miR-124 Regulates the Phase of Drosophila Circadian Locomotor Behavior

Abstract

Animals use circadian rhythms to anticipate daily environmental changes. Circadian clocks have a profound effect on behavior. In Drosophila, for example, brain pacemaker neurons dictate that flies are mostly active at dawn and dusk. miRNAs are small, regulatory RNAs (≈22 nt) that play important roles in posttranscriptional regulation. Here, we identify miR-124 as an important regulator of Drosophila circadian locomotor rhythms. Under constant darkness, flies lacking miR-124 (miR-124(KO)) have a dramatically advanced circadian behavior phase. However, whereas a phase defect is usually caused by a change in the period of the circadian pacemaker, this is not the case in miR-124(KO) flies. Moreover, the phase of the circadian pacemaker in the clock neurons that control rhythmic locomotion is not altered either. Therefore, miR-124 modulates the output of circadian clock neurons rather than controlling their molecular pacemaker. Circadian phase is also advanced under temperature cycles, but a light/dark cycle partially corrects the defects in miR-124(KO) flies. Indeed, miR-124(KO) shows a normal evening phase under the latter conditions, but morning behavioral activity is suppressed. In summary, miR-124 controls diurnal activity and determines the phase of circadian locomotor behavior without affecting circadian pacemaker function. It thus provides a potent entry point to elucidate the mechanisms by which the phase of circadian behavior is determined.

Significance statement: In animals, molecular circadian clocks control the timing of behavioral activities to optimize them with the day/night cycle. This is critical for their fitness and survival. The mechanisms by which the phase of circadian behaviors is determined downstream of the molecular pacemakers are not yet well understood. Recent studies indicate that miRNAs are important regulators of circadian outputs. We found that miR-124 shapes diurnal behavioral activity and has a striking impact on the phase of circadian locomotor behavior. Surprisingly, the period and phase of the neural circadian pacemakers driving locomotor rhythms are unaffected. Therefore, miR-124 is a critical modulator of the circadian output pathways that control circadian behavioral rhythms.

Keywords: Drosophila; circadian behavior; circadian rhythms; miRNAs.

Figure 1.

Loss of miR-124 advances circadian phase under constant darkness. A, Locomotor behavior under LD cycle and constant darkness. Representative double plotted actograms of w¹¹¹⁸, miR-124^KO, miR-124^KO/Df, and miR-124^KO rescue flies. White indicates the light phase, gray indicates the dark phase. B, Morning anticipation (small arrows) and lights-on startle response are eliminated in miR-124^KO flies under the LD cycle. Evening anticipation is indicated with large arrows. White bars represent activity during the day, gray bars at night. (C) Circadian behavior profile in DD. Circadian phase is dramatically advanced in miR-124^KO flies in constant darkness. Circadian time of peak activity is indicated on the graph. Gray shades indicate the subjective night. D, Phase of the evening peak observed in miR-124^KO flies is advanced by the per^S mutation and is thus under circadian control. E, Morning anticipation and lights-on startle response are restored in miR-124 ^KO /Df flies rescued with a genomic miR-124 construct.

Figure 2.

Loss of miR-124 advances circadian phase under and after temperature cycles. A, Locomotor behavior under TCs and constant darkness. White indicates the warm phase (29°C), gray the cold phase (20°C) or the release in constant conditions (25°C). B, Evening peak is advanced in miR-124^KO flies under the TC cycle. C, Phase is dramatically advanced in miR-124^KO flies after release in constant temperature. Gray shades indicate the subjective night (subjective cold phase).

Figure 3.

The molecular pacemaker is not affected in miR-124^KO flies. A, ERG recordings do not show any obvious light response defect in the visual photoreception cascade of miR-124^KO flies. Scale bar, 5 mV. B, sLNvs of brains from miR-124^KO and genomic rescue flies dissected at different time points (circadian time, CT) during the second day of DD and stained with anti-PDF (green) and anti-PER (red) antibodies. C, Quantification of PER staining in sLNvs, LNds, and DN1s at different circadian time points. Error bars indicate SEM.

Figure 4.

PDF neural network in wild-type and miR-124^KO flies. PDF staining (green) in w¹¹¹⁸ (A–C) and miR-124^KO (D–G) brains. A, D, G, Open arrows indicate the sLNv dorsal projections, OL the lLNv optic lobes projections, and closed arrows the lLNv contralateral projections. These projections were all present and normal in miR-124^KO brains (D), but a small fraction of mutant brains showed additional LNv projections, such as more ventral sLNv projections (G). Persistence of PDF-Tri projections (diamond arrows) was observed in most mutant brains (D, G). B, E, Terminal ends of PDF-positive sLNv projections in the dorsal protocerebrum at ZT1. C, F, Cell bodies of PDF-positive sLNvs (S) and lLNvs (L).