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Reference:Biol. Bull. 193: 343-400. (December, 1997) Limb Regeneration in the Eye Sockets of Crabs HSIAO-WEI KAO AND ERNEST S. CHANG* Bodega Marine Laboratory, UniversityofCalifornia, P.O. Box247, Bodega Bay, California 94923 Abstract. Theeyestalksofcrabswereremovedandvar- stumps of crab walking legs can induce the stumps to ious tissues ofthe limbs were autotransplanted into the regenerate claws or chimeras of claw and walking leg. emptyeyesocketstostudythecapacityofthelimbtissue Thepercentageoftheclaworclawlikestructuresregener- to regenerate in a heterotopic site. Autotransplantation ated from the walking leg stumps was higher when the of walking leg tissues into the eye sockets was able to autotransplantconsistedofacombinationofclawtissues regenerate complete walking legs in the new site. Auto- than when only a single type of tissue was used. With transplantationoftissuesofclawdigit(dactylandpollex) contralateral autotransplantation ofclaw tissues into the or more proximal claw segments (ischium and merus/ autotomized stumps ofthe walking leg, the stumps can carpusjoint)could regenerate complete claws in theeye regenerate claws with host-site handedness. sockets.Iftheautotransplantofclawtissuewascontralat- To further study whether claw tissues singly or in eral, claws could regenerate with host-site handedness. combination canregeneratecompleteclawsinahetero- Sham operations or autotransplantation of frozen claw topic site that lacks a regenerating limb field, we auto- tissue did not induce regeneration in the eye sockets. transplanted claw tissues into the carapace at sites from These results demonstrate that complete crab claws can which the eyestalks had been removed (these sites are regeneratefromtheeye socketsbyautotransplantationof hereaftercalled eye sockets). Crabs can regenerate their live limb tissue and that the regeneration is not due to autotomized limbs but not their eyestalks (reviewed by the traumatic effect oftransplantation. Hopkins, 1988). There are no reports ofcrabs found in Thestructureofthe limbsregeneratedin theeyesock- naturehavinglimbsintheplaceoftheeyestalks.Theeye ets was determined by the source ofthe transplantedtis- sockets thus provided an in vivo environment that was sue. Completeclawsresultedfromautotransplantationof isolated fromthe tissue ofthe autotomized limb stumps. thetissuesofthemostdistalclawsegments(clawdigits), In this study, we autotransplanted either single claw and the most distal claw segments regenerated first, fol- tissue or different combinations of claw tissues of the lowed by the proximal claw segments in subsequent distalormoreproximalclawsegmentsintotheeye sock- molts. Thus tissue from distal portions ofcrab claw can etstodeterminewhethertherewasanydifferentialregen- regenerateproximalportionsoftheclawintheeyesock- erative capacity. It hasbeen proposedthatthe distal por- ets. Such a mode of regeneration is not consistent with tionsofthelimbscannotregeneratetheproximalportions thedistalizationruleofthepolarcoordinatemodel,which of the limbs (distalization rule of the polar coordinate proposesthatdistalportionsofthelimbcannotregenerate model; Bryant et ai, 1981) and that different sizes or proximalportionsandthatthedirectionoflimbregenera- combinations of tissues can determine the regenerative tion is always from proximal to distal. capacity ofhydra (Shimizu etai, 1993) or the imaginal discsofDrosophila (KauffmanandLing, 1981). Inaddi- Introduction tion,wecontralaterallyautotransplantedlimbtissuesinto the eye sockets to study whether the handedness ofthe Ourpreviousstudy(KaoandChang, 1996)showedthat regenerated limbs would be changed by the host site. autotransplantation ofclaw tissues into the autotomized Materials and Methods Animalsanddissection escR*[email protected];ndaeccnecpetesdho2u2ldSepbte. a1d9d9r7e.ssed. E-mail: ofTcawroapsapceeciweisdotfhc=rab6s.9w-e2r7e.u9semdm:,CamnecaenrgraSciDli=s(1r2a.n0g8e 393 394 H.