Functional and genomic studies of tidal rhythmicity

在过去的40年里，关于从蓝藻到哺乳动物的各种模式生物是如何产生昼夜24小时节律的，已经积累了大量的知识。高等生物的生物钟由一组正调节因子CLK和BMAL1和负调节因子PER、TIM、CRY2组成。正向调节器激活负向调节器，负向调节器以24小时为周期反馈并抑制正向调节器。这种负反馈循环在细胞内产生24小时的分子循环。对生物钟的分子解剖是现代生物学的胜利之一。然而，反映潮汐涨落的潮汐12.4小时行为节律在许多潮间动物的运动行为中被完全忽略了。在过去的几年里，我们一直在开发一种小型甲壳类动物，等足目Eurydice Pulchra(海洋林虱)作为潮汐模型。这些动物表现出强劲的游泳潮汐周期以及其他昼夜节律。我们鉴定了Eurydice中的大多数昼夜节律基因，并发现通过干扰负调控因子之一PER，昼夜24节律被扰乱，但12.4小时的潮汐行为节律完全没有受到影响。然而，当我们从药物上扰乱了正性调节因子CLK时，潮汐周期和昼夜周期都被扰乱了。因此，正调节因子似乎同时控制潮汐和昼夜节律，而负调节因子仅控制昼夜节律行为。我们检查了大脑，发现四组细胞表达负调节因子PER和正调节因子CLK和BMA1的不同组合。一组细胞表达所有三种成分(背侧细胞)，而另一组细胞仅表达CLK和BMAL1(背侧细胞)。如果背侧细胞决定昼夜节律，而背外侧细胞控制潮汐周期，这将解释我们的结果，因为CLK干扰影响这两种节律，而PER干扰只影响昼夜振荡。这个模型如此简单，以至于以前从来没有人想到过它。我们将开发能够识别所有这些不同蛋白质的试剂--到目前为止，我们只有针对PER、CLK和BMAL1的良好抗体，后两种抗体来自果蝇。新的试剂将使我们能够准确地分辨哪些神经元表达哪些组合的时钟蛋白，并阐明我们的模型。然后，我们将破坏编码CRY2、TIM、BMAL1和CLK的基因，并检查昼夜节律和潮汐表型，再次看看我们的模型是否有效。我们的模型需要新的负潮汐调节器，其中至少一个必须以12.4周期循环来负调节CLK和BMAL1。对于我们在Eurydice中所知道的生物钟，Tim提供了24小时的循环分量。我们将如何找到这些难以捉摸的负面潮汐调节器？如果没有完整的注释基因组序列，我们无法在合理的时间内找到答案，事实上，基因组的缺乏已经在很大程度上阻碍了我们的努力。因此，我们将使用下一代测序方法来生成Eurydice的完整基因组，根据这些基因组，我们可以定位和识别其mRNAs周期为12.4小时的所有基因。从生物信息学分析中，我们将找到可能的潮汐调节剂，并产生试剂，告诉我们它们的蛋白是否在相关的时钟神经元中表达。我们还将干扰相应的基因，以观察潮汐行为是否受到影响。此外，我们将通过筛选与Eurydice正调控因子EPCLK-BEpMAL1相关的分子来专注于潮汐负调控因子。从进化的角度来看，潮汐节律有可能在动物成为陆地动物之前进化，因此我们感觉我们即将回答生物学中更大的问题之一。

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.