Pigment dispersing factor
|Chromosome||3R: 22.28 - 22.28 Mb|
Pigment dispersing factor (pdf) is a gene that encodes for the protein PDF, which is part of a large family of neuropeptides. Its hormonal product, pigment dispersing hormone (PDH), was named for the diurnal pigment movement effect it has in crustacean retinal cells, and was initially discovered in the central nervous system of arthropods. The movement and aggregation of the pigments in retina cells and extra-retinal cells is hypothesized to be under a split hormonal control mechanism. One hormonal set is responsible for concentrating chromatophoral pigment and responds to changes in the length of darkness presented to the organism whereas another set is responsible for dispersion and responds to the light cycle. However, insect PDF genes do not function in such pigment migration since they lack the chromatophore.
The gene was first isolated and studied in Drosophila by Jeffrey C. Hall's laboratory at Brandeis University in 1998, and has been found to function as a neuromodulator in controlling circadian rhythms.
In Drosophila, the pdf gene is intronless and is located at 97B on the third chromosome. It exists in a single copy per haploid genome and the approximately 0.8 kb transcript is expressed in the Drosophila's head. The cDNA clone in flies has 1080 base pairs with a single exon. Six alleles of this gene have been reported and are found in dorsal lateral neurons and the ventral lateral neurons in the Drosophila brain.
Pdf role in the circadian pathways
In the Drosophila brain, a group of cells called the lateral ventral neurons is thought to be the principal pacemaker regulating the circadian rhythm of Drosophila locomotion. Variation in PDF levels, which is expressed by some of these specialized cells, is believed to be the primary output of oscillations within these cells, coordinating fly circadian behavior.
Study on E and M cells
The 150 pacemaker neurons in Drosophila are organized into two groups of cells called M (morning) and E (evening) oscillators in the small and large lateral neurons (LNvs). These two groups of cells were first observed by Colin Pittendrigh in 1976. As indicated by their names, the two oscillators control circadian rhythm at different times of the day, yet the two must coordinate to synchronize circadian activity.
PDF synchronizes phase of M oscillators, while in E oscillators PDF delays their cycling and increases their amplitude. Stoleru et al. used mosaic (genetics) transgenic animals with different circadian periods to study the two oscillators. Their study showed that M-cells periodically send a "reset" signal which determines the oscillations of the E-cells. It is believed that the reset signal is PDF, because it is M-cell specific and plays a large role in maintaining normal rhythmicity.
PDF from s-LNv is responsible for the maintenance of a free-running rhythm, while PDF from large lateral ventral neurons is not required for normal behavior. Experiments at Brandeis University have shown that PDF neuropeptide is localized in small lateral ventral neurons (s-LNv) that specifically control morning anticipatory behavior. However, it has been found that large LNv working with other circadian neurons is sufficient to rescue the morning anticipation behavior and startle response in s-LNv-ablated flies. Thus, PDF's role in setting the free-running rhythm and the timing of light-dark cycles comes from both types of lateral ventral neurons.
Further evidence of distinct E and M peaks in Drosophila was provided by Grima et al. This work confirmed that the small lateral ventral neurons, which express PDF, are necessary for the morning peak in Drosophila circadian rhythms. Flies lacking functional s-LNv did not possess a lights-on anticipatory activity for the morning peak.
Other behavioral aspects of Drosophila such as eclosion activity have been monitored with ectopic expression of pdf, which in this case is concentrated in the dorsal central brain. These alterations in expression caused severely altered rhythmic behavior in eclosion of larvae, further substantiating the evidence that PDF modulates the rhythmic control of Drosophila behavior.
The PDF Receptor
The PDF receptor is necessary for rhythmicity since it acts a binding site for PDF on the pacemaker neurons. In a 12:12 light-dark cycle, normal flies exhibited locomotor behavior with a morning peak around dawn and an evening peak around dusk. Loss of PDF or loss of PDF-secreting LNvs resulted in weak or no morning peak, and an approximately 2-hour advance in the evening peak in a light-dark cycle. In constant conditions, loss of the PDF receptor or PDF secreting-cells resulted in desynchrony among the clock neurons.
