Cell, Vol. 118, 795–806, September 17, 2004, Copyright 2004 by Cell Press

C. elegans LIN-18 Is a Ryk Ortholog and Functionsin Parallel to LIN-17/Frizzled in Wnt Signaling

(related to tyrosine kinases) and Frizzled receptor LIN-17regulate the development of the P7.p vulval cell lineage.

The C. elegans vulva is formed from six vulval precur-

Takao Inoue,1,5 Helieh S. Oz,2,4 Debra Wiland,2

Shahla Gharib,1 Rashmi Deshpande,3

Russell J. Hill,3 Wendy S. Katz,1,2,5

sor cells (VPCs) called P3.p through P8.p (Figure 1A)and Paul W. Sternberg1,*(Sulston and Horvitz, 1977). During the L3 (third larval)1Division of Biology and Howard Hughesstage, interactions with an organizer cell in the gonadMedical Institute(the anchor cell; AC) and between VPCs induce theseCalifornia Institute of Technologycells to adopt an invariant pattern of cell fates (Kimble,Pasadena, California 911251981; Sternberg and Horvitz, 1986; Sternberg, 1988).2 Department of BiochemistryAmong the VPCs, P3.p, P4.p, and P8.p adopt the tertiaryUniversity of Kentucky College of Medicine(3�) fate, P5.p and P7.p adopt the secondary (2�) fate,800 Rose Streetand P6.p adopts the primary (1�) fate. These three fatesLexington, Kentucky 40536are distinguished by the pattern of cell divisions (number3 Department of Molecular Geneticsand axis of division) and by types of terminally differenti-The Ohio State Universityated cells produced (Sulston and Horvitz, 1977; Sharma-484 West 12th AvenueKishore et al., 1999). 3� cells divide once and fuse withColumbus, Ohio 43210the syncytial hypodermis, the default nonvulval cell fate.4 Department of Medicine2� cells divide to produce vulval cells vulA, vulB1, vulB2,Rush University Medical CentervulC, and vulD. The 1� cell divides to produce vulvalChicago, Illinois 60612cells vulE and vulF. The term “lineage” refers to thesepatterns of cell division and cell type specification.

The 2� VPCs P5.p and P7.p follow identical lineages,Summaryexcept that the patterns occur in opposite anterior-pos-terior orientations. The P5.p-like orientation is appar-Wnt proteins are intercellular signals that regulate var-ently the default state for both the P5.p and the P7.pious aspects of animal development. In Caenorhab-lineage (Ferguson et al., 1987; R. Deshpande et al., sub-ditis elegans, mutations in lin-17, a Frizzled-class Wntmitted). Mutations in lin-17 (which encodes a Frizzledreceptor, and in lin-18 affect cell fate patterning in theWnt receptor; Sawa et al., 1996) and lin-18 (cloning re-P7.p vulval lineage. We found that lin-18 encodes aported here) reverse the orientation of the P7.p lineagemember of the Ryk/Derailed family of tyrosine kinase-such that it resembles the P5.p lineage (Ferguson et al.,related receptors, recently found to function as Wnt1987). This reversal causes a characteristic morphologi-receptors. Members of this family have nonactive ki-cal defect (bivulva phenotype; Biv) in which an ectopicnase domains. The LIN-18 kinase domain is dispens-vulval lumen forms posterior to the main vulval lumenable for LIN-18 function, while the Wnt binding WIF(mid-L4 stage; Figure 1B). Analysis of marker expressiondomain is required. We also found that Wnt proteinssuggests that this morphological defect is a conse-LIN-44, MOM-2, and CWN-2 redundantly regulate P7.pquence of altered pattern of cell fates adopted by de-patterning. Genetic interactions indicate that LIN-17scendants of P7.p (R. Deshpande et al., submitted).and LIN-18 function independently of each other in

The involvement of LIN-17/Frizzled suggested that aparallel pathways, and different ligands display differ-cell signaling protein of the Wnt family controls P7.pent receptor specificities. Thus, two independent Wntorientation. The “canonical” Wnt pathway controls manysignaling pathways, one employing a Ryk receptor andaspects of development in various animals (Cadigan

the other a Frizzled receptor, function in parallel toand Nusse, 1997; Wodarz and Nusse, 1998) including

regulate cell fate patterning in the C. elegans vulva. C. elegans (Korswagen, 2002). In this conserved path-way, the Wnt ligand binds to a Frizzled seven transmem-

Introduction brane receptor and initiates a signaling cascade involv-ing Disheveled, APC, GSK3, Axin, �-catenin, and TCF

Organogenesis requires the generation of multiple cells (Cadigan and Nusse, 1997; Wodarz and Nusse, 1998).types in a specific spatial pattern, typically resulting The transcriptional regulation is achieved through thefrom multiple cell-cell interactions. The C. elegans vulva formation of a �-catenin/TCF transcription factor com-is a useful system for investigating molecular mecha- plex. A Wnt coreceptor Arrow/LRP5/LRP6 binds to Wntnisms controlling organogenesis and previously led to and Frizzled in a complex and in some cases is neces-the discovery of EGF (Ferguson and Horvitz, 1985; Hill sary for Wnt signaling (Wehrli et al., 2000; Tamai et al.,and Sternberg, 1992) and LIN-12/Notch (Greenwald et 2000; Pinson et al., 2000). A number of “noncanonical”al., 1983; Yochem et al., 1988) signaling pathways as Wnt signaling pathways are also known that differ in theimportant regulators of vulval cell fate specification. mechanism of signaling downstream of the receptor (forHere we report that parallel Wnt signaling pathways example, see McEwen and Peifer, 2000; Korswagen,involving tyrosine kinase-related receptor LIN-18/Ryk 2002). Many of these employ Frizzled as the receptor.

