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

The circadian clock enables anticipation of the day/night cycle in animals ranging from cnidarians to mammals. Circadian rhythms are generated through a transcription-translation feedback loop (TTFL or pacemaker) with CLOCK as a conserved positive factor in animals. However, CLOCK’s functional evolutionary origin and mechanism of action in basal animals are unknown. In the cnidarian Nematostella vectensis, pacemaker gene transcript levels, including NvClk (the Clock ortholog), appear arrhythmic under constant darkness, questioning the role of NvCLK. Utilizing CRISPR/Cas9, we generated a NvClk allele mutant (NvClkΔ), revealing circadian behavior loss under constant dark (DD) or light (LL), while maintaining a 24 hr rhythm under light-dark condition (LD). Transcriptomics analysis revealed distinct rhythmic genes in wild-type (WT) polypsunder LD compared to DD conditions. In LD, NvClkΔ/Δ polyps exhibited comparable numbers of rhythmic genes, but were reduced in DD. Furthermore, under LD, the NvClkΔ/Δ polyps showed alterations in temporal pacemaker gene expression, impacting their potential interactions. Additionally, differential expression of non-rhythmic genes associated with cell division and neuronal differentiation was observed. These findings revealed that a light-responsive pathway can partially compensate for circadian clock disruption, and that the Clock gene has evolved in cnidarians to synchronize rhythmic physiology and behavior with the diel rhythm of the earth’s biosphere.


Introduction
Throughout the history of life on Earth, organisms have had to adapt to a constantly changing environment, including the ~24-hour daily rhythm of light/dark, driving the development of endogenous biological clocks.The circadian clock, entrained by external stimuli such as light, enables the organism to anticipate the onset of the light and dark phases and synchronize its physiology and behavior in harmony with the environment.This, in turn, enhances the organism's fitness and survival [1][2][3] .From single-celled organisms to metazoans, circadian clocks have evolved multiple times, highlighting their importance to living organisms 1,2 .Despite the fundamental role circadian clocks play in regulating the rhythmicity of living organisms, their evolutionary origin and intricate molecular mechanisms remain ambiguous in early diverging animal lineages, such as cnidaria.
Rhythmic phenomena, including calcification, reproduction, and diel behavior patterns, have been examined in cnidarian species [3][4][5][6][7][8] .While environmental stimuli, such as light, directly triggered some of these patterns, others persist in the absence of external cues, suggesting the presence of an internally generated and self-sustaining circadian clock 6,9,10 .At the molecular level, cnidarians possess homologs of putative core pacemaker genes found in bilaterians [11][12][13] .Several studies have shown that most of these genes display diel expression patterns under light/dark cycles.However, unlike most animals, their oscillation generally ceases without light cues [14][15][16] .Thus, how the core pacemaker genes orchestrate rhythmic gene expression and circadian behaviors in cnidarians remains unclear.
One of the most studied cnidarian species in the field of chronobiology is the estuarine sea anemone, Nematostella vectensis.Few studies have shown that in diel lighting, the locomotor behavior of Nematostella has a ~24-h rhythm that is maintained under constant conditions, suggesting it is regulated by an endogenous circadian clock 6,17- 19 .By this, the Nematostella genome codes for conserved core pacemaker genes such as NvClk, NvCycle, and the cryptochromes NvCry1a and NvCry1b 12,13 .The proposed circadian clock model in Nematostella is composed of the positive transcription factors (bHLH-PAS family), NvCLK and NvCYCLE, that heterodimerize and upregulate lightdependent cryptochrome genes in the feedback loop, and NvPAR-bZIPs in the feedforward loop, which repress the transcription of the positive elements 11,12,19 .More recently, the NvCLK-interacting pacemaker, NvCIPC, was predicted to act as an additional repressor of the NvCLK:NvCYCLE dimer 19 .However, in contrast to the freerunning oscillation demonstrated for Nematostella behavior 6,17,19 , transcriptional expression profiles of most candidate genes implicated in the pacemaker do not retain their oscillation period without light 14,15,19 .
We employed the CRISPR/Cas9-mediated genome editing system to establish a NvClk mutant (NvClk  ) Nematostella.By combining behavioral monitoring and transcriptomic analysis, we aimed to elucidate the role of NvClk in regulating rhythmic locomotor activity and gene expression under varying light conditions.Our study revealed a robust light response pathway capable of compensation and a conserved function of CLOCK as a timekeeper without a light cue.

