Molecular and Cellular Endocrinology 361 (2012) 232–240

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Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce

PDF receptor signaling in Caenorhabditis elegans modulates locomotion

and egg-laying
Ellen Meelkop a,1, Liesbet Temmerman a,1, Tom Janssen a, Nick Suetens a, Isabel Beets a,
Liesbeth Van Rompay a, Nilesh Shanmugam b, Steven J. Husson a, Liliane Schoofs a,⇑
a
Research Group of Functional Genomics and Proteomics, Department of Biology, KU Leuven, Naamsestraat 59, 3000 Leuven, Belgium
b
Laboratory for Aging Physiology and Molecular Evolution, Department of Biology, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: In Caenorhabditis elegans, pdfr-1 encodes three receptors of the secretin receptor family. These G protein-
Received 20 January 2012 coupled receptors are activated by three neuropeptides, pigment dispersing factors 1a, 1b and 2, which
Received in revised form 26 March 2012 are encoded by pdf-1 and pdf-2. We isolated a PDF receptor loss-of-function allele (lst34) by means of a
Accepted 2 May 2012
mutagenesis screen and show that the PDF signaling system is involved in locomotion and egg-laying. We
Available online 11 May 2012
demonstrate that the pdfr-1 mutant phenocopies the defective locomotor behavior of the pdf-1 mutant
and that pdf-1 and pdf-2 behave antagonistically. All three PDF receptor splice variants are involved in
Keywords:
the regulation of locomotor behavior. Cell specific rescue experiments show that this pdf mediated
Pigment dispersing factor
PDF
behavior is regulated by neurons rather than body wall muscles. We also show that egg-laying patterns
Caenorhabditis elegans of pdf-1 and pdf-2 mutants are affected, but not those of pdfr-1 mutants, pointing to a novel role for the
Locomotion PDF-system in the regulation of egg-laying.
Reproduction Ó 2012 Elsevier Ireland Ltd. All rights reserved.
Egglaying

1. Introduction gamma-aminobutyric acid (GABA), and glutamate. Additionally,

the C. elegans genome contains a wide variety of predicted neuro-
The free-living nematode Caenorhabditis elegans is widely peptide precursor genes (113) which can be processed to over 250
used to study the neuronal origins of behavior thanks to the different neuropeptides (Nathoo et al., 2001; Li, 2005; Husson
existence of a detailed wiring diagram of the nervous system et al., 2005a,b, 2007b; Li and Kim, 2008). Mature, fully processed
(White et al., 1986) combined with a fully sequenced genome neuropeptides act as neurotransmitters and/or neuromodulators,
(The C. elegans Sequencing Consortium, 1998). Although the mainly through G protein-coupled receptors (GPCRs). GPCRs trans-
physical connections between neurons are known, many duce signals from the cell surface to the cytoplasm by means of
functional relationships still have to be unraveled. While the heterotrimeric G proteins consisting of a, b and c subunits and var-
nervous system of C. elegans appears simple and compact with ious second messenger pathways (Vanden Broeck, 2001; Schiöth
only 302 neurons in adult hermaphrodites, these cells make up and Fredriksson, 2005). The C. elegans genome is predicted to
about one-third of all 959 somatic cells. It is now clear that encompass approximately 1300 GPCR genes with the majority
behind this superficial simplicity, a complex network of neuronal (1000) encoding putative chemoreceptors (Bargmann, 1998;
circuits exists. Fredriksson and Schiöth, 2005; Thomas and Robertson, 2008). Both
Communication between neurons and target cells is primarily vertebrate and invertebrate GPCRs can be grouped into five main
established by the release of classical neurotransmitters like families according to the GRAFS system: Glutamate, Rhodopsin,
octopamine, tyramine, dopamine, serotonin, acetylcholine (ACh), Adhesion, Frizzled/Taste2 and Secretin type receptors are distin-
guished (Fredriksson and Schiöth, 2005). A database search based
⇑ Corresponding author. Tel.: +32 16 324577; fax: +32 16 323902. on putative transmembrane domains of the human secretin recep-
E-mail addresses: [email protected] (E. Meelkop), liesbet.temmer- tor family (also known as family B or II) genes predicted only six
[email protected] (L. Temmerman), [email protected] (T. Janssen), secretin receptor genes in C. elegans, one of which is the pdfr-1
[email protected] (N. Suetens), [email protected] (I. Beets), (C13B9.4) gene (Harmar, 2001; Cardoso et al., 2006). Through alter-
[email protected] (L. Van Rompay), liesbeth.vanrompay@ native splicing, pdfr-1 produces three pigment dispersing factor
bio.kuleuven.be (N. Van Rompay), [email protected] (N. Shanmugam),
[email protected] (S.J. Husson), [email protected]
(PDF) neuropeptide receptors (PDFR-1a, b and c) and is expressed
(L. Schoofs). in all body wall muscles, in several mechanosensory neurons,
1
These authors contributed equally to this work. chemosensory neurons, ring motor neurons and pharyngeal

0303-7207/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.mce.2012.05.001
 E. Meelkop et al. / Molecular and Cellular Endocrinology 361 (2012) 232–240 233

