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

Reviewer #1 (Public Review):

In this nice study, the authors set out to investigate the role of the canonical circadian gene Clock in the rhythmic biology of the basal metazoan Nematostella vectensis, a sea anemone, which might illuminate the evolution of the Clock gene functionality. To achieve their aims the team generated a Clock knockout mutant line (Clock-/- ) by CRISPR/Cas9 gene deletion and subsequent crossing. They then compared wild-type (WT) with Clock-/- animals for locomotor activity and transcriptomic changes over time in constant darkness (DD) and under light/dark cycles to establish these phenotypes under circadian control and those driven by light cycles. In addition, they used Hybridization Chain Reaction-In situ Hybridization (HCR-ISH) to demonstrate the spatial expression of Clock and a putative circadian clocl-controlled gene Myh7 in whole-mounted juvenile anemones.

The authors demonstrate that under LD both WT and Clock-/- animals were behaviourally rhythmic but under DD the mutants lost this rhythmicity, indicating that Clock is necessary for endogenous rhythms in activity. With altered LD regimes (LD6:6) they show also that Clock is light-dependent. RNAseq comparisons of rhythmic gene expression in WT and Clock-/- animals suggest that clock KO has a profound effect on the rhythmic genome, with very little overlap in rhythmic transcripts between the two phenotypes; of the rhythmic genes in both LD and DD in WT animals (220- termed clock-controlled genes, CCGS) 85% were not rhythmic in Clock-/- animals in either light condition. In silico gene ontology (GO) analysis of CCGS reflected process associated with circadian control. Correspondingly, those genes rhythmic in KO animals under DD (here termed neoCCGs) were not rhythmic in WT, lacked upstream E-box motifs associated with circadian regulation, and did not display any GO enrichment terms. 'Core' circadian genes (as identified in previous literature) in WT and Clock-/- animals were only rhythmic under entrainment (LD) conditions whilst Clock-/- displayed altered expression profiles under LD compared to WT. Comparing CCGs with previous studies of cycling genes in Nematostellar, the authors selected a gene from 16 rhythmic transcripts. One of these, Myh7 was detectable by both RNAseq and HCR-ISH and considered a marker of the circadian clock by the authors.

The authors claim that the study reveals insights into the evolutionary origin of circadian timing; Clock is conserved across distant groups of organisms, having a function as a positive regulator of the transcriptional translational feedback loop at the heart of daily timing, but is not a central element of the core feedback loop circadian system in this basal species. Their behavioural and transcriptomic data largely support the claims that Clock is necessary for endogenous daily activity but that the putative molecular circadian system is not self-sustained under constant darkness (this was known already for WT animals)- rather it is responsive to light cycles with altered dynamics in Clock-/- specimens in some core genes under LD. In the main, I think the authors achieved their aims and the manuscript is a solid piece of important work. The Clock-/- animal is a useful resource for examining time-keeping in a basal metazoan.

The work described builds on other transcriptomic-based works on cnidaria, including Nematostellar, and does probe into the molecular underpinnings with a loss-of-function in a gene known to be core in other circadian systems. The field of chronobiology will benefit from the evolutionary aspect of this work and the fact that it highlights the necessity to study a range of non-model species to get a fuller picture of timing systems to better appreciate the development and diversity of clocks.

Strengths:
The generation of a line of Clock mutant Nematostellar is a very useful tool for the chronobiological community and coupled with a growing suite of tools in this species will be an asset. The experiments seem mostly well conceived and executed (NB see 'weaknesses'). The problem tackled is an interesting one and should be an important contribution to the field.

Weaknesses:
I think the claims about shedding light on the evolutionary origin of circadian time maintenance are a little bold. I agree that the data do point to an alternative role for Clock in this animal in light responsiveness, but this doesn't illuminate the evolution of time-keeping more broadly in my view. In addition, these are transcriptomic data and so should be caveated- they only demonstrate the expression of genes and not physiology beyond that. The time-course analysis is weakened by its low resolution, particularly for the RAIN algorithm when 4-hour intervals constrain the analysis. I accept that only 24h rhythms were selected in the analysis from this but, it might be that detail was lost - I think a preferred option would be 2 or 3-hour resolution or 2 full 24h cycles of analysis.

The authors discount the possibility of the observed 12h rhythmicity in Clock-/- animals by exposing them to LD6:6 cycles before free-running them in DD. I suggest that LD cycles are not a particularly robust way to entrain tidal animals as far as we know. Recent papers show inundation/mechanical agitation are more reliable cues (Kwiatkowski ER, et al. Curr Biol. 2023, 2;33(10):1867-1882.e5. doi: 10.1016/j.cub.2023.03.015; Zhang L., et al Curr Biol. 2013, 23;19, 1863-1873 doi.org/10.1016/j.cub.2013.08.038.) and might be more effective in revealing endogenous 12h rhythms in the absence of 24h cues.