Phylogenetic analysis and spatial expression pattern of NvClk
Phylogenetic analysis of NvCLK protein sequences positioned NvClk within the cnidarian branch (Fig. 1a).It contains a basic helix-loop-helix (bHLH) DNA binding domain and two Per-Arnt-Sim (PAS) domains, similar to the protein structure found in other animals.PAS domains are crucial structural motifs in protein-protein interactions that drive the self-sustaining molecular mechanism underlying the circadian clock 20,21 .
In situ hybridization chain reaction (HCRv.3)was performed to localize NvClk expression at the polyp stage.Polyps were sampled at ZT10, i.e., peak expression of NvClk 6,12,22 .NvClk mRNA expression was observed throughout the animal tissue, and enriched expression was visible in the tentacle endodermis and mesenteries.In contrast, no signal was observed in the negative control (Fig. 1b, Extended Data Fig. 1a).This expression pattern resembled the expression observed at the larvae stage 22 .To date, functional manipulation of the NvClk gene has not been performed in basal animal lineages including, cnidarians, and its function is unknown in cnidarians 20,21 (Fig. 1a).

Generation of NvClk    Nematostella
To investigate the function of NvClk in Nematostella, we employed the CRISPR-Cas9 system to generate mutants.Based on existing knowledge from mouse and Drosophila models, we hypothesized that NvCLK:NvCYCLE dimer binds to the DNA motif CACGTG within the promoter of rhythmic target genes (Fig. 1c).Guide RNA (gRNA) was synthesized to target a region between the two PAS domains of the NvClk coding sequence (CDS).This gRNA and the Cas9 endonuclease were microinjected into zygotes (Methods).Subsequently, F0 animals were outcrossed with wild type (WT), and the F1 progeny were raised to adulthood.Genotyping of F1 polyps identified ten different mutated alleles, with six displaying a frame-shift mutation, including one with a 20 bp insertion (NvClk  ), resulting in a premature stop codon (Extended Data Fig. 1b).The predicted 203 amino acid truncated protein lacked 459 amino acids, including one co-factor dimerization PAS domain (Fig. 1c, Extended Data Fig. 1b).To obtain homozygous NvClk  polyps, we crossed heterozygous NvClk  F1 animals.
Genotyping of F2 polyps confirmed the expected 25% frequency of NvClk  mutants.Subsequently, we intercrossed NvClk  animals to obtain F3 NvClk  polyps for use in subsequent experiments aimed at assessing the role of NvClk in regulating behavioral and genetic rhythms.
NvClk is necessary to maintain circadian behavior in constant conditions.
To assess the impact of the NvClk  mutation on circadian rhythm, we monitored the locomotor behavior of WT and NvClk  polyps under different light conditions (Supp.  1) .While we could detect a 24hour rhythm for both genotypes, the delayed acrophase and reduced number of significant rhythmic polyps in the NvClk  suggest an alteration of the underlying rhythmicity mechanism.
We then investigated locomotor behavior under continuous conditions, namely continuous dark (DD) or continuous light (LL).WT polyps exhibited a 22-hour rhythmic behavior under both constant light conditions, with 17 out of 25 WT polyps displaying a 24-hour rhythm under DD and 7 out of 25 under LL (Fig. 1g-l).In contrast, a few NvClk  polyps displayed rhythmic behavior under constant conditions (1 out of 24 in DD and 1 out of 26 in LL) (Table 1).Additionally, we observed an intermediate phenotype in the locomotor behavior of heterozygous polyps for the NvClk  allele in DD (Extended Data Fig. 1c-f).These results revealed that NvClk  polyps could not maintain circadian rhythmicity without diel light cues.
A 24-hour rhythm of NvClk  polyps under LD conditions could be attributed to either direct light response or the partial functioning of the circadian clock due to the nature of the mutation.To distinguish between these two possibilities, we monitored locomotor activity under a 6-hour light: 6-hour dark (LD 6:6) cycle after a regular diel 72-hour entrainment under 12:12 LD.While WT polyps maintained a marginally significant periodicity of 22 hours, NvClk  polyps displayed a 12-hour rhythm at the population level (Fig. 1m-o).Specifically, we identified a clear difference of 12-hour rhythmic individual polyps between WT and NvClk  groups (1 out of 25 WT polyps vs. 13 out of 26 NvClk  polyps) (Table 1).Notably, entrainment with LD 6:6 did not lead to a 12-hour rhythm in DD for both WT and NvClk  polyps (Extended Data Fig. 1g-i).
These results support the hypothesis that the 24-hour rhythm observed in the NvClk  polyps in LD condition is due to the light-response pathway and not from an endogenous oscillator.

