Single-cell in vivo imaging of cellular circadian oscillators in zebrafish

PLoS Biol. 2020 Mar 13;18(3):e3000435. doi: 10.1371/journal.pbio.3000435. eCollection 2020 Mar.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Bacterial Proteins / genetics
  • Brain / cytology
  • Circadian Clocks / genetics*
  • Circadian Rhythm / genetics
  • Circadian Rhythm / physiology
  • Embryo, Nonmammalian / cytology
  • Luminescent Proteins / genetics
  • Nuclear Receptor Subfamily 1, Group D, Member 1 / genetics*
  • Photoperiod
  • Pineal Gland / cytology*
  • Pineal Gland / physiology
  • Promoter Regions, Genetic
  • Single-Cell Analysis
  • Time-Lapse Imaging
  • Zebrafish / embryology
  • Zebrafish / genetics*

Substances

  • Bacterial Proteins
  • Luminescent Proteins
  • Nr1d1 protein, zebrafish
  • Nuclear Receptor Subfamily 1, Group D, Member 1
  • yellow fluorescent protein, Bacteria

Grants and funding

This work was supported by National Science Foundation for Young Scientists of China grant (No. 31701029) and Natural Science Foundation of Shanghai grant (16ZR1448800) to HW; NSFC-ISF Joint Scientific Research Program grants (31861143035) to JY; and Natural Science Foundation of China grants (No. 31571209 to JY and No. 81401279 to ZY). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.