interneurons (Janssen et al., 2008). These three receptors are the Transgenic rescue strains generated in this work were accom-
only GPCRs of the secretin type family in C. elegans that are plished by re-introducing the wild type gene including its promoter.
deorphaned and they were shown to be activated by C. elegans The following primers were used to amplify these regions from
PDF neuropeptides (Janssen et al., 2008, 2009). The C. elegans PDF genomic DNA: pdf-2 forward primer 50 -TGCCCACAGATCGGCTTATG
receptors (PDFRs) are closely related to the fruit fly Drosophila ATTGC-30 and reverse primer 50 -ACTTTTACTTGCCAACGTCTCCAAGT
melanogaster PDFR and more distantly to the mammalian VIP CG-30 and pdfr-1 forward primer 50 -TCTGATGGAGTTACTCGGAGA-30
(VPAC2) and calcitonin receptors (Janssen et al., 2008). and reverse primer 50 -TGTCGTGTGATTTTCCCACT-30 . In order to
PDF neuropeptides belong to a conserved group of neuropep- rescue specific isoforms of the PDF receptor, we generated body wall
tides among insects, nematodes and crustaceans wherein they were muscle (Phlh-1) and neuron (Punc-119) specific fusion constructs (Ho-
originally discovered and named pigment dispersing hormones bert, 2002) whereby the hlh-1 promoter region (3 kb prior to the
(PDHs) (also reviewed by Rao and Riehm, 1993; Meelkop et al., ATG start codon) was amplified from the pKM1238 vector (a kind
2011). PDH is involved in the dispersion of pigment granules in gift from M. Krause, NIDDK, National Institutes of Health, Bethesda
the integument and compound eyes of crustaceans. Insect PDF con- MD, USA), the unc-119 promoter (2210 bp prior to the ATG start co-
tributes to the synchronization and transmission of signals from the don) was isolated from genomic DNA and the PDF receptor splice
circadian clock as was shown in the fruit fly D. melanogaster (Renn variants from the pcDNA3.1D vector (Janssen et al., 2008) with the
et al., 1999; Helfrich-Forster et al., 2000). Flies lacking the PDF following primers: Phlh-1 forward primer 50 -GCGTTTTTTGGGATTCTG
receptor exhibit increased circadian arrhythmicity under constant AATGAT-30 and reverse primer 50 -GAACGGTGACGTGGCATCCGCC
darkness and display altered geotactic behavior, similar to flies ATTTCTGGAAAATTATTGGAAAATTTGG-30 , Punc-119 forward primer
lacking the PDF neuropeptide (Renn et al., 1999; Helfrich-Forster 50 -GCAATTGTTTTGTGCCAAGCTTCA-30 and reverse primer 50 -GAAC
et al., 2000; Toma et al., 2002; Mertens et al., 2005). In addition, GGTGACGTGGCATCCGCCATATATGCTGTTGTAGCTGAAAATTTTGG-
based on immunocytochemical experiments in the blow fly, PDF 30 , pdfr-1 forward primer (for pdfr-1a, b and c) 50 -GAACGGTGACGTG
is suggested to be involved in reproduction (Hamanaka et al., 2005). GCATCCGCCAT-30 , pdfr-1a and pdfr-1b reverse primer 50 -TTATGGA-
C. elegans expresses more than one PDF-like neuropeptide gene: GATTTTGTGAGCGATTGG-30 and pdfr-1c reverse primer 50 -AATTTAT
pdf-1 and pdf-2, encoding three different PDF-like neuropeptides TCTTTGTTTTCTACTCTTCATAC-30 . Purified PCR products were micro-
named PDF-1a, b and PDF-2 (Janssen et al., 2009). According to injected at 10–50 ng/ll together with elt-2::gfp as a selection
their localization patterns, it is thought that PDF-1a, b and PDF-2 marker. At least three strains were analyzed for each rescue condi-
are mainly involved in the integration of environmental stimuli. tion. The strains described in this paper are: LSC59 = pdf-
The peptides already proved functional in locomotion as both the 2(tm4393); lstEx24 [pdf-2 +], LSC60 = pdfr-1(lst34); lstEx25 [pdfr-1
pdf-1 loss-of-function allele tm1996 and pdf-2 over-expression in- +], LSC64 = pdfr-1(lst34); lstEx112 [Punc-119::pdfr-1a], LSC67 = pdfr-
duce decreased locomotion speed, increased reversal frequency 1(lst34); lstEx117 [Punc-119::pdfr-1b] and LSC72 = pdfr-1(lst34);
and increased number of directional changes (Janssen et al., lstEx122 [Punc-119::pdfr-1c].
2008). Each of the three PDF neuropeptides is able to bind to each
of the three PDF receptors but with different affinities. While 2.2. Isolation of a pdfr-1 knockout allele
PDFR-1a and PDFR-1b signal through Gas and adenylate cyclase
and ultimately elevate cAMP levels, PDFR-1c inhibits cAMP levels We generated an in-house mutant C. elegans library of 230,400
through Gai/o (Janssen et al., 2008). haploid genomes through random mutagenesis with 0.5 mg/ml tri-
This work aims to determine whether the absence of functional oxsalen (Sigma–Aldrich) followed by UV radiation (Colibri 360 nm
C. elegans PDF receptors results in similar locomotion defects as ob- at 3%, DAPI filter, A-Plan 5x/NA 0.12 objective, 10 s for 50 J/m2 and
served in pdf-1 knockouts and to explore other possible pdfr-1 20 s for 100 J/m2) (Zeiss Axio Imager, Carl Zeiss MicroImaging
knockout phenotypes. In this study we report the isolation of a GmbH, Germany). Next, we distributed 50 mutagenized worms
pdfr-1 loss-of-function allele (lst34) by means of a mutagenesis per plate over 2304 NGM plates and carried out a PCR screen as
screen. pdfr-1(lst34) animals display aberrant locomotor behavior adapted from Lesa (2006). The PCRs were carried out according
confirming the involvement of these receptors in the regulation to the following PCR conditions: External PCR – 2 ll template
of locomotion. Our results show a novel role for the PDF-system DNA, 1.7 ll 10x rTaq PCR buffer (Invitrogen), 0.51 ll 50 mM MgCl2,
in the regulation of egg-laying. 0.34 ll 0.5 mM dNTPs, 0.34 ll 10 lM forward external primer,
0.34 ll 10 lM reverse external primer, 0.085 ll 5 U/ll rTaq (Invit-
rogen) and 11.685 MiliQ water. Step 1: 94 °C for 3 min; step 2:
2. Material and methods 94 °C for 10 s; step 3: 58 °C for 10 s; step 4: 72 °C for X seconds;
repeat steps two to four 29 times; step 5: 72 °C for 2 min; end at
2.1. Nematode strains and culturing 15 °C. X should be optimized so that there is no PCR product visible
as to limit the amplification of wild type amplicons and prioritize
All C. elegans strains were cultivated at 20 °C in constant dark- the amplification of the shorter deletion alleles. Internal PCR –
ness on nematode growth medium (NGM) agar seeded with 0.7 ll external PCR product, 1.7 ll 10x rTaq PCR buffer (Invitro-
Escherichia coli OP50 bacteria. Strains used were Bristol N2, gen), 0.51 ll 50 mM MgCl2, 0.34 ll 10 mM dNTPs, 0.34 ll 10 lM
LSC39 pdfr-1(lst34) (6x) also see Section 2.2, LSC27 pdf-1(tm1996) forward internal primer, 0.34 ll 10 lM reverse internal primer,
(6x), LSC40 pdf-2(tm4393) (1x), pdfr-1(tm4457), pdf-2(tm4780), 0.085 ll 5 U/ll rTaq (Invitrogen) and 11.685 MiliQ water. Step 1:
LSC10 = pdf-1(tm1996); lstIs1 [pdf-1 +] (Janssen et al., 2008), 94 °C for 3 min; step 2: 94 °C for 10 s; step 3: 58 °C for 10 s; step
LSC84 = N2; lstEx2[pdf-1 +], LSC54 = N2; lstEx3 [pdf-2 +], LSC85 = 4: 72 °C for Y seconds; repeat steps two to four 29 times; step 5:
N2; lstEx4 [pdfr-1 +] (Janssen et al., 2008) and BC11358 [Ppdfr-1::gfp]. 72 °C for 2 min; end at 15 °C. Y should be optimized so that there
JT191 daf-28(sa191) was cultured at 15 °C. is a PCR product visible and is typically two times X. Oligonucleo-
The vector containing Pmyo-3::mCherry (a kind gift from E. Berezi- tide primers were designed with AcePrimer 1.3 (http://ele-
kov and M. Isik, Hubrecht Institute, Utrecht, The Netherlands) was gans.bcgsc.bc.ca/aceprimer/aceprimer.shtml). The following gene-
used to microinject in wild type N2 and LSC39 pdfr-1(lst34) (6x) ani- specific primers (Sigma–Aldrich) were used to identify a deletion
mals generating the following expression strains: LSC55 = N2; in the pdfr-1 (C13B9.4) gene by two consecutive PCRs: forward
lstEx13 [Pmyo-3::mCherry], LSC56 = pdfr-1(lst34); lstEx14 [Pmyo-3:: external 50 -ATTCGGAACACCTAACAGCG-30 and reverse external
mCherry] and LSC57 = pdfr-1(lst34); lstEx15 [Pmyo-3::mCherry]. 50 -ACGTCATTATTGCCACGTCA-30 , 1784 bp apart; forward internal
234 E. Meelkop et al. / Molecular and Cellular Endocrinology 361 (2012) 232–240

