Ž .Brain Research 848 1999 26–34www.elsevier.comrlocaterbres

Interactive report

FMRFamide-related neuropeptide gene family in Caenorhabditis elegans 1

Chris Li ), Kyuhyung Kim, Laura S. Nelson 2

Department of Biology, Boston UniÕersity, 5 Cummington St., Boston, MA 02215, USA

Accepted 10 August 1999

Abstract

Neuropeptides are used as signaling molecules in the nervous system of most organisms, including mammals. The family ofŽ . Ž .FMRFamide Phe-Met-Arg-Phe-NH2 -like neuropeptides FaRPs all share an RFamide sequence at their C-termini and have been shown

to have diverse functions in the central and peripheral nervous systems. In the nematode Caenorhabditis elegans, FMRFamide-likeŽ .peptides FaRPs are expressed in at least 10% of the neurons, including motor, sensory, and interneurons that are involved in movement,

feeding, defecation, and reproduction. Twenty-two genes, designated flp-1 through flp-22, encode FaRPs in C. elegans, although thereare likely to be additional flp genes to be identified. Each flp gene encodes a different set of FaRPs, yielding a predicted total of 59distinct FaRPs; a few of the genes may also encode non-FaRPs. Inactivation of some of the flp genes indicates that at least one flp genehas unique functions, while at least two flp genes appear to have overlapping functions with other flp genes. These results suggest that acomplex family of FaRPs have varied roles through all stages of development and in adulthood in C. elegans. q 1999 Elsevier ScienceB.V. All rights reserved.

Keywords: Peptide function; Expression pattern; Model organism

1. Introduction

ŽThe neuropeptide FMRFamide Phe–Met–Arg–Phe–.NH was first isolated as a cardioactive agent on mollus-2

w xcan heart 50 . Since then, a large family of related pep-Ž .tides FaRPs , which share a common C-terminal Arg–

Phe–amide, have been found in the nervous system ofwanimals representing all major phyla 13–16,18,20,22,25,

x42,45,52,62 . These peptides have been shown to havewdiverse functions, including cardioexcitation 23,26,29,

x w x30,50 control of muscle contraction 3,21,35,41 , and neu-w xromodulation 6,11,57,60 in invertebrates as well as anti-

w xopioid effects in vertebrates 27,62,63 . FaRPs are oftenco-localized with the classical transmitters acetylcholineŽw x . w x28,53,54 ; J. Duerr, pers. comm. , serotonin 5,54 , and

w xg-aminobutyric acid 54 . Interestingly, the primary se-quence of FMRFamide is contained within an endogenous

Ž .mammalian opioid heptapeptide YGGFMRF , suggestingthat enkephalins and FaRPs may have co-evolved from a

w xcommon ancestral peptide 24 .

) Corresponding author.1 Published on the World Wide Web on 30 August 1999.2 Current address: Axys Pharmaceuticals, 180 Kimball Way, South San

Francisco, CA 94080, USA.

2. Pharmacology and physiology of FaRPs in inverte-brates

Despite structural similarities among different FaRPs,individual peptides have distinct and diverse effects. Forexample, in muscle preparations from the nematode As-caris suum, application of FaRPs such as SDPNFLR-

Ž . Ž .Famide PF1 and SADPNFLRFamide PF2 relaxes mus-cle strips; this hyperpolarization is independent of neuralinput, does not appear to cause a change in input conduc-tance, and may be through the opening of non-specific

w xcation channels in muscle 21,38 . By contrast, applicationŽ .of FaRPs such as AVPGVLRFamide AF3 and GDVPG-

Ž .VLRFamide AF4 causes robust, long-lasting contractionsof dorsal muscle strips. The effects of KSAYMRFamideŽ .AF8rPF3 on Ascaris muscle are more complicated, anddepend on whether the peptide is applied to dorsal orventral muscle strips: application to ventral muscles causescontraction while application to dorsal muscles cause re-

w xlaxation 38 . These effects are only seen in the presence ofw xneural input and external calcium 38 . KNEFIRFamide

Ž . Ž .AF1 and KHEYLRFamide AF2 also elicit complexresponses. Application of these peptides to muscle stripsresults in an initial hyperpolarization, followed by anexcitatory phase of rhythmic contractions; the excitatory

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 01972-1

( )C. Li et al.rBrain Research 848 1999 26–34 27

w xphase is abolished when neural input is removed 3,13 .The same FaRP can also have different effects on the sametissue from different species. For instance, FMRFamidehas excitatory effects on many molluscan hearts, but in atleast one species, the clam Lampsilis claibornensis, FMR-

w xFamide elicits cardioinhibitory responses 47 . Collec-tively, these results suggest that FaRPs act through multi-ple neural and non-neural mechanisms and receptors.

