A sleep-like state in Hydra unravels conserved sleep mechanisms during the evolutionary development of the central nervous system

Sleep behaviors are observed even in nematodes and arthropods, yet little is known about how sleep-regulatory mechanisms have emerged during evolution. Here, we report a sleep-like state in the cnidarian Hydra vulgaris with a primitive nervous organization. Hydra sleep was shaped by homeostasis and necessary for cell proliferation, but it lacked free-running circadian rhythms. Instead, we detected 4-hour rhythms that might be generated by ultradian oscillators underlying Hydra sleep. Microarray analysis in sleep-deprived Hydra revealed sleep-dependent expression of 212 genes, including cGMP-dependent protein kinase 1 (PRKG1) and ornithine aminotransferase. Sleep-promoting effects of melatonin, GABA, and PRKG1 were conserved in Hydra. However, arousing dopamine unexpectedly induced Hydra sleep. Opposing effects of ornithine metabolism on sleep were also evident between Hydra and Drosophila, suggesting the evolutionary switch of their sleep-regulatory functions. Thus, sleep-relevant physiology and sleep-regulatory components may have already been acquired at molecular levels in a brain-less metazoan phylum and reprogrammed accordingly

Hydra vulgaris exhibits day-night variation in behavior and gene expression levels

Abstract Background Day–night behavioral variation is observed in most organisms, and is generally controlled by circadian clocks and/or synchronization to environmental cues. Hydra species, which are freshwater cnidarians, are thought to lack the core clock genes that form transcription–translation feedback loops in clock systems. In this study, we examined whether hydras exhibit diel rhythms in terms of behavior and gene expression levels without typical clock genes. Results We found that the total behavior of hydras was elevated during the day and decreased at night under a 12-h light–dark cycle. Polyp contraction frequency, one component of behavior, exhibited a clear diel rhythm. However, neither total behavior nor polyp contraction frequency showed rhythmic changes under constant light and constant dark conditions. To identify the genes underlying diel behavior, we performed genome-wide transcriptome analysis of hydras under light–dark cycles. Using three different analytic algorithms, we found that 380 genes showed robust diel oscillations in expression. Some of these genes shared common features with diel cycle genes of other cnidarian species with endogenous clock systems. Conclusion Hydras show diel behavioral rhythms under light–dark cycles despite the absence of canonical core clock genes. Given the functions of the genes showing diel oscillations in hydras and the similarities of those genes with the diel cycle genes of other cnidarian species with circadian clocks, it is possible that diel cycle genes play an important role across cnidarian species regardless of the presence or absence of core clock genes under light–dark cycles

C-Terminal Binding Protein (CtBP) Activates the Expression of E-Box Clock Genes with CLOCK/CYCLE in <i>Drosophila</i>

<div><p>In <i>Drosophila</i>, CLOCK/CYCLE heterodimer (CLK/CYC) is the primary activator of circadian clock genes that contain the E-box sequence in their promoter regions (hereafter referred to as “E-box clock genes”). Although extensive studies have investigated the feedback regulation of clock genes, little is known regarding other factors acting with CLK/CYC. Here we show that Drosophila C-terminal binding protein (dCtBP), a transcriptional co-factor, is involved in the regulation of the E-box clock genes. <i>In vivo</i> overexpression of dCtBP in clock cells lengthened or abolished circadian locomotor rhythm with up-regulation of a subset of the E-box clock genes, <i>period</i> (<i>per</i>), <i>vrille</i> (<i>vri</i>), and <i>PAR domain protein 1ε</i> (<i>Pdp1ε</i>). Co-expression of dCtBP with CLK <i>in vitro</i> also increased the promoter activity of <i>per</i>, <i>vri</i>, <i>Pdp1ε</i> and <i>cwo</i> depending on the amount of dCtBP expression, whereas no effect was observed without CLK. The activation of these clock genes <i>in vitro</i> was not observed when we used mutated dCtBP which carries amino acid substitutions in NAD⁺ domain. These results suggest that dCtBP generally acts as a putative co-activator of CLK/CYC through the E-box sequence.</p></div

The actograms of <i>dCtBP</i>-knockdown and -overexpressing flies.

<p>Typical locomotor activity in the control (upper left), <i>dCtBP</i>-knockdown flies (upper right), and <i>dCtBP-</i>overexpressing flies (lower panels). The number in parentheses represents the free-running period of the corresponding flies. Adult flies were entrained to a 12-h light:12-h dark cycle (LD) for 3 days, and then kept in constant darkness (DD). Horizontal bars in white and black indicate times of light and dark, respectively, in LD. Vertical bar in white: LD; vertical bar in black: DD.</p

Temporal <i>dCtBP</i> expression in control and <i>dCtBP</i>-overexpressing flies.