NvClk regulates rhythmic gene expression differentially in response to light conditions.
We conducted transcriptional profiling to investigate the underlying molecular correlates of the behavioral phenotype found in NvClk  polyps.WT and NvClk  polyps were sampled seven times every 4 hours over 24 hours under LD and DD conditions (Fig. 2a).To identify rhythmic genes, we employed stringent statistical parameters, including Benjamini-Hochberg (BH.Q) for the JTK method 23 and adjusted p-value (p.adj) for the RAIN method 24 .This resulted in the identification of a minimal number of rhythmic genes.We detected only six rhythmic genes under LD conditions in WT polyps using the JTK method and 40 rhythmic genes using the RAIN method (Supp.Table 3).In DD condition, in the WT polyps, only two rhythmic genes were identified using the RAIN method (Supp.Table 3).Despite the risk of false positives, we opted not to use multiple testing but instead proposed to combine the JTK and RAIN algorithms to identify rhythmic genes, ensuring a robust approach to data analysis (p<0.01).We identified 119 rhythmic genes rhythmic under LD and 107 rhythmic genes under DD in WT polyps (Fig. 2b, Supp.Table 3).In NvClk  polyps, we detected 147 rhythmic genes under LD and only 37 under DD (Fig. 2b, Supp.Table 3).
The rhythmic genes in WT polyps displayed a delayed acrophase under DD compared to LD (17.20h vs. 12.93h, Fig. 2c).However, no differences were detected between LD and DD rhythmic genes in NvClk  polyps (Fig. 2d).Similarly, the relative amplitude (the gene amplitude divided by its baseline, rAMP) of DD rhythmic genes was higher in WT polyps compared to LD (0.61 vs. 0.43, Fig. 2e), but no rAMP difference was observed between LD and DD rhythmic genes in NvClk  polyps (Fig. 2f) .
Are rhythmic genes organized into "transcriptional time clusters"?Does the NvClk  mutation modify cluster recruitments, causing the loss of rhythmic behavior under DD conditions?We performed a clustering analysis on the rhythmic genes using the DPGP model (Dirichlet process Gaussian process mixture model).The number of genes per cluster between LD and DD conditions in WT polyps did not differ significantly (7.3 vs. 7.6, Extended Data Fig. 2a, Supp.Table 4).Interestingly, when clusters are organized by their acrophase, we observed clusters with higher numbers of genes peaking at subjective night in WT under DD conditions (Extended Data Fig. 2b, Supp.Table 4).
In NvClk  polyps, the number of genes per cluster was significantly reduced in DD compared to the LD condition (4.1 vs. 8.6, Extended Data Fig. 2c,d).We did not identify GO-term enrichment in any cluster.However, the overlap between clusters and behavior opens new directions for further functional analysis (Extended Data Fig. 3b,d and Supp.Table 4).Overall, the reduced number of rhythmic genes in NvClk  polyps under the DD condition and the reduced number of genes per cluster confirm the necessity of NvClk to recruit rhythmic genes in the DD condition and to organize them in transcriptional time clusters.