Recently, a family of proteins consisting of vertebrateRyk (Hovens et al., 1992), Drosophila Derailed (Callahan*Correspondence: pws@caltech.edu

5 These authors contributed equally to the work. et al., 1995), Drosophila Doughnut (Savant-Bhonsale et

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of the receptor tyrosine kinase (RTK) superfamily. Theyhave the typical RTK structure, with an extracellular do-main, a single transmembrane domain, and a kinasedomain. The kinase domain lacks some sequence motifsthat are conserved in most kinases and is believed tolack kinase activity (Katso et al., 1999). This suggeststhat Ryk signaling differs from classic RTK signaling.Several lines of evidence point to this family functioningas Wnt receptors. The extracellular domain of Ryk issimilar to the Wnt binding domain (the WIF domain) ofWIF-1 (Wnt Inhibitory Factor-1) (Hsieh et al., 1999; Pat-thy, 2000). During Drosophila axonal guidance, Wnt5disruption phenocopies Derailed disruption, and wnt5interacts genetically with derailed in a manner consis-tent with Wnt5 functioning as the Derailed ligand (Yoshi-kawa et al., 2003). Moreover, the extracellular domainof Derailed binds Wnt5 (Yoshikawa et al., 2003). So far,Drosophila axonal guidance is the only system in whicha Ryk family member has been shown to function in aWnt pathway. As no other component of this Wnt signal-ing process has been positively identified, it is unclearto what extent Ryk/Derailed signaling intersects withcanonical and noncanonical Wnt signaling pathways.

To determine the nature of the presumed Wnt signal-ing pathway controlling P7.p orientation, we studied thelin-18 gene, mutations in which cause a bivulva pheno-type similar to the lin-17/Frizzled mutant phenotype. Wecloned lin-18 and found it to encode the sole C. elegansmember of the Ryk family. P7.p development is the firstprocess demonstrated to require both Ryk and Frizzledreceptors. The expression patterns of lin-18::gfp and lin-17::gfp suggest that LIN-17 and LIN-18 are expressedtogether in the developing vulval tissue including P7.pdescendants. Surprisingly, LIN-18 protein lacking theentire kinase domain is functional. Genetic interactionsindicate that LIN-17/Frizzled and LIN-18/Ryk functionindependently in parallel pathways. We also identifyLIN-44, MOM-2, and CWN-2 as three Wnt ligands re-sponsible for P7.p orientation. Genetic interactions oflin-44, mom-2, and cwn-2 with lin-17 and lin-18 indicatethat different Wnt ligands show different receptor speci-

Figure 1. Overview of Vulval Development ficities. Our results suggest that LIN-18/Ryk and LIN-17/Anterior is to the left and ventral is to the bottom in this and all Frizzled operate in two parallel Wnt signaling pathwaysother figures. that together determine the orientation of the P7.p(A) At the start of the L3 stage, the six VPCs (P3.p P8.p) are arranged

lineage.in an anterior/posterior line along the ventral midline. LIN-3/EGFand LIN-12/Notch signaling direct VPCs to adopt either the 1�, 2�,or 3� fate in the pattern shown. VPCs that adopt the 1� and 2� fates Resultsundergo two to three rounds of cell divisions to produce seven celltypes (vulA, vulB1, vulB2, vulC, vulD, vulE, and vulF) in the order lin-18 Encodes a Ryk ReceptorABCD EFFE DCBA.

We cloned lin-18 using genetic mapping and germline(B) Phenotype of Wnt pathway mutants. Wnt pathway mutants dis-rescue. lin-18(e620) was mapped between RFLP mark-play a bivulva (Biv) phenotype with an ectopic posterior invaginationers mgP39 and syP8 (Figure 2; Experimental Proce-(black arrows) posterior to the main invagination (white arrows). The

ectopic invagination is produced by the descendants of P7.p and dures). Cosmid clones from the region were tested forresults from a reversal in the P7.p cell lineage that changes the rescue of lin-18(e620) and the cosmid T19D2 was foundorder in which cell types are produced. Nomarski images of the wild- to rescue the mutant phenotype. We tested subclonestype mid-L4 stage vulva and of the Biv phenotype in lin-18(e620), lin-

of T19D2 for the ability to rescue lin-18(e620) and found44(n1792); mom-2(RNAi), and lin-44(n1792); cwn-2(RNAi) are shown.that lin-18 corresponds to the predicted gene C16B8.1Scale bar � 20 �m.(Figure 2A; Experimental Procedures). To determine themRNA structure, we isolated cDNAs by RT-PCR andsequenced them. The sequence obtained is identicalal., 1999; Oates et al., 1998), and a C. elegans homolog

(this paper; Halford et al., 1999) has emerged as possible to a previously reported cDNA sequence (CeRYK [Hal-ford et al., 1999]; AF133217.1) and differs slightly fromalternative receptors for Wnt signals. These proteins are

not homologous to Frizzled and are instead members the gene prediction based on the genome sequence

C. elegans LIN-18/Ryk and Wnt Signaling797

(U41031.1). Sequencing of genomic DNA from lin-18 mu-tants identified sequence alterations in the geneC16B8.1, confirming the identification of C16B8.1 as lin-18 (Figure 2B; Experimental Procedures). lin-18 allelese620 and n1051 are nonsense mutations in the extracel-lular domain, consistent with genetic evidence thatthese alleles eliminate lin-18 activity. Consistent withthe earlier report that n1051 is suppressible by an ambersuppressor sup-5 (Ferguson and Horvitz, 1985), then1051 mutation changes a tryptophan codon to UAGStop/Amber. ga75 is a small deletion that begins in themiddle of exon 3 and terminates in intron 4. It is likelythat this allele causes a premature termination of theprotein. All three sequenced alleles exhibit similar mu-tant phenotypes.

The lin-18 gene encodes the C. elegans ortholog ofmammalian Ryk (Hovens et al., 1992), Drosophila De-railed (Callahan et al., 1995), and Drosophila Doughnut(Oates et al., 1998; Savant-Bhonsale et al., 1999) (Sup-plemental Figure S1 at http://www.cell.com/cgi/content/full/118/6/795/DC1). tBLASTn searches indicated thatLIN-18 is the only member of this family in C. elegans.tBLASTn searches of the draft Caenorhabditis briggsaegenome (Stein et al., 2003) indicated that the predictedprotein CBG14316 is the ortholog of LIN-18 and alsothe only member of the Ryk family in that genome. Theoverall structure of LIN-18 is similar to that of Ryk, De-railed, and Doughnut with an N-terminal extracellulardomain (WIF domain), a single transmembrane domain,and a tyrosine kinase-related domain. As in other mem-bers of the Ryk family, some of the motifs found in mostkinases are not conserved in LIN-18, suggesting thatLIN-18 proteins are not active as kinases (SupplementalFigure S1 on Cell website; Halford and Stacker, 2001).