-W. KAO AND E.S. CHANG 3.49mm, n = 211) and C.jordani (range ofcarapace claw (ischium + merus/carpus joint) were autotrans- width = 8.2-17.2mm, mean SD = 11.55 2.67mm. planted into eye sockets. To see whether the host site n = 12).Specimenswerecollectedsubtidallywithahand could regulate the morphology ofthe regenerated claw, netinBodegaHarbor,California.Thecrabswerekeptin different claw tissues (dactyl or pollex) orcombinations individualcompartmentstopreventcannibalism(Conklin ofclawtissues(dactyl + pollex.ischium + merus/carpus and Chang, 1993) and were fed brine shrimp, shrimp joint, ordactyl + pollex -I- ischium) werecontralaterally meat, or fish every other day. Crabs were acclimated in autotransplanted into eye sockets. To see whether dead the laboratoryforat leastone moltpriortoexperimenta- tissues had the ability to induce regeneration in the eye tion. Only postmolt animals (less than 3 days aftermolt) socket,frozenclawtissueswereautotransplantedintoeye were used. sockets. Animals were forced to autotomize one limb either Todeterminewhetherwalkinglegscanregeneratefrom a claw or the fourth walking leg. (Crab limbs are de- eye sockets, tissues from the dactyl, ischium, and the scribedand illustrated in Kao andChang [1996].) Autot- merus/carpusjointofthefourthwalkinglegswerecontra- omy was achievedbycrushingthe manus orcarpus seg- laterally autotransplantedintoeye sockets. Control sham mentofthelimbwithapairofforceps.Fordonortissues, operationswereconductedbyinsertinganinsectpinthree thedactyl, pollex, ischium, and merus/carpusjointofthe times into an eye socket. The transplantations were con- autotomized claw were used. In other experiments, the sidered failures ifno limb or limblike structure regener- dactyl, ischium. and merus/carpus joint of the autoto- ated from the eye sockets after the fourth postoperative mized walking leg were used. These segments were cut molt. offwithapairoffinescissorsandplacmedMintoadepressmioMn sK1lC0i1dm:eMw1i3tH.h3EPamEdMSr,opCpaoHCfl7c:.r4;ab2wi6stamhliNMneaO(M4Hg4;C0l1:0;02u3NnaimtCslM/;ml1Np1ae.n:3SicOil4-; ChaFroarcttehreisctoincvsenoifeanxceesoafnddeshcrainpdteidonn,estshreineCa.xegsraocfilcilsaws lin; 0.1 mg/ml streptomycin; Cooke et ai, 1989). The aredefined. Fortheproximodistalaxis,theproximalseg- slideswereplacedunderadissectingmicroscopeandfine ments are the segments closest to the crab body. The forcepswere usedtocarefully pull intacttissues (includ- distal segments are the segments farthest away from the ingepidermisandmuscle)outoftheexoskeletonofeach body. Thus,thecoxaandischiumareproximal segments segment. ofthe claw; thedactyl and pollex are the distal "digits" ofa claw. For the anteroposterior axis, the dactyl is the anteriormovable "digit" ofaclaw andthe pollex is the Removalofeyestalksandautotransplantation posterior fixed "digit" of a claw. For the dorsoventral oflimb tissues axis,thedorsalsurfaceoftheclawmanushasfourridges One eyestalk of each crab (C. gracilis, n = 211; C. (carinae. Fig. 1A) along theproximodistal direction. The jordani, n = 12)wasextirpatedasproximallyaspossible ventralsurfaceoftheclaw isrelativelysmoothand lacks withapairofforcepsunderadissectingmicroscope.This carinae (Fig. IB). The dorsal surface ofthe walking leg procedure leftan opening at the site ofentry ofthe eye- hassmallgranulosatuberclesanddarkerpurplecoloration stalk into the carapace (eye socket). Immediately after (Fig. 1C), and the ventral surface ofthe walking leg is eyestalk removal, limb tissues were autotransplanted or relativelysmoothandlighterincolor(Fig. ID).Thehand- shamoperations wereperformed. Theanimals werethen edness ofaclaw can be easily distinguished by bending returned to seawater. it.Whentheclawdactylisorientedabovetheclawpollex, Autotransplantation consisted of pushing limb tissue the right claw bends to the left and the left claw bends into the eye socket, using the bluntendofan insect pin. totheright. Handednessofthefourthwalking legcanbe umnitcirlotshceoptei.ssTueracnosuplldanntoswloenrgeerofbetwsoeetnypweis:thipasidliastsereaclt.inign iisdteinctsi.fWiehdebnyvcioembwiendadtoirosnaslloy,fbtheendfionugrthanrdigahxtiswaclhkairnacgtelre-g whichclawtissuewasremovedfromonesideofthecrab bendsinaclockwisedirectionandthefourthleftwalking body and placed into the eye socket on the same side leg bends counterclockwise. (e.g., left claw tissues autotransplanted to the left eye socket ofthe same animal); and contralateral, in which Results the claw tissues were placed in the eye socket on the Autotransplantation oflimb tissues into eye socketsof polpapnotseidteinstiodetoheftrhieghctraebye(es.go.c,keletftocfltahwetsiassmueesaanuitmoatlr)a.ns- C. gracilis To examine the regenerative ability ofdifferent claw Afterautotransplantationofcrablimb(claworwalking segments, singleclawdigits(dactylorpollex),bothclaw leg)tissuesintocrabeyesockets,limbsorlimblikestruc- digits (dactyl and pollex), or proximal segments of the tures regenerated from some eye sockets (22.1% ofsur- CRAB LIMB REGENERATION 395 Figure 1. CharacteristicsofCancerf-riii-ilislimbs.(A)Dorsalsurfaceofclawmanushasfourridges (carinae).(B)Ventralsurfaceofclawmanusdoesnothaveridges.(C)Dorsalsurfaceofthefourthwalking leghasgranulosatuberclesanddarkerpigmentation. (D) Ventral surfaceofthefourthwalking legdoes nothavegranulosatuberclesandhaslighterpigmentation. viving crabs that received live limb tissues, n == 104; ated per molt were variable among crabs. Regeneration TableI,Fig.2A-I).Accordingtoourdefinitions, "regen- ofclaws withcomplete segments (Figs. 2A, B, 3A) usu- erated" limbs contained at leasttwo segments, and their ally tooktwo to fourpostoperative molts. Inmostcases, handedness was recognizable; "limblike" regenerates crabs regenerated one or two distal segments after the consistedofone ormore segments, but theirhandedness secondpostoperativemolt(Fig.2A,B)andtherestofthe couldnot berecognized. Autotransplantationofclawtis- proximal segmentsafterthe thirdorfourthpostoperative sues resulted in regeneration ofclaws orclawlike struc- molts. Somecrabs,however,regeneratedcompleteclaws tures from eye sockets (20.6% of surviving crabs that afterthesecondpostoperativemolt(Fig.3A).Bothsham- received liveclaw tissues; Fig. 2A-H). Autotransplanta- operated and autotransplanted crabs regenerated normal tionoffourthwalkinglegtissuesresultedinregeneration limbsfromtheirautotomizedstumpsafterthefirstpostop- ofwalkinglegsorleglikestructuresfromtheeyesockets erative molt regardless of whether limbs (or limblike (33.3% of the surviving crabs; Fig. 21). Sham-operated structures) were regenerating in their eye sockets. The crabs or autotransplantation of frozen claw tissues into duration of the first postoperative molt interval of the eyesocketsdidnotregenerateanylimborlimblikestruc- sham-operated(22.44 l.OSd,n = 18)andexperimental ture fromthe sockets (Table I). (20.8 0.56d,n = 91)animalswasvariableamongindi- The number ofcrab molt cycles needed to regenerate viduals ofsimilar sizes and not statistically different be- thesestructuresandthenumberofclawsegmentsregener- tween the two groups (P = 0.094). 