Light-activation of the Archaerhodopsin H+-pump reverses age-dependent loss of vertebrate regeneration: sparking system-level controls in vivo.

0.65 MB·English

by  Adams, Dany Spencer

306 Research Article
+
Light-activation of the Archaerhodopsin H -pump
reverses age-dependent loss of vertebrate
regeneration: sparking system-level controls in vivo
Dany Spencer Adams*, Ai-Sun Tseng*,` and Michael Levin§
DepartmentofBiologyandTuftsCenterforRegenerativeandDevelopmentalBiology,TuftsUniversity,200BostonAvenue,Suite4600,Medford,MA
02155,USA
*Theseauthorscontributedequallytothiswork
`Presentaddress:SchoolofLifeSciences,UniversityofNevada,LasVegas,4505MarylandParkway,LasVegas,NV89154-4004,USA
§Authorforcorrespondence([email protected])
BiologyOpen2,306–313
doi:10.1242/bio.20133665
Received20thNovember2012
Accepted28thNovember2012
Summary
Optogenetics,theregulationofproteinsbylight,hasrevolutionized endogenous regenerative pathways. Electroneutral pH change,
the study of excitable cells, and generated strong interest in the inducedbyexpressionofthesodiumprotonexchanger,NHE3,did
therapeutic potential of this technology for regulating action notrescueregeneration,implicatingthehyperpolarizingactivityof
potentials in neural and muscle cells. However, it is currently Archaerhodopsin as the causal factor. The data reveal that
unknown whether light-activated channels andpumps will allow hyperpolarizationisrequiredonlyduringthefirst48hourspost-
control of restingpotential inembryonic or regenerating cells in injury,andthatexpressioninthespinalcordisnotnecessaryfor
vivo.Abnormalitiesinioncurrentsofnon-excitablecellsareknown theeffecttooccur.Ourstudyshowsthatcomplex,coordinatedsets
n
e to play key roles in the etiology of birth defects and cancer. ofstablebioelectriceventsthatalterbodypatterning—prevention
p
O Moreover, changes in transmembrane resting potential initiate ofbirthdefectsandinductionofregeneration—canbeelicitedby
Xenopus tadpole tail regeneration, including regrowth of a thetemporalmodulationofasingleioncurrent.Furthermore,as
y
g functioning spinal cord, in tails that have been inhibited by optogeneticreagentscanbeusedtoachievethatmanipulation,the
o
ol natural inactivity of the endogenous H+-V-ATPase pump. potential for this technology to impact clinical approaches for
Bi However, existing pharmacological and genetic methods allow preventive, therapeutic, and regenerative medicine is
neithernon-invasivecontrolofbioelectricparametersinvivonor extraordinary. We expect this first critical step will lead to an
theabilitytoabrogatesignalingatdefinedtimepoints.Here,we unprecedented expansion of optogenetics in biomedical research
showthatlightactivationofaH+-pumpcanpreventdevelopmental and in the probing of novel and fundamental biophysical
defects and induce regeneration by hyperpolarizing determinantsofgrowthandform.
transmembrane potentials. Specifically, light-dependent,
Archaerhodopsin-based, H+-flux hyperpolarized cells in vivo and (cid:2)2013.PublishedbyTheCompanyofBiologistsLtd.Thisis
thus rescued Xenopus embryos from the craniofacial and an Open Access article distributed under the terms of the
patterning abnormalities caused by molecular blockade of CreativeCommonsAttributionNon-CommercialShareAlike
endogenous H+-flux. Furthermore, light stimulation of Arch for License (http://creativecommons.org/licenses/by-nc-sa/3.0).
only 2 days after amputation restored regenerative capacity to
inhibited tails, inducing cell proliferation, tissue innervation, and Keywords: Bioelectricity, Optogenetics, Regeneration, Tail,
upregulationofnotch1andmsx1,essentialgenesintwowell-known Transmembrane voltage, Xenopus
Introduction can be facilitated by the flexibility of spatio-temporal control
The use of light-activated ion-transporting proteins to alter provided by optogenetics. Especially interesting is the potential
transmembrane voltage is an important part of the field of applicability of this technology to regenerative medicine, where
optogenetics and has led to remarkable breakthroughs in our bioelectrical signals have been shown to initiate regeneration of
abilityto elicit and inhibit action potentials in nerve and muscle complex, multi-tissue organs and appendages (Adams et al.,
cells (Kno¨pfel et al., 2010). However, it is unknown whether 2007;Beaneetal.,2011;Levin,2012;Paietal.,2012;Tsenget
these tools are adaptable for use in developing or otherwise al., 2010).
dynamic systems, especially during complex patterning and A powerful model for studying regeneration of complex
disease events (Levin, 2012). This is a critical issue as vertebratestructuresistheXenopustadpoletail,whichcomprises
endogenous bioelectrical signals are known to regulate cellular spinal cord, notochord, muscle, nerves, vasculature, and skin.
processes including proliferation, differentiation, migration, and Aftertailamputation,thetailstumpbecomeshighlydepolarized.
morphology (Levin, 2009). Thus biomedical applications that A bioelectrical hyperpolarizingsignal is then required to initiate