50 -CACCACAAGTAGCCAGAGCA-30 and reverse internal 50 -ACGAAATG bacteria. Every 12 h, the worms were transferred to a fresh NGM
CTGGAGAGATGG-30 , 1355 bp apart. Both amplicons cover exons plate. The number of offspring was determined when in L3/L4
5–9 of the pdfr-1 (C13B9.4) gene including the ligand binding stage. Incomplete offspring counts due to escaping or dying moth-
domain and several transmembrane domains (Wormbase WS215). ers were left out of the data analyses. This experiment was con-
ducted in eight separate runs.
2.3. Behavioral assays All data gathered from these experiments was analyzed with
the PROC MIXED procedure for linear models considering separate
2.3.1. Locomotion behavior runs as independent variable and random factor, and with a Dun-
2.3.1.1. Crawling: measuring locomotion speed with the Parallel Worm nett post hoc test in SAS 9.1 and Enterprise Guide 4 (SAS Institute
Tracker and counting reversals. Synchronized L1 nematodes (± 700) Inc.).
were put on 55 mm NGM plates seeded with OP50 bacteria and
kept at 20 °C for 48 h. Young adult worms were then filmed for 2.4. Microscopy
1 min using a video recording system consisting of a Stingray
F145B digital camera (ALLIED Vision Technologies) mounted on a Expression of the Pmyo-3::mCherry was visualized using a
stereomicroscope (Leica M165 FC, Leica Microsystems). The Mat- LSM510 confocal microscope (Carl Zeiss MicroImaging GmbH, Ger-
labÒ Image Acquisition Tool™ was used to control the camera many). Nomarski differential interference contrast (DIC) and epi-
and acquire AVI files suited for analysis by the Parallel Worm fluorescent images of the Ppdfr-1::gfp reporter were viewed on an
Tracker (Ramot et al., 2008) which we adapted to simplify batch Axio Imager Z1 (Carl Zeiss MicroImaging GmbH, Germany). Images
analysis. The Parallel Worm Tracker identifies worms according were captured with a Zeiss AxioCamMR3 digital camera.
to predefined parameters such as contrast and amount of pixels.
After positive identification of a worm, its centre is determined
2.5. CRE luciferase reporter assay
and centroid speed is calculated over a subsequent set of video
frames. The same movies were used to manually determine rever-
Human embryonic kidney cells (HEK293) were maintained and
sals. A reversal was scored when forward movement was switched
transfected as described in Janssen et al. (2008). Twenty-four hours
to backward movement for at least the length of the pharynx.
after transfection, HEK293 cells (50,000/well) were exposed to the
These data were generated in four independent experiments.
ligands for four hours in serum-free medium supplemented with
Each experiment involved at least three movies of one strain. Each
200 lM 3-isobutyl-1-methylxanthine (IBMX). Forskolin (Sigma)
movie generates multiple tracks. All data was analyzed with the
was added at a 10 lM concentration. The CRE luciferase reporter
PROC MIXED procedure for linear models considering independent
assay was also conducted as described in Janssen et al. (2008).
experiments as random factor and with a Dunnett post hoc test.
Movies from three of the four experiments were used to determine
reversals and these numbers were analyzed in a GLM with a neg- 3. Results
ative binomial distribution using SAS 9.1 and Enterprise Guide 4
(SAS Institute Inc.). 3.1. pdfr-1 knockout

2.3.1.2. Swimming: measuring swimming activity with the WormMi- In order to study the function of the C. elegans PDF receptors, a
crotracker. Ten staged L4 nematodes were picked to a well of a 96- library of randomly mutagenized nematodes was generated. The
well plate (U bottom or flat bottom) containing 100 ll of S Basal lst34 allele could be identified by means of a PCR screen targeting
(5.85 g NaCl, 1 g K2 HPO4, 6 g KH2PO4, 1 ml of 5 mg/ml cholesterol the pdfr-1 (C13B9.4) gene. Sequencing revealed that the resulting
in ethanol solution; added to 1 L with H2O) and 50 lM fluorode- 855 bp deletion spans exon 6 and a part of exon 7 of all three splice
oxyuridine (FUdR) to prevent self reproduction. After 5 h of recov- variants of pdfr-1. This covers a part of the ligand binding domain
ery, the swimming activity of the worms was measured every and the first two transmembrane domains which are likely to re-
minute and binned in 30-min blocks with the WormMicrotracker sult in a loss-of-function allele (Supplemental Fig. 1).
(Designplus) (Simonetta and Golombek, 2007) during 10 h. Activity
bins were reported every 30 min. The results were analyzed using 3.2. pdfr-1 mutants display altered locomotor behavior
the PROC MIXED procedure for linear models considering time
points as random factors. A Dunnett post hoc test was used to In D. melanogaster, the absence of either PDF or its receptor re-
determine significant deviations from wild type levels (SAS 9.1 sults in circadian arrhythmicity under constant darkness (Renn
and Enterprise Guide 4, SAS Institute Inc.). et al., 1999; Mertens et al., 2005). Janssen et al. (2008) already
demonstrated that the PDF peptides in C. elegans are involved in
2.3.2. The basal slowing response the control of locomotion as pdf-1(tm1996) loss-of-function and
The basal slowing response (BSR) was measured using ±100 pdf-2 over-expression both result in a severely altered locomotor
well fed young adult animals which were washed twice with M9 behavior (reduced speed, elevated turning and reversal frequency).
buffer and subsequently transferred to 55 mm NGM plates with a When regarding locomotion in C. elegans, one should actually dis-
central lawn of OP50 bacteria (worms were transferred to the part tinguish between two basic forms of locomotion outputs, which re-
without bacteria and allowed to crawl into the lawn). A control quire different patterns of neuromuscular activity and are
group was treated likewise but transferred to 55 mm NGM plates determined by the properties (solid or liquid) of the environment:
without a bacterial food source. The locomotion speed was mea- crawling and swimming. In order to analyze the locomotor behav-
sured using the Parallel Worm Tracker as described in Section ior of the PDF receptor and neuropeptide mutants pdfr-1(lst34),
2.3.1. This experiment was conducted four times independently pdf-1(tm1996), pdf-2(tm4393) and double mutants pdf-1(tm1996);
using at least two plates per condition. pdf-2(tm4393), we made video recordings of crawling animals
and analyzed these using the Parallel Worm Tracker (Ramot
2.3.3. Egg-laying et al., 2008). In addition, the WormMicrotracker was used to mea-
The progression of egg-laying and the overall brood size were sure swimming activity (Simonetta and Golombek, 2007). Mea-
determined by selecting 40 synchronized late L4 nematodes per surements of the locomotion speed of wild type N2, pdf-
strain and placing them each on a single NGM plate with OP50 1(tm1996) mutants and a pdf-2 over-expression strain coincide
 E. Meelkop et al. / Molecular and Cellular Endocrinology 361 (2012) 232–240 235