396 H.-W. KAO AND E.S. CHANG TableI Summuiyoflimbtu.tnriiiiti>triin.\/>liiiitiitii>iiintheeyesocketof Cancergracilis(carapacewidth = 12.05 3.4(1mini Donortissue CRAB LIMB REGENERATION 397 Figure 2. Limb or limblike structures regeneratedfrom Cancergraciliseye sockets. (A) Complete clawregeneratedfromtheeyesocketafterthefourthpostoperativemolt(M4).(B)Completeclawwitha mixture ofdonor and host-site handedness after M4. (C) Claw with dactyl, pollex. and partial manus segmentsafterthesecondpostoperativemolt(M2). (D)Y-shapedlimbafterM2.(E)Pollexlikestructure afterthethirdpostoperative molt. (F)ClawlikestructurewithmanusandcarpussegmentsafterM2. (G. H)DistallytaperedregeneratesafterM4.(I)CompletewalkinglegafterM2. Handedness ofregeneratedlimbs in C. gracilis of the claw. The claw digits regenerated first, and the proximal segments regeneratedduring subsequent molts. eyeCosnotcrkaeltasterreaglenaeurtaotterdanssipxlcalntaawtsiownithofreccloagwnitziasbslueeshainndt-o tIphsrieleatcerlaalwsautwoittrhanrsepcloangtnaitziaobnleofhcalnadwedtniessssu.esTrheegseneertahtreede edness.Amongthem,twoclawshadhost-sitehandedness claws retained the donor (same as the host-site) handed- o(Fnieg.c2lAa)w,htahdreeacmliaxwtsurheadofdohnoosrt-tainsdsuedohnaonrdehdannedsesd,naensds ness (Fig. 2C). Contralateral autotransplantation of the (nsFuailtgti.eodn2oBff)r.odimsTthcaoelntatrnawdloaptrceolrxaaliwmsaaulwtiocttlhraawnhsotspitls-assnuitetaset(ihdoaancntdoyfeldan+ecspoosmlblriee-x- ofnoenuserstwh(aFliwkga.ilnk2gi1)nl.geglweigthtisrseuceosgniinztaobleey,edosnoocrk-ettisssrueegehnaenrdaetde-d a++ndmisedcnohunisou/mrc;ahrFapinugd.sedj2onAie)ns,t)s.orrTehpseurlotcxeildmaawflrwocimtlhaawuattomitisrsxautneussrpel(ainostfcahhtioiusomtn ACu.tjootrrdaannsiplantation ofclaw tissues into eye sockets in ofacombinationofdistal andproximaltissues(dactyl + ContralateralautotransplantationofC.jordaniclawtis- pollex + ischium; Fig. 2B). It had donor handedness at sues into eye sockets regenerated one complete and one its distal partsand hosthandedness at the proximal parts incompleteclaw (Table II). Thecompleteclaw (Fig. 3A) 398 H.-W. KAO AND E.S. CHANG Figure3. Claw regenerated from Cancerjordanieye sockets. (A) Complete claw regeneratedfrom theeyesocketafterthesecondpostoperativemolt(M2).(B) Incompleteclawwithallsegmentsproximal totheclawdigitsregeneratedafterM2. resulted from contralateral autotransplantation of both clawdigitsandhadhost-sitehandedness.Likeanauthen- ticclaw,itcontainedfivesegments.Theincompleteclaw (Fig. 3B) resulted from contralateral autotransplantation oftheclawpollexandhaddonor-tissuehandedness.Ithad a fork-shaped structure at the distal tip and four normal proximalsegments.Thefork-shapedstructurewasregen- erated first, andthe proximal segmentswere regenerated insubsequentmolts. All sham-operatedanimals failedto regenerate limbs in eye sockets. Discussion Kim and Stocum (1986) autografted anteriororposte- riorhalves ofaxolotl forearms toeye sockets andampu- tatedthemdistally7dayslater.Thetransplantscouldnot regenerate proximal structures ofthe limbs. They either regresseduntilasmallfragmentoftheradiusremainedin the orbit orregenerated nomorethantwodigitsdistally. Pecorino el al. (1996) transplanted the hindlimb distal blastematotheforelimbproximal stumpsofnewtlimbs. Theproximalpartoftheregeneratewasmostlygenerated by the stump, and the transplanted cells made only a TableII Siiiiiniiiiyoflimbtissueautotransplantationintheeyesocketof Cancerjordani(carapacewidth = 10.79 4.05nun) CRAB LIMB REGENERATION 399 reportedforcrabs.Wealsodidnotobservesuchstructures clawtissuesintoeyesockets.Themechanismforchange