Evolutionary Biology

CLOCK evolved in cnidaria to synchronize internal rhythms with diel environmental cues

Raphael Aguillon author has email address
Mieka Rinsky author has email address
Noa Simon-Blecher
Tirza Doniger
Lior Appelbaum
Oren Levy author has email address

Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel
The Multidisciplinary Brain Research Center, Bar-Ilan University, Ramat Gan 52900, Israel

https://doi.org/10.7554/eLife.89499.2

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Figures and data

NvClk¹^-/- cannot maintain circadian behavior in non-diel light conditions.
(a) Phylogenetic tree showing the evolutionary relationship of CLK orthologs across different animal species. (b) In situ hybridization of NvClk in the WT juvenile with scale bars representing 0.1mm. (c) Schematic representation of the NvClk gene in grey, with the open reading frame (ORF) in dark grey and the conserved protein domains bHLH (yellow) and PAS1 and PAS2 (dark red). The CRISPR generated NvClk¹ allele has a +20nt insertion after the PAS1 domain, represented by a black arrowhead. NvCLK dimerizes via its three functional domain with NvCYCLE binding the CACGTG ebox to drive rhythmic transcription. (d-g-j-m). Normalized Movement (a.u), hourly binned over 72h, under different light conditions: 12h light:12h dark, continuous dark (Dark - Dark), continuous light (Light - Light), and 6h light:6h dark. The black line represents the WT and the red line represents the NvClk¹^-/- mutant. (e-h-k-n) Lomb-Scargle Periodograms for each corresponding light condition. The significant period value (p<0.01) is indicated for each genotype in the top left corner of each graph. (f-i-l-o) Phase detection (Cosinor) and genotype comparison of 24h rhythmic individuals. See number nrhythmic/ntotal on the x-axis indicating for each genotype the number of 24h-rhythmic animals over the total population.

Summary of rhythmic analysis of individual behavior.

NvClk¹^-/- shows rhythmic genes reduction in constant darkness with altered rhythmic features.
(a) Overview of the experimental design used to generate RNA-seq data. Polyps were entrained for 72 hours before sampling at 4-hour intervals over a 24-hour period (dark arrows) in both LD and DD cycles. (b) Venn diagram comparing the total number of 24h rhythmic genes identified in WT and NvClk¹^-/- in LD and DD cycles with a p <0.01 with RAIN and JTK. (c) Average acrophase comparison between rhythmic genes in LD and DD in WT polyps. Mann-Whitney test, p<0.001. (d) Average acrophase comparison between rhythmic genes in LD and DD in NvClk¹^- polyps. Mann-Whitney test, p>0.05. (3) Average relative amplitude comparison between rhythmic genes in LD and DD in WT polyps. Mann-Whitney test, p<0.05. (d) Average relative amplitude comparison between rhythmic genes in LD and DD in NvClk¹^-/- polyps. Mann-Whitney test, p>0.05. (c-f) sample size (n) indicated below each boxplots.

NvClk¹^-/- shows temporal gene clusters recruitment alteration in constant darkness.
(a) Gene per cluster count comparison between LD and DD in WT polyps. Number of cluster indicated below each boxplot. Mann-Whitney, p > 0.05. (b) Gene per cluster comparison between LD and DD in NvClk¹^-/-. Sample size (n) indicated below each boxplot. Mann-Whitney, p < 0.05. (c) Representative temporal gene cluster for each condition. For both light conditions, from top to bottom cluster peaking at day, then twilight, then at night. Number of gene for each specific cluster indicated on the x-axis.

NvClk¹^-/- alters temporal pacemaker genes expression.
(a) Four pacemaker gene are individually plotted showing the read count over 24h in LD and DD in WT (black) and NvClk¹^-/- (red). The continuous line represents significant rhythmicity (RAIN&JTK p<0.01) while the dashed line indicates no rhythmicity. (b) correlation matrix of candidate pacemaker genes expression in LD for WT on the left and NvClk¹^-/- on the right. (c) schematic representation of the promoter sequences analyses 5kb upstream the putative ATG. Black boxes represent canonical E-boxes while circadian E-boxes are green. Below the logo motif we used to identify canonical and circadian Ebox. (d) Circadian / Canonical ratio (in %) per condition. Kruskal-Wallis, multiple comparison, a vs b : p<0.05.

NvClk¹^-/- disrupts cell-cycle and neuronal pathways in constant darkness.
(a) Volcano plot showing the differential expression of genes (DEG) between WT and NvClk¹^-/- in LD (Left) and DD (right). Dashed line indicate the threshold used to detect DEG (p.adj<0.01). Red dots indicate down regulated genes and black dots up-regulated genes in NvClk¹^-/- compare to WT polyps (b) Gene Ontology (GO) terms with with significant fold-enrichment (Bonferroni corrected p-value or p.adjusted <0.01) for the DEG analysis in DD. Down regulated genes in Red while Up regulated genes in Black.

Summary of NvClk function in the regulation of Nematostella circadian rhythmicity.

(a) In situ hybridization HCRv.3 of control probe (zebrafish transcripts) in the WT juvenile with scale bars representing 0.1mm. (b) Nucleotide and translated amino acid sequences of wild-type and NVClk¹ alleles. sgRNA target site is boxed in green, PAM site is in bold, a black arrow indicates predicted site of editing. Predicted translation termination site is boxed in black. Insertion is labeled in red. Predicted immature peptide sequences in red. (c) Normalized Movement (a.u), hourly binned over 72h, under continuous dark (Dark - Dark), Blue line represents the NvClk^1+/- heterozygote. (d) Individual locomotor amplitude comparison between the three genotype. Kruskal-wallis, multitest comparison *** is p<0.001; * is p<0.05. (e) Lomb-Scargle Periodograms for each corresponding genotypes in constant darkness. The significant period value (p<0.01) is indicated for each genotype in the top left corner. (f) Phase detection (Cosinor) and genotype comparison of 24h rhythmic individuals. See number nrhythmic/ntotal on the x-axis indicating for each genotype the number of 24h rhythmic animals over the total number analyzed. (g) Normalized Movement (a.u), hourly binned over 72h, under continuous dark (Dark - Dark) after 72h of LD 6h: 6h entrainment, Black line represent the WT and red line represents the NvClk¹^-/-. (h) Lomb-Scargle Periodograms for each corresponding genotypes in constant darkness. The significant period value (p<0.01) is indicated for each genotype in the top left corner. (i). Phase detection (Cosinor) and genotype comparison of 24h rhythmic individuals. See number nrhythmic/ntotal on the x-axis indicating for each genotype the number of 24h rhythmic animals over the total number analyzed.

Venn diagram showing overlaps of rhythmic genes in LD (WT on the left and NvClk¹^-/- on the right) condition with rhythmic genes from Leach et al., 2019 and the candidate pacemaker genes (based on protein conservation).

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