Structure/Function Analysis of LIN-18To determine which domains are required for the func-tion of lin-18, we tested various versions of lin-18::gfpconstructs for the ability to rescue the lin-18(e620) mu-tant (Figure 3; Supplemental Table S1 online). In theseconstructs, the lin-18 genomic DNA, including 5 kb ofupstream regulatory region and lin-18 coding regions,are fused to gfp. Thus, LIN-18::GFP expression is drivenby the lin-18 promoter and regulatory sequence. Intronsupstream of the fusion point are also included inthese constructs.

The full-length LIN-18 protein fused to GFP at thecarboxyl terminus (“C-term” construct) rescued the mu-tant phenotype of lin-18(e620). We also found that thefusion of the extracellular and the transmembrane do-mains of LIN-18 to GFP (“ECD-TM” construct) rescuedthe lin-18 mutant as well as the full-length (C-term) fu-sion. This suggests that the kinase domain is not re-

Figure 2. lin-18 Sequence

(A) Cloning of the lin-18 gene (see Experimental Procedures). (B)Sequence of lin-18 cDNA. Locations at which gfp was fused andsites of lin-18 mutations e620 and n1051 are shown. A six base pairdeletion, which results in the loss of lin-18 function, is shown byasterisks. The resulting two amino acid deletion is shown by a box.The putative signal sequence and the transmembrane domain areindicated by the underline.

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A fusion of the extracellular domain (“ECD” construct)of LIN-18 to GFP did not rescue the lin-18(e620) mutant.Since the only difference between this and the rescuingECD-TM construct is the inclusion of a section of exon5 coding for the transmembrane domain, the lack ofrescue is not due to the absence of intronic sequenceelements regulating transcription. The ECD fusion is de-tectable on Western blots of C. elegans extracts. How-ever, in contrast to the other fusions, the ECD fusioncould not be detected by fluorescence on the surfaceof cells that normally express LIN-18. To test whetherthe LIN-18 transmembrane domain is required for signal-ing by LIN-18, we made a construct where the trans-membrane domain of LIN-18 was replaced by the trans-membrane domain of C. elegans DAF-1 (TGF-� type ISer/Thr kinase receptor; Georgi et al., 1990) (“ECD-

Figure 3. Molecular Structure of the lin-18 Gene and lin-18::gfp Con-DAF-1” construct). This fusion also rescued the mutantstructsphenotype of lin-18(e620), although not as well as the5� is to the left. Boxes indicate exons. Top: molecular structure ofECD-TM fusion protein. Thus, the main function of thethe lin-18 gene. Locations of mutations are indicated. Ferguson andtransmembrane domain of LIN-18 likely is to anchor theHorvitz (1985) report that n1051 (W70Amber) but not e620

(Q106Amber) is suppressible by amber tRNA-Trp suppressor sup-5. protein to the membrane.This suggests that a Q106W substitution in the extracellular domainresults in a nonfunctional protein as well. This residue is Q or S in

Expression Patterns of lin-17 and lin-18all Ryk and WIF family members (Supplemental Figure S1 on CellWe examined the expression pattern of lin-18::gfp usingwebsite) (Patthy, 2000). Bottom: molecular structures of lin-18::gfpthe rescuing full-length (C-term) fusion (Figures 4A andconstructs. The gfp gene also contains introns (not shown). The

transmembrane domain is shown in black, the kinase homology 4D). Expression from both extrachromosomal arraysdomain is shown in yellow, and the WIF domain is shown in red. and chromosomal integrants was observed, and the re-pTI03.56 replaces the gfp of pTI00.42 with a sequence encoding sults were fully consistent. LIN-18::GFP was expressedthe HA epitope tag, “YPYDVPDYA” (blue). pTI04.1 was made by

in neurons, body wall muscle, and vulval tissue. In theinserting a transmembrane domain-coding segment of the C. ele-vulva, LIN-18::GFP was expressed in P5.p, P6.p, andgans daf-1 gene into pTI03.35 (gray). “�” indicates rescue and “�”P7.p and all their descendants throughout the L3 andindicates nonrescue of the lin-18(e620) vulval phenotype by each

construct (see Supplemental Table S1 online). L4 stages. In most (�90%) L4 stage individuals, descen-dants of P5.p and P7.p expressed higher levels ofLIN-18::GFP than descendants of P6.p. No obvious dif-quired for the function of the LIN-18 protein, which isference in expression levels was found among differentsurprising considering the high level of conservation ofdescendants of a single VPC (e.g., between P7.paa,this domain across species (Supplemental Figure S1P7.pap, P7.ppa, and P7.ppp).online). GFP has the ability to dimerize (Zacharias et al.,

Previously, it was reported that LIN-17::GFP is ex-2002). Therefore, if the primary function of the kinasepressed in VPC granddaughters, and based on mutantdomain was to mediate dimerization, then a differentphenotypes, it was likely that LIN-17 is present in VPCdimerization domain such as GFP could potentially pro-lineages earlier in development (Sawa et al., 1996; Fer-vide this function. To test this, we made a construct inguson et al., 1987; R. Deshpande et al., submitted). Wewhich the extracellular and the transmembrane domainsanalyzed the expression of the lin-17 gene using a re-of LIN-18 were fused to the HA epitope tag (Figure 3).porter in which a nuclear localized variant of GFP wasThis construct also rescued the lin-18(e620) mutant.expressed under the control of the lin-17 promoter. ThisThus, the ability of GFP to dimerize does not accountconstruct expressed GFP in VPCs P5.p, P6.p, and P7.p,for the ability of the ECD-TM construct to rescue thetheir daughters, and granddaughters (Figures 4B, 4C,mutant. Because C. elegans transgenes express pro-and 4E), and was coexpressed with lin-18::DsRed2 (full-teins at high levels (Mello et al., 1991), our results dolength LIN-18::DsRed2 fusion under the control of thenot address whether the ECD-TM fusion is as active aslin-18 promoter). Thus, LIN-17 and LIN-18 proteins arethe C-term fusion on a per molecule basis.likely present in the same cells during vulval devel-A two amino acid deletion in a conserved region ofopment.the extracellular WIF domain abolished the activity of