in our experimental or sham-operated crabs. Some ani- ofhandednessofcrabclawsisunknown,butourobserva- mals regenerated tapered projections (Fig. 2G, H) or tions suggest that the axis of grafted tissues in the eye structuresdifferentfromtheoriginalgraftedtissuesinthe socket has been changed. eyes sockets (Fig. 2E, F). They were, however, neither Handednessoflimbsisdeterminedbythedorsoventral, an antennule nor an antenna, because both appendages proximodistal, and anteroposterior axes. Left and right had multiple segments. Autotransplantation of a single handshavethesamedorsoventralandproximodistalaxes claw digit into eye sockets resulted in a low percentage butareoppositeintheanteroposterioraxis.Oneexplana- ofregenerates in the eye socket, and all the regenerates tionforthechangeofclawhandednessisthatthegrafted were tapered ordistally incomplete. We believe that the clawtissuesintheeye sockets have been reorganized. It clawtissuesintheeyesockets underwent regressionand is known that limb regeneration does not take place by thatregeneration ofacomplete distal structure is depen- directoutgrowthbutbytheproductionofundifferentiated dentuponacompleteproximalclawstructure.Thisobeys blastemacells.Theblastemacellsarederivedfromdedif- the complete circle rule of the polar coordinate model ferentiation of stump tissues (Adiyodi, 1972; Stocum. (French etui. 1976; Bryant etai, 1981). 1991; Tsonis et al.. 1995). Since we grafted a piece or Autotransplantationofaclawpollexintoaneyesocket severalpiecesofclaw tissues intoeye sockets,thededif- regenerated a claw with incomplete distal segments but ferentiationprocessmightoccurinallpiecesofthetrans- complete proximal segments. A Y-shaped structure ini- plants. Dedifferentiation oflimb tissue might generate a tiallyemergedfromtheeyesocketafterthefirstpostoper- new limb primordiain which the axesare undetermined. ativemolt, andaclaw withcomplete proximal segments With acomplete dedifferentiation ofclaw tissues in eye but incomplete distal segments regenerated from theeye sockets,thehandednessofclawsmightbedeterminedby socket after the second postoperative molt. This result thehostsite.Withoutacompletededifferentiationofclaw suggests that claw proximalization, unlike distalization, tissuesineyesockets,thehandednessoftheregenerating neednothaveacompletestructureatthedistal tip. Limb claw might be inherited from the original donor tissues. tissues grafted into eye sockets did not regenerate eye A mixture ofdonorand host handedness may be due to structures. Instead,only limbstructuresregeneratedfrom the influence ofboth the donor tissues and the host site. eye sockets. Ourresults showedthat limbtissuesarenot Alternatively, it is possible that the handedness of the pluripotent;theirfateshavebeendeterminedtoregenerate clawsregeneratingintheeyesocketsmightbedetermined limbs even in the eye sockets. by a random process unrelated to the handedness ofthe Weobservedthatclaw regeneration in theeye sockets grafted tissues orhost sites. can proceed from the distal to the proximal parts ofthe claws(Fig. 2C,J).Thisprocesscanoccuroverfourmolt Acknowledgments cycles. Examination ofdissected animals and crab exu- viae revealed that this mode ofregeneration is produced Weappreciatethehelpfulcommentsoftheanonymous by continuously generating blastemas at the proximal reviewers. H.