An isoform-specific mutant reveals a role of PDP1 epsilon in the circadian oscillator

Abstract

The Drosophila PAR domain protein 1 (Pdp1) gene encodes a transcription factor with multiple functions. One isoform, PDP1epsilon, was proposed to be an essential activator of the core clock gene, Clock (Clk). However, a central clock function for PDP1epsilon was recently disputed, and genetic analysis has been difficult due to developmental lethality of Pdp1-null mutants. Here we report the discovery of a mutation that specifically disrupts the Pdp1epsilon isoform. Homozygous Pdp1epsilon mutants are viable and exhibit arrhythmic circadian behavior in constant darkness and also in the presence of light:dark cycles. Importantly, the mutants show diminished expression of CLK and PERIOD (PER) in the central clock cells. In addition, expression of PDF (pigment-dispersing factor) is reduced in a subset of the central clock cells. Loss of Pdp1epsilon also alters the phosphorylation status of the CLK protein and disrupts cyclic expression of a per-luciferase reporter in peripheral clocks under free-running conditions. Transgenic expression of PDP1epsilon in clock neurons of Pdp1epsilon mutants can restore rhythmic circadian behavior. However, transgenic expression of CLK in these mutants rescues the expression of PER in the central clock, but fails to restore behavioral rhythms, suggesting that PDP1epsilon has effects outside the core molecular clock. Together, these data support a model in which PDP1epsilon functions in the central circadian oscillator as well as in the output pathway.

Figure 1.

Circadian locomotor activities of Pdp1 mutant flies. While control flies are rhythmic, Pdp1 ³¹³⁵ mutant flies, as homozygotes and transheterozygotes over the null allele Pdp1 ^P205, are arrhythmic under LD and DD conditions. Heterozygous 3135 flies are rhythmic, indicating that the mutation is recessive.

Figure 2.

Molecular characterization of the Pdp1 ³¹³⁵ allele. A, Pdp1 has seven isoforms as annotated by Flybase. The RJ and RD isoforms are collectively referred to as the ε isoform in this study. The only difference between RJ and RD is a small 42 bp exon (supplemental Fig. 1B, available at www.jneurosci.org as supplemental material). The Pdp1 ³¹³⁵ mutation has a 4 bp deletion (indicated by an arrow) in the coding region of the second exon of RJ and RD. The triangle denotes a P-element insertion (d11071) in an intron common to all isoforms. B, mRNA levels of both the RD and RJ isoforms cycle in a circadian manner. Pdp1 mRNA levels were normalized to Actin mRNA levels. Data from four independent experiments for RJ and RD are shown. C, The RJ isoform, which is smaller than the RD isoform, is the predominant form in head extracts of control (CTL) wild-type flies. The second ZT18 sample is from the same wild-type strain carrying a UAS-Pdp1 transgene; the transgene is not expressed due to lack of a Gal4 driver. Protein levels of both RD and RJ are undetectable in Pdp1 ³¹³⁵ mutant flies. D, Compared with wild-type controls, PDP1 expression at ZT20 is markedly reduced in the lateral neurons of Pdp1 ³¹³⁵ mutants. CTL, Background control for Pdp1 ³¹³⁵.

Figure 3.

Rescue of Pdp1 ³¹³⁵ mutants using a UAS-Pdp1 transgene. A, Expression of a UAS-Pdp1-RD transgene rescued arrhythmic behavior when driven by tim-UAS-Gal4 (TUG) or cry-Gal4, but not by Pdf-Gal4. B, A UAS-Pdp1-RD transgene under the control of the TUG driver restores PDP1ε expression in Pdp1 ³¹³⁵ mutants (Resc). PDP1ε expression is increased above the normal levels when the transgene is driven by TUG in a wild-type background (Oxp). PDP1ε levels cycle even when overexpressed. CTL, TUG/+; mut, Pdp1 ³¹³⁵. C, PDP1 protein levels do not cycle when overexpressed at a high level with a stronger promoter (tim-Gal4, TG). CTL, UAS-Pdp1-RD/+.

Figure 4.

Circadian clock protein expression is reduced in the central clock cells of the Pdp1 ³¹³⁵ mutants. A, CLK protein levels at CT09 are reduced in both the dorsal and ventral lateral neurons of the Pdp1 ³¹³⁵ mutants. B, PER protein expression levels are low or undetectable in most lateral neurons of Pdp1 ³¹³⁵ mutants. In a minority of Pdp1 ³¹³⁵ brain hemispheres (3 of 10), 1–2 of the small lateral neurons have subnormal levels of PER expression at CT02. Most brain samples from Pdp1 ³¹³⁵ mutants have low to undetectable PDF expression in the small ventral lateral neurons. For the Pdp1 ³¹³⁵ mutants, samples with visible PDF in the small lateral neurons are shown, and PDF signals were enhanced to visualize the cell bodies. CTL, Background control for Pdp1 ³¹³⁵.

Figure 5.

PDF expression is reduced in the small ventral lateral neurons of the Pdp1 ³¹³⁵ mutants. Cell bodies of the lateral neurons were visualized by expression of UAS-gfp driven by TUG. Representative images of four brains examined at CT21 are shown.

Figure 6.

Altered expression of circadian clock genes in Pdp1 ³¹³⁵ mutants in the peripheral clock. A, mRNA levels of Clk are not dramatically affected in the Pdp1 ³¹³⁵ mutants in LD. In contrast, there are small reductions in peak mRNA levels of per and cry. Circadian clock gene mRNA levels were normalized to Actin mRNA levels. The error bars indicate SEM from four experiments. B, Clk mRNA levels are slightly lower in Pdp1 ³¹³⁵ mutants in DD. Data are averaged from two independent experiments, except for CT14, where data from one experiment are shown. C, CLK protein expression levels are not dramatically reduced in Pdp1 ³¹³⁵ mutants under LD conditions. However, peak expression of PER at ZT 20 is reduced. D, In DD, low-mobility forms of CLK is reduced in Pdp1 ^3135/P205 mutants relative to wild-type flies. Peak expression of PER is reduced at CT20. Similar results were obtained for Pdp1 ³¹³⁵ homozygotes. Representative Western blots of two independent experiments are shown in C and D. E, per-LUC expression is dampened over days in DD (n = 45). Similar effects were observed in Pdp1 ^3135/P205 mutants. CTL-background control for Pdp1 ³¹³⁵.

Figure 7.

Forced expression of Clk restores expression of PER in the lateral neurons of Pdp1 ³¹³⁵ mutants. While PER is constantly low in the Pdp1 ³¹³⁵ mutants, overexpression of Clk driven by cry-Gal4 (line 24) elevated PER expression and restored its cycling in the lateral neurons. Note that PDF labels the large LN_vs, but it is low to undetectable in the small LN_vs of Pdp1 ³¹³⁵ mutants. Thus a larger area is shown to cover the area where the small LN_vs normally localize. In contrast to PER, PDF expression is not restored by forced expression of Clk; instead, it is further reduced in the LN_vs. Representative images of 5 brains examined for each genotype and time point are shown.