Fundam.appl. NemalOl., 1997,20 (2), 173-186

InternaIlectin hindingpatternsin the nematodesCaenorhabditiselegans,Panagrolaimussuperbus

andAcrobeloidesmaximus

GaëtanBORGONIE*+o, EdilbertVAN DRlEsscHE**, ChristopherD. LrNK***,Myriam ClAEvs*, Dirk DE WAELE+o andAugustCOOMAt'ls*

* Institute oJZoology, University Cent, Ledeganckstraat35, 9000Cent, Belgium,+ Plant CeneticSystemsNV, J. Plateaustraat22, 9000Cent, Belgium,

"* InstituteJor MolecularBiology, Free University Brussels,Paardenstraat65, 1640St.-Cenesius-Rode,Belgium, and*** University oJDenver,DejJaTtmentoJBiological Sciences,2101 E. WesleyAve.,Denver, CO 80208, USA

Acceptedfor publication 18 March 1996.

Summary- Using ten different lectins, the binding patternswerestudiedin three[ree-living rhabditid nematodes: Caenorhabdiliselegam,Panagrolaimussuperbus,andAcrobeloidesmaximus.Although ail the nematodetissuesshowedbinding by oneor moreof thelectins used,considerabledifferenceswere noticed betweenthe three nematodespecies.Yolk, four of the coelomocytes,and theoocytesbind mostoftenwith the lectinsused.Although the intestinalbrushborderinteractswith a lot of foreign materials,it stainedorny with few lectins. The lectin binding patternof the yolk indicatedthat, at the rime of incorporationof the yolk in embryosorshorùythereafter,a processingof the yolk occursresuJtingin changesin lectin-bindingcharacteristicsof the yolk protein.

Résumé- Modalitésde liaison deslectineschezles nématodesCaenorhabditiselegans,PanagrolaiInussuperbusetAcrobeloidesmaximus- Les modalitésde liaisondeslectinessontétudiéescheztrois nématodesRhabditideslibres - Caenorhab-dilis elegans,Panagrolaimussuperbuset Acrobeloidesmaximus- en utilisant dix lectines différentes. Bien que tous les tissus desnématodesse lient à une ou plus des lectines utilisées, des différencesconsidérablessont observéesentre les trois espècesdenématodes.Le vitellus, quatredescoelomocyteset les oocytesse lient le plus souventavecles lectinestestées.Quoiqueréagissantàde nombreusessubstancesétrangères,la bordureen brossede l'intestin n'estcoloréeque par quelqueslectines.Les modalitésdeliaison du vitellus indiquentqu'aumomentde l'incorporationde ce dernierdansl'embryon- ou quelquepeu aprèscelle-ci - il seproduit dansle vitellus un processusqui modifie les caractéristiquesde liaison deslectinesavecson contenuprotéinique.

Key-words: Acrobeloidesmaximus,Caenorhabditiselegans,immunohistochemistry,lectin histochemistry,nematodes,Panagrolai-mussuperbus.

Lectinsare proteinsor glycoproteinsof non-immuneorigin extractedmostly from plantsand from sorneam-mals,which reversiblybind with high specificity to car-bohydrates.This properryhasled to theextensiveuseoflectins to study ceil and tissue glycoconjugates(Roth,1986).

In nematoJogy,lectins havemainly beenusedfor thestudy of carbohydrateson the exteriorsurfacesor exu-datesof mainly plant-parasiticnematodes(Spiegelet al.,1982; Bowman et al., 1988; Aumann & Wyss, 1989).Although the use of lectins to characterizeCaenorhab-ditis elegansproteinshasbeenreported(Sharrock,1983;Francis& Waterston, 1991), ta dateno systematicana-Iysis has beenperformedof the internai lectin-bindingsites in nematodes.

Considering the huge body of information alreadypresenton C. elegans,its usemay facilitate future moredetailed studies of lectin-glycoconjugateinteractions.

Furthermore,the application of information obtainedfrom this nematodeto other economicaily importantnematodeshasaiready beenindicated(Ward, 1988).Inthe presentstudy, the binding patternsof ten fluores-cent-Iabeledlectinswere analysedin threerhabditid ne-matodes,i.e., C. elegans (Rhabditidae),Panagrolaimussuperbus(Panagrolairrùdae),and Acrobeloidesmaximus(Cephalobidae).

Material andmethods

NEMATODES

C. elegansvar. Bristol was obtainedfrom T. Bogaert(MRC) andhim-8 (e1489IV CB1489)froml Hodgkinthroughl Vanfleteren(Belgium), P. superbuswas pro-vided by B. Sohlenius (Sweden) and A. maximusbyP. Baujard (Sénégal).

o Presentadresses:G. Borgonie: Max-Planck-Institutfiir Biochemie,Am Klopjerspitz 18A, 82152 Martinsried, Germany;D. DeWaele: LaboralOTYjor Tropical Plant CuitivatioTl, Kardinaal Mercierlaan 92, 3001 Heverlee,Belgium.

