Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China.
Shanghai Center for Brain Science and Brain-Inspired Intelligence Technology, Shanghai, China.
PLoS Biol. 2020 Mar 13;18(3):e3000435. doi: 10.1371/journal.pbio.3000435. eCollection 2020 Mar.
The circadian clock is a cell-autonomous time-keeping mechanism established gradually during embryonic development. Here, we generated a transgenic zebrafish line carrying a destabilized fluorescent protein driven by the promoter of a core clock gene, nr1d1, to report in vivo circadian rhythm at the single-cell level. By time-lapse imaging of this fish line and 3D reconstruction, we observed the sequential initiation of the reporter expression starting at photoreceptors in the pineal gland, then spreading to the cells in other brain regions at the single-cell level. Even within the pineal gland, we found heterogeneous onset of nr1d1 expression, in which each cell undergoes circadian oscillation superimposed over a cell type-specific developmental trajectory. Furthermore, we found that single-cell expression of nr1d1 showed synchronous circadian oscillation under a light-dark (LD) cycle. Remarkably, single-cell oscillations were dramatically dampened rather than desynchronized in animals raised under constant darkness, while the developmental trend still persists. It suggests that light exposure in early zebrafish embryos has significant effect on cellular circadian oscillations.
生物钟是胚胎发育过程中逐渐建立的细胞自主计时机制。在这里，我们生成了一种携带由核心时钟基因 nr1d1 启动子驱动的不稳定荧光蛋白的转基因斑马鱼系，以在单细胞水平报告体内的昼夜节律。通过对这条鱼系进行延时成像和 3D 重建，我们观察到报告基因表达从松果体中的光感受器开始，然后在单细胞水平上扩散到其他脑区的细胞，依次启动。即使在松果体中，我们也发现了 nr1d1 表达的异质性起始，其中每个细胞都经历了叠加在细胞类型特异性发育轨迹上的昼夜振荡。此外，我们发现单个细胞中 nr1d1 的表达在光暗（LD）循环下表现出同步的昼夜振荡。值得注意的是，在持续黑暗中饲养的动物中，单个细胞的振荡明显减弱而非去同步，而发育趋势仍然存在。这表明早期斑马鱼胚胎中的光照对细胞的昼夜振荡有显著影响。
