Diurnal transcriptome of adult zebrafish whole eye. (A) Schematic of sample collection time points and lighting duration (14 h light:10 h dark) used in zebrafish maintenance. (B) Density of novel transcripts across the genome; each horizontal line depicts a chromosome (1–25) and black boxes denote transcripts. (C) Heat map displays differentially expressed transcripts from RNA-seq analyses of adult zebrafish whole eye from ZT4 and ZT16 time points.

Differential expression of novel transcripts shows 24 h diurnal rhythm. (A) qRT-PCR validation of novel transcripts RNA-seq differential expression trend. The values, normalised to β-actin, are means±s.e.m. (n=5), with left and right y-axes representing qRT-PCR relative expression value and FPKM, respectively. The line graph indicates the FPKM trend of ZT4 and ZT16. (B) Heat map displays temporally resolved novel transcript expression from whole eye of adult zebrafish collected over three consecutive days. The values are normalised to β-actin and scaled (n=1/time point). Yellow line overlay depicts average expression profile of the group of genes marked by grey vertical lines. Asterisks indicate the transcript rhythm statistical significance (t-test: *P<0.05; **P<0.01; ***P<0.001) calculated using meta2d.

Ribo-seq status of retina enriched novel transcripts. (A) Novel transcript enrichment between whole eye and retina of adult zebrafish isolated at ZT4. The values, normalised to β-actin, are means±s.e.m. (n=5). (B) Relative expression (log₁₀ transformed) of novel transcripts across early developmental stages: 28 hpf, 48 hpf and 5 dpf of zebrafish. The values, normalised to β-actin, are means±s.e.m. (n=5). Asterisks indicate the statistical significance (t-test: *P<0.05; **P<0.01; ***P<0.001) calculated between 48 hpf and 5 dpf. (C) The table displays RNA-seq and Ribo-seq coverage from GWIPs-viz public datasets. Black tick indicates that the qRT-PCR trend coincides with RNA-seq coverage and grey tick indicates discordance. n/a, not applicable. (D) Ideogram of pc1 and pc10 transcripts indicating Ribo-seq coverage (red) and RNA-seq coverage (green). Ideogram was created using Gviz R packages.

RNA in situ hybridization shows that novel transcripts are enriched in the INL. (A) WISH on 5 dpf zebrafish larvae shows eye-specific expression (left). WISH followed by cryosectioning (20 µm thickness) revealed strong signal in the INL; indicated by an arrowhead (middle), while sense probe shows no signal (right). (B) WISH on 5 dpf larvae followed by cryosectioning indicates differential expression of transcripts pc1 and pc10 between ZT4 and ZT16. ONL, outer nuclear layer (photoreceptor layer); INL, inner nuclear layer; GCL, retinal ganglion cell layer. (C) RNA in situ of pc1 and pc10 transcripts; on adult eye cryosection (20 µm thickness) collected at ZT4 (left), ZT16 (middle), ZT4 section counterstained with DAPI to reveal nuclear layers (right). Arrows indicate RNA in situ signal in the INL of retina. Scale bars: 100 µm.

Cell type identity of novel transcripts using single-cell transcriptome data. (A,B) tSNE representation of pc2, pc10 and cabp5b expression in cell type clusters identified from 5 dpf whole zebrafish single cell transcriptome. The enlarged version highlights the co-localization between novel transcripts and cabp5b, a bipolar cell marker. (C) Dotplot indicates expression of novel transcripts (pc2 and pc10), core clock genes and marker genes in cell type clusters associated with the retina (mdka: horizontal cells; erg1: amacrine cells; rbpms: retinal ganglion cells; cabp5b: bipolar cells); dot size indicates percentage of cells expressing gene of interest. R1, R2, R3 represent functionally distinct clusters of cells of inner nuclear layer.

Expression of novel transcripts is unaffected by light and dopamine. (A) Schematic of ectopic light depletion regime. (B) Schematic of chemical ablation of Th⁺ amacrine cells by intraocular injection in adult zebrafish. (C) Th⁺ amacrine cells visualized in vehicle-injected and chemically ablated eye-cup by anti-Th immunolabelling on retinal whole mount preparation (3D constructed confocal micrograph). (D) per2 relative expression from ZT4 harvested eyecup under the following conditions: a, light; b, 3 h light depleted; c, light, Th⁺ amacrine cells ablated; d, 3 h light depleted, Th⁺ amacrine cells ablated; e, light, Th⁺ amacrine cells ablated, 200 µmol l⁻¹ dopamine injected at ZT0; f, 3 h light depleted, Th⁺ amacrine cells ablated, 200 µmol l⁻¹ dopamine injected at ZT0. The values, normalized to β-actin, are means±s.e.m. (n=5). (E) Relative expression of novel transcripts from ZT4 measured between light and light-deprived conditions. The values, normalized to β-actin, are means±s.e.m. (n=5). (F) Relative expression of novel transcripts from ZT4 measured between control (light/vehicle control) and A, light+Th⁺ amacrine cells ablated; B, light+Th⁺ amacrine cells ablated+200 µmol l⁻¹ dopamine injected at ZT0; C, light depleted+Th⁺ amacrine cells ablated+200 µmol l⁻¹ dopamine injected at ZT0; D, light+Th⁺ amacrine cells ablated+500 µmol l⁻¹ dopamine injected at ZT0. The values, normalized for beta-actin, are mean±s.e.m. (n=5). t-test: **P<0.01.