3. Identification of a gene family encoding FaRPs inCaenorhabditis elegans

Use of invertebrate systems to study neuropeptide func-tion and regulation has many advantages. First, inverte-brates have a relatively small number of cells in theirnervous systems, and specific neurons are readily identifi-able from organism to organism. Second, the neural cir-cuitry underlying many behaviors has been determined, soit is possible to directly test a particular function of aneuropeptide in the circuit. Lastly, two of the invertebratesystems, namely the fruitfly Drosophila melanogaster andthe nematode Caenorhabditis elegans, allow for the ge-netic dissection of the role of specific neuropeptides.

We have used C. elegans as a model system to examinethe function of neuropeptide gene families. C. elegans hasa small nervous system consisting of only 302 neurons andis the sole organism for which the entire neural circuitry

w xhas been determined 61 . Furthermore, the analysis ofneuropeptide gene families in C. elegans has been greatly

w xaided by the completion of its genomic sequence 10 .Through conventional screens of cDNA libraries,GENEFINDER predictions from the C. elegans GenomeConsortium, and our BLAST screens of the C. elegansgenome, we have identified 22 C. elegans genes that

Žencode FaRPs. The gene family has been named flp for.FMRFamide-like peptides and the genes have been num-

Žbered based on their chronological identification i.e., flp-1.through flp-22 . Despite extensive BLAST searches, all

flp family members have probably not been identified; twow xFaRPs isolated biochemically from C. elegans 17 are not

encoded by any of the identified flp genes. BecauseŽneuropeptide genes are relatively small the coding region

.is often less than 1 kbp , they are difficult to predict withstandard GENEFINDER programs.

The flp genes are distributed throughout the genomeŽ .Fig. 1 . Two clusters of flp genes are located on chromo-somes IV and X. The FaRPs encoded by genes withinthese clusters are no more similar to each other than toFaRPs encoded by genes not in the clusters, suggestingthat the clusters did not arise through simple gene duplica-

Žtions. Each flp gene encodes a unique set of FaRPs Table.1 , yielding a total of 59 possible distinct FaRPs. Six of the

flp genes, flp-6, flp-8, flp-9, flp-14, flp-20, and flp-22,encode multiple copies of only one FaRP; three genes,

Fig. 1. Chromosomal localizations of the flp genes in C. elegans. All locations are approximate and based on information from the C. elegans sequencew x w xdatabase 10 , except for flp-1, whose location was determined by three factor crosses 44 . Chromosomes, indicated by lines, are shown from y13 to q21

map units. flp genes are shown below the line; nearby genetic markers are shown above the line. The position of flp-14 is tentative.

( )C. Li et al.rBrain Research 848 1999 26–3428

Table 1Predicted FLP neuropeptidesa

d d bcflp-1 F23B2 IV flp-7 F49E10 X flp-15 ZK525 III

USADPNFLRFG x3 SPMQRSSMVRFG GGPQGPLRFG

USQPNFLRFG x2 TPMQRSSMVRFG RGPSGPLRFG

UASGDPNFLRFG SPMERSAMVRFG

U bcSDPNFLRFG SPMDRSKMVRFG flp-16 F15D4 IIU

AAADPNFLRFGU dPNFLRFG flp-8 F31F6 X x2 AQTFVRFGAGSDPNFLRFG GQTFVRFG

Ux3 KNEFIRFGd bflp-2 W07E11 X flp-17 C52D10 IV

dflp-9 C36H8 IVSPREPIRFG x2 KSAFVRFG

ULRGEPIRFG x2 KPSFVRFG KSQYIRFG

d d bflp-3 W07E11 X flp-10 T06C10 IV flp-18 Y48D7A X

SPLGTMRFG QPKARSGYIRFG DFDGAMPGVLRFGTPLGTMRFG EMPGVLRFG

UcdEAEEPLGTMRFG flp-11 K02G10 X x3 SVPGVLRFGNPLGTMRFG EIPGVLRFGASEDALFGTMRFG AMRNALVRFG SEVPGVLRFGEDGNAPFGTMRFG ASGGMRNALVRFG DVPGVLRFGSAEPFGTMRFG NGAPQPFVRFGSADDSAPFGTMRFG

