NvClkΔ/Δ 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 NvClkΔ allele has a +20nt insertion after the PAS1 domain, represented by a black arrowhead. NvCLK dimerizes via its three functional domains 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 NvClkΔ/Δ 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 the number rhythmic/total on the x-axis indicating the number of 24h-rhythmic animals over the total population for each genotype.

Summary of rhythmic analysis of individual behavior.

NvClkΔ/Δ shows rhythmic gene 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 NvClkΔ/Δ 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 NvClkΔ/Δ polyps. Mann-Whitney test, p>0.05. (e) 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 NvClkΔ/Δ polyps. Mann-Whitney test, p>0.05. (c-f) sample size (n) indicated below each boxplot.

NvClkΔ/Δ alters temporal pacemaker gene expression.

(a) Four pacemaker genes are plotted, showing the read counts over 24h in LD and DD in WT (black) and NvClkΔ/Δ (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 NvClkΔ/Δ on the right. (c) schematic representation of the promoter sequences analyses 5kb upstream of the putative ATG. Black boxes represent canonical E-boxes, while circadian E-boxes are green. Below is 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.

NvClkΔ/Δ disrupts cell-cycle and neuronal pathways in constant darkness.

(a) Volcano plot showing the differential expression of genes (DEG) between WT and NvClkΔ/Δ in LD (Left) and DD (right). Dashed line indicates the threshold used to detect DEG (p.adj<0.01). Red dots indicate down regulated genes and black dots up-regulated genes in NvClkΔ/Δ 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 NvClkΔ 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 +/Δ 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 NvClkΔ/Δ. (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.

(a) Gene per cluster counts comparison between LD and DD in WT polyps. The cluster number is indicated below each boxplot. Mann-Whitney, p > 0.05. (b) On the left Y-axis, each dot represents the count of genes per cluster acrophase, with the number associated indicating its number ID in supplementary Table 4. On right Y-axis, the normalized behavior over time in LD and DD conditions. Data from both Y-axis are organized over 24 hours (c) Gene per cluster counts comparison between LD and DD conditions in NvClkΔ/Δ polyps. The cluster number is indicated below each boxplot. Mann-Whitney, p < 0.05. (d) On the left Y-axis, each dot represents the count of genes per cluster acrophase, with the number associated indicating its number ID in supplementary Table 4. On right Y-axis, the normalized behavior over time in LD and DD conditions. Data from both Y-axis are organized over 24 hours.

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