Previous analyses suggested that lin-17 and lin-18both the C-terminal and the ECD-TM fusion proteins.mutations cause similar phenotypes based on morphol-By Western blot analysis and by direct observation ofogy and lineage analysis (Ferguson et al., 1987). MoreGFP fluorescence, these proteins were localized to therecently, this similarity was confirmed at the cellularplasma membrane and expressed at levels comparablelevel using cell fate markers and POP-1/TCF, a markerto analogous wild-type fusion proteins (Experimentalof tissue polarity (R. Deshpande et al., submitted). TheProcedures). Thus, the transmembrane domain and theprincipal defect found in lin-18 and lin-17 single mutantskinase domain are not by themselves sufficient for theappear to be the reversal of fates between P7.pa (ante-function of LIN-18 even when overexpressed. Residuesrior daughter of P7.p) and P7.pp (posterior daughter ofdeleted (Glu-Leu) are highly conserved in all Ryk/P7.p) lineages. To further corroborate this result, weDerailed family members (Glu-Leu or Glu-Val) and similar

(Asp-Ile) in WIF-1 (based on alignment in Patthy, 2000). examined the expression patterns of cell fate markers

C. elegans LIN-18/Ryk and Wnt Signaling799

egl-17::gfp (Burdine et al., 1998) and cog-1::gfp (Palmeret al., 2002) (Figure 5; Table 1). In the wild-type, both ofthese markers are expressed by some descendants ofP7.pa but not by descendants of P7.pp. We found thatin some lin-17 and lin-18 mutant animals, P7.pp descen-dants but not P7.pa descendants expressed thesemarkers, indicating that these mutations cause reversalsof fates between P7.pa and P7.pp. Quantitative differ-ences in frequencies of different expression patterns(Table 1) indicate that some aspects of cell type pat-terning may have different requirements for lin-17 andlin-18 functions. Nevertheless, these studies confirmthat lin-17 and lin-18 regulate a common process.

Wnts Regulate P7.p OrientationAlthough involvement of LIN-17/Frizzled Wnt receptorsuggested that P7.p orientation is controlled by a Wntsignal, the identity of the Wnt was not known. The C. ele-gans genome contains five Wnt genes, lin-44, mom-2,egl-20, cwn-1, and cwn-2 (Herman et al., 1995; Thorpeet al., 1997; Rocheleau et al., 1997; Maloof et al., 1999;Shackleford et al., 1993). Mutations in lin-44, mom-2,and egl-20 or RNAi treatment of cwn-1 and cwn-2 donot cause a bivulva phenotype.

To test whether Wnts act redundantly to orient P7.p,we examined various double mutant combinations usinggenetic mutations and RNAi. We found that simultane-ous disruptions of lin-44 and mom-2 or of lin-44 andcwn-2 caused a bivulva phenotype (Table 2; Figure 1B).The vulval morphology of such double mutants was in-distinguishable from that of lin-17(�) and lin-18(�) singlemutants. To confirm this phenotype at the cellular level,we examined the pattern of expression of ceh-2::yfpand cdh-3::cfp markers, which are expressed in specificsubsets of vulval cells (Inoue et al., 2002). Among P7.pdescendants, vulB cells express ceh-2::yfp and vulCand vulD cells express cdh-3::cfp. vulA cells can bedistinguished as sister-cell pairs that do not expresseither marker and remain adherent to the cuticle at themid-L4 stage. In wild-type animals, the posterior daugh-ter of P7.p (P7.pp) produces vulA cells. However, inmany lin-17 and lin-18 mutant animals, the anteriordaughter of P7.p (P7.pa) produces vulA cells instead(R. Deshpande et al., submitted). We found that lin-44; mom-2(RNAi) animals could also produce vulA cellsfrom the P7.pa cell (6 of 7 Biv animals). In addition, wefound that lin-44; cwn-2; mom-2 triple disruption caused

Figure 4. Expression of Wnt Pathway Components Controlling a Biv phenotype of higher penetrance than lin-44; cwn-2P7.p Orientation

or lin-44; mom-2 (Table 2). The mutant phenotype of lin-Corresponding Nomarski (bottom) and epifluorescence (top) images

44; cwn-2; mom-2 was weaker than the full (�100%are shown. The anchor cell is indicated by large arrowheads. Identi-penetrant) P7.p orientation reversal observed in lin-ties of VPC descendants are indicated below. Scale bar � 20 �m.

(A) Full-length (C-term) LIN-18::GFP fusion under the control of thelin-18 promoter. Expression in P5.p and P7.p granddaughters isshown. Although some puncta of GFP fluorescence were observed

Expression in P7.p daughters is shown. P5.p and P6.p daughterswith this and other fusions, all TM domain-containing GFP fusionsalso express lin-17::gfp and lin-18::gfp (or DsRed2) (not shown).were on the plasma membrane.(D) Membrane localized lin-18::gfp in VPCs, P6.p and P7.p.(B) Nuclear localized GFP under the control of the lin-17 promoter in(E) lin-17::gfp in VPCs, P6.p, and P7.p. P5.p also expresses lin-P5.p and P7.p granddaughters. Arrows point to ventral cord neurons17::gfp and lin-18::gfp (not shown). Downward arrows point to ven-that also express lin-17::gfp. Although animals photographed in (A)tral cord neurons expressing lin-17::gfp.and (B) display brighter expression in P5.p and P7.p compared to(F) GFP under the control of the mom-2 promoter in the anchor cellP6.p, many individuals express lin-17::gfp and lin-18::gfp in P6.pafter the first division of VPCs.granddaughters also.(G) GFP under the control of the mom-2 promoter in the anchor(C) Full-length LIN-18::DsRed2 fusion protein under the control ofcell and VPC granddaughters, P5.ppa, P5.ppp, P7.paa, and P7.papthe lin-18 promoter (shown in red and membrane localized) and(red arrows).nuclear localized GFP under the control of the lin-17 promoter.

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Figure 5. Marker Expression in lin-17 and lin-18 Mutants

All panels are in the same scale. Scale bar �

20 �m. A reversed pattern is observed in lin-17(n671) and lin-18(e620) animals.(A) In the mid-L4 stage, egl-17::gfp is ex-pressed in all P7.pa descendants (P7.paxx;P7.paa, P7.papl, P7.papr in the wild-type) butnot in P7.pp descendants (P7.ppxx; P7.ppaa,P7.ppap, P7.pppa, and P7.pppp). Ectopic in-vaginations are indicated by white arrows.(B) In the early L4 stage, cog-1::gfp is stronglyexpressed in P7.pa descendants P7.papland P7.papr but not expressed or only weaklyexpressed in P7.pp descendants. Ectopic in-vaginations are indicated by black arrows.Expression of cog-1::gfp in spermatheca-uterine junctions are indicated by white ar-rowheads. Expression of cog-1::gfp in vulvalcells is indicated by white arrows.

