PDP1epsilon 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 1epsilon (Pdp1epsilon) transcription, then sequential repression by VRI and activation by PDP1epsilon mediate rhythms in Clk transcription. Because VRI and PDP1epsilon bind the same regulatory element, the VRI/PDP1epsilon ratio is thought to control the level of Clk transcription. Thus, constant high or low PDP1epsilon levels in clock cells should eliminate Clk mRNA cycling and disrupt circadian oscillator function. Here we show that reducing PDP1epsilon levels in clock cells by approximately 70% via RNA interference or increasing PDP1epsilon levels by approximately 10-fold in clock cells does not alter Clk mRNA cycling or circadian oscillator function. However, constant low or high PDP1epsilon 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/PDP1epsilon ratio neither controls Clk mRNA cycling nor circadian oscillator function and argue that PDP1epsilon is not essential for Clk activation. PDP1epsilon is nevertheless required for behavioral rhythmicity, which suggests that it functions to regulate oscillator output.

Figure 1.

RNAi directed against PDP1 effectively reduces PDP1ε levels. A, Western blot of head extracts from cyc⁰¹, per⁰¹, and wild-type flies collected at the indicated times under LD conditions and probed with PDP1 antibody. B, Western blots containing head extracts from wild-type (wt), Clk^Jrk, w;UAS-PDP1i/+;UAS-PDP1i/+ (2xPDP1i), and w;UAS-PDP1i/+;UAS-PDP1i/timGal4 (2xPDP1i+tG4) flies (top) or 2xPDP1i and 2xPDP1i+tG4 flies (bottom) collected at the indicated times under LD conditions and probed with PDP1 antibody. The Western blot at the top contains a different transheterozygous combination of 2xUAS-PDP1i transgenes than that at the bottom. C, Quantification of PDP1ε levels in B and at least two independent repeats of each transheterozygous combination. PDP1ε levels were quantified as described in Materials and Methods. D, Brains dissected from 2xPDP1i+tG4 and control 2xPDP1i flies collected at ZT9 and ZT21 were probed with PDP1 and PDF antibodies. A representative 1 μm optical section of sLN_vs is visualized for PDP1 (left panels), PDF (middle panels), or PDP1 plus PDF (right panels). E, PDP1 immunostaining in 2xPDP1i+tG4 and control 2xPDP1i flies was quantified as the ratio of PDP1/PDF staining. Staining in sLN_vs was quantified from at least nine brains (i.e., at least 3 brains from 3 independent experiments).

Figure 2.

Constant low PDP1ε levels do not disrupt Clk mRNA or VRI cycling. A, qPCR of Clk mRNA from the heads of w;+/+;timGal4/+ (timGal4) and w;UAS-PDP1i/+;UAS-PDP1i/timGal4 (2xPDP1i+tG4) flies collected at the indicated times under LD conditions (ZT). Relative Clk mRNA levels were quantified as described in Materials and Methods. The black and white bars represent times when lights were on or off, respectively. B, Western blots of head extracts from 2xPDP1i+tG4 and wild-type, Clk^Jrk, timGal4 (tG4), and w;UAS-PDP1i/+;UAS-PDP1i/+ (2xPDP1i) control flies collected at the indicated times under LD conditions and probed with VRI antibody. C, Quantification of VRI levels from the Western blots in B and at least two independent repeats. VRI levels were quantified as described in Materials and Methods and normalized to wild type at ZT17. wt, Wild type.

Figure 3.