-w. Kao was a recipient of a scholarship ends ofthe previous regenerates. The proximal ends of from the Ministry ofEducation, Taipei, Taiwan, R.O.C. blastemas and future regenerated claws were not sus- ThispaperisfundedinpartbygrantsfromtheU.S.Dept. pendedinthecrabbody.Instead,theyfusedwithtendons of Agriculture (92-37206-7843) and the National Sea ofeyestalks (Fig. 3A), remnants ofeyestalks (Fig. 2F), Grant College Program, National Oceanic and Atmo- orthe carapace nearthe eye sockets (Fig. 2C). This ex- spheric Administration. U.S. Department ofCommerce, plains why they were not shed with the old exoskeleton undergrant numberNA36RG0537, project number R/F- duringmolt.Weobservedthatalthoughtheproximalseg- 160,throughtheCaliforniaSeaGrantCollegeSystem(to ments of the regenerated claws were capable of move- E.S.C.). The views expressed herein are those ofthe au- ment, thedactyl wasnot. Thustheywere not fully func- thors and do not necessarily reflect the views ofNOAA tional claws. or any ofits sub-agencies. The U.S. Government is au- In ourexperiments, a total of 12 limbs (11 claws and thorizedtoreproduceanddistributeforgovernmentalpur- 1 walkingleg)withrecognizablehandednessregenerated poses. fromthecrabeyesockets.Amongthem,8limbs(7claws and 1 walking leg) retained the donor-tissue handedness Literature Cited regardlessofwhethertheautotransplantationofclawtis- csuleaswswcahsanigpesidlaftreroamldoornocro-nttirsasluaetetroalh.osTt-hsrieteehraengdeendenreastse,d ABdaityePostdoirina,,teWRl..phGu1.s8a914h9.y7d2.rMoadtrWeoromiuoanlussd.fohIrentat.lhieRnegSvt.aundCdyytoorlfe.gVea3nr2ei:raat2ti5ioo7nn-2Ti8rne9a.ttheedcwriatbh. andoneregeneratedclawhadamixtureofdonorandhost SpecialRegardtoDiscontinuityintheOriginofSpecies.Macmillan, handednessfollowingcontralateralautotransplantationof London.598pp. 400 H.-W. KAO AND E.S. CHANG Bryant,S.V.,V.French,andP.J.Bryant.1981. Distalregeneration tary wing disc fragments ofDrosophila melanogaster. Dev. Bio/. andsymmetry.Science212:993-1002. 82: 238-257. Butler,E.G.1955. Regenerationoftheurodeleforelimbafterreversal Kim, VV.-S.,and D.L. Stocum. 1986. Retinoic acid modifiesposi- ofitsproximo-distalaxis.7.Morphol. 96: 265-281. tional memory in the anteroposterior axis ofregenerating axolotl Conklin, D.K..and E.S.Chang. 1993. Cultureofjuvenile lobster limbs.Dev. Biol. 114: 170-179. (Homarusamericanus).Pp.497-510inCRCHandbookofMaricul- Maynard,D.M.1965. Theoccurrenceandfunctionalcharacteristics ture. 2nded,J.P.McVey.ed.CRCPress.BocaRaton,FL. ofheteromorphantennules inanexperimental populationofspiny Cooke,I.,R.Graf,S.Grau,B. Haylett,D.Meyers,andP.Ruben. lobsters,Panuliritsargus.J.Exp.Biol.43:79-106. o1u9t8g9.rowtChruisntasciemapnlepempetdiiduermg.icPrnoecu.roNnastl.inAccauldt.urSecis.hUoSwAi8m0m:ed4i0a2t-e Nevimno,rpP.hAa.n,taennndulSe.Rin.aMacluelcthurae.d1f9r9e1s.hwaTtehreporcacwunr.renMcaecroofbarahcetheiruom- Dent4,06J..N. 1954. Astudyofregeneratesemanatingfromlimbtrans- m1s0e7.nbergii(DeMan)(Decapoda,Caridea). Crusiaceana60: 105- Rpelacn.ts11w8i:th84r1ev-e8r5s6e.dproximodistalpolarityintheadultnewt.Anat. Pecorino, L.T.,A. Entwistle,andJ.P. Brockes. 1996. Activation HForepnkincihne,sp.iVm.Po,.rPp.1h9iJ8.c8.BfrieylaCd.notnS,tcriaoelnndcoefS.1r9Ve3g.:enB9err6ya8atn-it9o.8n11.i9n7c6r.ustPaactetaenrsn.rPepg.ul3at2i7o-n Shimroieyfs.piaelc.siifHni.gc,laetYi.orne.StaiCnwtoaiirdcra.a,cBiiaodln.dre6cT:e.p5tS6ou3rg-i5iy6soa9fm.oar.m1m9e9d3i.ateMsipnriomxiummodtiisstsaule 340inEndocrinologyofSelectedInvertebrateTypes,H.Lauferand suerequiredforhydraregeneration.Dev. Biol. 155: 287-296. R.G.H.Downer,eds.A.R. Liss,New York. Stocum, D.L. 1991. Limb regeneration: a call to arms (and legs). Kao,H.-w.,andE.S.Chang.1996. Homeotictransformationoflimb Cell67: 5-8. by autotransplantation ofcrab claw tissue. Bio!. Bull. 190: 313 Tsonis, P.A., C.H. Washabaugh, and K. Del Rio-Tsonis. 1995. 321. Transditterentiation as a basis for amphibian limb regeneration. Kauffman,S.A.,and E.Ling. 1981. Regenerationbycomplemen- Semin. CellBiol.6: 127-135.