<p>A: Temporal expression profile of <i>dCtBP</i> (blue) and <i>Clk</i> (red) in the head of adult control flies measured by quantitative PCR assay (Q-PCR). ZT1 and ZT13 correspond to 1 h from the onset of light-on and -off conditions in LD, respectively. <i>dCtBP</i> expression reveals a circadian rhythm peaking at the end of night phase. Cross indicates significant difference with trough level of <i>Clk</i> at ZT17 (Tukey’s test, <i>P</i><0.05). Asterisks indicate a significant difference with the trough level of <i>dCtBP</i> at ZT9 (Tukey’s test, <i>P</i><0.05). RNAs were sampled three times at each point, and error bars represent S.E.M. B: The expression level of <i>dCtBP</i> at ZT1 and ZT13 in control flies (white) and <i>dCtBP-</i>overexpressing flies (black). <i>dCtBP</i> expression was higher in <i>dCtBP</i>-overexpressing flies than control flies at each phase (*: t test, <i>P<</i>0.05). RNAs were sampled three times at each point, and error bars represent S.E.M. (n  = 3).</p

Free-running periods of <i>dCtBP</i>-overexpressing and knockdown flies.

<p>N_R: Number of rhythmic flies recorded.</p><p>N_A: Number of arrhythmic flies recorded.</p>a<p>significantly different from the period of the flies carrying the <i>tim(UAS)-Gal4</i> as a control (t test, <i>P</i><0.05).</p>b<p>significantly different from the period of the flies carrying the <i>UAS</i> sequence as a control (t test, <i>P</i><0.05).</p

Mated Drosophila melanogaster females consume more amino acids during the dark phase.

To maintain homeostasis, animals must ingest appropriate quantities, determined by their internal nutritional state, of suitable nutrients. In the fruit fly Drosophila melanogaster, an amino acid deficit induces a specific appetite for amino acids and thus results in their increased consumption. Although multiple processes of physiology, metabolism, and behavior are under circadian control in many organisms, it is unclear whether the circadian clock also modulates such motivated behavior driven by an internal need. Differences in levels of amino acid consumption by flies between the light and dark phases of the day:night cycle were examined using a capillary feeder assay following amino acid deprivation. Female flies exhibited increased consumption of amino acids during the dark phase compared with the light phase. Investigation of mutants lacking a functional period gene (per0), a well-characterized clock gene in Drosophila, found no difference between the light and dark phases in amino acid consumption by per0 flies. Furthermore, increased consumption of amino acids during the dark phase was observed in mated but not in virgin females, which strongly suggested that mating is involved in the rhythmic modulation of amino acid intake. Egg production, which is induced by mating, did not affect the rhythmic change in amino acid consumption, although egg-laying behavior showed a per0-dependent change in rhythm. Elevated consumption of amino acids during the dark phase was partly induced by the action of a seminal protein, sex peptide (SP), on the sex peptide receptor (SPR) in females. Moreover, we showed that the increased consumption of amino acids during the dark phase is induced in mated females independently of their internal level of amino acids. These results suggest that a post-mating SP/SPR signal elevates amino acid consumption during the dark phase via the circadian clock

Expression level of an output gene, <i>takeout</i>, in <i>dCtBP</i>-overexpressing flies.

<p>Relative mRNA levels of <i>takeout</i> were measured at ZT1 and ZT13 using a quantitative PCR assay (Q-PCR). The blue, red and green bars represent the <i>tim(UAS)-Gal4</i>, <i>UAS-dCtBP-2</i> and <i>dCtBP</i> overexpression flies, respectively. The expression level in <i>dCtBP</i> overexpression flies was significantly different from that in <i>tim(UAS)-Gal4</i> (a: t test, <i>P<</i>0.05) and that of <i>UAS-dCtBP-2</i> (b: t test, <i>P<</i>0.05) at both phases. RNAs were sampled three times at each point and error bars represent S.E.M.</p

Expression levels of core clock genes in <i>dCtBP</i>-overexpressing flies.

<p>Relative mRNA levels of the indicated genes at the peak and trough phases were measured using a quantitative PCR assay (Q-PCR). Expression levels of <i>per, vri</i>, and <i>Pdp1ε</i> were higher in the <i>dCtBP</i> overexpression flies (black) than in control (white) at the peak phase. <i>dCtBP</i> overexpression decreased the expression levels of <i>cwo</i> at the trough phase. Asterisks indicate a significant difference from control values (t test, <i>P<</i>0.05). RNAs were sampled three times at each point, and error bars represent S.E.M.</p