Overexpression of PDP1e in oscillator cells. A, Western blot of head extracts from w;UAS-PDP1ε/+;+/+ (UPDP1ε) and w;UAS-PDP1ε/+;timGal4/+ (UPDP1ε+tG4) flies collected at the indicated times under LD conditions and probed with PDP1 antibody. B, Quantification of PDP1ε levels in A and at least four independent repeats using three different UAS-PDP1ε inserts. PDP1ε levels were quantified as described in Materials and Methods. C, Brains dissected from UPDP1ε+tG4 and control UPDP1ε flies collected at ZT9 and ZT21 were probed with PDP1 and PDF antibodies. A representative 1 μm optical section of sLN_vs is visualized for PDP1 (left panels), PDF (middle panels), or PDP1 plus PDF (right panels). D, PDP1 immunostaining in UPDP1ε+tG4 and control UPDP1ε flies was quantified as the ratio of PDP1/PDF staining. Staining in sLN_vs was quantified from at least four brains (i.e., at least 2 brains from 2 independent experiments).

Figure 4.

Overexpression of PDP1ε in oscillator cells does not disrupt Clk mRNA or VRI cycling. A, qPCR of Clk mRNA from the heads of w;UAS-PDP1ε/+;UAS-PDP1ε/+ (UPDP1ε) and w; UAS-PDP1ε/+;UAS-PDP1ε/timGal4 (UPDP1ε+tG4) flies collected at the indicated times under LD conditions (ZT). Relative Clk mRNA levels were quantified as described in Materials and Methods. The black and white bars represent times when lights were on or off, respectively. B, Western blot of head extracts from UPDP1ε and UPDP1ε+tG4 flies collected at the indicated times under LD conditions and probed with VRI antibody. The asterisk denotes the PDP1ε band that did not wash off completely from the blot in Figure 3A. C, Quantification of VRI levels in the Western blot in B and at least two independent repeats. VRI levels were quantified as described in Materials and Methods.

Figure 5.

PDP1 RNAi does not disrupt TIM cycling in sLN_vs during DD. A, Brains dissected from wild-type and w;UAS-PDP1iA/UAS-PDP1iB;timGal4/+ (2xPDP1i+tG4) flies collected during the second day of DD at CT9 and CT21 were probed with TIM and PDF antibodies. A representative 1 μm optical section of sLN_vs is visualized for TIM (left panels), PDF (middle panels), or TIM plus PDF (right panels). TIM (green) and PDF (red) coimmunostaining is visualized as yellow. B, TIM levels in control 2xPDP1i and 2xPDP1i+tG4 flies was quantified as the ratio of TIM/PDF immunostaining. Staining in sLN_vs was quantified from at least 12 brains (i.e., at least 4 brains from 3 independent experiments). wt, Wild type.

Figure 6.

PDP1ε overexpression does not disrupt PER cycling in sLN_vs during DD. A, Brains dissected from wild-type and w;UAS-PDP1ε/+;UAS-PDP1ε/timGal4 (UPDP1ε+tG4) flies collected during the second day of DD at CT1 and CT13 were probed with PER and PDF antibodies. A representative 1 μm optical section of sLN_vs is visualized for PER (left panels), PDF (middle panels), or PER plus PDF (right panels). PER (green) and PDF (red) coimmunostaining is visualized as yellow. B, PER immunostaining in wild-type (wt) and UPDP1ε+tG4 flies was quantified as the ratio of PER/PDF staining. Staining in sLN_vs was quantified from at least 12 brains (i.e., at least 4 brains from 3 independent experiments).

Figure 7.

Model for the function of PDP1ε. CLK-CYC heterodimers bind E-boxes to activate vri and Pdp1ε transcription. VRI accumulates in concert with its mRNA and binds V/P-boxes to inhibit Clk transcription (solid gray line). PDP1ε accumulates to peak levels several hours after VRI, activates output genes required for behavioral activity (solid black line), and possibly counteracts VRI inhibition (dashed black line). VRI may also contribute to rhythmic output gene transcription (dashed gray line). PER-TIM accumulates in parallel with PDP1ε and inhibits CLK-CYC-dependent transcription of vri and Pdp1ε (dashed blue line), thereby removing VRI inhibition of Clk transcription. An activator of Clk transcription (ACT) is constantly present, thus accounting for high levels of Clk mRNA in Clk^Jrk and cyc⁰¹ mutants and in wild-type flies when VRI is absent.