I55N 1164-5571/97/02 S 7.00/ © GauthIer-Villars - OR5TOM 173

G. Borgonieet al.

MONOXENIC AND AXENIC CULTURE

Ali nematodesweresterilisedfor monoxenicor axeniccultureusingalkalinehypochloritesolutionaccordingtoSulstonandHodgkin (1988).Nematodeswereculturedon Escherichiacali and generallyhandledaccordingtoBrenner (1974). Stock cultureswere kept at 20 oc. C.eleganswasculturedaxenica11yaccordingto Vant1eterenel al. (1990). A. maximuscannotbe sustainedindefi-nitely on this axenicmediumandrequiresan additional250 f.Li hemoglobinsolution/100 ml axenicmedium.Al-ternativch,haemecould be excludedif 30 %of the cul-ture t1uid' consistedof heatedliver extract (HLE) pre-pared based on Sayre el al. (1961). Commerciallyavailable liver was eut into small pieces,autolysedfor24 h at 4 oC, and homogenized1 : 1with waterat 4 oc.The homogenatewas heatedfor 4 min at 56 oC andcentrifuged at 8832g for 30 min at 4 oC in a Sorva11RC-5B RefrigeratedSuperspeedCentrifuge.After cen-trifugation the HLE supernatantwas fliter sterilized(0.2 f-lm), and frozen at - 20 oC until use. No suitableaxenic mediumwas identified which allowed indefiniteculture of P. superbus.

EMBRYO PREPARATION

Following Goh and Bogaert (1991) embryosof dif-ferent developmentalstageswere co11ectedby washingnematodes(gravid adults and eggs) from 5 cm plateswith 0.8 ml of M9 buffer (22 mM KH 2P04, 33 mMNa2HP04, 86 mM NaCI, 1mM MgS04). The suspen-sion was mixed with 0.4 ml of alkaline hypochlorite(4 vol. 2 M NaOH: 3 vol. 14 % (v/v) NaOCI) in anEppendorftube.After 8-10 min with occasionalmixing,the tubewas spunin an Eppendorfcentrifuge(1 min at3180g). Thepelletwas takenup in 0.4 ml of M9 bufferand rnixed with the samevolume of ice-cold 2 M su-crose/100mM NaCI. After 0.2 ml of distilled waterwasplacedon top of the sucrosecushionand the embryost10ated to the interface after 5 min centrifugation at3180 g. The embryoswere removedfrom the sucrosecushionwith a Pasteurpipetteandwashedoncein PBS(50 mM Na2HP04, 140mM NaCI, pH 7.2). En:bryoswereco11ectedby centrifugationandresuspendedln drs-tilled wateL

EMBRYO CENTRIFUGATION

Embryo centrifugationwas carried out accordingtoSchlicht and Schierenberg(1991). A drop of distilledwater containingeggs of a11 developmentalstageswasput on a poly-L-lysine (MW> 300 000, Sigma, St.Louis, MI, USA) coatedslide. The slide was put in aplastic holderwith two incisuresto tightly maintai.ntheslide.Theplasticholderwith theslide neatlyfitted ln themetal centrifugebeakerof a Sorva11RC 5B refrigeratedsuperspeedcentrifuge.Thebeakerwasfilled with disti11-ed wateL The slide was centrifuged at 14 926g for15 min at 10°C. After centrifugationthe eggswere put

174

under a coverslip, freeze-crackedand incubatedwithlectins as describedfurther in the text.

WHOLE NEMATODE/EMBRYO STAINING

Leclin binding : a detailedanalysisof thedifferent fixa-tion andtreatrnentof nematodetissuefor optimal bind-ing has beendescribedpreviously. Ali results reportedherewere carried out after freezecrackingfo11owedbyacetoneonly fixation (Borgonie el al., 1994). Nema-todeswere incubatedovemightat 4 oC with 100 f-lg/mlt1uorescent-Iabeledlectins,dissolvedin PBS containing0.5 % Triton X-100. For visualizationof nuclei, 4, 6-diamidino-2-phenylindole (DAPI, Sigma, St. Louis,MI, USA) was addedto the lectin at a final concentra-tion of 1 f-lg/ml. The slides were washedfive rimes inPBS pH 8.0 and mounted in 50 % glycerol-PBS (pH8.0). The coverslip was sealedwith clear nail polish.Controls were carried out by preincubatingthe lectinswith 0.3 M of complementarysugar for 1 h at roomtemperatureprior to incubation.Asialofetuin was usedat a final concentrationof 1 mg/ml. A secondcontrolwas done by preincubatingfreeze-crackednematodes/embryos overnight in the dark in 50 mM NaI04 in100mM potassiumacetatebuffer (pH 4.5) and sub-sequentwashingthreerimesin cold PBS (4 OC) prior tolectin incubation.

The lectins used were: the lectins from Canavaliaenslfarmis(Con A), Phasealus'vulgaris erythroagglutinin(PE) and P. vulgansleucoagglutinin(PHA-L), Lenscuh-naris (LCA), Ricinus cammunis (RCA-II), Ulex euro-paeus (UEA-I), Salanumtuberasum(STA), Arachis hy-pagaea (AHA) , Glycine max (SBA), and Limulus pal-yphemus(LPA) were obtainedfrom Sigma (St. Louis,MI, USA). Alilectins wereFITC labeledexceptUEA-Iwhich was TRITC labelled.Ali the lectin stainings,in-cluding the controls,weredonewith lectin comingfromone and the samepreparation.

Sugars and glycapraleins: used for controls were:(Con A) : o:-methyl-D-mannopyranoside,(SBA) : N-Acetyl-D-galactosamine,(AHA and RCA-II) : lactose,(STA): Triacetyl chitotriose and (PHA-E, PHA-L,LPA, UEA-I and LCA) : asialofetuinwere from Sigma(St. Louis, MI, USA). Table 1 summarizesthe sugarspecificity, inhibition and control experimentsperform-ed for eachof the lectins.