with previously published data (Fig. 1A) (Janssen et al., 2008). state that these two kinds of behavior differ from each other both in
While over-expression of pdf-2 results in a clear decline in speed, neuromuscular activity and kinematics (Pierce-Shimomura et al.,
the opposite is not true and pdf-2 mutants do not display an in- 2008). To assess whether the PDF signaling system is specifically in-
creased locomotion speed. The combination of pdf-1(tm1996) and volved in crawling or more generally in overall locomotion, we used
pdf-2(tm4393) knockout alleles, however, results in a slightly, the WormMicrotracker which measures swimming activity based
though not significantly, diminished locomotion speed compared on the interruption of an infrared laser beam (Simonetta and Gol-
to wild type speed. pdfr-1(lst34) mutants displayed an 83% de- ombek, 2007). This was assessed for pdf-1(tm1996), pdf-2(tm4393)
crease in centroid speed as compared to wild type N2. and pdfr-1(lst34) mutants, over-expression strains, general rescue
Previously, it has been shown that pdf-1(tm1996) mutants not strains and receptor/tissue specific rescue strains (Fig. 3).
only crawl slower, they also conduct more reversals and change Both pdf-1(tm1996) and pdfr-1(lst34) mutants are significantly
directions more often than wild type animals do (Janssen et al., less active compared to wild type C. elegans when swimming
2008). Footprints of individual animals on freshly seeded NGM (Fig. 3A). Activity of pdf-1(tm1996) animals could be restored by
plates after 24 h clearly show the severity of the locomotion de- re-introducing multiple copies of the pdf-1 promoter and gene,
fects of pdfr-1(lst34) mutants (Fig. 2). Their movement is confined while over-expression of pdf-1 using the same genomic region does
to a small area and comparable to, but more severe than, the loco- not result in any behavioral changes. The swimming activity of pdf-
motion defects of pdf-1(tm1996) animals (Janssen et al., 2008). In 1(tm1996);pdf-2(tm4393) double mutants is significantly reduced
comparison, wild type animals explore the entire bacterial lawn to approximately 2/3 of the wild type level. pdf-2(tm4393) mutants
(Fig. 2). pdf-1(tm1996);pdf-2(tm4393) double mutants and pdf- appear to have slightly elevated activity levels (103% compared to
2(tm4393) mutants generate superficially wild type footprints wild type; p = 0.06) when swimming. The rescue strain shows no
(data not shown). aberrant phenotype alterations and over-expression of pdf-2 re-
The possibility that these altered footprints are not solely due to sults in 12% less activity. The diminished pdfr-1(lst34) swimming
a decrease in locomotion speed was explored by determining the activity can be rescued by re-introducing copies of the pdfr-1 gene
amount of reversals during one minute (Fig. 1B). pdf-1(tm1996) and its promoter and over-expression of this gene does not seem to
neuropeptide mutants conduct almost two times more reversals influence activity levels (Fig. 3A).
than wild type animals (1.9 ± 0.2 vs. 1.0 ± 0.1) while pdf- Because pdfr-1 is particularly expressed in all 95 body wall mus-
2(tm4393) mutants reverse less often (0.6 ± 0.2). The pdfr-1(lst34) cles (Janssen et al., 2008) and pdfr-1(lst34) mutants show such se-
receptor mutants also display more reversals (1.7 ± 0.1). In con- vere locomotion defects, the question arose whether or not the
trast, pdf-1(tm1996);pdf-2(tm4393) double mutants behave like muscle structure in these mutants is intact. In addition, a proteo-
wild types (1.2 ± 0.2) (Fig. 1B). mic experiment which compared the proteome of pdf-1(tm1996)
As a consequence of living in the soil, C. elegans regularly mutants with wild type N2 animals, revealed that the triose phos-
encounters water where it will exert another type of behavior phate isomerase TPI-1 was less abundant in the pdf-1(tm1996) mu-
named swimming or thrashing. While some groups argue that tants (Temmerman et al., 2011). Knock down of tpi-1 results in
these are the same types of locomotion but look different in differ- defective body wall muscle myosin organization (Meissner et al.,
ent media (solid or liquid medium) (Majmudar et al., 2012), others 2009). Inspection of Normaski DIC images and transgenic strains
using the myosin heavy chain marker Pmyo-3::mCherry failed to re-
veal any signs of muscle deterioration such as disorganized sarco-
meres, indicating that the sluggish phenotype of pdfr-1(lst34)
animals is unlikely to be a result of anatomical defects caused by
the absence of PDF receptor signaling (Supplemental Fig. 2).
In order to gain information on where and which specific PDF
receptor splice variants are involved in locomotion, we generated
a battery of splice variant-specific rescue strains. These express
only one of the receptor splice variants in either body wall muscles
(Phlh-1) or neurons (Punc-119). Expression of either Phlh-1::pdfr-1a, b or
c did not result in restoration of activity (not shown). Pan-neuronal
expression of either pdfr-1a, b or c suffices to restore pdfr-1(lst34)
movement to wild type levels (Fig. 3B). The experiments displayed
in Fig. 3A–C were conducted in U-bottom 96-well plates while the
experiment displayed in Fig. 3D was conducted in a flat bottom 96-
well plate. The swimming activity of worms in U-bottom wells is
generally higher than in flat bottom wells since the shape of the
well causes the worms to accumulate exactly in the path of the la-
ser beam which detects activity. In flat bottom wells, the nema-
todes are more spread out and interfere less often with the laser
beam. The decreased swimming activity of pdfr-1(lst34) is there-
fore more pronounced in flat bottom wells as is the case for other
locomotion defective strains (not shown).
The basal slowing response (BSR) is a mechanism that decreases
the locomotion speed of crawling C. elegans after encountering bac-
Fig. 1. Locomotion speed and reversals. (A) Speed of wild type N2 (WT), pdf- terial food resources (Sawin et al., 2000). The BSR is initiated in
1(tm1996), pdf-2(tm4393), pdfr-1(lst34) and pdf-1(tm1996);pdf-2(tm4393) double well fed animals through a dopaminergic pathway after the per-
mutants. The number of tracks analyzed is indicated at the base of each column. The ception of mechanical stimulation by bacteria. In order to deter-
analysis of one single movie generates multiple tracks. (B) Number of reversals mine if pdf-1, pdf-2 and/or pdfr-1 are involved in this kind of
during one minute of wild type N2 (WT), pdf-1(tm1996), pdf-2(tm4393), pdfr-
1(lst34) and pdf-1(tm1996);pdf-2(tm4393) double mutants. Number of animals
behavior, we examined their responses to food. Particularly, we
tested is indicated at the base of each column. Error bars indicate S.E.M. ⁄p 6 0.05, examined the BSRs of pdf-1(tm1996), pdfr-1(lst34) and pdf-2 over-
⁄⁄
p 6 0.005 and ⁄⁄⁄p < 0.0005. expression strains since these already move slower than wild type
236 E. Meelkop et al. / Molecular and Cellular Endocrinology 361 (2012) 232–240

Fig. 2. Footprints. Footprints after 24 h of wild type N2 (WT) and pdfr-1(lst34) animals. Scale bars indicate 1 mm.

Fig. 3. Swimming activity. (A) Swimming activity of wild type N2 (WT), pdf-1(tm1996), pdf-1 rescue, pdf-1 over-expression (pdf-1 +), pdf-2(tm4393), pdf-2 rescue, pdf-2 over-
expression (pdf-2 +), pdf-1(tm1996); pdf-2(tm4393), pdfr-1(lst34), pdfr-1 rescue and pdfr-1 over-expression (pdfr-1 +) strains measured in U-bottom wells. (B) Swimming
activity of wild type N2 (WT), pdfr-1(lst34) and Punc-119::pdfr-1a, Punc-119::pdfr-1b and Punc-119::pdfr-1c rescue strains measured in flat bottom wells. Error bars indicate S.E.M.
⁄
p 6 0.05, ⁄⁄p 6 0.005, ⁄⁄⁄p < 0.0005.

animals. Nevertheless, the BSR is maintained and well fed pdf-

1(tm1996), pdfr-1(lst34) and pdf-2 over-expression strains become
even slower when encountering food (Fig. 4).