d bcNPENDTPFGTMRFG flp-12 C05E11 X flp-19 CEM79 X

dflp-4 C18D1 II RNKFEFIRFG WANQVRFGASWASSVRFG

dPTFIRFG flp-13 F33D4 IVbASPSFIRFG flp-20 E01H11 X

SDRPTRAMDSPLIRFGUcdflp-5 C03G5 X AADGAPLIRFG x2 AMMRFGUAPEASPFIRFG

bGAKFIRFG ASPSAPLIRFG flp-21 C26F1 VAGAKFIRFG SPSAVPLIRFGAPKPKFIRFG ASSAPLIRFG GLGPRPLRFG

d bc bflp-6 F07D3 V flp-14 Y37D8A III flp-22 F39H2 I

U Ux6 KSAYMRFG x4 KHEYLRFG x3 SPSAKWMRFG

UIsolated biochemically from C. elegans. See text for references.

a For each flp gene, the cosmid or YAC containing the genomic region,the linkage group of the gene, and the peptides encoded by the gene areindicated.b Exonrintron boundaries for some of the genes are not confirmed;encoded peptides may change. Number of copies of encoded peptides areindicated; common amino acids are bolded. The C-terminal glycine islikely to donate an amide group.c EST isolated.d cDNA isolated.

flp-10, flp-12, and flp-21, encode one copy of one FaRP.The remaining flp genes encode multiple FaRPs. With theexception of flp-17, the peptides encoded by each flpgene are of the same class and share a few amino acidsŽ .from one to four in common in addition to the RFamidemoiety at the C terminus. For instance, the flp-1 gene

encodes seven distinct peptides all terminating in PNFLR-w xFamide 51 . The flp-17 gene encodes two classes of

FaRPs, KSAFVRFamide and KSQYIRFamide. A few ofthe genes, such as flp-1 and flp-20, encode FaRPs as wellas peptides not related to FaRPs.

4. Expression of flp genes

To determine whether the flp genes are transcribed andwhether the exonrintron boundaries predicted by the C.elegans Genome Consortium are correct, two strategieswere used. First, primers against the predicted transcriptswere used to amplify reverse-transcribed cDNA; the prod-ucts were sequenced and compared to the predictions madeby GENEFINDER. Second, the C. elegans Expressed

Ž .Sequence Tag EST database was screened for EST se-quences corresponding to the different flp genes. Comple-mentary DNAs were isolated for flp-1 through flp-13,indicating that these genes are expressed. Alternative tran-

w x w xscripts were isolated for flp-1 51 , flp-2 43 , and flp-11w x43 . The alternative transcripts of flp-2 do not differ inthe number or type of FaRPs encoded. By contrast, theFaRPs encoded by the alternative transcripts of flp-1 andflp-11 differ by one peptide. Seven FaRPs are encoded byone flp-1 transcript, while the second transcript encodesonly six of the seven FaRPs. Similarly, one flp-11 tran-script encodes three FaRPs while the other flp-11 tran-script encodes only two of these FaRPs. The functionalconsequences of the alternative transcripts are unknown.

EST sequences were found for flp-5, flp-11, flp-14through flp-16, flp-18, and flp-19. At least 18 of the 22flp genes, therefore, are expressed in C. elegans. Unfortu-nately, many of the cDNAs represented by the EST se-quences are only partial cDNAs, so that the exact genomicorganization of flp-14, flp-16, flp-18, and flp-19 is uncer-tain. In addition, flp-20 through flp-22 were not predictedby the C. elegans Genome Consortium, nor have any ESTsequences for these genes been isolated. We are currentlyusing GENEFINDER programs to predict open readingframes for these genes, and will determine whether ourpredictions are correct by designing primers for amplifica-tion of predicted cDNAs.

To determine when flp genes are expressed, primersspecific for flp-1 through flp-13 were used to amplifycDNA reverse-transcribed from RNA isolated from eggs,animals from each of the four larval stages, and adultsw x43 . Most of the flp genes are expressed throughout thelife cycle of the animal. For three genes, flp-8, flp-9, andflp-13, no products could be amplified from adult cDNA,suggesting that these genes are either expressed at lowlevels or not expressed at all in adult animals. An antibody

Ž . w xagainst the FLP-8 peptide also known as AF2 13 ,however, stains cells in adult animals, indicating that thelevel of flp-8 transcription is lower in adults than indeveloping animals.