L\.1.IViUNOHISTOCHEMISTRY

After freeze-cracking,the slide was dipped in pre-cooledmethanol(- 20 OC) for 5 min and thenin acetone(- 20 oC) for 5 min and air dried. The slides werewashedfour rimes for 15 min in buffer (PBS, 0.01 %(v/v) Tween-20,5 % (v/v) dried milk). Monoclonalan-ti-yolk antibodies (generously provided by SusanStrome, Indiana University, Bloomington, IN, USA)werediluted in thesamebuffer 1:20 andincubatedover-night at 4 oc. The slides were washedthree rimes for15 min in buffer. The worms were then incubatedin

Pundam.appl. NemalOl.

Lectin binding pallerns in nemaLOdes

Table 1. Sugarsandglycoproleinsusedin control expen'menlsLO black lectin staining.

Lectin

CONARCA-IIPHA-EPHA-LLCASTAUEA-IAHASBALPA

Affmity

cx-D-man,cx-D-gJcI3-D-galNAc, cx-D-galoligosaccharidesoligosaccharidescx-D-mantriacetyl chitotriosecx-L-fucI3-D-gal (l-3)-D-galNACD-gaINAcfetuin > NeuNAc

Inhibitingsugar(s)used

cx-memanlacasialoferuinasialoferuincx-D-man,asialoferuintriacetyl chitotriosecx-L-fuc & asialoferuinlacD-galNAcasialoferuin

Sugargivingcompleteinhibition

cx-memanlacasialofetuinasialoferuin--- (*)triacetyl chitotrioseasialoferuinlacD-galNAcasialoferuin

50 mMNaI°4

++++

+++++

Sugaraffiniry as reportedby the manufacrurer(SIGMA) (Man: mannose;glc: Glucose;a-meman: a-methyl-D-mannopyranoside;galNac:N-Aceryl-D-galactosamine;lac: laclOse;gJcNAc : N-Aceryl-D-gJucosamine;fuc : fucose;NeuNAc : N-Acetyl neuraminicacid). (+) : Completeinhibition of slaining; (-) : no inhibition of slaining. Data presenledare from acelOne-onlyfixed nemalodes,using overnighl incubational 4 oc.(*) asialoferuininhibited ail staining,exceplanteriorbrush borderslaining in C. eiegam.

FITC-Iabeledgoat anti-mouseIgG (Sigma) at a 1:50dilution for 4 h at room temperature. Slides werewashedthreetimes 15 min eachin buffer and one timein PBS for 15 min before being mountedas describedabove. Three anti-yolk monoclonal antibodies wereused;PIla A3 C9 (recognizesyolk protein (yp) 170B),or A5 D9 (recognizesyp170) and or Cl D3 (recog-nizesyp170A, yp115andyp88) (Sharrockel al., 1990).

To identify neurons,a goat anti-horseradishperox-idase(Type VI, Sigma,St. Louis, MI, USA) antibodywasusedsincethis antibodyhasbeenreportedto identi-fy cells of the nervous system (among others) in C.elegansand Drosophi/ame/anogasler(Siddiqui & Culotti,1991;Wangel al., 1994).Previouslyit wasreportedthatthis antibodyrecognizes27 of the 302 neurons,besidesseveralnon-neuronalcells in C. elegans(Siddiqui & Cu-loni, 1991). Preparationof the nematodetissuewas asdescribedaboveexceptthat thewashingand incubationbuffer contained0.1 % (v/v) Triton X-I00 insteadofTween-20.Theprimaryantibodywasdiluted 1:25.Thesecondaryantibody was a rabbit anti-goat IgG FITCconjugate(Sigma,St. Louis, Ml, USA) diluted 1:50.

COLLAGENASE DIGESTION

Collagenasedigestion of freeze cracked nematodeswasperformedby incubatingthe nematodeswith 2 mg!ml collagenase(SigmatypeIV, 460 units/mg,Sigma,St.Louis, MI, USA) in 100 mM Tris HCI pH 7.5, 1 mMCaCI2at 37 oC for 1 h. After washingfive rimes in PBS,nematodeswere incubatedwith lectin overnightas de-scribedabove.

For lectins and irnrnunostainings,mixed cultures(containingeggs,ail four juvenile stages,youngand oldadults)of ail threenematodespecieswerestained.Sincemales in A. maximusare rare, none were used for thestudy.

Vol. 20, n° 2 - 1997

Observationsweremadeusinga Leitz Diaplan (Wet-zlar, Germany)microscopeequippedfor fluorescencemicroscopy. Photographswere taken on Kodak ekta-chrome160T fum and processedcommercially.

Results

A summaryof the binding patternsobtainedis givenin Table2. Sorne additional or in depth analysesaredescribedin the next paragraphs.

CON AIdentificationof thevesicles(Fig. lA, B) wasaidedby

centrifugationof singlecell embryosof the threenema-todesand subsequentstainingof the embryowith ConA. Centrifugation results in three cytoplasmic bands(yolk, granule-freeand lipids) in the embryo as de-scribed by Schlicht and Schierenberg(1991). UsingTEM, they observedthat yolk accumulates in the cen-trifugai pole. This approachled to concentrationof thefluorescentstainin the centrifugaipole in C. elegansandP. superbusbut led to stainingof the two polar bandsinA. ma.x;imuswith the strongeststainingin the centrifugaipole (Fig. 1C).

