Molecular genetics of biological rhythms in an intertidal custacean

昼夜节律是行为和生理的24小时周期，是在30亿年无情的昼夜循环中进化而来的。值得注意的是，在果蝇中运行生物钟的基因，如周期、时钟、双倍时间，也在哺乳动物中编码生物钟。然而，对于生活在海边的动物来说，最重要的环境节律不是白天和黑夜，而是每12.4小时的涨潮和退潮。生活在潮间带的动物根据涨潮和退潮的机械搅动来调整它们的行为。在退潮时，它们通过在沙子中挖洞来躲避捕食者，而在涨潮时，螃蟹则会游泳并寻找食物。陆生动物是从海洋生物进化而来的，所以潮汐节律甚至可能早于24小时的昼夜周期。我们对潮汐节律的分子基础一无所知。一种观点认为，决定昼夜节律的基因可能也与潮汐周期有关。由于许多生物钟基因产物（mRNA和蛋白质）在动物大脑中以24小时为周期循环，我们可以预期，如果相同的基因控制潮汐周期，那么它们的mRNA在控制潮汐节律的大脑部分也应该显示12.4小时的表达周期。另外，一组完全不同的基因可能决定潮汐周期，但我们仍然预计其中一些基因在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类动物Eurydice pulchra（“海虱”）的分子基础。欧律狄刻表现出非常强劲的昼夜节律和潮汐周期行为。我们已经确定了几乎所有的Eurydice的生物钟基因，这些基因将在产生24小时周期中发挥主要作用，并且我们确实在一些特定的Eurydice神经元中发现了表达PER蛋白的节律性表达。在我们知道潮汐周期行为被表达的条件下，我们发现另外两个神经元开始表达PER。可能是两组表达PER的神经元都有节律性的24 h周期，但相互作用形成了~12 h的潮汐周期。为了了解哪些基因以12.4小时的mRNA节奏循环，而不需要事先猜测它们的身份，我们开发了一种Eurydice微阵列，一种玻璃载玻片，上面有成千上万个Eurydice基因对应的序列。通过将该芯片与不同时间从Eurydice大脑中收集的mRNA进行比对，将其与芯片上相应的DNA序列杂交，我们获得了大约80个候选潮汐基因，这些基因的表达周期为~12 h。这项拨款提案寻求使用潮汐基因的那些部分（启动子），负责它们的循环，并分离控制这种循环的蛋白质。然后，这些蛋白质将被分析它们自己的基因和启动子，通过这种方式，我们将逆向研究潮汐时钟。我们还将使用我们从欧律狄刻（Eurydice）鉴定出的标准生物钟基因，并使用标记其蛋白质的抗体，看看表达神经元的生物钟基因群是否可能形成一个网络，从这个网络中可以出现~12小时的潮汐节律。我们还将尝试在欧律狄刻个体中敲除这些时钟基因，如果这破坏了它们的潮汐行为，这将意味着潮汐时钟可能是由昼夜节律振荡产生的。最后，我们将用从动物身上提取的RNA挑战微阵列，这些动物已经严重暴露于主要的环境刺激下，这些环境刺激会引起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响应基因。振动响应基因在帮助我们找到潮汐时钟的解剖输入途径方面特别有用。

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.