Light Entrained Rhythmic Gene Expression in the Sea Anemone Nematostella vectensis: The Evolution of the Animal Circadian Clock

Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, USA.
PLoS ONE (Impact Factor: 3.23). 09/2010; 5(9):e12805. DOI: 10.1371/journal.pone.0012805
Source: PubMed

ABSTRACT Circadian rhythms in behavior and physiology are the observable phenotypes from cycles in expression of, interactions between, and degradation of the underlying molecular components. In bilaterian animals, the core molecular components include Timeless-Timeout, photoreceptive cryptochromes, and several members of the basic-loop-helix-Per-ARNT-Sim (bHLH-PAS) family. While many of core circadian genes are conserved throughout the Bilateria, their specific roles vary among species. Here, we identify and experimentally study the rhythmic gene expression of conserved circadian clock members in a sea anemone in order to characterize this gene network in a member of the phylum Cnidaria and to infer critical components of the clockwork used in the last common ancestor of cnidarians and bilaterians.
We identified homologs of circadian regulatory genes in the sea anemone Nematostella vectensis, including a gene most similar to Timeout, three cryptochromes, and several key bHLH-PAS transcription factors. We then maintained N. vectensis either in complete darkness or in a 12 hour light: 12 hour dark cycle in three different light treatments (blue only, full spectrum, blue-depleted). Gene expression varied in response to light cycle and light treatment, with a particularly strong pattern observed for NvClock. The cryptochromes more closely related to the light-sensitive clade of cryptochromes were upregulated in light treatments that included blue wavelengths. With co-immunoprecipitation, we determined that heterodimerization between CLOCK and CYCLE is conserved within N. vectensis. Additionally, we identified E-box motifs, DNA sequences recognized by the CLOCK:CYCLE heterodimer, upstream of genes showing rhythmic expression.
This study reveals conserved molecular and functional components of the circadian clock that were in place at the divergence of the Cnidaria and Bilateria, suggesting the animal circadian clockwork is more ancient than previous data suggest. Characterizing circadian regulation in a cnidarian provides insight into the early origins of animal circadian rhythms and molecular regulation of environmentally cued behaviors.