Reviewer #2 (Public Review):

This manuscript addresses an important question: what is the role of the gene Clock in the control of circadian rhythms in a very primitive group of animals: Cnidaria. Clock has been found to be essential for circadian rhythms in several animals, but its function outside of Bilaterian animals is unknown. The authors successfully generated a severe loss-of-function mutant in Nematostella. This is an important achievement that should help in understanding the early evolution of circadian clocks. Unfortunately, this study currently suffers from several important weaknesses. In particular, the authors do not present their work in a clear fashion, neither for a general audience nor for more expert readers, and there is a lack of attention to detail. There are also important methodological issues that weaken the study, and I have questions about the robustness of the data and their analysis. I am hoping that the authors will be able to address my concerns, as this work should prove important for the chronobiology field and beyond. I have highlighted below the most important issues, but the manuscript needs editing throughout to be accessible to a broad audience, and referencing could be improved.

Major issues:

1. Why do the authors make the claim in the abstract that CLOCK function is conserved with other animals when their data suggest that it is not essential for circadian rhythms? dCLK is strictly required in Drosophila for circadian rhythms. In mammals, there are two paralogs, CLOCK and NPAS2, but without them, there are no circadian rhythms either. Note also that the recent claim of BMAL1-independent rhythms in mammals by Ray et al., quoted in the discussion to support the idea that rhythms can be observed in the absence of the positive elements of the circadian core clock, had to be corrected substantially, and its main conclusions have been disputed by both Abruzzi et al. and Ness-Cohn et al. This should be mentioned.

2. The discussion of CIPC on line 222 is hard to follow as well. How does mRNA rhythm inform the function of CIPC, and why would it function as a "dampening factor"? Given that it is "the only core clock member included in the Clock-dependent CCGs," (220) more discussion seems warranted. Discussing work done on this protein in mammals and flies might provide more insight.

3. The behavioral arrhythmicity seen with their Clock mutation is really interesting. However, what is shown is only an averaged behavior trace and a single periodogram for the entire population. This leaves open the possibility that individual animals are poorly synchronized with each other, rather than arrhythmic. I also note that in DD there seem to be some residual rhythms, though they do not reach significance. Thus, it is also possible that at least some individual animals retain weak rhythms. The authors should analyze behavioral rhythms in individual animals to determine whether behavioral rhythmicity is really lost. This is important for the solidity of their main conclusions.

4. There is no mention in the results section of the behavior of heterozygotes. Based on supplement figure 2A, there is a clear reduction in amplitude in the heterozygous animals. Perhaps this might be because there is only half a dose of Clock, but perhaps this could be because of a dominant-negative activity of the truncated protein. There is no direct functional evidence to support the claim that the mutant allele is nonfunctional, so it is important to discuss carefully studies in other species that would support this claim, and the heterozygous behavior since it raises the possibility that the mutant allele acts as a dominant negative.

5. I do not understand what the bar graphs in Figure 2E and 3B represent - what does the y-axis label refer to?

6a. I note that RAIN was used, with a p<0.05 cut-off. I believe RAIN is quite generous in calling genes rhythmic, and the p-value cut-off is also quite high. What happens if the stringency is increased, for example with a p<0.01.
b. It would be worth choosing a few genes called rhythmic in different conditions (mutant or wild-type. LD or DD), and using qPCR to validate the RNAseq results. For example, in Figure 3D, Myh7 RNAseq data are shown, and they do not look convincing. I am surprised this would be called a circadian rhythm. In wild-type, the curve seems arrhythmic to me, with three peaks, and a rather large difference between the first and second ZT0 time point. In the Clock mutants, rhythms seem to have a 12hr period, so they should not be called rhythmic according to the material and methods, which says that only ca 24hr period mRNA rhythms were considered rhythmic. Also, the result section does not say anything about Myh7 rhythms. What do they tell us? Why were they presented at all?

1. The authors should explain better why only the genes that are both rhythmic in LD and DD are considered to be clock-controlled genes (CCGs). In theory, any gene rhythmic in DD could be a CCG. However, Leach and Reitzel actually found that most genes in DD1 do not cycle the next day (DD2)? This suggests that most "rhythmic" genes might show a transient change in expression due to prolonged obscurity and/or the stress induced by the absence of a light-dark cycle, rather than being clock controlled. Is this why the authors saw genes rhythmic under both LD and DD as actual CCGs? I would suggest verifying that in DD the phase of the oscillation for each CCG is similar to that in LD. If a gene is just responding to obscurity, it might show an elevated expression at the end of the dark period of LD, and then a high level in the first hours of DD. Such an expression pattern would be very unlikely to be controlled by the circadian clock.

2. Since there are still rhythms in LD in Clock mutants, I wonder whether there is a paralog that could be taking Clock's place, similar to NPAS2 in mammals.

3. I do not follow the point the authors try to make in lines 268-272. The absence of anticipatory behavior in Drosophila Clk mutants results from disruption of the circadian molecular clock, due to the loss of Clk's circadian function. Which light-dependent function of Clock are the authors referring to, then? Also, following this, it should be kept in mind that clock mutant mice have a weakened oscillator. The effect on entrainment is secondary to the weakening of the oscillator, rather than a direct effect on the light input pathway (weaker oscillators have increased response to environmental inputs). The authors thus need to more clearly explain why they think there is a conservation of circadian and photic clock function.