NvClk regulates the temporal expression pattern of pacemaker genes.
In line with previous findings in Nematostella 12,14 , candidate pacemaker genes showed arrhythmic expression under DD conditions (Fig. 3a, Supp.Table 3).However, the altered expression patterns observed in NvClk  compared to WT polyps in LD condition showed increased transcripts for some genes (i.e., NvClk and NvPar-bzipd).
In contrast, others (NvCipc and NvPar-bzipc) exhibited a reduction in transcript numbers (Fig. 3a, Supp.Table 3).If we hypothesize that the first two genes (NvClk and NvPar-bzipd) act as positive factors and the latter two (NvCipc and NvPar-bzipc) potentially serve as negative regulator of the former, the lack of functionality of the NvClk  allele would explain the observed difference in transcript levels between NvClk  and WT polyps.
To systematically assess the mutation's impact on all the potential pacemaker genes, we utilized a correlation matrix based on their temporal transcript number levels, offering a comprehensive overview of their temporal organization.In WT polyps under LD conditions, the clustering categorized genes into two groups: one exhibiting a daytime peaking, containing NvClk, and another peaking at night comprising NvParbzipc and NvCipc.Notably, in LD NvClk  polyps, this second cluster contained two additional genes and displayed a weakened anticorrelation with the NvClk cluster (Fig. 3b).These observations suggest that the pacemaker oscillation, generated by the interplay of positive and negative feedback loops, relies on the precise temporal organization of these potential pacemaker factors into distinct clusters.The disruption of this organization by the NvClk  allele underscores the central role of NvClk in pacemaker function.
To go further into the regulatory mechanisms downstream of the pacemaker, we examined the presence of circadian E-box motifs (CACGTG) within 5kb upstream of the predicted ATG of rhythmic genes.We calculated circadian/canonical E-box enrichment to account for the total variation in the number of canonical E-boxes (Fig. 3c).Notably, only the candidate pacemaker genes exhibited a significant enrichment in circadian E-boxes in their promoters (15.9%) compared to the WT (5.6%), NvClk  (4.8%) rhythmic genes, and non-rhythmic genes (6.8%) (Fig. 3d).

NvClk coordinates cell division and neuronal pathways in constant darkness .
In addition to the transcriptomic rhythmic analysis, we aimed to identify processes regulated by NvClk that may not necessarily exhibit rhythmicity.We conducted a differential gene expression analysis on the total transcriptome between genotypes under each light condition to achieve this.Under LD conditions, NvClk  polyps exhibited 457 down-regulated genes and 646 up-regulated genes, with no significant enrichment in GO terms observed (Fig. 4a, Supp.Table 4 and 5).However, in DD conditions, NvClk  displayed 2450 down-regulated genes and 1770 up-regulated genes (Fig. 4b, Supp.Table 4).Notably, we identified enrichment in down-regulated genes in processes related to mitosis, microtubules, and ciliary/flagellar motility.
Conversely, the up-regulated genes showed significant enrichment in processes such as the modulation of another organism's processes, axonal guidance, and sensory perception (Fig. 4b, Supp.Table 5).