3.3. Egg-laying

Although PDF is not known for its involvement in reproduction,

Hamanaka et al. (2005) have suggested that PDF is involved in the
induction of reproductive diapause in the blow fly Protophormia terra-
enovae. The involvement of PDF signaling in egg-laying in C. elegans
hermaphrodites was determined by observing the progression of
egg-laying during four and a half day of the reproductive stage. Syn- Fig. 4. The basal slowing response (BSR). The BSR of wild type N2 (WT), pdf-
chronized L4 nematodes were selected and transferred to freshly 1(tm1996), pdfr-1(lst34) and animals over-expressing pdf-2 (pdf-2 +). = Animals
seeded NGM plates every 12 h. Progeny was counted in L3/L4 stage transferred from plates with food to plates without food. + = Animals transferred
from plates with food to other plates with food. Error bars indicate S.E.M. ⁄p 6 0.05,
as to determine the total amount of progeny produced (Fig. 5) as well ⁄⁄⁄
p 6 0.0005, when comparing food conditions for each strain.
as the progression of egg-laying (Fig. 6). While the total amount of
progeny is not effected in pdf-1(tm1996), pdf-2(tm4393), pdf-
1(tm1996);pdf-2(tm4393) or pdfr-1(lst34) mutants (Fig. 5), the tim- 2(tm4393) and pdf-1(tm1996);pdf-2(tm4393) (Fig. 6A–C). This effect
ing of the egg-laying peak is delayed in pdf-1(tm1996), pdf- can be rescued in pdf-1(tm1996) animals by re-introducing wild type
 E. Meelkop et al. / Molecular and Cellular Endocrinology 361 (2012) 232–240 237

copies of the pdf-1 gene (Fig. 6A). The delay of the reproductive peak GPCRs remain orphan receptors, with ligands yet to be identified
of pdf-2(tm4393) animals could be confirmed in a second loss- (Janssen et al., 2010). Of the six predicted secretin receptor family
of-function strain pdf-2(tm4780) (Fig. 6B). Compared to the single GPCR genes in C. elegans, only pdfr-1 has been deorphaned (Harmar,
mutants, double pdf-1(tm1996);pdf-2(tm4393) mutants show a 2001; Cardoso et al., 2006; Janssen et al., 2008; Janssen et al., 2009).
similar delay (Fig. 6C). Elimination of properly functioning PDF pdfr-1 codes for three different receptor isoforms which in vitro all
receptors does, however, not result in a shift off the egg-laying peak bind the three PDF peptides: PDF-1a, PDF-1b and PDF-2. Although
(Fig. 6D) and this could be confirmed with a second knockout allele it was already known that a deletion in the pdf-1 gene (encoding
for pdfr-1(tm4457). PDF-1a and PDF-1b) results in locomotion defects, the exact role of
Although the expression patterns of pdf-1, pdf-2 and pdfr-1 have pdf-2 and pdfr-1 in locomotion remained elusive so far, due to the
been largely defined (Janssen et al., 2008), we now looked for inexistence of knockout mutants. Our isolation of a pdfr-1 mutant,
specific expression in relation to the egg-laying apparatus. A trans- followed by the generation of two pdf-2 mutants and an additional
genic GFP-expressing strain indicates that pdfr-1 expression is pdfr-1 mutant by the Mitani group (Department of Physiology, To-
present in the toroid vulD cell (Supplemental Fig. 3). This cell encir- kyo Woman’s Medical University School of Medicine, Japan) allowed
cles the vulval passageway and is attached to vulva muscles regu- us to characterize the other molecular players of the PDF signaling
lating the passage of eggs (Inoue et al., 2005). Expression of pdf or system in C. elegans.
pdfr genes could not be observed in the vulva- or uterine muscles
themselves or in other vulva-associated cells such as the HSN mo- 4.1. Locomotion
tor neuron nor anywhere else in the hermaphrodite reproductive
system. The Parallel Worm Tracker was used to analyze the crawling
speed of pdf-1(tm1996), pdf-2(tm4393), pdf-1(tm1996);pdf-
2(tm4393) double mutants and pdfr-1(lst34) mutants and confirmed
3.4. A constitutively active receptor
the previously reported locomotion defects of pdf-1(tm1996) mu-
tants. They are slower than wild type animals while conducting
In general, GPCRs of the secretin receptor family regulate intra-
more forward/backward transitions (Janssen et al., 2008). The recep-
cellular concentrations of cAMP by signaling through the heterotri-
tor (pdfr-1(lst34)) mutants move similarly though are more severely
meric G protein Gas and adenylyl cyclase (Harmar, 2001; Mertens
disrupted in their crawling behavior, indicating that the PDF-1a and/
et al., 2005). By means of an in vitro CRE-luciferase cAMP reporter
or PDF-1b peptides and PDF receptors function in the same signaling
assay, Janssen et al. (2008) have shown that any of the three PDF
pathway in vivo. The role of PDF-2 in locomotion seems to be limited
neuropeptides elicited increased cAMP levels in HEK293 cells that
to stimulating reversals as pdf-2 mutants conduct fewer reversals
express either the PDFR-1a or PDFR-1b receptor. In contrast, PDFR-
but display a wild type-like speed. The proposed antagonistic effect
1c acts trough a Gai/o type of G protein and causes cAMP levels to
of pdf-1 and pdf-2 in Janssen et al. (2008) is supported by the present
decrease (Janssen et al., 2008). Additional experiments with for-
findings, at least when considering the amount of reversals as these
skolin induced cAMP release show that PDFR-1c is constitutively
are elevated in pdf-1(tm1996), reduced in pdf-2(tm4393) and normal
active without addition of any of the PDF peptides (Supplemental
in pdf-1(tm1996);pdf-2(tm4393) double mutants.
Fig. 4).
In addition to crawling, C. elegans is able to swim. The same mu-
tants were challenged in a swimming assay in order to determine
4. Discussion whether their mutations induce similar changes in activity. From
our results, it can be assumed that PDF signaling is involved in
GPCRs play an important role in modulating neuron and muscle the general modulation of locomotion and not specific for a certain
activity, and represent a major group of pharmacological targets gait since both pdf-1(tm1996) and pdfr-1(lst34) mutants are less ac-
contributing to 50% of all modern drugs (Roman and Traynor, tive, while pdf-2(tm4393) mutants show no aberrant activity
2011). Various C. elegans GPCRs showed to have an important bio- changes. pdf-1(tm1996);pdf-2(tm4393) double mutants, however,
logical function in for example egg-laying, reproduction, male mat- display a decreased activity level, similar to pdf-1(tm1996) mu-
ing behavior and social behavior (Segalat et al., 1995; de Bono and tants, implying that pdf-2 is not involved in the regulation of
Bargmann, 1998; Janssen et al., 2008; Roman and Traynor, 2011). swimming.
Despite the obvious significance of these receptors, most C. elegans Taken together, crawling behavior is normal in double pdf-
1(tm1996);pdf-2(tm4393) mutants, but swimming activity is re-
duced in these animals. This could be explained as follows: the ab-
sence of PDF-2 decreases the reversal frequency and thus
increases speed on top of the decrease caused by absence of PDF-
1a and/or PDF-1b peptides, resulting in wild type speed in double
neuropeptide mutants. Swimming animals, however, do not reverse
in a way that crawling animals do and this might explain the low-
ered activity levels of pdf-1(tm1996);pdf-2(tm4393) mutants. This
explanation, however, contradicts the decreased activity levels of
animals bearing additional pdf-2 copies but one should note that
pdf-2 transcript levels in these worms are over 15 times upregulated
(Janssen et al., 2009) which might create such a strong negative ef-
fect on locomotion or indirect (toxic) side effects of the transgenes
which result in the observed phenotype.
It is interesting to note that the pdfr-1(lst34) locomotion pheno-
type is stronger than that of pdf-1(tm1996). Previous in vitro exper-
iments have shown that only the PDFR-1a and PDFR-1b receptors
Fig. 5. Total brood size of hermaphrodites. The total brood size of wild type N2
are able to bind the PDF neuropeptides in physiologically relevant
(WT), pdf-1(tm1996), pdf-2(tm4393), pdf-1(tm1996);pdf-2(tm4393) and pdfr-1(lst34). doses. This suggests that locomotion is not only regulated by the
Numbers at the base of each column indicate number of animals tested. PDF peptides through PDFR-1a and PDFR-1b, but also governed
238 E. Meelkop et al. / Molecular and Cellular Endocrinology 361 (2012) 232–240

