The Drosophila Receptor Protein Tyrosine Phosphatase LAR Is Required for Development of Circadian Pacemaker Neuron Processes That Support Rhythmic Activity in Constant Darkness But Not during Light/Dark Cycles

Abstract

InDrosophila, a transcriptional feedback loop that is activated by CLOCK-CYCLE (CLK-CYC) complexes and repressed by PERIOD-TIMELESS (PER-TIM) complexes keeps circadian time. The timing of CLK-CYC activation and PER-TIM repression is regulated post-translationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Although kinases that control PER, TIM, and CLK levels, activity, and/or subcellular localization have been identified, less is known about phosphatases that control clock protein dephosphorylation. To identify clock-relevant phosphatases, clock-cell-specific RNAi knockdowns ofDrosophilaphosphatases were screened for altered activity rhythms. One phosphatase that was identified, the receptor protein tyrosine phosphatase leukocyte-antigen-related (LAR), abolished activity rhythms in constant darkness (DD) without disrupting the timekeeping mechanism in brain pacemaker neurons. However, expression of the neuropeptide pigment-dispersing factor (PDF), which mediates pacemaker neuron synchrony and output, is eliminated in the dorsal projections from small ventral lateral (sLNv) pacemaker neurons whenLarexpression is knocked down during development, but not in adults. Loss ofLarfunction eliminates sLNvdorsal projections, but PDF expression persists in sLNvand large ventral lateral neuron cell bodies and their remaining projections. In contrast to the defects in lights-on and lights-off anticipatory activity seen in flies that lack PDF,LarRNAi knockdown flies anticipate the lights-on and lights-off transition normally. Our results demonstrate thatLaris required for sLNvdorsal projection development and suggest that PDF expression in LNvcell bodies and their remaining projections mediate anticipation of the lights-on and lights-off transitions during a light/dark cycle.

Significance statement: In animals, circadian clocks drive daily rhythms in physiology, metabolism, and behavior via transcriptional feedback loops. Because key circadian transcriptional activators and repressors are regulated by phosphorylation, we screened for phosphatases that alter activity rhythms when their expression was reduced. One such phosphatase, leukocyte-antigen-related (LAR), abolishes activity rhythms, but does not disrupt feedback loop function. Rather,Lardisrupts clock output by eliminating axonal processes from clock neurons that release pigment-dispersing factor (PDF) neuropeptide into the dorsal brain, but PDF expression persists in their cell bodies and remaining projections. In contrast to flies that lack PDF, flies that lackLaranticipate lights-on and lights-off transitions normally, which suggests that the remaining PDF expression mediates activity during light/dark cycles.

Keywords: circadian rhythms; clock genes; locomotor activity; neuropeptides; pacemaker neurons; phosphatase.

Figure 1.

Locomotor activity analysis of clock-cell-specific Lar RNAi knockdown and Lar mutant flies. Adult males of the indicated genotypes were entrained in LD for 3 d and transferred to DD (gray asterisk) for at least 7 d. Analysis of activity in DD and fly genotypes are as described in Materials and Methods. Representative double-plotted actograms for single flies of each genotype are shown. White boxes, lights-on period; black boxes, lights-off period; vertical bars, fly activity. The height of vertical bars indicates relative level of activity.

Figure 2.

Rhythms in clock gene expression are intact in Lar knockdown flies. Flies were entrained for 3 d in LD conditions and collected at the indicated times on the first day of DD for Western analysis or the third day of DD for PER immunostaining. A, Western blots of head extracts from w¹¹¹⁸;UAS-LarRNAi/+ and UAS-Dicer2/+;UAS-LarRNAi/timGal4 (UAS-LarRNAi/timGal4) flies were probed with CLK, PER, and TIM antisera. Bands corresponding to hyperphosphorylated CLK, PER, and TIM (hyperCLK, hyperPER, and hyperTIM, respectively) and hypophosphorylated CLK, PER, and TIM (hypoCLK, hypoPER, hypoTIM, respectively) are shown. Actin serves as a loading control. B–G, Brains dissected from adult flies collected at CT22 or CT10 were immunostained with PER antisera and imaged by confocal microscopy. B, E, Projected Z-series images of right brain hemispheres from w¹¹¹⁸;UAS-LarRNAi/+ flies. C, F, Projected Z-series images of right brain hemispheres from UAS-Dicer2/+;UAS-LarRNAi/timGal4 (UAS-LarRNAi/timGal4) flies. D, G, Projected Z-series images of right brain hemispheres from Df(2L)E55/Lar^13.2 (Lar Df/Lar^13.2) flies. PER staining is detected in all three groups of dorsal neurons (DN₁+DN₂, DN₃); LN_d; and LN_v. All images are representative of nine or more fly brains.

Figure 3.

