PROJECT ID: PRJNA554650

Data source SRA: PRJNA554650
Description we report a transgenic zebrafish line using destabilized fluorescent protein, Venus-NLS-PEST (VNP), driven by the promoter of a key circadian clock gene, nr1d1. This system allows us to monitor the development of single-cell circadian rhythm in live zebrafish larva in a cell-type specific manner. To identify the cell types expressing nr1d1:VNP in the whole brain, we conducted single cell RNA-seq (scRNA-seq) of ~15,000 cells dissociated from the brain of Tg(nr1d1:VNP) larval fish at 6.5dpf. Among them, 6514 cells were identified with number of genes > 500 and used for the following analysis. 26 cells clusters were classified from scRNAseq, and manually annotated by comparing the marker genes with the adult zebrfiash whole brain single cell RNA-seq data. The mRNA of nr1d1:VNP was enriched in photoreceptors in pineal gland, granule cells and purkinje cells in cerebellum, habenula cells as well as non-neuron cell.
Key word clock; expression; differentiation; platform; reveals; rhythms; protein; period2; genes; onset
Publication Wang H, Yang Z, Li X, Huang D et al. Single-cell in vivo imaging of cellular circadian oscillators in zebrafish. PLoS Biol 2020 Mar;18(3):e3000435. PMID: 32168317
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.


Dataset Information

Dataset ID Species Tissue / Organ Experiment type Sample Source dataset ID
1. PRJNA554650 (6.5dpf) Danio rerio brain baseline 6.5dpf, whole brain of nr1d1:VNP fish SRA: SRR9685040

Clustering Result

Cluster Cell type Gene id (symbol) Marker class Evidence
1 Midbrain Glut+ ENSDARG00000009311 (pvalb6) marker DOI:10.1038/nbt.4103
2 Optic Tectum Gaba+++ ENSDARG00000015537 (gad2) marker DOI:10.1038/nbt.4103
3 Hyp ENSDARG00000068691 (kctd4) marker DOI:10.1038/nbt.4103
4 Pallium (Glut+) ENSDARG00000023771 (gabrb3) marker DOI:10.1038/nbt.4103
4 Pallium (Glut+) ENSDARG00000044098 (egr2a) marker DOI:10.1038/nbt.4103
5 Nascent neurons ENSDARG00000037997 (tubb5) marker DOI:10.1038/nbt.4103
6 Nascent neurons ENSDARG00000037997 (tubb5) marker DOI:10.1038/nbt.4103
7 Nascent neurons ENSDARG00000037997 (tubb5) marker DOI:10.1038/nbt.4103
8 Progenitors ENSDARG00000038205 (her2) marker DOI:10.1038/nbt.4103
8 Progenitors ENSDARG00000054560 (her15.2) marker DOI:10.1038/nbt.4103
8 Progenitors ENSDARG00000054562 (her15.1) marker DOI:10.1038/nbt.4103
9 Dorsal habenula ENSDARG00000042970 (gng8) marker DOI:10.1038/nbt.4103
9 Dorsal habenula ENSDARG00000078859 (g0s2) marker DOI:10.1038/nbt.4103
9 Dorsal habenula ENSDARG00000069843 (kctd12.1) marker DOI:10.1038/nbt.4103
10 Nascent neurons ENSDARG00000037997 (tubb5) marker DOI:10.1038/nbt.4103
11 Progenitors ENSDARG00000038205 (her2) marker DOI:10.1038/nbt.4103
11 Progenitors ENSDARG00000054560 (her15.2) marker DOI:10.1038/nbt.4103
11 Progenitors ENSDARG00000054562 (her15.1) marker DOI:10.1038/nbt.4103
12 Progenitors ENSDARG00000038205 (her2) marker DOI:10.1038/nbt.4103
12 Progenitors ENSDARG00000054560 (her15.2) marker DOI:10.1038/nbt.4103
12 Progenitors ENSDARG00000054562 (her15.1) marker DOI:10.1038/nbt.4103
13 Radial glia ENSDARG00000036427 (slc3a2a) marker DOI:10.1038/nbt.4103
13 Radial glia ENSDARG00000041623 (mt2) marker DOI:10.1038/nbt.4103
13 Radial glia ENSDARG00000045979 (zgc:153704) marker DOI:10.1038/nbt.4103
14 Granule cells ENSDARG00000097478 (qkia) marker DOI:10.1038/nbt.4103
15 Endothelium ENSDARG00000104068 (gstp1) marker DOI:10.1038/nbt.4103
16 Progenitors ENSDARG00000038205 (her2) marker DOI:10.1038/nbt.4103
16 Progenitors ENSDARG00000054560 (her15.2) marker DOI:10.1038/nbt.4103
16 Progenitors ENSDARG00000054562 (her15.1) marker DOI:10.1038/nbt.4103
17 Endothelium ENSDARG00000104068 (gstp1) marker DOI:10.1038/nbt.4103
18 Microglia ENSDARG00000040295 (apoeb) marker DOI:10.1038/nbt.4103
19 Purkinje cells ENSDARG00000057661 (aldoca) marker DOI:10.1038/nbt.4103
19 Purkinje cells ENSDARG00000039098 (ca8) marker DOI:10.1038/nbt.4103
19 Purkinje cells ENSDARG00000062448 (skor1b) marker DOI:10.1038/nbt.4103
20 Hyp ENSDARG00000035832 (pyyb) marker DOI:10.1038/nbt.4103
20 Hyp ENSDARG00000023181 (pcp4l1) marker DOI:10.1038/nbt.4103
20 Hyp ENSDARG00000017174 (dlx2b) marker DOI:10.1038/nbt.4103
21 Oligodendrocyte precursor cells ENSDARG00000036670 (aplnrb) marker DOI:10.1038/nbt.4103
21 Oligodendrocyte precursor cells ENSDARG00000002172 (aplnra) marker DOI:10.1038/nbt.4103
21 Oligodendrocyte precursor cells ENSDARG00000040948 (olig1) marker DOI:10.1038/nbt.4103
21 Oligodendrocyte precursor cells ENSDARG00000058821 (sema5a) marker DOI:10.1038/nbt.4103
22 Cranial ganglion ENSDARG00000004930 (lmo7a) marker DOI:10.1038/nbt.4103
23 Pineal gland ENSDARG00000035798 (gngt1) marker DOI:10.1038/nbt.4103
23 Pineal gland ENSDARG00000009637 (rcvrn3) marker DOI:10.1038/nbt.4103
23 Pineal gland ENSDARG00000111827 (rcvrnb) marker DOI:10.1038/nbt.4103
23 Pineal gland ENSDARG00000044199 (gnat1) marker DOI:10.1038/nbt.4103
23 Pineal gland ENSDARG00000012610 (saga) marker DOI:10.1038/nbt.4103