Table Of ContentReference: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.
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