PDF expression is absent in sLN_v dorsal projections of PDF neuron-specific Lar RNAi knockdown flies. Brains dissected from adult flies collected at ZT2 were immunostained with CLK and PDF antibodies and imaged by confocal microscopy. Each image shows a left brain hemisphere, where lateral is right and dorsal is at the top. A–C, Projected Z-series image (76 μm) of a UAS-LarRNAi/UAS-Dicer2 brain. D–F, Projected Z-series image (90 μm) of a UAS-LarRNAi/UAS-Dicer2;pdf-Gal4/+ brain. G–I, Projected Z-series image (76 μm) of a Lar Df/+ brain. J–L, Projected Z-series image (78 μm) of a Lar^13.2/+ brain. M–O, Projected Z-series image (88 μm) of a Lar Df/Lar^13.2 brain. P–R, Projected Z-series image (82 μm) of a Lar Df/Lar^13.2;timGal4/UAS-Lar brain. Colocalization of PDF (green) and CLK (red) is seen as yellow. LN_v denotes the position of LN_v cell bodies. White arrowhead denotes the sLN_v dorsal projection. All images are representative of nine or more fly brains.

Figure 4.

Both lLN_vs and sLN_vs are present in PDF neuron-specific Lar RNAi knock down and Lar Df/Lar^13.2 mutant flies. Brains dissected from adult flies collected at ZT2 were immunostained with CLK and PDF antibodies and the region containing LN_vs was imaged by confocal microscopy. A–C, Projected Z-series (18 μm) from a UAS-LarRNAi/UAS-Dicer2 brain. D–F, Twelve micrometer projected Z-series image from a UAS-LarRNAi/UAS-Dicer2;pdfGal4/+ brain. G–I, Fourteen micrometer projected Z-series image from a Lar Df/Lar^13.2 brain. Colocalization of PDF (green) and CLK (red) is seen as yellow. All images are representative of eight or more fly brains.

Figure 5.

Lar is required during development, but not in adults, for PDF accumulation in sLN_v dorsal projections. A–F, Flies were raised at 18°C to block Gal4 activation, shifted to 30°C after eclosion to permit Gal4 activation, and collected at ZT2. A–C, Seventy-four micrometer projected Z-series image of a tubGal80^ts/+;pdfGal4/+ brain. D–F, Eighty-two micrometer projected Z-series image of a UAS-LarRNAi/tubGal80^ts;pdfGal4/+ brain. G–L, Flies were raised at 30°C to permit Gal4 activation, shifted to 18°C after eclosion to block Gal4 activation, and collected at ZT2. G–I, Eighty-six projected Z-series image of a tubGal80^ts/+;pdfGal4/+ brain. J–L, Eighty-two micrometer projected Z-series image of a UAS-LarRNAi/tubGal80^ts;pdfGal4/+ brain. Brains were dissected, immunostained with CLK and PDF antibodies, and imaged by confocal microscopy. A left brain hemisphere is shown in each image, where lateral is right and dorsal is top. LN_v, sLN_v, and lLN_v cell bodies; white arrowhead, sLN_v dorsal projection. All images are representative of 10 or more brain hemispheres.

Figure 6.

PDF neuron-specific Lar RNAi knockdown eliminates the sLN_v dorsal projection. Brains were dissected from adult flies collected at ZT2, immunostained with GFP antibody, and imaged by confocal microscopy. Projected Z-series images of whole brains are shown, where lateral is left and dorsal is at the top. A, Seventy-six micrometer projected Z-series image from a control UAS-mCD8GFP/+;pdfGal4/+ fly brain. B, Seventy-eight micrometer projected Z-series image from a UAS-mCD8GFP/UAS-LarRNAi;pdfGal4/+ fly brain. White arrowhead, sLN_v dorsal projection; blue arrowhead, lLN_v POT projection; yellow arrowhead, lLN_v medulla projection; red arrowhead, sLN_v and lLN_v aMe projections; gray arrowhead, lLN_v aMe ventral elongation projection. All images are representative of 12 or more brain hemispheres.

Figure 7.

Activity profiles of clock-cell-specific Lar RNAi knockdown, pdf⁰¹ and control flies during LD cycles. Average activity histograms indicating relative levels of locomotion for adult males of the indicated genotypes during LD days 2–7 are shown. A, UAS-LarRNAi/+ flies (n = 16). B, UAS-LarRNAi/+;pdfGal4/+ flies (n = 14). C, UAS-LarRNAi/+;timGal4/+ flies (n = 16). D, pdf⁰¹ mutant flies (n = 14). ZT time in hours during an LD cycle where lights-on is ZT0 and lights-off is ZT12; white bars, daytime activity; black bars, night time activity; lights-on anticipation, gray arrow; lights-off anticipation, black arrow.