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.
Highlights
Circadian rhythm evolves to align animal behaviors to periodic daily environmental changes
To monitor circadian rhythm at the single-cell level in live animals, we have screened for in vivo circadian reporters among various combinations of destabilized fluorescent proteins driven by core clock gene promoters in larval zebrafish
We examined the spatial distribution of fluorescence-labeled cells in nr1d1:VNP at 7.5 dpf using an in vivo two-photon imaging system and found nr1d1:VNP-positive cells in many brain regions, including the pineal gland, the optic tectum, and the cerebellum (Fig 1C, S1 and S2 Movies)
Summary
Circadian rhythm evolves to align animal behaviors to periodic daily environmental changes. The vertebrate circadian clock is mainly generated through transcriptional/translational feedback loops of core clock genes [1]. Two transcription factors (TFs), BMAL1 ( known as ARNTL or MOP3) and CLOCK form heterodimers to bind to E-boxes in the promoters and initiate the transcription of their target genes [2–4], including Per family genes (Per, Per, and Per3) and Cry family genes (Cry and Cry). Two transcription factors (TFs), BMAL1 ( known as ARNTL or MOP3) and CLOCK form heterodimers to bind to E-boxes in the promoters and initiate the transcription of their target genes [2–4], including Per family genes (Per, Per, and Per3) and Cry family genes (Cry and Cry2) The activation of these genes results in the formation of the PER/CRY complex and thereby inhibits CLOCK/.
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