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

Article (PDF Available)inPLoS ONE 5(9):e12805 · September 2010with37 Reads
DOI: 10.1371/journal.pone.0012805 · Source: PubMed
Abstract
Background: 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. Methodology/principal findings: 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. Conclusions/significance: 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.
    • "Nematostella has a primitive CYC orthologue that likely resulted from ARNT duplication (Simionato et al 2007;2010). CLK and CYC form a heterodimer and act as positive regulators (Reitzel et al. 2010). It is clear that Nematostella does not have a PER orthologue, but it does have a CRY 2 (Figure 18). "
    [Show abstract] [Hide abstract] ABSTRACT: ABSTRACT The circadian rhythm is important to all organisms and plays a key role in physiology. Although much is known about the genetic regulation of the circadian clock in Drosophila melanogaster and in Mus musculus, less is known about the circadian clock in cnidarians, let alone in the lower Bilateria. This study attempts to fill the information gap between Cnidaria and higher metazoans by characterizing the core circadian clock of Isodiametra pulchra (Acoelomorpha). These animals live in between sand grains above the sulfide zone on low wave- action beaches, exposing them to a wide array of daily environmental changes. I. pulchra circadian core-clock proteins were identified by comparing known Drosophila and Mus orthologues. Final orthology assignments were chosen based on protein domain characteristics, sequence similarities, and phylogenetic analyses. By comparing known orthologues of clock proteins found in insects and vertebrates, three full sequences and two incomplete sequences were identified. I examined the mRNA expression of two of these proteins (IPUL1_3616 and IPUL1_2961) using a semi-quantitative RT_PCR approach. I found that IPUL1_3616 varied in a circadian fashion where expression was highest one hour before lights on, but IPUL1_2961 did not vary significantly in expression. This study is the first study of the circadian clock in a basal bilaterian.
    Full-text · Thesis · Apr 2015 · EvoDevo
    • "Levy et al. [107] showed that in corals, there is a night time preference for DNA replication. Furthermore, orthologs of various photoreceptors and many core circadian genes common to mammals and Drosophila are present in the coral Acropora millepora and in the sea anemone Nematostella vectensis [25, 28, 108, 109]. The expression of several opsins (acropsins 1–3) in planulae of Acropora palmata and the demonstration that specific Acropora G proteins can be activated by acropsins in a light-dependent manner in vitro [110] indicates that functional photoreceptors can be formed that may play a role in color preference during settlement, vertical positioning, and other light-guided behaviors observed in coral larvae. "
    [Show abstract] [Hide abstract] ABSTRACT: Ecological developmental biology (eco-devo) explores the mechanistic relationships between the processes of individual development and environmental factors. Recent studies imply that some of these relationships have deep evolutionary origins, and may even pre-date the divergences of the simplest extant animals, including cnidarians and sponges. Development of these early diverging metazoans is often sensitive to environmental factors, and these interactions occur in the context of conserved signaling pathways and mechanisms of tissue homeostasis whose detailed molecular logic remain elusive. Efficient methods for transgenesis in cnidarians together with the ease of experimental manipulation in cnidarians and sponges make them ideal models for understanding causal relationships between environmental factors and developmental mechanisms. Here, we identify major questions at the interface between animal evolution and development and outline a road map for research aimed at identifying the mechanisms that link environmental factors to developmental mechanisms in early diverging metazoans.Also watch the Video Abstract.
    Full-text · Article · Dec 2014
    • "The analysis of the genes likely involved in the circadian clock in the adults showed that 4 days of constant darkness were adequate to abolish the circadian expression for virtually all of them. Our data are nearly identical to the pattern previously observed, although that study kept N. vectensis for 30 days in constant darkness [35]; however, our results show that quenching of cyclical gene expression occurs within days of removal of light cues. Data from corals have similarly shown loss of the rhythmicity of some clock genes within 24 hours (Acropora millepora [57]) or 72 hours (Favia fragum [45]). "
    [Show abstract] [Hide abstract] 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.
    Full-text · Article · Aug 2014
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