Fig. 6. Progression of egg-laying. The number of offspring in wild type N2 (WT), (A) pdf-1(tm1996) and pdf-1 rescue, (B) pdf-2(tm4393) and pdf-2(4780), (C) pdf-1(tm1996);pdf-
2(tm4393) double mutants and (D) pdfr-1(lst34) and pdfr-1(tm4457) mutants. Error bars indicate S.E.M. p 6 0.05, p 6 0.005 and p < 0.005, compared to WT.

by constitutively active PDFR-1c receptor. Although in vitro exper- sensation of bacteria and expresses pdf-1 (Sawin et al., 2000;
iments do not necessarily reflect in vivo receptor activity, the Janssen et al., 2009). We show that the BSR is conserved in pdf-
rescue experiments which involve the reintroduction of the indi- 1(tm1996) and pdfr-1(lst34) mutants and in pdf-2 over-expression
vidual receptor splice variants confirm that each of the three recep- animals and although all tested strains are generally slower than
tors is capable of restoring swimming activity in pdfr-1(lst34) wild types, the worms are capable of an additional deceleration.
mutants. Moreover, despite the prominent pdfr-1 expression in Based on these results, the possibility that the PDF signaling path-
body wall muscles, only pan-neuronal expression of the receptors way exerts its effects on the (mechanosensory) input level, should
rescues swimming activity. Therefore, it can be assumed that all not be completely abandoned as both PLM and ALM mechanosen-
three PDF receptor isoforms are involved in the regulation of loco- sory neurons express pdfr-1 as well these cells were not shown to
motion and that expression in neurons rather than in muscles is be involved in the BSR or ESR (Sawin et al., 2000; Janssen et al.,
important. We propose that PDF-1a and/or PDF-1b neuropeptides 2008). Stimulation of ALM and PLM induces backward and forward
signal through PDFR-1a and PDFR-1b in neurons stimulating loco- movement respectively through a complicated system of interneu-
motion, while the PDFR-1c receptor exerts its actions through loco- rons and motor neurons (de Bono and Maricq, 2005). Note that
motion-inhibiting neurons. Of course this does not rule out the among these interneurons, AVD also expresses pdf-2 (Janssen
possibility that PDFR signaling in body wall muscles is used to fine et al., 2008). In addition to mechanosensory neurons, several
tune movement in particular circumstances. No severe disruptions chemosensory neurons such as the amphids ASI and ASK express
in muscle morphology of pdfr-1 mutants could be detected by vi- pdf-1 and the plasmids PHA and PHB express pdf-2 and pdfr-1
sual inspection of the body wall muscles using Nomarski DIC or (Janssen et al., 2008, 2009). In addition to mechanical cues, chemo-
the Pmyo-3::mCherry marker. This also excludes a major function sensory signals could contribute to the C. elegans exploratory
in muscle development and supports the presumed neurotrans- behavior while the reversal-inhibiting interneuron RIM expressing
mitting/modulating nature of the PDF receptor. pdf-2, might modulate chemosensory controlled turning (Gray
et al., 2005).
4.2. The basal slowing response
4.3. Egg-laying
C. elegans modulates its speed according to food conditions:
well fed animals will lower their speed when encountering bacte- In the past, PDF has been suggested to be involved in the timing
ria and exert a basal slowing response (BSR) in order to maintain of reproductive diapause in the blow fly Protophormia terraenovae,
proximity to the newly found food source (Sawin et al., 2000). Dis- based on immunohistochemistry data (Hamanaka et al., 2005).
ruptions in this behavior are caused by failure to detect the bacte- Several other neuropeptides and GPCRs already have been shown
ria or by disruptions in the dopaminergic networks passing on to modulate reproduction (Ringstad and Horvitz, 2008; Lindemans
these signals. The BSR assay was used to determine whether the et al., 2009). Knockdown of the C. elegans adipokinetic hormone-
pdf-1(tm1996) and pdfr-1(lst34) mutants experience a disrupted like neuropeptide Ce-AKH-GnRH and/or its receptor Ce-GnRHR is
perception of feeding conditions, possibly through the mechano- known to induce a delayed timing of egg-laying (Lindemans
sensory neurons expressing these genes. The mechanosensory et al., 2009). The EGL-6 GPCR inhibits egg-laying through Gai/o as
ADE neuron for example, was shown to be involved in BSR related do its ligands, FLP-10 and FLP-17 (Ringstad and Horvitz, 2008).
 E. Meelkop et al. / Molecular and Cellular Endocrinology 361 (2012) 232–240 239