17(�); lin-18(�) double mutants. This difference may lin-18 double mutants (Table 2). This enhancement wasconfirmed by marker expression (R. Deshpande et al.,reflect an incomplete disruption of cwn-2 by RNAi. Muta-

tions in egl-20 or RNAi against cwn-1 did not result submitted). The mutual enhancement indicates thatLIN-17 can function in the absence of LIN-18, and LIN-18in obvious enhancement of the Biv phenotype in any

background tested. We conclude that Wnt proteins reg- can function in the absence of LIN-17.To determine which Wnt signaling molecules actulate the orientation of the P7.p cell lineage.

through which receptor, we examined the effect of elimi-nating each Wnt in lin-17(null) and lin-18(null) geneticExpression Pattern of Wnt Genesbackgrounds (Table 2). We expect that a mutation in aTo determine the cellular source of Wnt signals orientingligand would not enhance a null mutation in its receptor,the P7.p lineage, we constructed mom-2::gfp and lin-provided that the ligand does not signal through an44::gfp reporters. In the vicinity of the developing vulva,alternative receptor. In contrast, null mutations that dis-consistent mom-2::gfp expression was observed in therupt parallel pathways often enhance. We found that theanchor cell (Figures 4F and 4G). Some transgenic linesloss of lin-44 had little effect on lin-17(null) but enhancedalso expressed GFP in ventral uterine cells near thethe Biv phenotype of lin-18(null). Conversely, loss ofanchor cell (VU and descendants), HSN neurons, ventralmom-2 had little effect on lin-18(null) but enhanced thecord neurons, and VPC granddaughters P5.ppa, P5.ppp,Biv phenotype of lin-17(null). Thus, lin-44 and mom-2P7.paa, and P7.pap (Figure 4G). For lin-44::gfp, as re-differ in the pattern of interactions with receptor muta-ported previously (Herman et al., 1995), the lin-44 pro-tions. This is also consistent with the genetic evidencemoter drove expression of GFP in the tail region. Also,that LIN-17 and LIN-18 can act independently of eachfaint and inconsistent expression was sometimes ob-other. These genetic studies did not identify a specificserved in the anchor cell (not shown). Images of lin-receptor for CWN-2, as disruption of cwn-2 did not sig-44 expression from the systematic in situ hybridizationnificantly enhance either lin-17 or lin-18 mutants.project are consistent with expression of lin-44 in or

near the anchor cell (http://nematode.lab.nig.ac.jp/,clone yk120c7) (Y. Kohara, personal communication). DiscussionThus, it is possible that lin-44 is coexpressed withmom-2 in the ventral uterus and/or the anchor cell to Wnt Signaling Pathways Regulate the Orientationorient the P7.p lineage. of the P7.p Lineage

The C. elegans P7.p lineage is an example of a patterningevent occurring at single-cell resolution. During this pro-Genetic Analysis of Wnt and Receptor Genes

Reveals Parallel Wnt Pathways cess, seven nuclei of five distinct cell types are producedin a specific invariant spatial arrangement. We foundTo understand the relationship between components of

signaling pathways that control the P7.p orientation, we that the Wnt signals LIN-44, MOM-2, and CWN-2 actthrough two types of Wnt receptors, LIN-17/Frizzled andanalyzed various receptor-ligand and receptor-receptor

double mutants. lin-17(null) and lin-18(null) mutations LIN-18/Ryk, to determine the anterior-posterior orienta-tion of the P7.p lineage. This is the second demonstratedwere found to mutually enhance each other in lin-17;

C. elegans LIN-18/Ryk and Wnt Signaling801

Table 1. Phenotypes of lin-17 and lin-18 Mutants

Phenotype

Mutation Marker Normal Abn Biv Biv Biv Biv

(P7.pa �)a P7.pa� P7.pa� P7.pa� P7.pa�

(P7.pp �)a P7.pp� P7.pp� P7.pp� P7.pp�

� egl-17::gfp 11 0 0 0 0 0lin-18(e620) egl-17::gfp 15 0 4 0 1 4lin-17(n671) egl-17::gfp 12 3 12 0 19 3� cog-1::gfp 13 0 0 0 0 0lin-18(e620) cog-1::gfp 14 0 2 2 0 9lin-17(n671) cog-1::gfp 4b 2 3 3 3 5

Animals were classified based on the morphology (normal, Abn, Biv) and then based on marker expression for Biv animals. The number ofanimals in each category is shown. “P7.pa �” indicates that one or more P7.pa descendants expressed the gfp marker. “P7.pa �” indicatesthat none of the P7.pa descendants expressed the gfp marker. The last column (Biv, P7.pa �, P7.pp �) represents the most extreme casesof P7.p lineage reversals, and examples of animals in this category are shown on Figure 5.a Most (68/69) morphologically wild-type (“normal”) animals exhibited the wild-type marker expression pattern (P7.pa�, P7.pp�).b Includes one animal in which cog-1::gfp appeared to be expressed in both P7.pa and P7.pp descendants (P7.pa�, P7.pp�).

case of Ryk function in Wnt signaling (following Yoshi- sections. This gradient may cause sister cells to adoptdifferent fates, or alternatively, directionally polarize par-kawa et al., 2003) and confirms that Ryk proteins func-

tion in general as Wnt receptors. A novel aspect of P7.p ent cells prior to cell division. Either way, the functionof MOM-2 signaling is to orient the lineages with respectorientation is that this is the first process requiring both

a Frizzled and a Ryk receptor and thus provides an to the anchor cell. The analysis of POP-1/TCF expres-sion pattern in a lin-17; lin-18 double mutant (R. Desh-opportunity to investigate the relationship between

these two types of receptors. pande et al., submitted) suggests that P5.p and P7.plineages are polarized to the P5.p-like orientation byWe propose the following model of Wnt signaling

function in P7.p lineage orientation (Figure 6A). mom- default. Thus, in the absence of LIN-17 and LIN-18 sig-naling, the P7.p adopts a P5.p-like orientation, while the2::gfp is expressed from the anchor cell and its vicinity.