Discussion
Conserved behavioral CLOCK function through animal evolution.Our study provides valuable insights into the evolution of circadian clocks by characterizing the effects of the first Clock mutation in a cnidarian, the sea anemone Nematostella vectensis.Our behavioral assays showed that NvClk is essential for maintaining rhythmic locomotor activity without an entraining light cue.Although the rhythmicity of the NvClk + heterozygote polyps was affected in DD, our results could not discriminate a dominant-negative from a total loss of function to identify the nature of this mutation (Extended Data Fig. 1g-i).Studies in various model organisms further support the importance of CLOCK in regulating circadian locomotion.For instance, both DmClk Jrk/Jrk and DmClk ar/ar mutant flies exhibit a loss of circadian locomotion in constant darkness 25,26 .Interestingly, the heterozygote for the allele DmClk Jrk , a dominantnegative mutation, had similar consequences on fly's behavior to our observation of NvClk + polyps behavior under DD conditions 26 suggesting that shortened CLOCK protein have the potential to be dominant-negative (Extended Data Fig. 1g-i).Within the vertebrate, the DnClk1a dg3/dg3 zebrafish mutant displayed a shortened period under the same conditions 27 .The dominant-negative mutant MmClock Δ5-6/Δ5-6 mice showed a loss of circadian locomotion in constant darkness, however the complete deletion of the MmClock gene did not affect the circadian behavior rhythm in constant darkness suggesting compensation by a paralog [28][29][30] .Overall, these findings support a conserved role of CLOCK in preserving circadian behavioral rhythms in the absence of light cues across the distant Nematostella, flies, zebrafish, and mice.
Moreover, the conservation of a 24-hour locomotion rhythm in LD of the NvClk  polyps with a delayed acrophase revealed a light-response pathway independent of the circadian circuit, consistent with observations in other animal models 25,26,28 (Fig. 1f).NvClk  polyps exposed to a 12:12h LD cycle exhibited a 24-hour period.In contrast, those exposed to a 6:6h LD cycle displayed a 12-hour period.Notably, nearly no WT polyps exhibited a 12-hour rhythm under this condition, suggesting that the circadian clock overrides the light-response pathway (Fig. 5a).While some of the circadian factors can directly sense the light, such as CRY proteins 31 , 29 putative NvOpsin have been identified in the genome which could be involved in the lightresponse pathway 32 .Behavioral tracking of NvClk  polyps exposed to different wavelengths could help to identify candidates for further functional studies of the lightresponse pathway.
Transcriptional rhythmicity plasticity downstream NvClk.At the transcriptomic level, previous studies in Nematostella have shown large changes in the transcriptional profile of many genes after a single day of constant darkness, including the candidate pacemaker genes that were found arrhythmic despite sustaining circadian locomotion 12,14,22 .Consistent with previous transcriptomic analysis in cnidarian 7,14,16,33 , most of the rhythmic genes identified in LD differed from those identified in DD in the WT polyps.Notably, they displayed higher mean acrophase and larger mean amplitude in DD, suggesting a differential regulation in response to light conditions, which had not been investigated in previous cnidarian studies.Furthermore, the overlap observed between our LD rhythmic genes and those identified by Leach et al. 14 underscores the robustness of pacemaker rhythmic transcription in LD conditions (Extended data Fig. 3).However, the lack of overlap for rhythmic genes downstream of the pacemaker raises intriguing questions.Differences in experimental conditions, including genetic backgrounds, light system (Neon vs. LED), salinity (12ppt vs. 15ppt), and temperature (17°C vs. 25°C), may contribute to these discrepancies.Further investigations are necessary to determine if the lack of overlap of rhythmic genes downstream of the potential pacemaker genes results from an organism's adaptation to its environment and, therefore, reflects the plasticity of the pacemaker in regulating its downstream rhythmic genes.
Our study identified 24-hour rhythmic behavior in NvClk  polyps under LD conditions, suggesting an alternative mechanism for generating molecular rhythmicity via the lightsensing pathway.However, it is crucial to note minimal overlap between the rhythmic genes identified in NvClk  and WT polyps under LD conditions.This discrepancy indicates that the light-response pathway may not fully replicate the normal pacemaker functions observed in WT polyps, highlighting the need for further investigation into the recruitment and function of these genes.Additionally, the reduced number of rhythmic genes identified in NvClk  polyps under the DD condition underscores the crucial role of NvClk in maintaining molecular rhythm without light cues.
The clustering analysis revealed that rhythmic genes can be categorized into "transcriptional time clusters" (aka synexpression clusters) 16,34 by group of seven/eight genes in average in the WT (Fig. 5b).Their existence raises a fundamental question that has yet to be answered: How is a group of genes co-regulated in time and space (cell types) by the pacemaker?Their recruitment is disrupted in the DD NvClk  polyps suggesting an essential function of NvClk in absence of light.The combination of published scAtlas 35 and multiplexed FISH techniques 36 will be essential to further investigate the biological regulation and function of these transcriptional time clusters.

