The circadian clock gene bmal1 is necessary for co-ordinated circatidal rhythms in the marine isopod Eurydice pulchra (Leach).
The circadian clock gene bmal1 is necessary for co-ordinated circatidal rhythms in the marine isopod Eurydice pulchra (Leach).
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DOI：
10.1371/journal.pgen.1011011
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发表时间：
2023-10
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影响因子：
4.5
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陆生动物的生物钟是由分子反馈回路编码的，包括负调节因子PERIOD、TIMELESS或CYPTOCHROME 2和正转录因子CLOCK和BMAL 1/CYCLE。在沿海生物中广泛表达的昼夜（~12.4小时）或其他月球介导的周期（~15天，~29天）的分子基础尚不清楚。破坏昼夜节律时钟似乎不会影响栖息在海岸线上的几种生物的月球节律，这表明两个周期的分子独立性。然而，药理学抑制酪蛋白激酶1（CK 1），目标周期稳定的哺乳动物和苍蝇，影响昼夜节律和昼夜节律表型在Eurydice pulchra（EP），斑点海虱。在这里，我们表明，这些药物抑制剂CK 1也影响EpCLK和EpBMAL 1的磷酸化，并破坏EpCLK-BMAL 1介导的转录在果蝇S2细胞，揭示了这两个积极的昼夜节律调节和昼夜节律行为之间的潜在联系。因此，我们在取自野生动物的Eurydice中进行Epbmal 1以及主要负调节因子Epcry 2的dsRNAi敲低。Epcry 2和Epbmal 1敲低破坏了Eurydice的昼夜表型的色素细胞分散，tim mRNA循环和昼夜节律调节的昼夜游泳，正如预期的那样。然而，昼夜节律的行为是特别敏感的Epbmal 1敲低与一致的影响的功率，幅度和节奏的昼夜游泳周期。因此，除了EpPER和EpTIM（来自先前的研究）之外，三个Eurydice负昼夜节律调节剂EpBMAL 2似乎不需要表达稳健的昼夜节律行为，与正调节剂EpBMAL 1相反。我们提出了一个神经发生模型，即积极的昼夜节律调节EpBMAL 1-CLK之间共享的昼夜节律和昼夜节律的机制在Eurydice，但昼夜节律需要一个新的，迄今未知的负调节。分子反馈环是陆生动物昼夜~24小时行为和生理节律表达的基础，其中PERIOD、TIMELESS和CYPTOCHROME 2代表负调控因子，CLOCK和BMAL 1/CYC代表正调控因子，酪蛋白激酶1（CK 1）则充当调节因子。在沿海海洋动物中，月球介导的~12.4小时昼夜节律代表了主要的生物周期，但其分子基础尚不清楚。现有的证据表明，昼夜节律和昼夜节律的机制是分子独立的，但在斑点海虱，Eurydice pulchra（EP），药理学操纵CK 1产生相关的变化，这两种类型的周期，这表明一个共享的组件。在这里，使用果蝇细胞系统，我们表明，CK 1抑制剂改变EpCLOCK-BMAL 1的翻译后修饰，导致其转录能力的降低，并表明这些功能的变化EpCLOCK-BMAL 1可能产生的CK 1通道介导的昼夜节律表型。为了直接测试这一点，我们使用dsRNAi敲低从野生捕获的成年Eurydice中EpBMAL 1的表达。我们还下调了主要的负调节因子Epsilon 2。我们观察到一致的干扰昼夜节律EpBMA 11，但没有EpBMA 12敲低，这表明积极的昼夜节律调节参与昼夜节律和月球介导的行为。
Circadian clocks in terrestrial animals are encoded by molecular feedback loops involving the negative regulators PERIOD, TIMELESS or CRYPTOCHROME2 and positive transcription factors CLOCK and BMAL1/CYCLE. The molecular basis of circatidal (~12.4 hour) or other lunar-mediated cycles (~15 day, ~29 day), widely expressed in coastal organisms, is unknown. Disrupting circadian clockworks does not appear to affect lunar-based rhythms in several organisms that inhabit the shoreline suggesting a molecular independence of the two cycles. Nevertheless, pharmacological inhibition of casein kinase 1 (CK1) that targets PERIOD stability in mammals and flies, affects both circadian and circatidal phenotypes in Eurydice pulchra (Ep), the speckled sea-louse. Here we show that these drug inhibitors of CK1 also affect the phosphorylation of EpCLK and EpBMAL1 and disrupt EpCLK-BMAL1-mediated transcription in Drosophila S2 cells, revealing a potential link between these two positive circadian regulators and circatidal behaviour. We therefore performed dsRNAi knockdown of Epbmal1 as well as the major negative regulator in Eurydice, Epcry2 in animals taken from the wild. Epcry2 and Epbmal1 knockdown disrupted Eurydice’s circadian phenotypes of chromatophore dispersion, tim mRNA cycling and the circadian modulation of circatidal swimming, as expected. However, circatidal behaviour was particularly sensitive to Epbmal1 knockdown with consistent effects on the power, amplitude and rhythmicity of the circatidal swimming cycle. Thus, three Eurydice negative circadian regulators, EpCRY2, in addition to EpPER and EpTIM (from a previous study), do not appear to be required for the expression of robust circatidal behaviour, in contrast to the positive regulator EpBMAL1. We suggest a neurogenetic model whereby the positive circadian regulators EpBMAL1-CLK are shared between circadian and circatidal mechanisms in Eurydice but circatidal rhythms require a novel, as yet unknown negative regulator. Molecular feedback loops underlie expression of circadian ~24-hour behavioural and physiological rhythms in terrestrial animals, with PERIOD, TIMELESS and CRYPTOCHROME2 representing the negative, and CLOCK and BMAL1/CYC the positive regulators, with casein kinase 1 (CK1) acting as a modulator. In coastal marine animals, the lunar-mediated ~12.4 hour circatidal rhythm represents the dominant biological cycle but its molecular basis is unknown. The available evidence indicates that circadian and circatidal mechanisms are molecularly independent yet in the speckled-sea louse, Eurydice pulchra (Ep), pharmacological manipulation of CK1 generates correlated changes in both types of cycles, suggesting a shared component. Here, using a Drosophila cell system, we show that CK1 inhibitors alter post-translational modifications of EpCLOCK-BMAL1 leading to a reduction in their transcriptional ability and suggesting that these functional changes in EpCLOCK-BMAL1 may have produced the CK1 inhibitor-mediated circatidal phenotypes. To test this directly we used dsRNAi to knockdown the expression of EpBMAL1 in adult Eurydice captured from the wild. We also downregulated the main negative regulator EpCRY2. We observe consistent disruptions to circatidal rhythms in EpBMAl1 but not EpCRY2 knockdowns suggesting that the positive circadian regulator is involved in both circadian and lunar-mediated behaviour.