Dataset Information

RNA-interference knockdown of Drosophila pigment dispersing factor in neuronal subsets: the anatomical basis of a neuropeptide's circadian functions.

ABSTRACT:

Background

In animals, neuropeptide signaling is an important component of circadian timekeeping. The neuropeptide pigment dispersing factor (PDF) is required for several aspects of circadian activity rhythms in Drosophila.

Methodology/principal findings

Here we investigate the anatomical basis for PDF's various circadian functions by targeted PDF RNA-interference in specific classes of Drosophila neuron. We demonstrate that PDF is required in the ventro-lateral neurons (vLNs) of the central brain and not in the abdominal ganglion for normal activity rhythms. Differential knockdown of PDF in the large or small vLNs indicates that PDF from the small vLNs is likely responsible for the maintenance of free-running activity rhythms and that PDF is not required in the large vLNs for normal behavior. PDF's role in setting the period of free-running activity rhythms and the proper timing of evening activity under light:dark cycles emanates from both subtypes of vLN, since PDF in either class of vLN was sufficient for these aspects of behavior.

Conclusions/significance

These results reveal the neuroanatomical basis PDF's various circadian functions and refine our understanding of the clock neuron circuitry of Drosophila.

SUBMITTER: Shafer OT

PROVIDER: S-EPMC2788783 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Publications

RNA-interference knockdown of Drosophila pigment dispersing factor in neuronal subsets: the anatomical basis of a neuropeptide's circadian functions.

Shafer Orie T OT Taghert Paul H PH

PloS one 20091214 12

<h4>Background</h4>In animals, neuropeptide signaling is an important component of circadian timekeeping. The neuropeptide pigment dispersing factor (PDF) is required for several aspects of circadian activity rhythms in Drosophila.<h4>Methodology/principal findings</h4>Here we investigate the anatomical basis for PDF's various circadian functions by targeted PDF RNA-interference in specific classes of Drosophila neuron. We demonstrate that PDF is required in the ventro-lateral neurons (vLNs) of ...[more]

PMID: 20011537

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Project description:Dissociation between the output of the circadian clock and external environmental cues is a major cause of human cognitive dysfunction. While the effects of ablation of the molecular clock on memory have been studied in many systems, little has been done to test the role of specific clock circuit output signals. To address this gap, we examined the effects of mutations of Pigment-dispersing factor (Pdf) and its receptor, Pdfr, on associative memory in male and female Drosophila Loss of PDF signaling significantly decreases the ability to form associative memory. Appetitive short-term memory (STM), which in wild-type (WT) is time-of-day (TOD) independent, is decreased across the day by mutation of Pdf or Pdfr, but more substantially in the morning than in the evening. This defect is because of PDFR expression in adult neurons outside the core clock circuit and the mushroom body (MB) Kenyon cells (KCs). The acquisition of a TOD difference in mutants implies the existence of multiple oscillators that act to normalize memory formation across the day for appetitive processes. Interestingly, aversive STM requires PDF but not PDFR, suggesting that there are valence-specific pathways downstream of PDF that regulate memory formation. These data argue that the circadian clock uses circuit-specific and molecularly diverse output pathways to enhance the ability of animals to optimize responses to changing conditions.SIGNIFICANCE STATEMENT From humans to invertebrates, cognitive processes are influenced by organisms' internal circadian clocks, the pace of which is linked to the solar cycle. Disruption of this link is increasingly common (e.g., jetlag, social jetlag disorders) and causes cognitive impairments that are costly and long lasting. A detailed understanding of how the internal clock regulates cognition is critical for the development of therapeutic methods. Here, we show for the first time that olfactory associative memory in Drosophila requires signaling by Pigment-dispersing factor (PDF), a neuromodulatory signaling peptide produced only by circadian clock circuit neurons. We also find a novel role for the clock circuit in stabilizing appetitive sucrose/odor memory across the day.