Thus, there is probably a gradient in which anchor cell- orientation of the P5.p lineage is unaffected. Sourcesof LIN-44 and CWN-2 signals are not clear. One possibil-proximal sections of P7.p and P5.p lineages receive

stronger Wnt signals in comparison to anchor cell-distal ity is that they are coexpressed with MOM-2 and func-

Table 2. Phenotypes of Wnt, Receptor Double Mutants

Phenotype

Genotype Normal Abn Biv % Biv

Wild-type many 0 0 0lin-17(n671) 75 5 207 72lin-18(e620) 172 2 131 43lin-17(n671); lin-18(e620) 0 0 63 100lin-44(n1792) 120 0 0 0mom-2(or42)a 81 1 1 1mom-2(RNAi) 68 0 0 0cwn-2(RNAi) 89 0 0 0lin-44(n1792); mom-2(or42) 52 0 75 59lin-44(n1792); mom-2(RNAi) 140 0 21 13lin-44(n1792); cwn-2(RNAi) 125 0 5 4mom-2(or42); cwn-2(RNAi) 28 0 0 0lin-44(n1792); mom-2(or42); cwn-2(RNAi) 15 0 57 79lin-17(n671) 75 5 207 72lin-17(n671) lin-44(n1792) 62 16 108 58c

lin-17(n671); mom-2(or42) 0 0 103 100b

lin-17(n671); mom-2(RNAi) 2 2 96 96b

lin-17(n671); cwn-2(RNAi) 30 6 124 78lin-18(e620) 172 2 131 43lin-18(e620); lin-44(n1792) 9 5 100 88b

lin-18(e620); mom-2(or42) 55 0 44 44lin-18(e620); mom-2(RNAi) 49 2 73 59lin-18(e620); cwn-2(RNAi) 44 1 22 33

aAll mom-2(or42) strains also contain dpy-11(e224). dpy-11(e224) did not enhance the penetrance of the Biv phenotype in lin-44(n1792), lin-17(n671), or lin-18(e620) (Experimental Procedures).b p .0001 (comparison with lin-17 and lin-18 single mutants, Fisher’s exact test).c lin-44 lin-17 double mutants show a decreased rate of Biv animals compared to lin-17 single mutants (p � .0019). This decrease is largelydue to the increase in the frequency of animals with an abnormal anatomy (p � .0008), which may indicate a distinct genetic interaction. Thefrequency of phenotypically wild-type animals is not significantly increased (p � .09).

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(Ryk/Derailed) synergize, indicating that they functionindependently of each other. Also, genetic interactionswith Wnt mutations indicate pathway specificity; if thespecificities of LIN-44 and MOM-2 were similar, muta-tions in lin-44 and mom-2 would enhance lin-17 and lin-18 mutations in a qualitatively similar manner. However,lin-17; mom-2 shows a stronger phenotype than lin-17;lin-44, and lin-18; lin-44 shows a stronger phenotypethan lin-18; mom-2. This pattern of interactions couldnot be explained if the functions of LIN-44 and MOM-2were identical.

The simplest interpretation of our results is shown inFigure 6B. Since lin-44(null) enhances lin-18(null) butnot lin-17(null), lin-44 must function in parallel to lin-18. Similarly, since mom-2(null) enhances lin-17(null),mom-2 must function in parallel to lin-17. Based onthese results, we propose that LIN-44 preferentiallyfunctions as the ligand for LIN-17/Frizzled and MOM-2preferentially functions as the ligand for LIN-18/Ryk.Since lin-44 and mom-2 single mutant phenotypes areweaker than those of lin-17 and lin-18, each receptorlikely transduces additional signals (including LIN-44/LIN-18 and MOM-2/LIN-17 combinations as well asCWN-2). A weak enhancement of lin-18(e620) by mom-2(RNAi) supports this possibility (Table 2). Our resultsdo not rule out the possibility that LIN-44 or MOM-2signals through a third pathway. However, the completereversal of the P7.p orientation observed in the lin-17;lin-18 double mutant suggests that the two receptorsaccount for most of the P7.p orienting activity (R. Desh-pande et al., submitted). LIN-17 and LIN-44 are alsorequired for other fate specifications in C. elegans, sug-gesting that LIN-17 acts as a LIN-44 receptor in multipletissues (Sternberg and Horvitz, 1988; Herman and Hor-vitz, 1994; Chamberlin and Sternberg, 1995; Jiang andSternberg, 1998). Sequence analysis (Prud’homme et

Figure 6. Models al., 2002) suggests that CWN-2 is the ortholog of Wnt5,(A) A model of Wnt signaling function in P7.p orientation. mom- the ligand for Derailed in Drosophila. Therefore, the2::gfp expression pattern (Figures 4F and 4G) suggests that one or

involvement of CWN-2 is consistent with it functioningmore Wnt ligands are expressed from the anchor cell and the vicinity.as a LIN-18 ligand, although we were not able to resolveExpression of lin-17 and lin-18 reporters in P7.p descendants (Fig-the receptor specificity for this ligand. The orthologyures 4A–4E) suggests that this signal directly controls tissue orienta-

tion in P7.p and its descendants. The Wnt signal would likely act relationship of MOM-2 is not clear. MOM-2/Wnt andon P5.p as well, but we hypothesize that it is not necessary for MOM-5/Frizzled are required for endoderm inductionproper P5.p development because P5.p has the correct tissue orien- (Rocheleau et al., 1997; Thorpe et al., 1997). However,tation by default. The Wnt signal could act on the P7.p daughters

we found no evidence of MOM-5 involvement in P7.pas shown or at other stages.orientation (not shown), and LIN-18 is not required for(B) A model of the Wnt signaling pathways that regulate the P7.pendoderm induction.cell lineage. Our results suggest that LIN-44 and MOM-2 signal

preferentially through LIN-17 and LIN-18, respectively (bold lines), Since LIN-17 and LIN-18 are partially redundant re-and are consistent with lower levels of signaling through other Wnt ceptors for related signals, loss of one receptor mightsignal/receptor combinations (dashed lines). be compensated for by overexpression of another. How-

ever, we did not observe a rescue of the lin-17 mutantphenotype by overexpression of GFP-tagged LIN-18

tion to orient the P7.p lineage with respect to the anchor (Experimental Procedures).cell. It is likely that LIN-17 and LIN-18 function in thesame cell at the same time based on their coexpression

LIN-18/Ryk Is Not a Coreceptorand similarity of phenotypes. However, it remains possi-for LIN-17/Frizzledble that LIN-17 and LIN-18 function in different cells,LIN-18 and LIN-17 are membrane-spanning proteinse.g., in different cells within the P7.p lineage.with different types of Wnt binding domains, raising thepossibility that they function together as obligatory sub-units in a Wnt receptor complex. In such a case, theGenetic Interactions Suggest Parallel Pathways

