态的细胞行为及其潜在分子机制提供了重要方法。基因编码报告基因表达小鼠品系是实时成像实验中的重要工具。此类报告基因品系可通过放置感兴趣的顺式调控元件来指导所需报告基因的表达进行构建。如果这些顺式调控元件是下游靶点，并因此在信号通路激活时被激活，那么此类报告基因可提供细胞信号状态的读数。Notch信号通路是一条在多个发育过程中起作用的进化保守通路，也是多种先天性疾病的基础。转录因子CBF1是Notch信号通路中一个核心的进化保守成分。它与Notch受体的活性形式（NICD）结合，随后与Notch反应基因启动子中的顺式调控区域（CBF1结合位点）结合。这样，CBF1结合位点是设计Notch信号报告基因的良好靶点。
为了生成单细胞分辨率的Notch信号报告基因，我们使用了CBF反应元件来指导核定位荧光蛋白的表达。为此，我们将4个拷贝的共有CBF1结合位点与猿猴病毒40（SV40）基本启动子相连，将此盒式结构置于由与黄色荧光蛋白（YFP）Venus相连的人组蛋白H2B组成的荧光蛋白融合体之前，Venus是现有的最亮的YFP之一。我们使用CBF:H2B-Venus构建体生成了转基因胚胎小鼠干细胞（ES）系和一个能报告Notch信号活性的转基因小鼠品系。
通过将多个CBF1结合位点与亚细胞定位的、基因编码的荧光蛋白H2B-Venus一起使用，我们生成了一个转基因小鼠品系，该品系能在单细胞分辨率下忠实地重现Notch信号。这是第一个能使转导Notch信号的单个细胞可视化的小鼠报告基因品系。该报告基因提高的分辨率使其成为实时成像由Notch信号通路调控的发育过程以及由于荧光报告基因的持久性而成为Notch表达细胞的短期谱系示踪剂的理想选择。总之，CBF:H2B-Venus小鼠品系是研究和理解由Notch信号通路调控的形态发生事件的独特工具。
