Functional and genomic studies of tidal rhythmicity

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Over the past 40 years a considerable amount of knowledge has accumulated on how circadian 24 hour rhythms are generated in a variety of model organisms, from Cyanobacteria to mammals. The circadian clock in higher organisms is composed of a set of positive regulators CLK and BMAL1 and the negative regulators, PER, TIM, CRY2. The positive regulators activate the negative regulators, and the negative regulators feed back in a 24 hour cycle and inhibit the positive regulators. This negative feedback loop generates molecular cycles of 24 hours within a cell. The molecular dissection of the circadian clock is one of the triumphs of modern biology.However, tidal 12.4 hour behavioural rhythms that reflect the ebb and flow of the tides and are observed in the locomotor behaviour of many intertidal animals have ben completely ignored molecularly. Over the past few years we have been developing a small crustacean, the isopod Eurydice pulchra (marine woodlouse) as a tidal model. These animals show robust tidal cycles of swimming as well as other circadian rhythms. We identified most of the circadian clock genes in Eurydice, and found that by disrupting one of the negative regulators, PER, circadian 24 rhythms were disrupted but the 12.4 hour tidal rhythm of behaviour was completely unaffected. However, when we pharmacologically disrupted the positive regulator CLK, both tidal and circadian cycles were disrupted. Thus the positive regulator appears to control both tidal and circadian rhythms whereas the negative regulator controls only circadian behaviour.We examined the brain and found four groups of cells that expressed different combinations of the negative regulator PER and the positive regulators CLK and BMA1. One group of cells expressed all three components (the Dorsal cells), whereas another expressed only CLK and BMAL1 (the Laterodorsal cells). If the Dorsal cells determine circadian rhythms and the Laterodorsal cells run the tidal cycle, that would explain our results, in that CLK disruption affects both rhythms but PER disruption affects only circadian oscillations. This model is so simple it's disturbing that nobody has ever thought of it before. We will develop reagents that recognise all these different proteins - so far we only have good antibodies against PER, CLK and BMAL1 and the latter two come from the fruitfly. The new reagents will allow us to resolve exactly which neurons express which combinations of clock proteins and illuminate our model. We will then disrupt the genes that encode CRY2, TIM, BMAL1 and CLK and examine the circadian and tidal phenotypes, again to see if our model works. Our model requires novel negative tidal regulators, at least one of which must cycle with a 12.4 period to negatively regulate CLK and BMAL1. For the circadian clock we know in Eurydice that TIM provides that 24 h cycling component. How will we find these elusive negative tidal regulators? We cannot find out within a reasonable time without a full annotated genome sequence and indeed the lack of a genome has hindered our efforts considerably. Consequently, we shall use next generation sequencing methods to generate the complete genome of Eurydice, against which we can map and identify all the genes whose mRNAs cycle with a 12.4 hour period. From bioinformatic analyses we shall home in on putative tidal regulators, and generate reagents that will inform us whether their proteins are expressed in the relevant clock neurons. We shall also disrupt the corresponding genes to see whether tidal behaviour is affected. In addition, we will focus in on tidal negative regulators by screening for molecules that sssociate with the Eurydice positive regulators EpCLK-BEpMAL1 In evolutionary terms, it is possible that tidal rhythms evolved before animals became terrestrial so we feel we are on the verge of answering one of the bigger questions in biology.