Abstract
BackgroundCircadian 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 FindingsWe 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/SignificanceThis 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.
Highlights
Most organisms exhibit daily physiological and behavioral rhythms that are regulated by molecular circadian clocks
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
Through bioinformatic queries and phylogenetic analyses we identified a number of Nematostella vectensis orthologs to core circadian clock genes shared by vertebrates and insects
Summary
Most organisms exhibit daily physiological and behavioral rhythms that are regulated by molecular circadian clocks. In the only study of circadian-like gene expression in a cnidarian, Levy et al [20] identified two cryptochromes from the coral Acropora millepora that displayed rhythmic gene expression in response to a light:dark treatment. We tested for conserved protein-protein interactions between N. vectensis’ orthologs to Clock and Cycle and identified canonical E-box motifs in the promoters of NvClock and one cryptochrome, two genes that showed rhythmic gene expression.
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