Genetic interactions among Wnt pathway components coreceptor might enhance the affinity of the ligand tothe receptor or present the ligand in a manner accessiblesuggest that distinct Ryk and Frizzled pathways act in

parallel. Null mutations in lin-17 (Frizzled) and lin-18 to the receptor. While our structure function results are

C. elegans LIN-18/Ryk and Wnt Signaling803

consistent with this possibility, our genetic results are for the intracellular domain of LIN-18, the high level ofsequence conservation in the kinase domains of C. ele-not. In particular, the enhancement shown by lin-17 and

lin-18 null mutations indicates that LIN-17 and LIN-18 gans LIN-18, C. briggsae LIN-18, and other Ryk familymembers suggests that this domain retains some func-can each act independently of the other. Also, several

developmental processes that are mediated by lin-17 tionality within the context of nematode biology (Supple-mental Figure S1 on Cell website). One possibility isoccur normally in lin-18 mutants (e.g., P11/P12 specifi-

cation; P.W.S. and R.J.H. unpublished data). Therefore, that the kinase domain enhances the function of LIN-18protein, for example by localizing the protein to the cor-LIN-17 can function in the absence of LIN-18, the sole

Ryk receptor in C. elegans, and thus, LIN-18/Ryk is not rect subcellular location or by enhancing the binding ofsignaling partners. Alternatively, the kinase domain mayan obligatory coreceptor for LIN-17/Frizzled. The possi-

bility that Frizzled proteins are obligatory coreceptors be required for nonvulval functions of LIN-18. It is possi-ble that transphosphorylation by another receptor tyro-for LIN-18 cannot be rigorously ruled out in the absence

of null mutations for other members of the Frizzled fam- sine kinase plays a role in these functions.ily. However, genetic mutations and RNAi for all otherFrizzled genes cause no obvious P7.p phenotype (not Downstream Components of LIN-17shown). and LIN-18 Signaling

It is unclear to what extent the signaling pathways thatModels of Downstream Signaling by LIN-18 act downstream of LIN-17 and LIN-18 in the vulva resem-The mechanism of signaling by Ryk/Derailed family of ble canonical or noncanonical Wnt signaling pathways.receptors is unclear. Based on the alteration of con- LIN-17 and LIN-18 each regulate the subcellular localiza-served residues and the absence of detectable in vitro tion of POP-1/TCF (R. Deshpande et al., submitted),kinase activity (Katso et al., 1999; Halford and Stacker, suggesting that LIN-18 and LIN-17 signaling pathways2001), kinase domains of these proteins are believed to converge at some point downstream of receptors butbe inactive. Also, a mutation affecting the active site upstream of POP-1. So far, none of the intermediateof the kinase (which should completely eliminate any components of the canonical Wnt signaling pathwaycatalytic activity if present) does not affect the function (e.g., BAR-1 �-catenin) has been demonstrated to playof Derailed (Yoshikawa et al., 2001). Thus, signaling by a role in P7.p lineage orientation. However, this may beRyk differs from classic RTK signaling (e.g., by EGFR) due to redundancy (multiple genes encoding �-cateninsin which homodimerization allows the kinase domain and Dishevelleds exist in C. elegans; Natarajan et al.,of each receptor to phosphorylate the other receptor 2001; Korswagen, 2002). Also, Wnt signaling has been(transphosphorylation). The function of such kinase- implicated in a distinct function earlier in vulval develop-dead receptors is an interesting problem (Kroiher et al., ment. Specifically, mutations in bar-1 (�-catenin), pry-12001). In addition to members of the Ryk family, several (axin), and apr-1 (APC) affect VPC cell fate (Eisenmannother putative kinase-dead tyrosine kinase receptors et al., 1998; Gleason et al., 2002). It is possible that forare known, for example, ErbB3, a member of the EGFR some genes, an effect of the mutation on this processfamily in vertebrates (Pinkas-Kramarski et al., 1996). masks a later role of the gene in P7.p orientation.Upon ligand binding, ErbB3 forms a heterodimer with

Experimental Proceduresanother kinase-active member of the EGFR family, re-sulting in transphosphorylation of ErbB3 by the active

Morphology and Marker Expressionkinase and interactions of the phosphorylated ErbB3For Table 2 and Supplemental Table S1, vulval morphology waswith downstream components. A similar mechanism has scored in the mid-L4 larval stage. Animals that had a single ectopic

been proposed for Ryk. Although no kinase-active mem- posterior invagination (developing lumen) distinct from the mainbers of the Ryk family appear to exist, mouse Ryk has vulval invagination were scored as bivulva (Biv). Any animal that

appeared abnormal but could not be classified as Biv was scoredbeen shown to bind to and be phosphorylated by recep-as abnormal (Abn). Since the Biv phenotype correlates with and istor tyrosine kinases EphB2 and EphB3 (Ephrin recep-likely a direct result of the transformation of presumptive vulC ortors) (Halford et al., 2000).vulD to vulA, this phenotype is evidence for a reversal in the P7.pOur structure function experiments argue against lineage after the first round of division (R. Deshpande et al., submit-

transphosphorylation of LIN-18 in a heteromeric com- ted). For Table 1, morphology and expression pattern of markersplex as the mechanism of LIN-18 function. A variant of were scored in the mid-L4 stage for egl-17::gfp and in the early L4

stage for cog-1::gfp.LIN-18 lacking the entire kinase domain retains activityin vivo. Moreover, mutations in vab-1, which encodes

Cloning of lin-18the sole Ephrin receptor in C. elegans, do not cause aRecombination mapping placed lin-18 between the N2-BergeracP7.p phenotype (George et al., 1998; our data not shown).RFLPs mgP39 (Greenstein et al., 1994) and syP8 (a HindIII RFLPTherefore we consider other models of LIN-18 functiondetected on a Southern blot by cosmid W01H2). Sixteen cosmids in

relying on extracellular and transmembrane domains. this 300 kb region were tested for rescue: C55E12, C07F2, W08F10,Because mutations in the WIF domain abolish LIN-18 W07F4, T06F4, T18B7, W01D12, T22A4, K11F1, F09F9, R02E4,