Using theanti-yolkmonoclonalantibodies,stainingofthe yolk in the nematodeC. elegansgavea slightly differ-ent binding pattern.Stainingwith a mixture of the anti-bodiesresultedin strongstainingin the developingoo-cytes and embryos, far less in the intestinal cells andpseudocoelomaticcavity (Fig. ID). None of the anti-bodies, raised againstC. elegansyolk proteins,bind toyolk proteinseither from P. superbusor A. maximus.

Binding is alsovisible at thepositionof thedevelopingpharynxlumenand proceedsfrom the invaginatingan-terior end towardsthe middle (Fig. 2F, G). In A. maxi-mustwo additionalthin processeswerestainedoriginat-ing dorsally and ventrally and running paraUelwith the

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G. Borgonieet al.

Table 2. Summaryof interior leelln b-inding.

Lectin Structure C. elegans P. superbus A. maximus(1)

E F M E F M E F

CONA oocytesurface na na na na na +++yolk ++ ++ (2) na ++ ++ (2) na +++ +++

UEA-II yolk + ++ na + ++ na ++ +++epidermis + +

PHA-E yolk + + ++ +++ (3) + ++PHA-L yolk + + ++ +++ + +

sperm na na na + na naRCA-II oocytesurface na +++ (4) na na +++ (4) na na +++ (4)

oocytecytoplasm na ++ (5) na na na na ++ (5)intest. lumen embryo + na na +++ na na naintest. brushborder + +++ (6) +++ (6) +++ +++ +++developingpharynx +++ na na +++ na na +++ nadevelopingrectum ++ (7) na na ++ (7) na na ++ (7) nasecr./excr.duet +++ (7) na na +++ (7) na na +++ (7) naadult pharynx na +++ +++ na + + na +++nervoussystem na ++ +++ +++ +++ +++outer layer nervering (8) + +++ +++ + +++ +++outercuticle lining pharynx(8) ++ ++ ++ ++ ++coelomocYtes(9) + + + + ++ + + ++

LCA yolk ++ na ++ naoocyteyolk na +++ na na +++ na na +outernervering (8) ++ ++ ++ ++ ++ ++ ++ ++pharynx(8) ++ ++ ++ ++ ++ ++ ++ ++rectum +++ + + +++ + + +++ +coelomocytes(9) + +++ +++ + +++ +++ + +++

STA pharynxprecursors? + na na + na na + naouter intestinelayer na na na na + nasecr./excr.system +++ +++coelomocytes + +++ +++ + +++ +++ + +++intest. brushborder + +++intest. cytoplasm +++coelomocytes(9) ++ ++ ++ ++ ++

ARA yolk +surfaceepid. cells +headneurons +coelomocytes(9) + + + + ++ +++uteruslining +++ na na na naepidermalnerves +++ +microtubulecells AVM, PYM +++

SBA yolk +++ + +++oocytesurface na +++ na na + na na +++gonadprecursors na na na na na ++rectum ++ + ++sperm na ++ ++ na + navulva na +++ na na + na na

LPA no specifiestaining

Datapresenredarefrom acerone-onlyflxed nemarodes,usingovernightincubationar 4 oC. E :embryo;F: female;M : male;+ : weakbinding; ++ :moderarebinding; +++ . srrongbinding;- - : no binding;NA : norapplicable.(1) A. maximllS malesarerare,nonewereusedfor chisanalysis_(2) Noyolk stainingin ceUs immediarelyposreriorro the pharynx. (3) Srrongsrainingin ceUs immediarelyposreriorto the pharynx.GraduaIdecreaseinsrainingfrom firsr ro fourth juveniJesrage.(4) SraininggraduaUydisappearsastheoocyteenrerstheurerusandis complerelyabsenrwhentheeggoheUis formed. (5) Srainingdisappearswhen the eggoheUis formed. (6) Excepr the anrerior ring of four ceUs. (7) Variable sraining obtained,nor aUspecimensexibitedsraining.(8) BindiogabolishedafrercoUageoaserrearrneor_(9) Only four coelomocytessrainin anyof thethreenematodespecies

176 Fundam.appl. Nemalol.

LeClin binding pallemsin nemalodes

Fig. 1. A : SlrongsLainingofimeslinalyolkvesiclesin lhe nemaLOdePanagrolaimussuperbususingPHA-E; LO demonSlTalelhe slrenglhoflhe sLaining, lhis epifluoresencephoLOgraphwas Laken wilh lhe microscopelighl on, al ils lowesl selling; amerior LOp; B: Slaining of anAcrobeloidcsmaximusspecimenby Con A showinga concenlralionofsLainingnear lhe basalsidesof the inteslinein lhe pseudocoeÙJm;amerior top; C: CentnfugedA. maximus single cel! embryo, showing lWO distinct bands(arrowheads)after staining with Con A; D :Caenorhabditiselcgansembryostil!znsidelhe uterusafter swiningwùh monoclonalami-yolkamibodies;E: PeripheralsLainingofyolksphereswùhLCA revealedin A. maximusaftersquashingembryos;F: SameasE, bul at higherznagnification(BB : Basalbulb, 1 : intestine,NU: nucleus,Y: yolk vesicle). (A-B: bar = 2{) JLm; C-D: bar =10 JLm, E-F: bar =5 JLm).