Seol Hee Im and Paul H. Taghert used pdfr mutant flies (pdfr3369 and pdfr5304) to engineer pdfr-GAL4 lines to show that Gal4 mediated rescue of pdfr phenotypes is insufficient to provide complete behavioral rescue. A series of GAL4 driver experiments found that any rescue experiments using Gal4-UAS system always produced incomplete rescue. Unlike pdfr-GAL4 lines however, the 70-kB pdfr-myc transgene is capable of fully rescuing the circadian behavioral deficiencies of the pdfr mutant flies. Thus, a 70 kN PDF receptor transgene leads to complete rescue of the circadian behavioral deficiencies of the pdfr mutant flies. This transgene is widely expressed among pacemakers and also found in a limited number of non pacemaker cells.
In a series of experiments done at Washington University School of Medicine and Brandeis University, pdf was shown to be critical for circadian output coordination. Flies mutant at the pdf gene locus displayed arrhythmic circadian oscillation. Wild type flies, over the 24-hour LD cycle, are active at dawn, quieter at midday and active again at the evening, and their rhythmic behavior persists in constant dark (DD). Flies with the pdf-null (pdf01) mutation displayed disrupted circadian behavior. In locomotor activity rhythms of pdf01, homozygous and hemizygous pdf01 flies were well entrained during LD cycles, and their evening activity peak was advanced by approximately 1 hr. However, in constant darkness free running rhythms were much less rhythmic than in wild type flies.
Further research was conducted on selective ablation of the lateral ventral neurons that express the pdf gene. Fly lines with ablated PDF neurons were created using Gal4-UAS-regulated transgenes and crossing two fly lines: UAS-rpr control group or UAS-hid. Ablation did not affect the flies' ability to entrain to LD cycles, but their evening locomotor phases showed a 0.5 hour advance. This indicates the rpr and hid ablation individuals that were persistently rhythmic in DD showed shorter period length. In addition, utilizing time-series immunostainings, Lin et al. showed that PDF does not function to maintain circadian rhythmicity in protein levels, but rather that it is required to coordinate rhythms among the various Drosophila pacemakers. These experiments thereby confirmed the importance of the coordination role pdf expression plays in regulating circadian locomotor activity in Drosophila.
In 2014, Li et al showed that PDF synchronizes circadian clock neurons by increasing levels of cAMP and cAMP-mediated protein kinase A (PKA). Increasing cAMP and PKA stabilized levels of the period protein PER in Drosophila, which slows the clock speed in PDF receptor (PDFR) containing neurons. A light pulse caused more PER degradation in flies with pdf-null neurons than flies with wild-type neurons, indicating that PDF inhibits light-induced PER degradation. These experiments demonstrated that PDF interacts with secondary messenger components to coordinate circadian output.
PDF is also sufficient to induce high levels of Timeless (gene) protein (TIM), another essential protein that regulates circadian rhythm. Studies had found that flies with mutated ion channels at the posterior dorsal neurons 1 (DN1(p)s) showed reduced anticipatory behavior and free-running rhythms. This deficit can be rescued by synapsing PDF-expressing neurons onto mutated DN1(p)s, as the elevated TIM level is enough to rescue circadian rhythm.
Regulation through glia
In 2011, Ng et al demonstrated that glial-neural signaling may physiologically modulate pdf in a calcium dependent manner. The glial cells, specifically astrocytes, in the adult Drosophila brain physiologically regulate circadian neurons, and affect the output PDF. Separate experiments using Gal4-UAS-regulated transgenes to alter glial release of internal calcium stores, glial vesicle trafficking, and membrane gradients all produced arrhythmic locomotor activity. Immunohistochemistry staining for the peptide in the LNv dorsal projections showed a significant reduction after disruption of glial functions, suggesting that PDF transport and release are affected by glial cells.
Conservation of PDF
Pdf is conserved across Bilateria and homologs have been identified in organisms such as mosquitos and C.elegans. A common misconception is that the PDF gene is found in vertebrates, such as rodents, chimpanzees, and humans.
Pdf has also been studied in the cricket Gryllus bimaculatus; studies proved that Pdf is not necessary for generating the circadian rhythm, but involved in control of nocturnal behavior, entrainment, and the fine-tuning of the free-running period of the circadian clock.
