PDP1ε Functions Downstream of the Circadian Oscillator to Mediate Behavioral Rhythms

Abstract

The Drosophila circadian oscillator is composed of autoregulatory period/timeless (per/tim) and Clock (Clk) feedback loops that control rhythmic transcription. In the Clk loop, CLOCK-CYCLE heterodimers activate vrille (vri) and PAR domain protein 1 (Pdp1) transcription, then sequential repression by VRI and activation by PDP1 mediate rhythms in Clk transcription. Because VRI and PDP1 bind the same regulatory element, the VRI/PDP1 ratio is thought to control the level of Clk transcription. Thus, constant high or low PDP1 levels in clock cells should eliminate Clk mRNA cycling and disrupt circadian oscillator function. Here we show that reducing PDP1 levels in clock cells by ϳ70% via RNA interference or increasing PDP1 levels by ϳ10-fold in clock cells does not alter Clk mRNA cycling or circadian oscillator function. However, constant low or high PDP1 levels in clock cells disrupt locomotor activity rhythms despite persistent circadian oscillator function in brain pacemaker neurons that extend morphologically normal projections into the dorsal brain. These results demonstrate that the VRI/PDP1 ratio neither controls Clk mRNA cycling nor circadian oscillator function and argue that PDP1 is not essential for Clk activation. PDP1 is nevertheless required for behavioral rhythmicity, which suggests that it functions to regulate oscillator output.

The circadian output gene takeout is regulated by Pdp1ε

“…Depending on which Pdp1 allele/transgene was used and which tissue was tested, Clk levels and the core circadian oscillator are either not affected (10,11) or moderately impacted (12). However, interestingly, flies expressing high or low levels of PDP1ε were consistently arrhythmic in a locomotor activity assay, indicating that PDP1ε levels regulate oscillator output and thus control circadian output genes (10)(11)(12). takeout has been identified in several screens as an output gene of the circadian oscillator.…”

“…CLK/CYC activate vrille (vri) and Pdp1ε, a transcriptional repressor and activator respectively, that have been shown to bind the Clock promoter competitively (8,9). Although it was thought that this competition accounts for the oscillatory regulation of Clk mRNA, the fact that Clk mRNA levels are still high in Clk JRK mutants (4) and that the core oscillator is only minimally impacted in flies with increased or decreased PAR domain protein 1 (Pdp1ε) levels (depending on the allele/transgene and tissue examined) (10)(11)(12), indicate that an as yet unknown activator is required for Clk transcription. It has recently been demonstrated that CLK protein levels are constant in cells and that it is the phosphorylation state of the protein that determines its binding to DNA and its transcriptional activator function (7).…”

“…The UAS-PDP1i and UAS-PDP1 transgenic strains were as described in ref. 10. The timGal4 driver (39), the fat body driver 3.1 kb Lsp2-Gal4 (29) and the to-Gal4 driver (28,29) have been described before.…”

“…Generation of Clk JRK (CS):Clk JRK , ry flies were crossed to CS flies. Individual recombinant progeny of Clk JRK (CS), ry/CS females were then screened for the absence of ry and the presence of Clk JRK by arrhythmicity in a locomotor assay (10). (Location of genes: Clk 66A, ry 87D, to 96C).…”

Identifying Specific Light Inputs for Each Subgroup of Brain Clock Neurons inDrosophilaLarvae

“…All experiments, performed at least twice with similar results, were done on whole-mounted third-instar larval brains, which were dissected and labeled as previously described (Malpel et al, 2002). Antibodies were as previously described (Malpel et al, 2002;Picot et al, 2009), except for the use of the GP47 guinea pig anti-CLOCK (Houl et al, 2006), at 1:5000 dilution, or a guinea-pig anti-PDP1 used at 1:10,000 dilution (Benito et al, 2007). Although GP47 cross-reacts with DACHSHUND (Houl et al, 2008), the clock neurons were on average more strongly stained, and could also be distinguished based on size, shape, and location (see Figs.…”