Molecular genetics of biological rhythms in an intertidal custacean

昼夜节律是24小时的行为和生理周期，是对30亿年来无情的昼夜循环的反应。值得注意的是，控制果蝇生物钟的相同基因，如周期、时钟、双重时间，也对哺乳动物的生物钟进行编码。然而，对于生活在海边的动物来说，最重要的环境节律不是昼夜节律，而是每12.4小时涨落潮的环境节律。居住在这一潮间带的动物调整自己的行为，以适应来潮和退潮的机械搅动。在退潮时，它们通过在沙子里挖洞来躲避捕食者，而在涨潮时，例如螃蟹，会游泳和觅食。陆地动物是从海洋生物进化而来的，因此潮汐节律甚至可能早于昼夜24小时的周期。人们对潮汐节律的分子基础一无所知。一种想法是，决定昼夜节律的相同基因也可能参与潮汐周期。由于许多生物钟基因产物(mRNAs和蛋白质)在动物大脑中以24小时为周期循环，我们可能会认为，如果同样的基因控制潮汐周期，它们的mRNA在大脑中控制潮汐节律的部分也应该表现出12.4小时的表达周期。或者，一组完全不同的基因可能正在决定潮汐周期，但我们仍然预计其中一些基因在其mRNA水平上有12.4小时的表达周期。考虑到这一点，我们研究了潮间带甲壳动物Eurydice Pulchra(海虱)的分子基础。Eurydice显示了非常强大的昼夜和潮汐行为周期。我们已经鉴定了几乎所有Eurydice的生物钟基因，这些基因有望在24小时周期的产生中发挥主要作用，事实上，我们在一些表达PER蛋白的特定Eurydice神经元中发现了节律性表达。在我们知道行为的潮汐周期被表达的条件下，我们发现另外两个神经元开始表达PER。这可能是两组表达PER的神经元都是有节律性的24小时周期，但相互作用给出了~12小时的潮汐周期。为了了解哪些基因以12.4h的mRNA节律循环，而不需要事先猜测它们的身份，我们开发了一种Eurydice微阵列，在玻璃玻片上已经发现了对应于数千个Eurydice基因的序列。通过用不同时间从Eurydice脑中收集的mRNA与微阵列上相应的DNA序列杂交，我们获得了大约80个候选潮汐基因，表现出~12h的表达周期。这项拨款提案寻求使用潮汐基因(启动子)中负责其循环的部分，并分离控制这种循环的蛋白质。然后，这些蛋白质将对它们自己的基因和启动子进行分析，这样我们就可以倒退到潮汐时钟。我们还将利用我们从Eurydice获得的典型的生物钟基因，并使用标记其蛋白的抗体，来观察表达时钟基因的神经元是否可能形成一个网络，从中出现~12小时的潮汐节律。我们还将尝试敲除单个尤里迪斯的这些时钟基因，如果这扰乱了它们的潮汐行为，这将意味着潮汐时钟可能是由昼夜振荡产生的。最后，我们将用从动物身上提取的RNA来挑战微阵列，这些动物已经强烈地暴露在主要的环境刺激下，这些刺激包括昼夜节律和潮汐行为，即光和振动。这将使我们能够识别光和振动反应基因。振动响应基因将在帮助我们找到进入潮汐时钟的解剖学输入路径方面特别有用。

Circadian rhythms are 24 h cycles of behaviour and physiology that evolved in response to three billion years of relentless cycles of day and night. Remarkably, the same genes that run the circadian clock in the fruitfly, such as period, Clock, doubletime, also encode the clock in mammals. However, the most important environmental rhythms for animals that inhabit the seashore are not those of day and night, but those of high and low tide that ebb and flow every 12.4 h. Animals that inhabit this intertidal zone adjust their behaviour to the mechanical agitation of the incoming and outgoing tide. At low tide, they hide from predators by burrowing into the sand, and at high tide, crabs, for example, swim and forage for food. Terrestrial animals evolved from marine organisms so tidal rhythms may even have predated the circadian 24 h cycle. Nothing is known about the molecular basis for tidal rhythms. One idea is that the same genes that determine circadian rhythmicity may also be involved in tidal cycles. As many of the circadian clock gene products (mRNAs and proteins) cycle in the animals' brains with a 24 h period, we might expect that if the same genes controlled tidal cycles, their mRNA should also show 12.4 h cycles of expression in the part of the brain which controls tidal rhythmicity. Alternatively, a completely different set of genes might be determining tidal cycles, but we would nevertheless expect some of these to have12.4 h cycles of expression at the level of their mRNA. With this in mind, we have studied the molecular basis of the intertidal crustacean, Eurydice pulchra (the 'sea louse'). Eurydice shows very robust circadian and tidal cycles of behaviour. We have already identified almost all of Eurydice's circadian clock genes that would be expected to play the major role in generating 24 h cycles, and indeed we find rhythmic expression in some specific Eurydice neurons that express the PER protein. Under conditions in which we know that tidal cycles of behaviour are expressed, we find that two other neurons begin to express PER. It could be that the two groups of PER expressing neurons are both rhythmic with 24 h periods, but interact to give the ~12 hour tidal cycle. In order to see which genes cycle with 12.4 h mRNA rhythms, without any prior guesswork as to their identity, we have developed a Eurydice microarray, a glass slide on which the sequences corresponding to many thousands of Eurydice genes, have been spotted. By interrogating this microarray with mRNA collected from Eurydice brains at different times, which will hybridise to their corresponding DNA sequences on the microarray, we have obtained about 80 candidate tidal genes that show ~12 h cycles of expression. This grant proposal seeks to use those parts of tidal genes (promoters), that are responsible for their cycling and isolate the proteins that control this cycling. These proteins will then have their own genes and promoters analysed, and in this way we will work backwards into the tidal clock. We shall also use the canonical circadian clock genes from Eurydice that we have identified and use antibodies which label their proteins, to see whether groups of clock gene expressing neurons might form a network from which a ~12 h tidal rhythm could emerge. We shall also attempt to knock out these clock genes in individual Eurydice, and if this disrupts their tidal behaviour, it will mean that the tidal clock is probably generated by circadian oscillations. Finally we shall challenge the microarray with RNA taken from animals that have been acutely exposed to the major environmental stimuli that entrain circadian and tidal behaviour, namely light and vibration. This should allow us to identify light and vibration responsive genes. The vibration responsive genes would be particularly useful in helping us find the anatomical input pathways into the tidal clock.