Unimodal and bimodal behavior patterns in animals housed in 12 hr light: 12 hr dark (LD).

<p>24-hr profile of locomotor behavior in individual <i>N. vectensis</i> showing A) unimodal or B) bimodal patterns of activity. Dark bars indicate lights off and white bars indicate lights on. The red line indicates a calculated moving average of 10 bins. C) 24 hr profile of locomotor behavior averaged from 18 animals (group data) showing unimodal pattern of activity. D, E) FFT analysis of the same individuals showing a strong frequency component at approximately 24 hours. E) Example of FFT analysis from an individual showing a prominent bimodal pattern of activity with a higher frequency peak at approximately 11 hours. F) FFT analysis of group data showing a strong frequency component at approximately 24 hours. All FFT analyses were conducted on raw data over the 4 days in LD.</p

Characterization of Circadian Behavior in the Starlet Sea Anemone, <em>Nematostella vectensis</em>

<div><h3>Background</h3><p>Although much is known about how circadian systems control daily cycles in the physiology and behavior of <em>Drosophila</em> and several vertebrate models, marine invertebrates have often been overlooked in circadian rhythms research. This study focuses on the starlet sea anemone, <em>Nematostella vectensis</em>, a species that has received increasing attention within the scientific community for its potential as a model research organism. The recently sequenced genome of <em>N. vectensis</em> makes it an especially attractive model for exploring the molecular evolution of circadian behavior. Critical behavioral data needed to correlate gene expression patterns to specific behaviors are currently lacking in <em>N. vectensis</em>.</p> <h3>Methodology/Principal Findings</h3><p>To detect the presence of behavioral oscillations in <em>N. vectensis</em>, locomotor activity was evaluated using an automated system in an environmentally controlled chamber. Animals exposed to a 24 hr photoperiod (12 hr light: 12 hr dark) exhibited locomotor behavior that was both rhythmic and predominantly nocturnal. The activity peak occurred in the early half of the night with a 2-fold increase in locomotion. Upon transfer to constant lighting conditions (constant light or constant dark), an approximately 24 hr rhythm persisted in most animals, suggesting that the rhythm is controlled by an endogenous circadian mechanism. Fourier analysis revealed the presence of multiple peaks in some animals suggesting additional rhythmic components could be present. In particular, an approximately 12 hr oscillation was often observed. The nocturnal increase in generalized locomotion corresponded to a 24 hr oscillation in animal elongation.</p> <h3>Conclusions/Significance</h3><p>These data confirm the presence of a light-entrainable circadian clock in <em>Nematostella vectensis</em>. Additional components observed in some individuals indicate that an endogenous clock of approximately 12 hr frequency may also be present. By describing rhythmic locomotor behavior in <em>N. vectensis</em>, we have made important progress in developing the sea anemone as a model organism for circadian rhythm research.</p> </div

Locomotor periodicity persists in <i>N. vectensis</i> exposed to constant light.

<p>A) Actogram and B) FFT analysis of a single <i>N. vectensis</i> over the course of a 10-day experiment. Entrained animals were monitored in LD for 2 days prior to transfer to LL for a subsequent 8 days as indicated on the actogram. Locomotor activity is double plotted and a red line approximates average of activity onsets over the 8 days in LL. B) FFT analysis of this animal indicating a circadian period of 22 hours. C) FFT analysis was conducted on averaged raw activity data from all 9 animals and shows a prominent rhythm frequency approximately 22.5 hrs.</p

Locomotor profile shifts in response to an 8 hr advance in photoperiod.

<p>A) Actogram of averaged locomotor activity of <i>N. vectensis</i> (N = 27) over the course of the experiment. Shaded area indicates time of lights off. B) Locomotor activity profile of animals housed for 3 days in the original LD photoperiod (top) and after 7 days in an 8-hour advanced photoperiod (bottom). Seven days following the 8-hour advance in photoperiod, animals show clear entrainment to the new photoperiod. Animals exhibit an anticipatory increase in activity prior to lights off and a shift of activity to occur during the scotophase of the new LD cycle. Shaded bar represents time of lights off.</p

Average body length of <i>N. vectensis</i> increases in darkness.

<p>A) Average body length of 9 <i>N. vectensis</i> over a 24 hr period. Animals were housed in a 12 hr: 12 hr LD photoperiod (shaded area indicates period of lights off). Body length was significantly greater during early night as compared to time points during the day (*Newman-Keuls; p<0.05). Bars represent standard error. B) Images of an individual animal housed in a petri dish taken at various time points throughout the day. Animals remain constricted during the day and increase in length significantly during early night to an elongated, more active state. Oral and aboral ends of the animal are indicated by arrows. While peak length occurs just after lights off, animals begin to anticipate the onset of darkness by initiating elongation several hours earlier. This anticipatory behavior is characteristic of endogenous clock control.</p

Locomotor periodicity persists in individual <i>N. vectensis</i> exposed to constant darkness.

<p>A and C) Locomotor activity of individual animals over the course of 4 days. On day 1, animals were maintained on a 12 hr: 12 hr LD photoperiod and released to constant darkness for days 2–4 of the experiment. B and D) FFT analysis was conducted on raw activity data during the DD days for each individual. B) An example of an animal with a prominent circadian component showing a period of approximately 24 hours. D) An example of an animal with a primary frequency component of approximately 12 hours. This component is not seen on Day 1 when the animal is in LD (only a nocturnal peak is evident), but emerges as the animal is transferred to constant darkness.</p

Differential stability of beta-catenin along the animal-vegetal axis of the sea urchin embryo mediated by dishevelled.

beta-Catenin has a central role in the early axial patterning of metazoan embryos. In the sea urchin, beta-catenin accumulates in the nuclei of vegetal blastomeres and controls endomesoderm specification. Here, we use in-vivo measurements of the half-life of fluorescently tagged beta-catenin in specific blastomeres to demonstrate a gradient in beta-catenin stability along the animal-vegetal axis during early cleavage. This gradient is dependent on GSK3beta-mediated phosphorylation of beta-catenin. Calculations show that the difference in beta-catenin half-life at the animal and vegetal poles of the early embryo is sufficient to produce a difference of more than 100-fold in levels of the protein in less than 2 hours. We show that dishevelled (Dsh), a key signaling protein, is required for the stabilization of beta-catenin in vegetal cells and provide evidence that Dsh undergoes a local activation in the vegetal region of the embryo. Finally, we report that GFP-tagged Dsh is targeted specifically to the vegetal cortex of the fertilized egg. During cleavage, Dsh-GFP is partitioned predominantly into vegetal blastomeres. An extensive mutational analysis of Dsh identifies several regions of the protein that are required for vegetal cortical targeting, including a phospholipid-binding motif near the N-terminus.</p