Also, proprotein convertase EGL-3 and the carboxypeptidase E movement. Whether neuronal control is situated at the sensory
ortholog EGL-21, which are necessary for proper processing of and/or interneuron level remains to be determined. In addition,
FMRFamide-like (FLP) precursors and neuropeptide-like protein PDF signaling turns out to be involved in egg-laying and we propose
(NLP) precursors, stimulate egg-laying, indicating that various pep- that this is also under influence of the PDF system at the level of
tides are likely to be involved in the regulation of egg-laying (Kass sensory input. pdfr-1 is clearly a multifunctional locus and the
et al., 2001; Jacob and Kaplan, 2003; Husson et al., 2006, 2007a). chances are that in the future, additional functions can be allocated
This is the first time that the PDF system could be functionally to the PDF receptors and their ligands.
linked to egg-laying as pdf-1(tm1996), pdf-2(tm4393) and pdf-
1(tm1996);pdf-2(tm4393) mutants show a delayed timing of the Acknowledgements
reproductive peak. These effects are gene-specific as the pdf-1 ef-
fect could be rescued by re-introducing copies of the pdf-1 gene We would like to thank the Caenorhabditis Genetics Center,
and the pdf-2(tm4393) delay was also observed in mutants with which is funded by the NIH National Centre for Research Resources
another pdf-2 allele (tm4780). The magnitude of the pdf-1 and (NCRR) for the wild type strains and Dr. Shohei Mitani (Tokyo Wo-
pdf-2 deletions’ effect is similar: both cause a delay in the egg- men’s Medical University, Tokyo) for the tm1996, tm4393 and
laying peak of roughly twelve hours. A combination of these two tm4457 allele strains. We also thank Prof. Dr. E. Cuppen, dr. H.C.
mutations in a pdf-1;pdf-2 double mutant does not add up as the Korswagen and M. Harterink for sharing their experience with
egg-laying peak is not additionally delayed, suggesting that all mutagenesis and knockout screens, E. Vanvlasselaer and W. De
PDF ligands modulate the same pathway in this biological process. Haes for statistical support, L. Vanden Bosch, J. Gijbels and S.
Two different pdfr-1 deletion alleles, lst34 and tm4457 did not re- Simonetta for their excellent technical support and D. Van Meensel
sult in a clear shift of the reproductive peak. The fact that knocking (Zeiss) for the confocal images. EM is supported by the Flemish
out the PDF receptors does not have the same effect as knocking government agency for Innovation by Science and Technology
out the PDF neuropeptides seems contradictory but can be ex- (IWT-vlaanderen) and a faculty research fund of the KU Leuven
plained as follows: While pdf-1 and pdf-2 promote egg-laying (FLOF). LT, TJ, SJH and IB are supported by the FWO Flanders.
through PDFR-1a and PDFR-1b, PDFR-1c is constitutively active
and inhibits egg-laying as it does not signal through the cAMP
Appendix A. Supplementary data
stimulating Gas protein, but the inhibiting Gai/o protein. This
way, elimination of all three receptors does not have an effect on
Supplementary data associated with this article can be found, in
the timing of egg-laying. An alternative explanation would be that
the online version, at http://dx.doi.org/10.1016/j.mce.2012.05.001.
the PDF neuropeptides are able to signal through another receptor.
In vitro experiments with a variety of C. elegans GPCRs conducted in
our laboratory have, however, not revealed any suitable References
candidates.
Bargmann, C.I., 1998. Neurobiology of the Caenorhabditis elegans genome. Science
The involvement of the PDF signaling system in egg-laying 282, 2028–2033.
behavior can to some extent be supported by pdfr-1 expression Cardoso, J.C., Pinto, V.C., Vieira, F.A., Clark, M.S., Power, D.M., 2006. Evolution of
data. According to a previous localization study, pdfr-1 is expressed secretin family GPCR members in the metazoa. BMC Evol. Biol. 6, 108.
Chalfie, M., Sulston, J., 1981. Developmental genetics of the mechanosensory
in all 95 body wall muscle cells, mechanosensory neurons (PLM, neurons of Caenorhabditis elegans. Dev. Biol. 82, 358–370.
ALM, FLP, OLQD and OLQV), chemosensory neurons (PHA and De Bono, M., Bargmann, C.I., 1998. Natural variation in a neuropeptide Y receptor
PHB), ring motor neurons (RMED and RMEV) and in the pharyngeal homolog modifies social behavior and food response in C. elegans. Cell 94, 679–
689.
interneuron pair I1 (Janssen et al., 2008). We here show that, in De Bono, M., Maricq, A.V., 2005. Neuronal substrates of complex behaviors in C.
addition, pdfr-1 is expressed in the vulD cell of the hermaphrodite elegans. Annu. Rev. Neurosci. 28, 451–501.
vulva. This toroid cell encircles the vulval passageway and is at- Fredriksson, R., Schiöth, H.B., 2005. The repertoire of G-protein-coupled receptors in
fully sequenced genomes. Mol. Pharmacol. 67, 1414–1425.
tached to vulva muscles regulating the passage of eggs (Inoue Gray, J.M., Hill, J.J., Bargmann, C.I., 2005. A circuit for navigation in Caenorhabditis
et al., 2005). The exact function of the vulD cell is unknown and elegans. Proc. Natl. Acad. Sci. USA 102, 3184–3191.
it is unsure if the observed phenotypes can be explained by PDF Hamanaka, Y., Yasuyama, K., Numata, H., Shiga, S., 2005. Synaptic connections
between pigment-dispersing factor-immunoreactive neurons and neurons in
signaling in the vulD cell. pdfr-1 expression could not be detected
the pars lateralis of the blow fly Protophormia terraenovae. J. Comp. Neurol. 491,
in the vulva- or uterine muscles or in other vulva-associated cells 390–399.
such as the HSN motor neuron or VC neurons, nor anywhere else Harmar, A.J., 2001. Family-B G-protein-coupled receptors, Gen. Biol. 2.
in the reproductive system. It is also possible that PDF signaling Helfrich-Forster, C., Tauber, M., Park, J.H., Muhlig-Versen, M., Schneuwly, S.,
Hofbauer, A., 2000. Ectopic expression of the neuropeptide pigment-
does not influence egg-laying on the level of the egg-laying appa- dispersing factor alters behavioral rhythms in Drosophila melanogaster. J.
ratus itself but rather through a sensory pathway. As stated above, Neurosci. 20, 3339–3353.
pdfr-1 expression has been determined in several mechano- and Husson, S.J., Clynen, E., Baggerman, G., De, L.A., Schoofs, L., 2005a. Discovering
neuropeptides in Caenorhabditis elegans by two dimensional liquid
chemosensory neurons. Of those, mechanosensory neuron PLM chromatography and mass spectrometry. Biochem. Biophys. Res. Commun.
provides an indirect synaptic input to the vulva muscle through 335, 76–86.
HSN (Chalfie and Sulston, 1981). Husson, S.J., Clynen, E., Baggerman, G., De, L.A., Schoofs, L., 2005b. Peptidomics of
Caenorhabditis elegans: in search of neuropeptides. Commun. Agric. Appl. Biol.
Sci. 70, 153–156.
5. Conclusions Husson, S.J., Clynen, E., Baggerman, G., Janssen, T., Schoofs, L., 2006. Defective
processing of neuropeptide precursors in Caenorhabditis elegans lacking
proprotein convertase 2 (KPC-2/EGL-3): mutant analysis by mass
As opposed to other known PDF systems such as the one in spectrometry. J. Neurochem. 98, 1999–2012.
D. melanogaster, the C. elegans system contains multiple PDF neuro- Husson, S.J., Janssen, T., Baggerman, G., Bogert, B., Kahn-Kirby, A.H., Ashrafi, K.,
peptides and receptors, making it a challenge to dissect these nem- Schoofs, L., 2007a. Impaired processing of FLP and NLP peptides in
carboxypeptidase E (EGL-21)-deficient Caenorhabditis elegans as analyzed by
atode PDF signaling pathways. We have demonstrated that PDF
mass spectrometry. J. Neurochem. 102, 246–260.
receptor deletion mutants (pdfr-1(lst34)) phenocopy the sluggish Husson, S.J., Mertens, I., Janssen, T., Lindemans, M., Schoofs, L., 2007b.
locomotor behavior of pdf-1(tm1996) mutants and that all three Neuropeptidergic signaling in the nematode Caenorhabditis elegans. Prog.
PDF receptors are involved in the regulation of this behavior. More- Neurobiol. 82, 33–55.
Inoue, T., Wang, M., Ririe, T.O., Fernandes, J.S., Sternberg, P.W., 2005. Transcriptional
over, this is mainly regulated at the level of the signal-transducing network underlying Caenorhabditis elegans vulval development. Proc. Natl.
neurons rather than the body wall muscle, which finally execute Acad. Sci. USA 102, 4972–4977.
240 E. Meelkop et al. / Molecular and Cellular Endocrinology 361 (2012) 232–240