Vol. 20, n° 2 - 1997 177

G. Borgonieet al.

Fig. 2. A : Slaining of disseetedovary from Acrobeloidesma.ximusby RCA-fI. Suifaceslaining of oocytesincreasesas developmemproceeds(arrow); B: Slaining by RCA-Ilof lhe layer surroundingpharynx, lhe excreLOryduel and slrong siaining of lhe inleSlinal brushborderin Caenorhabditiselegans;notelhe absenceofslaining in lhe cells immedialelyposlerior10 lhe basalbulb (belweenarrowheads);lheadjacenl cells slain weak; anterior lop; C: SLainingof C. elegansafler collagenasedigeslion; nole absenceof pen·-pharyngealsLaining;amerior IOp; D-E: Differenceof SlrucLUres slained in A. maximus by LCA; (D arrow) and a polyclonal anli-honeradishperoxidaseanlibody; (E arrows); amerior IOp; F-G: ProgressivesLainingof the developingphmynxin C. elegansembryos;lhe slaining of the reClumwasnOl a/wayspresent(0: oocytes;D : excrelOryduel; BB : basalbulb; J: imestine;PH : pharyrLx; R : reclum). (A-E: bar = 20 /Lm; F-G:bar =10 /Lm).

178 Fundam.appl. NemalOl

Leetin bindingpallerns in nematodes

Fig. 3. A : Stainingof the invaginatingpharynxandrectumearly in developmentin AcrobeloidesmaximususingLCA; B : LCAfaintlystains the developingnerve ring; in comparisonto RCA-II, the pharynxstains muchweakerin A. maximus; C: Stainingof epidermisassociatednervesin A. maximus(smaltarrowheads)and neurons(large arrowhead)by LCA; D : Stainingof thecoelomocytesandintestinein A. maximusby STA; besidesstain beingpresentin the entire lumen, a large numberof smaltcylOplasmicvesiclesstained,close to theintestinalbrush border (CC: coelomocyte;1: intestine;PH: pharynx:; R : rectum; NR : nerven'ng; LU: intestinal lumen; anten'or top).(A-B: bar =10 IJ-m; C-D : bar =20 IJ-m).

Vol. 20, n° 2 - 1997 179

G. Borgonie et al.

Fig. 4. A : Stainingof the secl'ewry/excretorysystemin a squashedCaenorhabditiselegansby STA; the Large arrowheadindicates lhetransversedUCl, the smaLLarrowheadsshowpart ofthe duct that hadpartLy dzsLocatedas a resull of the staining protocoL; amenortop; B :StainingofAcrobeloidesmaximusby SBA,massiveslainingofintestinaLyoLkandsurfaceswiningoftheoocytes;C-D : Disappearanceofyolk staining during embryonicdevelopmemin three A. maximusembryos(large arrows); thepositionof the embryocan be wmparedbynucLearstaining (comparesmaLLarrowheads)using DA?! in C versusthe weakyolk slaining in D usingAHA (G: Gonad; 0 : oocytes).(A-B: bar =20 J.Lm; C-D: bar = lO J.Lm).

180 Fundam. appl. Nematol.

Leclin binding pallerns in nemalOdes

Fig. 5. A .' Slainingoflhe nervering andassocialednerves(small a1Towheads)in Acrobeloidesmaximusby AHA; B : Surfaceslainingoflhe epidennalceUsin A. maximusduângde:velopmemby AHA; lhis slainingwasonly presentfor a shorl lime; no slainingis evidentin lheheadarea (a1Towhead)(BB .' basalbulb; NR .' nervering). (A: bar = 20 !J.m; B . bar = 10 !J.m).

stainedpharynx.Theposteriorendof that processgrewconsiderablyas developmemproceededand was pre-sentin adult wormsasa large,sometimesbiJobedtrian-gular structure.Furthermore,the anti-horseradishanti-body stainedthis structureas weil.

LCAIn ail threenematodespecies)binding patternsin em-

bryos vary considerablyduring development.In C. ele-gans and P. superbus)yolk is sIained in the developingoocytesand early embryonicdevelopment.In squashedembryosin which the yolk sphereswere releasedon theslide, it \Vas observedthat the lectin-binding of theseyolk sphereswas strong at the periphery (Fig. 1E, F)while no staining was presentin the center.Using themonoclonalanti-yolk antibodies,no such difference inbinding patternscould be observedin C. elegans.Yolkstainingrapidly disappearsand the lumen of the devel-oping pharynx) the layer surroundingthe rectum) andthe nervering arestainedsubsequently.In A. maximus)yolk is weakly stainedinitiaily, strongerbindinggradual-

Vol. 20, n° 2 - 1997

ly appearsin the intestinalareain the commastage,butin later stagesof development)yolk stainingdisappearsand the samestructuresare stainedas in the other wospecies(Fig. 3A, B). In adult C. elegans)strongbindingto the basallayerof the pharynx.and the layersurround-ing the nervering, also susceptibleto collagenasediges-tion, is evident. Binding ta the nerves in C. elegansislimited to the cephalic sensilla and amphid-associatedstructuresand sorne staining could be observedalongthe nervescoming from theseStructures.The intestinalyolk, sperm,and the lining of the reproductivesystemshowed weaker binding. The intestinal brush borderstainsintensivelyalong the entire tract but this stainingof the ring of four ceUs immediately posterior to thepharynx cannotbe inhibited by NaI04 pre-incubationand is thereforeconsideredto be non-specifie.

LPASorneweakstainingwas observedalongthe intestinal

brushborderof C. elegansandP. superbus)andalongthebuccalcavity of A. maximus.However)this bindingpat-

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G. Borgonieet al.

tern was very variableand inconsistentbetweendiffer-ent replicasandthereforeconsideredta be non-specific.When in the axenicmediumof C. elegansandA. maxi-mus the haemewas replacedby heatedliver extract,nospecific binding couId be observed.