( )C. Li et al.rBrain Research 848 1999 26–34 29

5. Biochemical characterization of FaRPs in C. elegans

As previously reported for other neuropeptide genesw x19 , all predicted FaRPs may not be produced in C.elegans if putative cleavage sites in the propeptides are notused. To determine whether any or all of the FaRPs areproduced, several teams are collaborating on the biochemi-cal isolation of FaRPs from C. elegans. To date, 13 FaRPs

w w x w xderived from seven flp genes flp-1 52 , flp-6 34 , flp-8Ž .N. Marks, A. Maule, and C. Shaw, pers. commun. , flp-9w x w x w x Ž36 , flp-13 33 , flp-14 37 , and flp-18 N. Marks, A.

.xMaule, and C. Shaw, pers. commun. have been isolatedfrom C. elegans. Two FaRPs encoded by flp-16 and

w xflp-21 have been isolated from Ascaris 14,17 and arepresumably also produced in C. elegans. Several addi-tional FaRPs isolated from Ascaris or encoded by theafp-1 gene in Ascaris have sequence similarity to FaRPs

w xencoded by flp-4 and flp-5 14,17 . These results suggestthat many, if not all of the predicted FaRPs are indeedproduced.

6. Cell-specific expression of flp genes in C. elegans

Are multiple flp genes expressed in the same cells orare different flp genes expressed in distinct cells? Tobegin to address this question, we examined the cellularlocalization of FaRPs in C. elegans using an anti-FMRFamide antibody specific for the C-terminal Arg–

w xPhe–NH moiety of FMRFamide 54 . About 10% of the2

302 neurons, consisting of mainly motor and interneurons,

were found to be immunoreactive. This number of FaRP-expressing neurons in C. elegans, however, appears to bean underestimate. In Ascaris, a closely related nematode,about 60% of the neurons are FMRFamide-like immunore-

w x Ž .active 12 , and our current data see below indicates thatthe expression of flp genes in C. elegans is significantlymore widespread than initially reported.

Most polyclonal antibodies generated against variousFaRPs do not readily distinguish among the different

w xFaRPs 32,46,49 . Generation of specific FaRP antibodiesis complicated by the small size of the peptides and the

w xstructural similarities among class members. Nichols 39has successfully generated antibodies against specificFaRPs by capitalizing on the N-terminal differences among

w xsimilar peptides. Stretton and co-workers 56 used crudeC. elegans extracts as antigens to generate monoclonalantibodies, and a few were found to recognize FaRPs.However, only one of these monoclonal antibodies, 2438,recognizes a single FaRP, the FLP-8 peptide. Four cellswere detected in C. elegans with the 2438 antibody; noneof these cells were stained with our anti-FMRFamide

w xantibody 54 .The generation of antibodies that recognize specific

FaRPs or specific classes of FaRPs is both labor-intensiveand expensive. We have taken a molecular approach todetermine gene-specific expression patterns of the differentflp genes in C. elegans. Reporter constructs in which thepromoter region of each flp gene is amplified and placedupstream of either the lacZ or green fluorescent protein( )GFP reporter genes are used for germline transformationto generate transgenic animals in which the expression

Ž .Fig. 2. Cell-specific expression of flp genes in C. elegans. Transgenic animals expressing a green fluorescent protein gfp reporter construct under thecontrol of four flp different promoters show overlapping patterns of expression. Cells expressing specific flp genes are indicated. For flp-3, flp-5, andflp-6, cells are only seen in the anterior head region. flp-8-expressing cells are seen in the anterior head and posterior tail regions. The head region ofadults are shown, except for flp-8, where the entire length of a second stage larval animal is shown. Anterior is to the left. The intestine has endogenousautofluorescence in C. elegans. Scale bars20 mm.

( )C. Li et al.rBrain Research 848 1999 26–3430

patterns can be visualized. Thus far, the expression patternof nine flp genes has been analyzed. Many of the flp-ex-pressing cells were not detected with the anti-FMRFamideantisera, confirming that flp gene expression is moreprevalent than initially reported. With the exception of

w xflp-1 44 , the flp genes have distinct, but overlappingŽ .expression patterns for example, see Fig. 2 ; flp-1 is not

co-expressed with any of the other flp genes examined todate. These results suggest that some FaRPs have overlap-ping functions and some FaRPs have unique functions.