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Available from: Lars Behrendt, Sep 27, 2015
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    • "Studies utilizing cnidarians as experimental models are therefore useful for determining the antiquity of molecular functions for shared proteins and hormones, as well as for identifying potentially novel features for homologous molecules when compared to bilaterians. Studies of several anthozoan cnidarians has revealed that most of the core circadian clock genes are conserved in cnidarians, and they are differentially expressed in diel lighting conditions [35,36], Despite these similarities, no research to date has addressed whether other mechanisms of the circadian clock may be conserved in cnidarians, particularly the role of hormones in signaling and resetting of the clock. This information is of importance in the understanding of the origin and evolution of melatonin/circadian cycles. "
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    ABSTRACT: Background The primary hormone of the vertebrate pineal gland, melatonin, has been identified broadly throughout the eukaryotes. While the role for melatonin in cyclic behavior via interactions with the circadian clock has only been reported in vertebrates, comparative research has shown that the transcription-translation loops of the animal circadian clock likely date to the cnidarian-bilaterian ancestor, leaving open significant questions about the evolutionary origin of melatonin signaling in circadian behavior by interacting with the molecular clock. Results Expression of melatonin in adult anemones showed peak expression at the end of light period (zeitgeber time (ZT) = 12) when cultured under diel conditions, coinciding with expression of genes and enzyme activity for members of the melatonin synthesis pathway (tryptophan hydroxylase and hydroxyindol-O-methyltransferase), which also showed rhythmic expression. During embryogenesis and juvenile stages, melatonin showed cyclic oscillations in concentration, peaking in midday. Spatial (in situ hybridization) and quantitative (real-time PCR) transcription of clock genes during development of N. vectensis showed these ‘clock’ genes are expressed early in the development, prior to rhythmic oscillations, suggesting functions independent of a function in the circadian clock. Finally, time-course studies revealed that animals transferred from diel conditions to constant darkness lose circadian expression for most of the clock genes within 4 days, which can be reset by melatonin supplementation. Conclusions Our results support an ancient role for melatonin in the circadian behavior of animals by showing cyclic expression of this hormone under diel conditions, light-dependent oscillations in genes in the melatonin synthesis pathway, and the function of melatonin in initiating expression of circadian clock genes in the cnidarian N. vectensis. The differences in expression melatonin and the circadian clock gene network in the adult stage when compared with developmental stages of N. vectensis suggests new research directions to characterize stage-specific mechanisms of circadian clock function in animals.
    EvoDevo 08/2014; 5(26). DOI:10.1186/2041-9139-5-26 · 3.03 Impact Factor
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    • "We have previously identified five predicted N. vectensis genes within the HSP70 family (A.M.R. and A.M.T., unpublished data). Among these, one (JGI 234533, referred to here as HSP70) is most strongly induced by exposure to either cadmium or extreme temperatures, and a second (JGI 195315, referred to here as HSC71) exhibits relatively constant expression throughout a normal 24 h period (Reitzel et al., 2010) and following exposure to cadmium or extreme temperatures (A.M.R. and A.M.T., unpublished data). Three genes were tested as potential normalizer genes in this study: elongation factor 1a (EF1), heat shock cognate protein 71 (HSC71) and the 18S ribosomal protein (18S). "
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    ABSTRACT: Organisms are continuously exposed to reactive chemicals capable of causing oxidative stress and cellular damage. Antioxidant enzymes, such as superoxide dismutases (SODs) and catalases, are present in both prokaryotes and eukaryotes and provide an important means of neutralizing such oxidants. Studies in cnidarians have previously documented the occurrence of antioxidant enzymes (transcript expression, protein expression, and/or enzymatic activity), but most of these studies have not been conducted in species with sequenced genomes or included phylogenetic analyses, making it difficult to compare results across species due to uncertainties in the relationships between genes. Through searches of the genome of the sea anemone Nematostella vectensis Stephenson, one catalase gene and six SOD family members were identified, including three copper zinc SODs (CuZnSODs), two manganese SODs (MnSODs), and one copper chaperone of SOD (CCS). In 24-hour acute toxicity tests, juvenile N. vectensis showed enhanced sensitivity to combinations of ultraviolet radiation (UV) and polycyclic aromatic hydrocarbons (PAHs, specifically pyrene, benzo[a]pyrene, and fluoranthene) relative to either stressor alone. Adult N. vectensis exhibited little or no mortality following UV, benzo[a]pyrene or crude oil exposure but exhibited changes in gene expression. Antioxidant enzyme transcripts were both up- and downregulated following UV and/or chemical exposure. Expression patterns were most strongly affected by UV exposure but varied between experiments, suggesting that responses vary according to the intensity and duration of exposure. These experiments provide a basis for comparison with other cnidarian taxa and for further studies of the oxidative stress response in N. vectensis.
    Journal of Experimental Biology 01/2014; 217(9). DOI:10.1242/jeb.093690 · 2.90 Impact Factor
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    • "While many key elements of the bilaterian circadian clock (e.g., homologs of Clock and Cycle) have not been identified in sponges, characterization of cryptochromes and nocturnin provides unique insight into the origins of the animal clock. While studies of circadian regulation in cnidarians are still at an early stage, it is now clear that several elements of the bilaterian circadian clock are present in cnidarians, particularly anthozoans (sea anemones and corals) (Levy et al. 2007; Reitzel et al. 2010; Hoadley et al. 2011). Anthozoans contain homologs of Clock and Cycle, as well as several cryptochromes. "
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    ABSTRACT: Diurnal and seasonal cues play critical and conserved roles in behavior, physiology, and reproduction in diverse animals. The circadian clock is a transcription-translation feedback loop that represents the molecular mechanism underlying many of these periodic processes, frequently through responses to light. Although much of the core regulatory machinery is deeply conserved among diverse animal lineages, there are also many examples of innovation in the way the clock either is constructed at the molecular-level or deployed in coordinating behavior and physiology. The nine articles contained within this issue address aspects of circadian signaling in diverse taxa, utilize wide-ranging approaches, and collectively provide thought-provoking discussion of future directions in circadian research.
    Integrative and Comparative Biology 05/2013; 53(1). DOI:10.1093/icb/ict062 · 2.93 Impact Factor
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