The two different culture techniquesemployedgavedifferent results.WhenusingSTA on nematodesfed onE. coli the bacteriawere strongly stained as weil andappearedas numerousspots in the intestinal lumen. Itwas observedthat, upon using RCA-II, the intestinalbrushborderstaining\Vas strongerin animaisgrown onaxenic mediumthan thosegrown on bacteria.

Discussion

PHARYNX

The outerlayerwhich lines the cuticle of the pharynxbinds with severallectins at a very early time in devel-opment. InitiaUy the entire length is stainedbut, as de-velopmentproceeds,the anterior half is stained mostconsistentJy.Themostposteriorhalf is stainedirregular-ly. Whether this irregularity is the result of changesincarbohydratecomposition during developmentis un-known. Severallectinsstainedthe layer aroundthe pha-rynx intensely. Only one lectin (STA) was binding tothe surfaceof the embryonicceUsthat form the pharynxin A. maximus.More lectins stainedthe layer surround-ing the pharynxin adults.Supportfor the identificationof this layeras thebasallaminacornesfrom the observa-tion that, when lectin incubationis done after collage-nase(Type IV) treatment,the stainingof the layer sur-rounding the pharynx disappears.Since the C. elegansbasallaminamostlikely containscollagenIV (Kingston,1991), its disappearanceafter digestion is as would beexpected.

EP1DERMIS

No strongbinding was observedwith any of the lec-tins used, with the exception of epidermis associatednerves(seenervoussystem).

INTESTINE

Of the ten lectins used,RCA-II, STA and LCA bindto the glycocalyx and/or the microvilli of sorne of thespecies.ThebindingpatternofRCA-II in C. elegansandSTA bindingpatternsin P. superbusandA. maximusaremost likely linked to feeding. As long as the embryo isencasedin an eggshell,harcily any stain is visible in theintestine;hO\,vever,ail hatchednematodesexhibit sU'ongbinding. The carbohydraterecognizedis most likely se-creted, maybe resulting from feeding. In P. superbus,RCA-II binding is also strong in embryos inside theeggshell.The binding of LCA ta the intestinal brushborderof C. elegansis considerednon-specificsincepartof the binding at the brushbordercannotbe inhibited.

182

GONAD

Stainingof the gonadtissueitself is limited. AHA didstain the interior lining of the uterusin C. elegansandA.maximus,LCA did bind embryonicto precursorcells ofthe gonad.Most of the binding patternin the gonadisassociatedwith the outersurfaceof early oocytes.Thisuniform superficial membranestaining disappearsasthe oocyte progressesin the gonad. Repeatecily, nomembranebinding was observedonce the eggshellwasformed. The presenceof binding, predominantly inearly oocytes, may not be surprising considering themany interactions oocytes undergo at that stage. Inmales,spermis recognizedby severallectins.Unlike theasymmeu'icalbinding of the sperm surface by WheatGermAgglutinin (Roberts& Ward, 1982a,b), we onlyobserveduniform surfacebinding of the spermsurface.

NERVOUS SYSTEM

The lectins AHA, LCA and RCA-II bind to severalneuronsand/orneuronalprocesses.In the caseof AHAthis had a1ready been reported (E. HedgecockandM. Chalfie, pers. comm.). Although the binding tonerves in the head area in C. elegans is extensive,farmorebindingcanbeobservedin A. maximus.As a resultof the paucityof dataavailableon the nervousstructureof that species, comparison with the better knownC. elegansnervoussystemis presentlynot possible.Be-sidesthe nerves,amphidassociatedstructures(nerves?)arestainedaswell and mostprominentlyin A. maximus.Despite its large size and consistentstaining, we havebeenunable to identify the structurethat is recognizedin Fig. 2D. In A. maximus, the structurestainedwithRCA-II is alsostainedwith the anti-horseradishperoxi-daseantibody,unlike the structurestainedwith AHA, orLCA. This might indicate that the RCA-II is staininglarge c10selyspacedneuronsand that the stainedstruc-ture is not a part of the amphid sensustricto despiteitsposition. Both other lectins bind effectively to the am-phidial sacor associatednon-nervousstructures.

SECRETORY/EXCRETORYSYSTEM

The secretary/excretorysystemwas only stained inC. elegansusingSTA. In the embryoshoweverRCA-IIand SBA did bind occasionallyto the excretory duct(duct = the shortcanalrunningfrom theexcretoryporeto the sinusconnectingboth lateralcanalsof thesecreto-ry/excretory system) in ail three species.In the adultsthis binding was highly variable.With the RCA-II aftercollagenasetreatment, the excretory duct remainedstained(Fig. 2B), indicating that the lectin did not bindto the membranesurroundingthe duct and that thelectin boundeitherto the lining of theductor to the fluidinsidetheduct itself. We assumethat thelectin boundtofluid presentin theductratherthantheductitself, whichwould explain the transientnatureof the staining pat-tern. For that samereasonwe doubt that STA bindingto the entire secretory/excretarysystem (except the

Fundam.appl. NemaLOI.

duet) in C. elegansis due ta the fluid inside the tubularnetworkbut representseffectivebinding of the lectin taa componentof the secretary/excretarysystem.Suchabinding would explain the consistentbinding patternobservedin the severalreplicasversusthe aberrantbind-ing ta the dUCL