7. Function of flp genes

Why are there so many flp genes and so many FaRPsexpressed in C. elegans? How much functional redun-dancy is there among the different genes? To address thesequestions, we have begun a general screen to inactivateeach of the flp gene family members. Screens to isolateknockout animals in C. elegans are based on polymerasechain reaction methods that make no a priori assumptionsabout possible phenotypes. Thus far, animals carryingdeletions in four flp genes, flp-1, flp-3, flp-8, and flp-10,have been isolated. Three of these deletion lines will bediscussed below.

7.1. Function of flp-1

The flp-1 gene appears to be expressed only in in-w xterneurons in the head region of the animal 44 . TwoŽstrains carrying independent deletions in flp-1 yn2 and

.yn4 have been isolated. Both strains show several sensoryand motor deficits, suggesting that flp-1-expressing neu-rons are important intermediates in sensory and motor

w xpathways 44 . The phenotypes are more severe in yn2than in yn4 animals, suggesting that the yn2 allele repre-sents a complete loss-of-function mutation, while the yn4allele is a partial loss-of-function mutation.

Two sensory behaviors are altered in flp-1 deletionw xanimals 44 . Wild-type animals avoid regions of high

osmolarity. By contrast, flp-1 deletion animals lose theirability to sense regions of high osmolarity, although theyretain their ability to sense soluble and volatile odorants.Second, wild-type animals recoil and move backwardswhen their nose runs against an object. flp-1 deletionanimals do not respond to nose touch, although theirresponses to other touch stimuli, such as body touch, arenormal. flp-1 is expressed in an interneuron that receivesinput from the ASH neuron, a multi-modal sensory neuronthat mediates sensitivity to osmolarity and nose touch.Loss of flp-1 in this interneuron presumably interrupts theinformation flow from the ASH neuron to the motorneurons.

The most striking phenotype of flp-1 deletion animals,w xhowever, is their uncoordinated movement 44 . Wild-type

animals move in a sinusoidal waveform and move at

certain frequencies depending on their stage of develop-ment. flp-1 deletion animals have a very exaggerated

Ž .waveform referred to as loopy; Fig. 3 and are hyperactiveŽas assayed by counting the number of body bends per

.minute . These phenotypes can be rescued by germlinetransformation with a wild-type copy of flp-1. Further-

Ž .more, overexpression xs of flp-1 has the reciprocal ef-fects on movement; transgenic animals carrying a flp-1cDNA under the control of a heat shock promoter becomesluggish and their waveform is barely discernible after heat

Ž .shock Fig. 3 . flp-1 is expressed in interneurons thatsynapse directly on motor neurons controlling movement,and presumably lack of flp-1 disrupts the modulatoryeffects of the interneurons on the motor neurons. Levels offlp-1 peptides, therefore, must be tightly regulated to getcoordinated movement in C. elegans.

flp-1 deletion animals also show a wandering behaviorw x44 . When placed on a bacterial food source in an agar-filled petri dish, wild-type animals will remain on the foodsource until it is depleted, whereupon they will crawlbeneath the agar surface to search for food. flp-1 deletionanimals will wander off their food source, most frequentlyonto the sides of the plates, where they dessicate and die.It is unclear which pathway is affected in this behavior.The deletion animals can sense the bacterial food sourceand chemotax towards it; perhaps the animals are lackingthe signal to stop and feed when they reach their foodsource, and instead they continue to move until they hit theside of the plate.

Because some of the interneurons that express flp-1synapse onto neurons of the egg-laying system, the numberof progeny from flp-1 deletion hermaphrodites was exam-ined. Wild-type animals lay about 300 viable eggs during

Žtheir lifetime. In yn4 animals the brood size about 235.eggs is significantly lower than wild type; because most

yn2 animals wander off the plate before their reproductivestage is over, their brood size could not be accuratelydetermined. FLP-1 peptides, therefore, may modulate egglaying in wild-type animals. Such a role is supported bypharmacological data in C. elegans. Wild-type animals,when placed in liquid culture, are transiently inhibitedfrom egg laying; this inhibition can be overridden by

w xaddition of serotonin 59 . Addition of FLRFamide, thefour C-terminal amino acids in FLP-1 peptides, has noeffect on egg laying in liquid culture. However, FLR-Famide can potentiate the effects of serotonin, causing an

w xincreased number of eggs to be laid 54 . These resultssuggest that FLRFamide, and presumably FLP-1 peptides,act in the same pathway as serotonin to affect egg laying.