Using Ca2+ imaging studies, researchers found two types of pacemaker cells which contained PDF in the accessory medulla, the circadian pacemaker of the cockroach, Rhyparobia maderae. Type 1 cells showed that PDF signaled via elevation of intracellular cAMP levels. In contrast, in type 2 cells PDF transiently raised intracellular Ca2+ levels even after blocking adenylyl cyclase activity. The researchers hypothesized that in type 1 cells PDF-dependent rises in cAMP concentrations block primarily outward K+ currents. This PDF-dependent depolarization could be the underlying cause of PDF-dependent phase advances of pacemaker in the cockroach. The authors proposed that PDF-dependent modulation of K+ and Na+ ion channels in coupled pacemakers causes ultradian membrane potential oscillations for efficient synchronization of pacemaker cells.
The neuropeptide VIP is a homolog of PDF instrumental for cellular and behavioral 24-hour rhythms in mammals. It is expressed in 10 percent of neurons in the SCN. In a study of VIP and VIP receptor 2 (VIPR2) knockout mice, both mutants displayed entrained activity rhythms in light dark condition. However, in constant darkness both models displayed poor rhythmicity (very short period), and half of the animals tested were arrhythmic.
VIP and PDF are functional homologs which play a role in synchronizing and supporting rhythmicity by diverse SCN pacemakers. Loss of PDF and VIP in free-running conditions resulted in similar behavioral phenotype: dampened behavioral rhythm with a portion of the knockout mutants showing arrhythmicity. The molecular basis of these phenotypes was a loss in synchrony between pacemaker cells. Both knockout mutants show damped molecular oscillations; VIP knockouts show reduced mRNA levels, while PDF knockouts show reduced protein. Similar behavioral and molecular phenotypes are observed in loss of PDF and VIP receptors.
- Rao KR, Riehm JP (May 1993). "Pigment-dispersing hormones". Annals of the New York Academy of Sciences. 680: 78–88. doi:10.1111/j.1749-6632.1993.tb19676.x. PMID 8512238.
- The Interactive Fly  2011 Apr 28.
- Park JH, Hall JC (June 1998). "Isolation and chronobiological analysis of a neuropeptide pigment-dispersing factor gene in Drosophila melanogaster". J. Biol. Rhythms. 13 (3): 219–28. doi:10.1177/074873098129000066. PMID 9615286.
- Helfrich-Förster C, Täuber M, Park JH, Mühlig-Versen M, Schneuwly S, Hofbauer A (May 2000). "Ectopic expression of the neuropeptide pigment-dispersing factor alters behavioral rhythms in Drosophila melanogaster". J. Neurosci. 20 (9): 3339–53. PMID 10777797.
- National Center for Biotechnology Information: Pdf Pigment-dispersing factor (Drosophila melanogaster).  2011 Mar 29.
- Flybase: A Database of Drosophila Genes & Genomes.  2011 Apr 27.
- Renn SC, Park JH, Rosbash M, Hall JC, Taghert PH (December 1999). "A pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in Drosophila". Cell. 99 (7): 791–802. doi:10.1016/S0092-8674(00)81676-1. PMID 10619432.
- Im SH, Taghert PH (June 2010). "PDF receptor expression reveals direct interactions between circadian oscillators in Drosophila". J. Comp. Neurol. 518 (11): 1925–1945. doi:10.1002/cne.22311. PMC . PMID 20394051.
- Duvall, Laura B.; Taghert, Paul H.; Bellen, Hugo J. (5 June 2012). "The Circadian Neuropeptide PDF Signals Preferentially through a Specific Adenylate Cyclase Isoform AC3 in M Pacemakers of Drosophila". PLoS Biology. 10 (6): e1001337. doi:10.1371/journal.pbio.1001337. PMC . PMID 22679392.
- Stoleru D, Peng Y, Nawathean P, Rosbash M (November 2005). "A resetting signal between Drosophila pacemakers synchronizes morning and evening activity". Nature. 438 (7065): 238–242. doi:10.1038/nature04192. PMID 16281038.
- Shafer OT, Taghert PH (2009). Nitabach, Michael N., ed. "RNA-interference knockdown of Drosophila pigment dispersing factor in neuronal subsets: the anatomical basis of a neuropeptide's circadian functions". PLoS ONE. 4 (12): e8298. doi:10.1371/journal.pone.0008298. PMC . PMID 20011537.
- Lear BC, Zhang L, Allada R (2009). "The neuropeptide PDF acts directly on evening pacemaker neurons to regulate multiple features of circadian behavior". PLoS Biol. 7 (7): e1000154. doi:10.1371/journal.pbio.1000154. PMC . PMID 19621061.