NvClk temporally organizes pacemaker gene expression to drive rhythmic gene
recruitment.Our study reveals that NvClk plays a crucial role in regulating the temporal transcription of pacemaker candidate genes (Fig. 3a) .Our analysis identified two clusters of pacemaker genes: One containing NvClk and a second one containing a potential NvClk inhibitor (NvCipc) 37,38 .These two clusters suggest the organization of the potential pacemaker genes transcription into interlock feedback loops with antiphase peaks, probably at the origin of the pacemaker oscillator function 21,39,40 .The alteration of cluster composition with a weaker anticorrelation in LD NvClk  polyps might generate a desynchronization of the pacemaker factors' availability.Indeed, regulation of rhythmic transcription involved a complex protein-protein-DNA timing interaction.Furthermore, we did not identify any circadian E-boxes enrichment in rhythmic genes between conditions, except for the candidate pacemaker genes.
Altogether, this supports the function of NvClk in orchestrating the timing interaction of pacemaker factors to select downstream rhythmic genes, indicating a more complex regulatory landscape at play.However, one significant unanswered question in our study is the reason for the arrhythmic transcription of putative pacemaker genes in DD.Using whole animals for sampling material might mask oscillating gene expression signals, especially if signals are present in a small number of cells or if tissues exhibit rhythmic gene expression in different phases.Furthermore, we must acknowledge a limitation in our interpretation, which is common in chronobiology: using RNA oscillation as a proxy for protein oscillation and function.The development of tools to study the pacemaker factors at the protein level in Nematostella will leverage this limitation in the field.
NvClk regulates processes involved in cell proliferation and the neural system in the absence of light.Our study of NvClk suggests coordination of cellular processes, especially in the absence of light.Our rhythmic transcriptomic analysis results (Fig. 2 and Extended data Fig. 2) raised questions regarding indirect effects and the non-rhythmic function of NvClk.We performed a differential gene expression analysis on the total transcriptome for each light condition.Under LD conditions, while NvClk  polyps exhibited significant changes in gene expression, we could not identify any GO term enrichment (Fig. 4a, Supp.Table 5), revealing multiple altered processes we cannot yet identify.
In contrast, under DD conditions, NvClk  polyps displayed more pronounced alterations, with more DEGs and enriched GO-terms for down-regulated genes related to mitosis, microtubule organization, and ciliary/flagellar motility, while the up-regulated genes showed enrichment in processes such as the modulation of other organism's processes, axonal guidance, and sensory perception (Fig. 4b, Supp.Table 5).These results imply that NvClk has non-circadian functions dependent on light availability.This is particularly noteworthy considering the expression of core pacemaker genes, known to be arrhythmic during larvae stages, potentially involved in developmental processes 22 .
This study provides novel insights into circadian regulation in Nematostella vectensis and sheds light on the evolutionary origin of circadian time maintenance.Our findings indicate that CLOCK function is conserved from cnidaria to mammals to maintain rhythmicity without diel light cues.Furthermore, it revealed a light-response pathway able to compensate at both behavioral and molecular levels using light cues.This circadian clock mutant opens new avenues for investigating cell-type-specific mechanisms of the circadian clock that drive the molecular and phenotypical oscillations of cnidarians.By further exploring the circadian clock mechanisms in cnidarians, we can gain deeper insights into the evolutionary origins of this critical aspect of biology, enhancing our understanding of how organisms have evolved to keep track of time and adapt to their environment.

Fig. 1
Fig.1 NvClk    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 NvClk  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 NvClk  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.

Fig. 2
Fig.2 NvClk    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 NvClk  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 NvClk  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 NvClk  polyps.Mann-Whitney test, p>0.05.(c-f) sample size (n) indicated below each boxplot.

Fig. 3
Fig.3 NvClk    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 NvClk  (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 NvClk  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 Eboxes 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.

Fig. 4
Fig.4 NvClk    disrupts cell-cycle and neuronal pathways in constant darkness.(a) Volcano plot showing the differential expression of genes (DEG) between WT and NvClk  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 upregulated genes in NvClk  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.

Fig. 5
Fig.5 Summary of NvClk function in the regulation of Nematostella circadian rhythmicity.