7.2. Function of other flp genes

The flp-3 gene is expressed in three pairs of neurons inthe anterior region of the animal: the OL1, IL1D, and theURB neurons. OL1 and IL1D neurons innervate the outerand inner labial sensilla, respectively, although their exact

( )C. Li et al.rBrain Research 848 1999 26–34 31

( )Fig. 3. Changing the levels of flp-1 gene expression affects locomotion. A. Wild-type animals move with a distinct sinusoidal waveform. B. flp-1 yn2deletion animals have an exaggerated, loopy waveform. C. Overexpression of flp-1. Transgenic animals carrying a flp-1 cDNA under the control of a heatshock promoter show a flattened waveform. Scale bars0.1 mm.

functions are unknown. URB cells are interneurons down-stream of several sensory neurons, including the IL1 cells.Animals in which the coding region of flp-3 has beencompletely deleted have been isolated. Thus far, the flp-3deletion animals have no phenotype, suggesting that flp-3has overlapping functions with another flp gene.

flp-8 is expressed in two sensory neurons, ASE andPVM, and the interneurons URX. This expression patternoverlaps with that of the flp-5, flp-6, and flp-12 genes.

w xASE mediates the response to soluble odorants 2 ; PVMshares the distinctive features of the mechanosensory neu-rons mediating body touch, but does not appear to play a

w xnecessary role in touch 7 . Animals carrying deletions ofthe flp-8 coding region have no discernible phenotypes,

suggesting that the function of flp-8 overlaps with that ofanother flp gene, such as flp-5, flp-6, andror flp-12.

8. Regulation of flp genes

What controls the regulation of the flp genes? We haveapproached this question using two methods. The firstapproach is to do standard promoter deletion analyses withreporter genes to delineate enhancer regions. Through thismethod, we have identified an enhancer region between107 and 232 bp upstream of the start of flp-1 transcriptionthat is necessary for expression of a reporter gene. Thesecond approach is to use the transgenic animals contain-

Ž .Fig. 4. Animals that show modified green fluorescent protein GFP expression. Transgenic animals carrying a construct containing a GFP reporter geneunder the control of a flp-1 promoter show expression in a subset of flp-1-expressing cells, the paired AVK neurons. This transgenic line was mutagenized

( ) ( ) ( )and screened for disrupted GFP expression. Several mutant animals were isolated. mof-1 yn11 , mof-3 yn14 , and mof-2 yn12 animals show decreased,increased, and no GFP expression, respectively. All mutations are recessive. Adult animals are shown. The intestine has endogenous autofluorescence in C.elegans. Scale bars20 mm.

( )C. Li et al.rBrain Research 848 1999 26–3432

Ž .ing the different promoter–reporter constructs see abovefor mutagenesis screens to isolate animals that have alteredexpression patterns. In particular, since GFP can be visual-ized in living animals, this screen can be performed rela-tively rapidly. Such screens using transgenic animals con-taining a flp-1 reporter construct have yielded animalswith no GFP expression, decreased expression, increased

Ž .expression, and altered expression Fig. 4 . The mutations,named mof for modifier of f lp, are currently beingcharacterized.

9. Pathways through which the flp genes signal

9.1. FaRP receptors

The multitude of FaRPs in C. elegans and the phar-w xmacology of FaRPs on muscle in Ascaris 4 suggest that

multiple types of FaRP receptors are present in C. elegans.Unlike other neuropeptides, two types of FaRP receptorshave been isolated, both from molluscs: a ligand-gated

w x w xchannel 31 and a G-protein coupled receptor 58 . NoFaRP-binding receptor has been identified thus far in C.elegans. In BLAST searches of the C. elegans database,over 1000 G-protein coupled receptors were identified; 54of these receptors had similarity to vertebrate neuropeptide

w xreceptors 1 and may include FaRP receptors.To identify FLP-1 peptide receptors, we are taking two

genetic approaches. The first is to perform a directedŽ .screen with the flp-1 xs strain. These animals are mutag-