R02E12, ZC464, W09F1, C16B8, and T19D2. Only C16B8 and T19D2function, LIN-18 probably binds a Wnt protein and trans-rescued the lin-18(e620) mutant to over 90% wild-type. Variousduces the signal into the P7.p lineage cells. A differentsubclones were tested for rescue; details are available upon request.membrane protein is likely required for this process. WeThe sequence of the junction for the ga75 deletion is AAA CAT TTT//propose that Wnt binding to LIN-18 allows LIN-18 toCCG ATT ATT. Assuming the reading frame continues into intron 4

associate with (or release from) a second membrane through the junction, a stop codon is encountered after 24 aminoprotein that mediates signal transduction. acids. BLAST searches were carried out using the NCBI (http://

www.ncbi.nlm.nih.gov/) and WormBase (http://www.wormbase.org/)While our rescue experiments do not reveal a function

Cell804

web sites. Clustalw alignment was carried out using MacVector expressing double-stranded RNA, and their progeny scored at theL4 stage. RNAi vectors were constructed by subcloning KpnI-SacIsoftware.fragments of cDNA clones yk657b5 (mom-2) and yk343h8 (cwn-2)into the L4440 vector (A. Fire, personal communication).lin-18, lin-17, and wnt Fusion Transgenes

lin-18 fusions were made by subcloning lin-18 DNA into a gfp vectorAcknowledgments(A. Fire personal communication) or a vector containing DsRed2

(Clontech); details are available upon request. All fusion constructsWe thank J. Goodwin for technical assistance, X.Z.S. Xu for DsRed2contain 5 kb of upstream sequence and no downstream sequence.plasmids, and B.P. Gupta for lin-17 plasmids. We thank A. Fire, S.The fusion junction of pTI01.2 is “C TGC CTG GCC AAT GTG AATXu, J. Ahnn, and G. Seydoux for gfp vectors, D. Eisenmann and S.ATG//gfp.” pTI03.20 and pTI03.44 are identical to pTI00.42 andKim for ga75, and Y. Kohara for cDNA clones and in situ data. SomepTI00.43, except that they contain a 6 bp deletion in the secondstrains were obtained from Caenorhabditis Genetics Center, fundedexon. The mutation does not visibly interfere with protein productionby NIH, National Center for Research Resources. P.W.S. is an Inves-as determined by a Western blot analysis. pTI04.1 is identical totigator with and W.S.K. was an Associate with the HHMI, whichpTI03.35, except sequence “SPGAR PAASS TWLIL TILAL LTFIVsupported this work. T.I. was supported by fellowship DRG-1646LLGIA IFLTR KSWEA KFDS” containing the transmembrane regionfrom the Damon Runyon Cancer Research Foundation. W.S.K. wasof C. elegans DAF-1 (Georgi et al., 1990) is inserted betweenalso supported by grants: March of Dimes 5-FY97-0674, ACS IRG-LIN-18ECD and GFP.163H, and EPSCoR OSR-9452895. R.J.H. was supported by MarchExtrachromosomal transgenic arrays were made by coinjectingof Dimes 5-FY00-550.lin-18::gfp constructs with an unc-119(�) clone (Maduro and Pilgrim,

1995) and pBluescript IIKS� (Stratagene) as described (Mello et al.,Received: March 16, 20041991). Extrachromosomal arrays were either made in unc-119(ed4)Revised: July 14, 2004background and crossed into an unc-119(ed4); lin-18(e620) strainAccepted: July 24, 2004or directly made by injection into unc-119; lin-18. Non-Unc-119 L4Published: September 16, 2004animals were scored for the rescue of the lin-18 phenotype. To test

for the expression of nonrescuing lines, Western blots of wormReferenceslysates were probed with a polyclonal anti-GFP antibody (Molecular

Probes). syEx592 and syEx600 (Supplemental Table S1 online) wereBurdine, R.D., Branda, C.S., and Stern, M.J. (1998). EGL-17(FGF)made from 60 ng/�l and 42 ng/�l of the lin-18::gfp construct, respec-expression coordinates the attraction of the migrating sex myo-tively. syEx516, syEx520, syEx521, syEx523, syEx524, syEx581,blasts with vulval induction in C. elegans. Development 125, 1083–syEx590, syEx606, syEx607, syEx608, syEx653, syEx657, sEx658,1093.and syEx659 were made from injection of 20 ng/�l. All other linesCadigan, K.M., and Nusse, R. (1997). Wnt signaling: a commonwere made by injection of 5 ng/�l of lin-18::gfp. Representative linestheme in animal development. Genes Dev. 11, 3286–3305.(syEx519, syEx520, and syEx521) failed to rescue the Biv phenotype

of lin-17(n671), indicating that the rescue is specific to lin-18 and that Callahan, C.A., Muralidhar, M.G., Lundgren, S.E., Scully, A.L., andoverexpression of full-length GFP-tagged LIN-18 (syEx519) does not Thomas, J.B. (1995). Control of neuronal pathway selection by arescue lin-17. Drosophila receptor protein-tyrosine kinase family member. Nature

mom-2::gfp was made by cloning 2 kb of mom-2 5� region into 376, 171–174.the GFP vector pPD95.75 (A. Fire personal communication). The Chamberlin, H.M., and Sternberg, P.W. (1995). Mutations in themom-2::gfp plasmid (at 25 or 40 ng/�l) was coinjected with unc- Caenorhabditis elegans gene vab-3 reveal distinct roles in fate spec-119(�) (10 or 15 ng/�l) and HinfI-digested complex DNA (55 or 35 ification and unequal cytokinesis in an asymmetric cell division. Dev.ng/�l) into unc-119(ed4) animals. Some variations in expression Biol. 170, 679–689.patterns were observed among different transgenic lines. However,

Eisenmann, D.M., Maloof, J.N., Simske, J.S., Kenyon, C., and Kim,anchor cell expression was observed in four out of eight lines ana-S.K. (1998). The beta-catenin homolog BAR-1 and LET-60 Ras coor-lyzed. These four include lines from three injected animals. No an-dinately regulate the Hox gene lin-39 during Caenorhabditis eleganschor cell expression was observed in lines made by injection ofvulval development. Development 125, 3667–3680.mom-2::gfp without complex carrier DNA. lin-44::gfp was made byFerguson, E.L., and Horvitz, H.R. (1985). Identification and charac-cloning 2 kb of lin-44 promoter into a GFP vector.terization of 22 genes that affect the vulval cell lineages of thelin-17::4XNLSgfp contains 6 kb of lin-17 5� region inserted intonematode Caenorhabditis elegans. Genetics 110, 17–72.the gfp vector pPD121.83 (A. Fire personal communication). GFP

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