Jacob, T.C., Kaplan, J.M., 2003. The EGL-21 carboxypeptidase E facilitates Pierce-Shimomura, J.T., Chen, B.L., Mun, J.J., Ho, R., Sarkis, R., McIntire, S.L., 2008.
acetylcholine release at Caenorhabditis elegans neuromuscular junctions. J. Genetic analysis of crawling and swimming locomotory patterns in C. elegans.
Neurosci. 23, 2122–2130. Proc. Natl. Acad. Sci. USA 105, 20982–20987.
Janssen, T., Husson, S.J., Lindemans, M., Mertens, I., Rademakers, S., Ver, D.K., Ramot, D., Johnson, B.E., Berry Jr., T.L., Carnell, L., Goodman, M.B., 2008. The Parallel
Geysen, J., Jansen, G., Schoofs, L., 2008. Functional characterization of three G Worm Tracker: a platform for measuring average speed and drug-induced
protein-coupled receptors for pigment dispersing factors in Caenorhabditis paralysis in nematodes. PLoS ONE 3, e2208.
elegans. J. Biol. Chem. 283, 15241–15249. Rao, K.R., Riehm, J.P., 1993. Pigment-dispersing hormones. Ann. NY Acad. Sci. 680,
Janssen, T., Husson, S.J., Meelkop, E., Temmerman, L., Lindemans, M., Verstraelen, K., 78–88.
Rademakers, S., Mertens, I., Nitabach, M., Jansen, G., Schoofs, L., 2009. Discovery Renn, S.C., Park, J.H., Rosbash, M., Hall, J.C., Taghert, P.H., 1999. A pdf neuropeptide
and characterization of a conserved pigment dispersing factor-like gene mutation and ablation of PDF neurons each cause severe abnormalities of
neuropeptide pathway in Caenorhabditis elegans. J. Neurochem. 111, 228–241. behavioral circadian rhythms in Drosophila. Cell 99, 791–802.
Janssen, T., Lindemans, M., Meelkop, E., Temmerman, L., Schoofs, L., 2010. Ringstad, N., Horvitz, H.R., 2008. FMRFamide neuropeptides and acetylcholine
Coevolution of neuropeptidergic signaling systems: from worm to man. Ann. synergistically inhibit egg-laying by C. elegans. Nat. Neurosci. 11, 1168–1176.
NY Acad. Sci. 1200, 1–14. Roman, D.L. Traynor, J.R., 2011. Regulators of G-protein signaling (RGS) proteins as
Kass, J., Jacob, T.C., Kim, P., Kaplan, J.M., 2001. The EGL-3 proprotein convertase drug targets: modulating G-protein-coupled receptor (GPCR) signal
regulates mechanosensory responses of Caenorhabditis elegans. J. Neurosci. 21, transduction. J Med. Chem. [Epub ahead of print].
9265–9272. Sawin, E.R., Ranganathan, R., Horvitz, H.R., 2000. C. elegans locomotory rate is
Lesa, G.M., 2006. Isolation of Caenorhabditis elegans gene knockouts by PCR modulated by the environment through a dopaminergic pathway and by
screening of chemically mutagenized libraries. Nat. Protoc. 1, 2231–2240. experience through a serotonergic pathway. Neuron 26, 619–631.
Li, C., 2005. The ever-expanding neuropeptide gene families in the nematode Schiöth, H.B., Fredriksson, R., 2005. The GRAFS classification system of G-protein
Caenorhabditis elegans. Parasitology 131 (Suppl), S109–S127. coupled receptors in comparative perspective. Gen. Comp. Endocrinol. 142, 94–
Li, C., Kim, K., 2008. Neuropeptides. WormBook, 1–36. 101.
Lindemans, M., Liu, F., Janssen, T., Husson, S.J., Mertens, I., Gade, G., Schoofs, L., 2009. Segalat, L., Elkes, D.A., Kaplan, J.M., 1995. Modulation of serotonin-controlled
Adipokinetic hormone signaling through the gonadotropin-releasing hormone behaviors by Go in Caenorhabditis elegans. Science 267, 1648–1651.
receptor modulates egg-laying in Caenorhabditis elegans. Proc. Natl. Acad. Sci. Simonetta, S.H., Golombek, D.A., 2007. An automated tracking system for
USA 106, 1642–1647. Caenorhabditis elegans locomotor behavior and circadian studies application. J.
Majmudar, T., Keaveny, E.E., Zhang, J., Shelley, M.J., 2012. Experiments and theory of Neurosci. Methods 161, 273–280.
undulatory locomotion in a simple structured medium. J. R. Soc. Interf. [Epub Temmerman, L., Bogaerts, A., Meelkop, E., Cardoen, D., Boerjan, B., Janssen, T.,
ahead of print]. Schoofs, L., 2011 A proteomic approach to neuropeptide function elucidation.
Meelkop, E., Temmerman, L., Schoofs, L., Janssen, T., 2011. Signalling through Peptides [Epub ahead of print].
pigment dispersing hormone-like peptides in invertebrates. Prog. Neurobiol. 93, The C. elegans Sequencing Consortium, 1998. Genome sequence of the nematode C.
125–147. elegans: a platform for investigating biology. Science 282, 2012–2018.
Meissner, B., Warner, A., Wong, K., Dube, N., Lorch, A., McKay, S.J., Khattra, J., Thomas, J.H., Robertson, H.M., 2008. The Caenorhabditis chemoreceptor gene
Rogalski, T., Somasiri, A., Chaudhry, I., Fox, R.M., Miller 3rd, D.M., Baillie, D.L., families. BMC Biol. 6, 42.
Holt, R.A., Jones, S.J., Marra, M.A., Moerman, D.G., 2009. An integrated strategy Toma, D.P., White, K.P., Hirsch, J., Greenspan, R.J., 2002. Identification of genes
to study muscle development and myofilament structure in Caenorhabditis involved in Drosophila melanogaster geotaxis, a complex behavioral trait. Nat.
elegans. PLoS Genet. 2009 (5), e1000537. Genet. 31, 349–353.
Mertens, I., Vandingenen, A., Johnson, E.C., Shafer, O.T., Li, W., Trigg, J.S., De, L.A., Vanden Broeck, J.V., 2001. Insect G protein-coupled receptors and signal
Schoofs, L., Taghert, P.H., 2005. PDF receptor signaling in Drosophila contributes transduction. Arch. Insect Biochem. Physiol. 48, 1–12.
to both circadian and geotactic behaviors. Neuron 48, 213–219. White, J.G., Southgate, E., Thomson, J.N., Brenner, S., 1986. The structure of the
Nathoo, A.N., Moeller, R.A., Westlund, B.A., Hart, A.C., 2001. Identification of nervous-system of the nematode Caenorhabditis elegans. Philosophical
neuropeptide-like protein gene families in Caenorhabditis elegans and other Transactions of the Royal Society of London Series B-Biological Sciences 314,
species. Proc. Natl. Acad. Sci. USA 98, 14000–14005. 1–340.