enized, and progeny that suppress the sluggish phenotypedue to flp-1 overexpression are selected. Mutations ingenes that act downstream of flp-1, including receptors,proteases, and signaling molecules, can be isolated in thisscreen. The second approach is to do a general screenwhere wild-type animals are mutagenized and progeny thatare loopy, hyperactive, andror wandering are selected.Many different types of genes, including genes that actdownstream of flp-1, will be isolated in this screen. Sev-eral mutants have been isolated from the two screens andare currently being characterized. To determine whetherany of these isolated mutants are in the flp-1 signalingpathway, they will be crossed with different flp-1 mutants.

9.2. G-protein signaling

Physiological and pharmacological studies suggest thatmany FaRPs act through a G protein-coupled second mes-

w xsenger system 8,9,48 . C. elegans animals carrying loss-of-function and gain-of-function mutations in a G protein

Ž .subunit G a goa-1 show similar uncoordination andow xhyperactivity phenotypes 40,55 as the flp-1 loss-of-func-

tion and gain-of-function animals, respectively, suggestingthat flp-1 may signal through a G a pathway. Loss-of-o

Ž .function lf mutations in the G a subunit encoded by theo

C. elegans goa-1 gene results in hyperactivity and wander-ing behavior and animals with loopy waveforms; overex-

Ž .pression of goa-1 xs results in sluggishness and a flat-w xtened waveform 40,55 . To test whether flp-1 signals

Ž .through G a , we generated two double mutants: 1 flp-1oŽ . Ž . Ž . Ž . Ž .xs ; goa-1 lf ; and 2 flp-1 lf ; goa-1 xs . In bothcases, the double mutants showed uncoordination and hy-peractivity phenotypes similar to the goa-1 single mutants;

Ž . Ž .that is, a goa-1 lf ; flp-1 xs double mutant is hyperac-Ž . Ž .tive, while a goa-1 xs ; flp-1 lf double mutant is

w xsluggish 44 . FLP-1 peptides, therefore, appear to signalthrough a G protein-coupled pathway to coordinate move-ment. It is tempting to speculate that the FLP-1 peptidereceptor may be a G-protein coupled receptor.

Serotonin has also been implicated in locomotion in C.elegans, and its effects are also mediated through a G-pro-

w xtein pathway 55 . Wild-type animals placed in high con-centrations of exogenous serotonin become paralyzed.

Ž . w x Ž . w xgoa-1 lf 55 and flp-1 lf 44 animals are partially orfully resistant to exogenous serotonin, respectively, andcontinue moving in the presence of serotonin. FLP-1 pep-tides, therefore, act downstream or parallel to serotonin inmodulating movement. Recently, a serotonin-deficient ani-

Ž . Žmal tph-1 has been isolated J. Sze and G. Ruvkun, pers..comm. . The tph-1 mutant can be used to construct double

mutants with flp-1 mutants to further characterize thelocomotory pathway.

10. Summary

The presence of FaRPs in primitive organisms such asw xcoelenterates 25 suggests that FaRPs are among the first

molecules used for chemical communication in the ner-vous system. The flp gene family in C. elegans is thelargest neuropeptide family identified to date in any organ-ism. The multitude of FaRPs in C. elegans and the overlapin flp gene expression indicates that this family of pep-tides has complex actions and regulatory mechanisms. Thenumber of genes encoding peptides not related to FaRPsroughly equals the number of flp genes. In addition, fewhomologues to vertebrate neuropeptides have been foundin C. elegans. The flp genes, therefore, appear to beresponsible for the majority of neuropeptide functions inC. elegans. With the completion of its genomic sequenceand the detailed information available about its nervoussystem, C. elegans is one of the most tractable systems inwhich neuropeptide gene families can be examined. Un-raveling the functions of the different FaRPs and themechanisms whereby these neuropeptides exert their ac-tions in C. elegans is likely to give insights into the role ofneuropeptide gene families in other organisms as well.

Acknowledgements

We thank Sarah Craven and John Memmott for assis-tance with some of the antibody experiments, Anne Hart

( )C. Li et al.rBrain Research 848 1999 26–34 33

and Art Edison for helpful discussions, and Tom Gilmorefor constructive criticisms on the manuscript. This workwas supported by NSF IBN-9320262 and 9808861 andNIH K02 AG00708.

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