A sensitive and bright single-cell resolution live imaging reporter of Wnt/ß-catenin signaling in the mouse.
A sensitive and bright single-cell resolution live imaging reporter of Wnt/ß-catenin signaling in the mouse.
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DOI：
10.1186/1471-213x-10-121
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发表时间：
2010-12-21
影响因子：
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通讯作者：
Hadjantonakis AK
中科院分区：
文献类型：
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作者：
Ferrer-Vaquer A;Piliszek A;Tian G;Aho RJ;Dufort D;Hadjantonakis AK
了解动态细胞行为和驱动形态发生的潜在分子机制是生物学中的一个持续挑战。实时成像提供了必要的方法来解开塑造胚胎的协同和刻板的细胞和分子事件。基因编码的报告基因是实时成像的重要工具。可以通过放置感兴趣的顺式调节元件以指导所需报告基因的表达来工程化报告菌株。在典型Wnt信号传导（也称为Wnt/β-连环蛋白信号传导）的情况下，由于下游转录反应已得到充分理解，因此可以设计反映活性Wnt信号传导位点的报告基因，而不是基因转录位点，正如许多荧光报告基因的情况一样。然而，即使已经产生了几种转基因Wnt/β-连环蛋白报告菌株，迄今为止，没有一种提供有利于活成像研究的单细胞分辨率。我们已经将六个拷贝的TCF/Lef响应元件和hsp 68最小启动子置于包含人组蛋白H2 B与GFP的荧光蛋白融合物之前，并使用它来产生将报告Wnt/β-连环蛋白信号传导活性的小鼠品系。所得TCF/Lef：H2 B-GFP菌株的发育和成体阶段的表征揭示了转基因在先前表征的Wnt/β-连环蛋白信号传导位点的离散和特异性表达。为了支持TCF/Lef：H2 B-GFP报告基因的灵敏度增加，观察到了其他报告基因未记录但通过遗传和胚胎学分析鉴定的Wnt/β-连环蛋白信号传导的其他位点。此外，报告子的亚细胞定位使报告子持久性最小化，并允许可视化和跟踪组群内的单个细胞，从而促进形态发生期间细胞行为和信号传导活性的详细分析。通过将多聚化TCF/Lef DNA结合位点的Wnt活性读出效率与亚细胞定位的荧光融合蛋白（如H2 B-GFP）提供的高分辨率成像相结合，我们创建了一种小鼠转基因系，该转基因系在单细胞分辨率下忠实地再现了Wnt信号传导活性。TCF/Lef：H2 B-GFP报告基因代表了一种独特的工具，用于对Wnt/β-catenin信号传导触发的体内过程进行实时成像，因此应该有助于对定义由该信号传导途径调节的形态发生过程的系列事件进行高分辨率理解。
Understanding the dynamic cellular behaviors and underlying molecular mechanisms that drive morphogenesis is an ongoing challenge in biology. Live imaging provides the necessary methodology to unravel the synergistic and stereotypical cell and molecular events that shape the embryo. Genetically-encoded reporters represent an essential tool for live imaging. Reporter strains can be engineered by placing cis-regulatory elements of interest to direct the expression of a desired reporter gene. In the case of canonical Wnt signaling, also referred to as Wnt/β-catenin signaling, since the downstream transcriptional response is well understood, reporters can be designed that reflect sites of active Wnt signaling, as opposed to sites of gene transcription, as is the case with many fluorescent reporters. However, even though several transgenic Wnt/β-catenin reporter strains have been generated, to date, none provides the single-cell resolution favored for live imaging studies. We have placed six copies of a TCF/Lef responsive element and an hsp68 minimal promoter in front of a fluorescent protein fusion comprising human histone H2B to GFP and used it to generate a strain of mice that would report Wnt/β-catenin signaling activity. Characterization of developmental and adult stages of the resulting TCF/Lef:H2B-GFP strain revealed discrete and specific expression of the transgene at previously characterized sites of Wnt/β-catenin signaling. In support of the increased sensitivity of the TCF/Lef:H2B-GFP reporter, additional sites of Wnt/β-catenin signaling not documented with other reporters but identified through genetic and embryological analysis were observed. Furthermore, the sub-cellular localization of the reporter minimized reporter perdurance, and allowed visualization and tracking of individual cells within a cohort, so facilitating the detailed analysis of cell behaviors and signaling activity during morphogenesis. By combining the Wnt activity read-out efficiency of multimerized TCF/Lef DNA binding sites, together with the high-resolution imaging afforded by subcellularly-localized fluorescent fusion proteins such as H2B-GFP, we have created a mouse transgenic line that faithfully recapitulates Wnt signaling activity at single-cell resolution. The TCF/Lef:H2B-GFP reporter represents a unique tool for live imaging the in vivo processes triggered by Wnt/β-catenin signaling, and thus should help the formulation of a high-resolution understanding of the serial events that define the morphogenetic process regulated by this signaling pathway.