COELOMOCYTES

In the embryo of the three speciesstudied, strongbinding to four coelomocyteswas observed. In theadults of ail three nematodes,only four coelomocytesarestained.This is surprisingsince therearemore coe-lomocytesin adults than in embryos.In C. eleganstwomore coelomocyresare presentin the adult hermaph-rodite (onemorein the male) (Sulston& Horvitz, 1977;SuJstanet al., 1980). In A. maximus there are sevencoelomocyresin the femaJe(De Ley & Siddiqi, 1991)andwe found the samenumberin adultsof P. superbus.The absenceof stainingin post-embryonicallydorsallyformedcoelomocyres(Mdlpa andMdrla) , indicatesthatin ail three families thereare at leasttwo different typesof coelomocyres,basedon their gJycoconjugatecompo-sition. No lectin wasfound ta bind to ail coelomocytesinanyof the threenematodespecies.Furthermore,not oneof the lectins used stained the post-embryonicallyformed coelomocytes.It is impossible ta explain thisdifferencenor to advanceany sensibleexplanationforthe differencesin staining observed,since the functionand structureof coelomocytesin free-living nematadesis still unknown.Severalhypotheseshavebeenproposedvarying from their implication in cellular defense(phagocyrosis)to ceils coilecting undigestableor poi-sonousmaterial or stains (for review seeWood, 1988;Bird & Bird, 1991). Other possiblefunctions proposedinclude coelomocytesas importantcentersfor interme-diatemetabolism(Turpeenniemi,1993) or involvementin the correct positioning of the gonad during devel-opment(De Ley & Siddiqi, 1991).

YOLK

Severallectins bind strongly to the yolk or yolk pre-cursors in the intestine, yolk proteins secretedin thepseudocoelomiccavity and in embryos. The stainingof yolk by severaJlectins is not exceptionalsince thebinding of Con A ta yolk from C. elegansand Doli-chorhabditis sp. had previously been demonstratedbySharrock (1983) and Wimer (1992). However, thestrongbinding with severallectins cOnltrms the heavilyglycosylatednatureof the nematodeyolk proteins.

Processingof the yolk proteins is clearly evidentfromthe changing lectin-binding capabilities during devel-opment.Furthermore,if yolk stainsin onegiven speciesit doesnot necessarilystain in any of the other speciestested,which indicatesdifferencesin yolk glycosylationbetweenspeciesof the same family. Several bindingpatternswere observed;although the various patternswere presentin ail three nematodespecies,the most

Vol. 20, n° 2 - 1997

Lectin binding pallemsin nernatodes

distinct patterns,with the lectins used,were in A. maxi-mus: i) with the lectins Con A, UEA-ll, PHA-A andPHA-L, the binding is stable throughoutdevelopmentand is presentin ail developmentalstages;iz) a secondpattern is evident using SBA; strong binding in adultnematades,weakbindingin embryosandnonein theJI;iii) by contrast,LCA and AHA lectins barely bind tayolk in the adult, but bind ta yolk strongly in the devel-oping oocyte;binding hasdisappeared,however,in theJI stage. AJthough both binding patterns Ｈ ｩ ｺ ｾ iiz) aredifferent, they indicate sorne processingmust occur atthe rime or shortJy after incorporation of yolk or itsprecursorsinta the maturingoocyte,causingeither lossofbinding (SBA) or theappearanceof newbindingsites(LCA, AHA).

Several authors describe yolk spheresas modified" Iysosome-like" organelles containing unusual andconsiderableenzymaticactivity (Wall & Meleka, 1985;Fagotto,1990a,b, 1991) necessaryin yolk formation byprocessingcarbohydratesof the yolk precursor,viteilo-genin (Wall & Meleka, 1985). Removalof the carbo-hydratesreleasedwas consideredessentialfor this pro-cessing reaction ta proceed in membrane-boundorganelles.It has been proposedthat the endogenousyolk lectins regulate this reaction (Yoshizaki, 1992).This wouId lead ta a high concentrationof sugarresi-duesat the surfaceof the yolk vacuolewherethey bindthe endogenouslectin. The appearanceof the lectinbindingat the peripheryof the yolk spheresin squashedembryosand the changingbinding patternsis reminis-centof resultsobtainedby Yoshizaki (1990, 1992).Theperipherallectinbinding and the changingbinding pat-ternscould be explainedby a similar processingmecha-nism during incorporationof yolk in the oocyte. Theprocessingof carbohydratesin nematodeyolk spheresand their subsequentremovalcould eitherlead to exist-ing binding sites disappearingor new binding sites ap-pearing. Ultrastructuralanalysisof yolk vesicles in theintestineand of developingoocytesof A. maximuspro-vide support for this interpretation (Borgonie et al.,1995). In intestinal cells, only yolk vesicleswere foundwhich appearuniformily black. However, identical ves-icles are found, in the developingoocytes,but also ves-icles wherethe enclosedyolk materialexhibits randomlystriatedorientedtubular cavities,consideredby Yoshi-zaki (1990) in Xenopus,ta be indicativeof yolk cystaili-zation. With one exception,thesestriatedvesicleswereexclusivelyfound in the oocytes(in 147 randomlytakenmicrographsof A. maximussections,only one caseofstriatedvesiclesappearsin the intestine;seeBorgonieetal., 1995).

We believeit is very likely that the " yolk " presentinthe intestine is the yolk precursorviteilogenin and thatthe final crystallizationto yolk occursin the oocyte.Onecanwonderwhatwould bethe usefullnessof first addingcarbohydrate,only to remove it at a later stage. Onepossibility might be that the addedcarbohydrateson the

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vitellogeninserveasbindingsitesto ensurecorrectrout-ing to, and incorporationin the gonad,after which theywould becomeobsolete.