- Sheeba V, Fogle KJ, Holmes TC (2010). "Persistence of morning anticipation behavior and high amplitude morning startle response following functional loss of small ventral lateral neurons in Drosophila". PLoS ONE. 5 (7): e11628. doi:10.1371/journal.pone.0011628. PMC . PMID 20661292.
- Grima, Brigitte; Chélot, Elisabeth; Xia, Ruohan; Rouyer, François (14 October 2004). "Morning and evening peaks of activity rely on different clock neurons of the Drosophila brain". Nature. 431 (7010): 869–873. doi:10.1038/nature02935. PMID 15483616.
- Lin Y, Stormo GD, Taghert PH (September 2004). "The neuropeptide pigment-dispersing factor coordinates pacemaker interactions in the Drosophila circadian system". J. Neurosci. 24 (36): 7951–7957. doi:10.1523/JNEUROSCI.2370-04.2004. PMID 15356209.
- Li, Yue; Guo, Fang; Shen, James; Rosbash, Michael (2014). "PDF and cAMP enhance PER stability in Drosophila clock neurons". Proc Natl Acad Sci U S A. 111 (13): E1284–E1290. doi:10.1073/pnas.1402562111. PMC . PMID 24707054.
- Seluzicki, Adam; Flourakis, Matthieu; Kula-Eversole, Elzbieta; Zhang, Luoying; Kilman, Valerie; Allada, Ravi; Blau, Justin (18 March 2014). "Dual PDF Signaling Pathways Reset Clocks Via TIMELESS and Acutely Excite Target Neurons to Control Circadian Behavior". PLoS Biology. 12 (3): e1001810. doi:10.1371/journal.pbio.1001810.
- Zhang L, Chung BY, Lear BC, Kilman VL, Liu Y, Mahesh G, Meissner RA, Hardin PE, Allada R (April 2010). "DN1(p) circadian neurons coordinate acute light and PDF inputs to produce robust daily behavior in Drosophila". Curr. Biol. 20 (7): 591–599. doi:10.1016/j.cub.2010.02.056. PMC . PMID 20362452.
- Ng FS, Tangredi MM, Jackson FR (April 2011). "Glial cells physiologically modulate clock neurons and circadian behavior in a calcium-dependent manner". Curr Biol. 21 (8): 625–634. doi:10.1016/j.cub.2011.03.027. PMC . PMID 21497088.
- Hassaneen E, El-Din Sallam A, Abo-Ghalia A, Moriyama Y, Karpova SG, Abdelsalam S, Matsushima A, Shimohigashi Y, Tomioka K (February 2011). "Pigment-dispersing factor affects nocturnal activity rhythms, photic entrainment, and the free-running period of the circadian clock in the cricket gryllus bimaculatus". J. Biol. Rhythms. 26 (1): 3–13. doi:10.1177/0748730410388746. PMID 21252361.
- Hamasaka Y, Mohrherr CJ, Predel R, Wegener C (2005). "Chronobiological analysis and mass spectrometric characterization of pigment-dispersing factor in the cockroach Leucophaea maderae". J. Insect Sci. 5: 43. doi:10.1093/jis/5.1.43. PMC . PMID 17119625.
- Wei, Hongying; Yasar, Hanzey; Funk, Nico W.; Giese, Maria; Baz, El-Sayed; Stengl, Monika; Yamazaki, Shin (30 September 2014). "Signaling of Pigment-Dispersing Factor (PDF) in the Madeira Cockroach Rhyparobia maderae". PLoS ONE. 9 (9): e108757. doi:10.1371/journal.pone.0108757.
- Im, Seol Hee; Taghert, Paul H. (1 June 2010). "PDF receptor expression reveals direct interactions between circadian oscillators in Drosophila". The Journal of Comparative Neurology. 518 (11): 1925–1945. doi:10.1002/cne.22311. PMC . PMID 20394051.
- Vosko, Andrew M.; Schroeder, Analyne; Loh, Dawn H.; Colwell, Christopher S. (June 2007). "Vasoactive intestinal peptide and the mammalian circadian system". General and Comparative Endocrinology. 152 (2-3): 165–175. doi:10.1016/j.ygcen.2007.04.018.