Synergistic Interactions between the Molecular and Neuronal Circadian Networks Drive Robust Behavioral Circadian Rhythms in Drosophila melanogaster

Abstract

Most organisms use 24-hr circadian clocks to keep temporal order and anticipate daily environmental changes. In Drosophila melanogaster CLOCK (CLK) and CYCLE (CYC) initiates the circadian system by promoting rhythmic transcription of hundreds of genes. However, it is still not clear whether high amplitude transcriptional oscillations are essential for circadian timekeeping. In order to address this issue, we generated flies in which the amplitude of CLK-driven transcription can be reduced partially (approx. 60%) or strongly (90%) without affecting the average levels of CLK-target genes. The impaired transcriptional oscillations lead to low amplitude protein oscillations that were not sufficient to drive outputs of peripheral oscillators. However, circadian rhythms in locomotor activity were resistant to partial reduction in transcriptional and protein oscillations. We found that the resilience of the brain oscillator is depending on the neuronal communication among circadian neurons in the brain. Indeed, the capacity of the brain oscillator to overcome low amplitude transcriptional oscillations depends on the action of the neuropeptide PDF and on the pdf-expressing cells having equal or higher amplitude of molecular rhythms than the rest of the circadian neuronal groups in the fly brain. Therefore, our work reveals the importance of high amplitude transcriptional oscillations for cell-autonomous circadian timekeeping. Moreover, we demonstrate that the circadian neuronal network is an essential buffering system that protects against changes in circadian transcription in the brain.

PDF Signaling Is an Integral Part of the Drosophila Circadian Molecular Oscillator

“…The UAS-CLKGR transgene directs the expression of a fusion between CLK and the glucocorticoid receptor ligand-binding domain. This fusion protein acts as a dominant negative of CLK (Weiss et al., 2014), but addition of the artificial glucocorticoid analog dexamethasone (Dex) activates the fusion provoking a quick and large increases in CLK-dependent transcription (Kadener et al., 2007, McDonald and Rosbash, 2001, Weiss et al., 2014). We drove expression of CLKGR with the tim GAL4 driver ( tim CLKGR- tim Tomato flies) and observed a strong upregulation in signal from the reporter, within the circadian cells, in brain of flies incubated with Dex (Figure S2G).…”

“…Since ZT19 is the peak of TOMATO expression in the s-LNvs (Figure 1E, white circle), upregulation at this ZT cannot be a consequence of a phase shift; thus, it must reflect an increase in the amplitude of TOMATO oscillation. This demonstrate that in the absence of PDF signaling, the LNvs lose control of their own circadian molecular clockwork (Collins et al., 2014, Peng et al., 2003), likely because it is overridden by signals from the LNds and/or the DNs (Weiss et al., 2014, Yao and Shafer, 2014). …”

“…Tim GAL4, pdf GAL4,pdfR Han5 , UAS-CLKGR; UAS- Dcr2, Hr38 RNAi , sr RNAi , Clk RNAi ,UAS- t-PDF , UAS-TrpA1, and Clk SV40 fly lines were previously described (Choi et al., 2009, Choi et al., 2012, Dietzl et al., 2007, Hyun et al., 2005, Kaneko and Hall, 2000, Lerner et al., 2015, Perkins et al., 2015, Renn et al., 1999, Sivachenko et al., 2013, Weiss et al., 2014). …”

“…This fits well with studies performed in mammals (Nagoshi et al., 2004, Welsh et al., 2004), as well as in Neurospora and cyanobacteria (Brunner and Káldi, 2008, Kitayama et al., 2008). In this context, the main function of the circadian neuronal network is readjusting individual circadian oscillators, hence facilitating resonance or coherence in the network (Abraham et al., 2010, Busza et al., 2007, Depetris-Chauvin et al., 2011, Peng et al., 2003, Tang et al., 2010, Weiss et al., 2014). However, several studies provided evidence for a role of neuronal connectivity in the timekeeping process per se, in flies (Peng et al., 2003, Weiss et al., 2014) (Nitabach et al., 2002, Nitabach et al., 2005 but also see Depetris-Chauvin et al., 2011) and mammals (Bernard et al., 2007, Takahashi et al., 2010).…”

Clk post-transcriptional control denoises circadian transcription both temporally and spatially

“…These layers of regulation extend even beyond the single-cell level. Circadian neurons in the brain are organized in a network that synchronizes and likely amplifies the individual neuronal oscillators thereby contributing to a coherent and robust behavioural output 2,[37][38][39] . Circadian clocks must also control or buffer transcriptional noise and its consequences, especially for genes that are expressed at limiting levels (that is, Clk), as it is well known that transcriptional noise is inversely correlated with transcriptional rate 40 .…”