Project description:Daily and annually cycling conditions manifested on the Earth have forced organisms to develop time-measuring devices. Circadian clocks are responsible for adjusting physiology to the daily cycles in the environment, while the anticipation of seasonal changes is governed by the photoperiodic clock. Circadian clocks are cell-autonomous and depend on the transcriptional/translational feedback loops of the conserved clock genes. The synchronization among clock centers in the brain is achieved by the modulatory function of the clock-dependent neuropeptides. In insects, the most prominent clock neuropeptide is Pigment Dispersing Factor (PDF). Photoperiodic clock measures and computes the day and/or night length and adjusts physiology accordingly to the upcoming season. The exact mechanism of the photoperiodic clock and its direct signaling molecules are unknown but, in many insects, circadian clock genes are involved in the seasonal responses. While in Drosophila, PDF signaling participates both in the circadian clock output and in diapause regulation, the weak photoperiodic response curve of D. melanogaster is a major limitation in revealing the full role of PDF in the photoperiodic clock. Here we provide the first description of PDF in the linden bug, Pyrrhocoris apterus, an organism with a robust photoperiodic response. We characterize in detail the circadian and photoperiodic phenotype of several CRISPR/Cas9-generated pdf mutants, including three null mutants and two mutants with modified PDF. Our results show that PDF acts downstream of CRY and plays a key role as a circadian clock output. Surprisingly, in contrast to the diurnal activity of wild-type bugs, pdf null mutants show predominantly nocturnal activity, which is caused by the clock-independent direct response to the light/dark switch. Moreover, we show that together with CRY, PDF is involved in the photoperiod-dependent diapause induction, however, its lack does not disrupt the photoperiodic response completely, suggesting the presence of additional clock-regulated factors. Taken together our data provide new insight into the role of PDF in the insect's circadian and photoperiodic systems.

Project description:Pigment-dispersing factor neuropeptides (PDFs) occur in a wide range of protostomes including ecdysozoans (= molting animals) and lophotrochozoans (mollusks, annelids, flatworms, and allies). Studies in insects revealed that PDFs play a role as coupling factors of circadian pacemaker cells, thereby controlling rest-activity rhythms. While the last common ancestor of protostomes most likely possessed only one pdf gene, two pdf homologs, pdf-I and pdf-II, might have been present in the last common ancestors of Ecdysozoa and Panarthropoda (Onychophora + Tardigrada + Arthropoda). One of these homologs, however, was subsequently lost in the tardigrade and arthropod lineages followed by independent duplications of pdf-I in tardigrades and decapod crustaceans. Due to the ancestral set of two pdf genes, the study of PDFs and their receptor (PDFR) in Onychophora might reveal the ancient organization and function of the PDF/PDFR system in panarthropods. Therefore, we deorphanized the PDF receptor and generated specific antibodies to localize the two PDF peptides and their receptor in the onychophoran Euperipatoides rowelli. We further conducted bioluminescence resonance energy transfer (BRET) experiments on cultured human cells (HEK293T) using an Epac-based sensor (Epac-L) to examine cAMP responses in transfected cells and to reveal potential differences in the interaction of PDF-I and PDF-II with PDFR from E. rowelli. These data show that PDF-II has a tenfold higher potency than PDF-I as an activating ligand. Double immunolabeling revealed that both peptides are co-expressed in E. rowelli but their respective levels of expression differ between specific cells: some neurons express the same amount of both peptides, while others exhibit higher levels of either PDF-I or PDF-II. The detection of the onychophoran PDF receptor in cells that additionally express the two PDF peptides suggests autoreception, whereas spatial separation of PDFR- and PDF-expressing cells supports hormonal release of PDF into the hemolymph. This suggests a dual role of PDF peptides-as hormones and as neurotransmitters/neuromodulators-in Onychophora.

Dataset Information

RNA-interference knockdown of Drosophila pigment dispersing factor in neuronal subsets: the anatomical basis of a neuropeptide's circadian functions.

Background

Methodology/principal findings

Conclusions/significance

Publications

RNA-interference knockdown of Drosophila pigment dispersing factor in neuronal subsets: the anatomical basis of a neuropeptide's circadian functions.

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