Using lectin binding we observed likc Sharrock(1983) and Bossingerand Schierenberg(1992) thatconsiderableamountsof yolk are still presentin welldevelopedembryosand newly hatchedfust stagejuve-nile. Both reportsproposedthat this yolk reservewouldsupport post-embryonicdevelopment.BossingerandSchierenberg(1992) also indicatedthat the yolk reservein the intestinecould be partof a backupenergysourceallowing survival in periodsof starvation.Sorneof ourdata support both views but stressthe role of yolk instarvationsurvival. Axenically cultured C. eleganscon-tain less yolk since they stain considerablylesswith themonoclonalanti-yolk antibodiesor lectins,and theysur-vive for a shorter rime when starved than nematodescultured on E. coll (unpubl. data). Theseobservationssupportthe view of Vanileteren(pers. comm.) that ax-enically cultured C. elegansare in a continuedstateofnutritional restriction. By containing less yolk than C.eleganscultured on E. coli, they have less backupwhenstarvedand die sooner. Axenically cultured C. elegansare probablyunableto producesimilar amountsof yolkas whenculturedmonoxenicallybecausethe axenicme-dium is suboptimal as a food source or as an envi-ronrnent. It has beenobservedthat axenically culturedC. elegans produce Jess offspring when compared tothose cultured on E. coli (Sulston & Hodgkin, 1988).However,we consistentlyobservedweakeryolk bindingin male juvenile specimenas developmentproceeded.This might further support the view that the surplusyolk is indeeda necessityfor completingpostembryonicdevelopment.

Comparisonof the yolk binding patternbetweenlec-tins and the anti-yolk monoclonalantibodiesgives sorneinformationaboutthe targetrecognizedby the antibod-ies. Relevant is the consistentcompleteyolk vacuolebinding usingantibodiesin C. elegansirrespectiveof thedevelopmentalstage,versusthe shifting, circumferential,age-dependentshift in staining obtainedwith sorne ofthe lectins. This indicatesthat the epitopesrecognizedby the antibodiesare either polypeptidesor carbohy-drates of the yolk protein and its precursorand thatthesearenot involved in the processingoccurringin theembryo.Theseepitapesarenot sharedby the two othernematadespecies,as evidencedby the lack of bindingwith theseantibodies.

Specific binding of LPA was not observedwhen hae-moglobin was replacedwith liver extract in the axenicculture medium. The absenceof binding with LPA isnot surprising, given that the results of Batic el al.(1990)hadalreadyindicatedtheabsenceof sialic acid inC. elegansand Panagrellusredivivus.However,theseau-thors did obtain evidencefor sialic acid in both speciesusing 1251-Limaxflavus agglutinin in competitive dis-placementexperiments,but only if liver extract was

184

usedin the culturemedium.We were unableto confirmthe observationsof difference in binding patterns asresult of diet of Baéic et al. (1990), possiblybecauseofthe reducedsensitivity of direct labeling.

The importanceof the two differencesin bindingpat-terns observedbetweenanimaIsgrown on bacteriaandon axenicmediumis intriguing but unclearta date.Thestronger binding observedwith RCA-Il could be theresult of the presenceof bacteria.Nematodesculturedon bacteriahave shorterintestinal microvilli than thoseculturedaxenically (unpubl. data).As such therewouldbe fewer binding sites availablein the former case.

ln comparisonwith results reported earlier (Link etal., 1988) aboutexternalbinding patternsin C. elegans,therearemuchmoredifferent binding patternswith thelectins usedthan reportedfor externallectin binding.

In conclusion,considerabledifferencesexist in lectin-binding patternsbetweenthe threefree-li\ring nematodespecieswith the samelectin. Furthermore,al! tissuesinthe three speciesare stained with one or more of thelectins used. In some instanceslectins can be used asrapid (directstaining)andcheapalternativesto antibod-ies (indirect staining) as markersduring embryonicde-velopment(pharynx,secretory/excretorysystem,repro-ductive systemin A. maximus,yolk). Besidesthe yolk,four of the coelomocytesand the oocytesarestructureswhich stain most often with the lectins used. In retro-spect,this may not be surprisingsinceconsideringtheirfunction, both receivea lot of externalcuesor interactrepetitively with other tissuesor substances.Althoughthe intestinal brush border interactswith considerableamountsof foreign material, it is somewhatsurprisingthat the intestinal brush border binds with only a fewlectins. However, since acetonewas Ù1e only fixativewith which optimal binding and minimal backgroundstaining could be obtained,loss of glycolipids is ta beexpectedunder theseconditions, it is bl-cly that morebinding sitesare presentand that sornedatamight havebeenlost during the processingof the tissue.Thereforepoorerstainingof any given tissuedoesnot necessarilyreflect paucityof binding sites.

Acknowledgements

We wish to thankJ Vanfleterenfor use of his fluoresencemicroscope.We are indebtedto SusanStromefor generouslymakingcopiousamountsof anti-yolk antibodiesavailableandto M. Chalfie, E. Hedgecockand].Vanfleterento allow theuseof unpublisheddata. Some of the nematodesused were ob-tained from the CaenorhabditisGeneticsCenter, .S.A, un-der contractwith the NJ.H. Financial supportwas providedby the Fund for Joint Basic Research to AC (FKFO32.0083.92) and a pre-doctoral grant to GB from therWONL.

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