Single-cell in vivo imaging reveals light-initiated circadian oscillators in zebrafish

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, we observed the sequential initiation of the reporter expression starting at photoreceptors in pineal gland then spreading to cells in other brain regions. Even within pineal gland, we found heterogeneous onset of nr1d1 expression in which each cell undergoes circadian oscillation superimposed over cell-type specific developmental trajectory. Furthermore, we found that single-cell expression of nr1d1 showed synchronous circadian oscillation under light-dark cycle. Remarkably, single-cell oscillations were lost in animals raised under constant darkness while developmental trend still persists. It suggests that light exposure in early clock development initializes cellular clocks rather than synchronizes existing individual oscillators as previously believed.