Real-time in vivo monitoring of circadian E-box enhancer activity: A robust and sensitive zebrafish reporter line for developmental, chemical and neural biology of the circadian clock
Real-time in vivo monitoring of circadian E-box enhancer activity: A robust and sensitive zebrafish reporter line for developmental, chemical and neural biology of the circadian clock
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DOI：
10.1016/j.ydbio.2013.04.035
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发表时间：
2013-08-15
影响因子：
2.7
通讯作者：
Dickmeis, Thomas
中科院分区：
文献类型：
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作者：
Weger, Meltem;Weger, Benjamin D.;Dickmeis, Thomas
生物钟协调生理和行为与日/夜周期。它由一个转录-翻译反馈环组成，该反馈环通过E-box增强子元件在大约24小时的时间内产生转录活性的自我维持振荡。核心时钟反馈回路功能的许多体内方面仍然不完全清楚，包括其在发育过程中的成熟，组织特异性活性和疾病状态的扰动。斑马鱼是很有前途的生物医学研究模型，由于其高的再生能力和适用于在体内药物筛选，和转基因斑马鱼线是有价值的工具，以研究在体内转录活性在development.To监测在体内的核心时钟反馈环的活动，我们创建了一个转基因斑马鱼线表达的荧光素酶报告基因的最小启动子和四个E-box的调节下。该Tg（4xE-box：Luc）系在光-暗循环下和释放到恒定黑暗中时均显示出稳健的振荡报告基因表达。荧光素酶活性在发育的第一天开始振荡，表明核心时钟循环在早期阶段已经起作用。为了测试Tg（4xE-box：Luc）线是否可用于旨在鉴定靶向体内生物钟的化合物的药物筛选，我们检查了药物对昼夜节律周期的影响。我们能够很容易地检测到低至0.7小时的治疗期间延长药物氯化锂和longdaysin的测定设置适合于大规模屏幕的变化。报告基因mRNA的表达也在成人大脑中检测到，并揭示了整个大脑的差异时钟活动，与内源性时钟基因的表达重叠。值得注意的是，核心时钟活动与神经发生的大脑区域密切相关，并且可以在几种类型的神经progeniters.Our结果表明，Tg（4xE-box：Luc）线是一个很好的工具，用于研究生物钟的调节及其在体内和真实的时间成熟。此外，它非常适合于考虑完整生物体复杂性的靶向核心时钟机制的体内筛选。最后，它允许映射的时钟活动在脊椎动物模型生物体的大脑中具有突出的成年神经发生和高再生能力。(c)2013 Elsevier Inc. All rights reserved.
The circadian clock co-ordinates physiology and behavior with the day/night cycle. It consists of a transcriptional-translational feedback loop that generates self-sustained oscillations in transcriptional activity with a roughly 24 h period via E-box enhancer elements. Numerous in vivo aspects of core clock feedback loop function are still incompletely understood, including its maturation during development, tissue-specific activity and perturbation in disease states. Zebrafish are promising models for biomedical research due to their high regenerative capacity and suitability for in vivo drug screens, and transgenic zebrafish lines are valuable tools to study transcriptional activity in vivo during development.To monitor the activity of the core clock feedback loop in vivo, we created a transgenic zebrafish line expressing a luciferase reporter gene under the regulation of a minimal promoter and four E-boxes. This Tg(4xE-box:Luc) line shows robust oscillating reporter gene expression both under light-dark cycles and upon release into constant darkness. Luciferase activity starts to oscillate during the first days of development, indicating that the core clock loop is already functional at an early stage. To test whether the Tg(4xE-box:Luc) line could be used in drug screens aimed at identifying compounds that target the circadian clock in vivo, we examined drug effects on circadian period. We were readily able to detect period changes as low as 0.7 h upon treatment with the period-lengthening drugs lithium chloride and longdaysin in an assay set-up suitable for large-scale screens. Reporter gene mRNA expression is also detected in the adult brain and reveals differential clock activity across the brain, overlapping with endogenous clock gene expression. Notably, core clock activity is strongly correlated with brain regions where neurogenesis takes place and can be detected in several types of neural progenitors.Our results demonstrate that the Tg(4xE-box:Luc) line is an excellent tool for studying the regulation of the circadian clock and its maturation in vivo and in real time. Furthermore, it is highly suitable for in vivo screens targeting the core clock mechanism that take into account the complexity of an intact organism. Finally, it allows mapping of clock activity in the brain of a vertebrate model organism with prominent adult neurogenesis and high regeneration capacity. (c) 2013 Elsevier Inc. All rights reserved.