Pigment dispersing factor

Pigment dispersing factor (pdf) is a gene that encodes for the protein PDF, which is part of a large family of neuropeptides. The analogous hormone, 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 under a hypothesized split hormonal control. 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.

Gene characteristics
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 length of the cDNA clone in flies is 1080 base pairs and there is 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 are thought to be the principle 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.

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. Further research demonstrated that selective ablation of the lateral ventral neurons that express the pdf gene did not affect flies' ability to entrain to light, however, these flies were arrhythmic in constant conditions, indicating that PDF is not required for response to light, and is probably a part of the output pathway. In addition, utilizing time-series immunostainings, Lin et. al. showed that PDF does not function in the maintenance of 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.

As for localization of PDF, experiments at Brandeis University have shown that PDF neuropeptide is expressed in small lateral ventral neurons (s-LNv) that specifically control morning anticipatory behavior. Since the Drosophila circadian clock is governed by a morning and an evening oscillator (M and E, respectively), Stoleru et al. used mosaic 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, plays a large role in maintaining normal rhythmicity, and functions similarly to the mammalian neuropeptide, VIP.

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. In another study on Pdf expression in lateral ventral neurons, it was discovered that 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. Furthermore, it has been found that large LNv when 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.

In addition to the LNv, another study has found that a subset of the posterior dorsal neurons 1 (DN1(p)s) modulate the startle response to the onset of light and morning anticipatory behavior. The PDF-expressing neurons synapse onto DN1(p)s neurons and this combined (DN1(p)Pdfr) expression is sufficient to rescue part of the lack of morning anticipation and arrhythmicity.

There is new evidence 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.

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.

Homologs
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 liquid chromotography in conjunction with several biological assays, PDF, was also isolated in the insect Leucophaea maderae, a cockroach.

A study on the larval Rhodnius prolixus brain showed that a clock exists even in the early stages of morphological and neurological development. The larval brain secretes neurohormones to control locomotor rhythmicity. While the lateral neurons in the optic lobe contain PDF, dorsal neurons lack the gene but are synapsed onto by their lateral counterparts, thus synchronizing the clock system. This study shows that insect PDF not only controls rhythmicity in behavior, but also hormone secretion and shows analogies to the mammalian SCN.