A tidal clock

The molecular basis of 24 hour circadian rhythms in terrestrial organisms is well understood and represents one of the major advances in the study of gene regulation of complex characters. However, the predominant rhythms in marine species that live on the coast in the intertidal zone is 12. 4 hours, reflecting the ebb and flow of the tides which are determined by the gravitational pull of the Moon and Sun on the Earth. For decades, scientists have speculated whether tidal rhythms are also related to circadian rhythms and whether they share some or all of the underlying molecular components of the 24 hour clock. We have been studying the specked sea louse, Eurydice pulchra, which shows both circadian rhythms in pigment dispersion and clear tidal rhythms in its swimming behaviour. We have identified all the main circadian clock genes in Eurydice and we can divide them up into their function. There are the genes that encode the positive regulators CLOCK and BMAL1, and these activate the genes for the negative regulators TIM, CRY2 and PER, which then feed back in a loop to deactivate the function of the positive regulators in a 24 hour cycle. This is called the negative feedback loop and explains how rhythms in gene transcription and translation of clock gene products can generate 24 hour molecular cycles. We have discovered that tidal rhythms share the positive factors but not the negative factors of the circadian clock. Furthermore we have identified putative circadian neurons and putative tidal cells in the Eurydice brain. These are major insights into how tidal clocks work.

We have assembled a draft genome for Eurydice and it contains several additional genes whose products act to regulate the positive factors and the negative factors. We would expect to find these in the corresponding tidal and circadian cells, so we shall localise the expression of these additional clock genes in the brain to see whether they are found in tidal or circadian cells, or both, or even other neurons, using a very sensitive technique called RNAscope. We shall also knock down the expression of these genes in Eurydice and examine whether they show changes in tidal or circadian behaviour thereby associating specific clock genes with specific types of rhythmic behaviour, tidal or circadian, or both. One of the positive factors that is important for tidal rhythms is BMAL1. This protein is known as the circadian transcription factor and with its partner CLOCK, binds to genes and activates them (see above). Consequently whatever DNA sequence BMAL1 binds, is potentially a control region for a circadian or tidal gene. We shall use a technique called ChIPseq to identify the DNA sequences and corresponding genes to which BMAL1 binds by referring to our Eurydice draft genome. Some of the genes under BMAL1 control may be switched on rhythmically with a tidal period and we shall compare these genes to those we already know are activated in 12 h cycles. Any that cross-match are candidates for being tidal output genes or the tidal regulators. We imagine that the crucial tidal regulators will also physically interact with BMAL1 in the same way that the circadian regulators like PER-TIM-CRY2 interact with the positive factors to generate circadian rhythms, so we shall compare the identity of proteins that interact with BMAL1 (using two techniques called co-IP and yeast-two hybrid) and again crossmatch any interactors with the genes we know bind BMAL1 or cycle with 12 h periods. In this way we hope to generate candidate genes for the elusive tidal regulators. When we have these candidate genes, we shall study where they are expressed in the brain and also knock down their expression levels to see whether they disrupt tidal behaviour. Our strategies will converge on the important genes that generate tidal rhythms and will perhaps provide a general model as to how these lunar-related rhythms are regulated in the animal kingdom.