harnesstheseprocessesbymanipulatingrestingpotentialinvivo regeneration and is one of the earliest signals identified in this
Optogenetic regeneration 307
process(Adamsetal.,2007).Startingat6 hourspost-amputation
(hpa),theprotonpumpH+-V-ATPaseisstronglyexpressedinthe
plasmamembraneoftheregenerationbudcellsattheinjurysite.
The H+-V-ATPase-dependent H+-efflux acts to repolarize the
regeneration bud and this event is required to initiate
proliferation, directed innervation, and induction of
regenerative signaling pathways, leading to successful
replacement of the missing tail (Tseng and Levin, 2012).
Conversely, molecularly or chemically inhibiting H+-V-ATPase
activity blocks regeneration. Interestingly, Xenopus tadpoles
exhibit age-dependent changes in regenerative ability similar to
humans (Beck et al., 2003). The absence of endogenous H+-
effluxandtheunrelieveddepolarizationofthetailstumpinhibits
regeneration during this age-specific physiological non-
regenerative (also known as the ‘‘refractory’’) period of
development (Adams et al., 2007).
We explored the ability of optogenetics to address two
important unknowns. First, can a minimally invasive method be
developedforprecisecontrolofbioelectricstatesofcellsoutside
of the neuromuscular system in vivo? Second, because
pharmacological reagents cannot be reliably washed out of 3-
dimensional tissues, itis difficulttoput a temporal boundary on Fig.1. PhenotypescausedbyinhibitionoftheH+-V-ATPase.(A)Dorsal
the activity of membrane voltage in regenerative pathways. viewofnormalstage47tadpole.Whitearrowsindicatenormaleyes,olfactory
bulbsandbranchialarches.(B–D)Craniofacialphenotypescommonlyseen
Given the ability of light-gated ion pumps to be regulated
afterinjectionattheone-cellstagewithmRNAencodingYCHE78,adominant
temporally, we hypothesized that using such a tool in negativeH+-V-ATPasesubunit.Bshowsabnormallysmallbranchialarches;
regenerating cells could allow us to control the external thearrowinCpointstoectopicpigmentsintheopticnerve;thearrowsinD
n
manipulation of V at precisely defined time points. As this pointtoamissingeyeandanabnormalolfactorybulb.ScalebarinC(forCand
e mem
p approach has not yet been experimentally investigated, a D)50.5mm.(E)Profileofanormalstage42embryo.Arrowsforcomparison
O withF.(F)Stereotypicalswellingscausedbyincubationoftadpolesin10nM
successful result would: represent an important additional use
y concanamycinfor24hoursfollowingtailamputationatstage40.Evenif
g for optogenetic methods, identify novel schemes for clinical removedfromconcanamycinatthisstage,alltadpolessubsequentlydie.
o therapies, and reveal new aspects of the mechanisms of Anterioristotheleftanddorsalisup.(G)Aregeneratedtailshown
ol bioelectrical regulation during development and regeneration. approximately8dayspost-amputation.Theamputationplanewasjustanterior
Bi to(totheleftof)theleftmostwhitearrow.(H)Anon-regeneratingtail.After
To test the ability of a light-gated ion translocator to alter
amputationatstage47,thistaildidnotgrowback,acharacteristicoftailscutat
endogenous voltage and thereby induce regeneration in non-
thisagethatdefinestherefractorystage.Theredarrowpointstothearea
regenerative tadpole tails, and to further probe the temporal referredtoastheregenerationbud;theamputationplaneisimmediately
requirements of ion flows during regeneration, we employed anterior.Exceptwherenoted,scalebars51mm.
Archaerhodopsin (Arch), a light-activated, hyperpolarizing H+-
pump (Chow et al., 2010) to manipulate H+-flux-dependent chemicalH+-V-ATPaseinhibitor,atanearlytadpolestagecauses
initiationofregeneration.Here,weshowthatArchactsinalight- edemas in the headand trunk andsubsequent lethality (compare
dependent manner in larval Xenopus tissues in vivo to Fig. 1E,F). H+-V-ATPase function is also required for initiating
hyperpolarize cells, and thus can functionally replace the tadpole tail regeneration. During the refractory period, the
endogenous V-type proton pump. Light-activated Arch, absence of endogenous H+-V-ATPase activity after tadpole tail
functioning outside of the spinal cord, rescues both amputationpreventsregenerativeregrowth(compareFig. 1G,H).
developmental and regenerative defects caused by age- Together, these observed phenotypes demonstrate essential
dependent (endogenous) or experimental inhibition of normal requirements for stable H+ efflux in normal craniofacial
H+-efflux,anditactsthroughNotchandMSX1.Light-activation development, osmotic balance, and tail regeneration.
for only 48 hours post-injury, followed by continuous darkness, The multiple roles of H+ efflux during development and
is sufficient to initiate the entire program of regeneration of this regenerationsuggestthatthisioncurrentshouldbeanimportant
complex neuromuscular appendageat agesthat otherwisewould target for therapeutic manipulation of tissues. We therefore
not regenerate. sought to identify optogenetic tools that induce stable,
controllable H+ efflux in vivo and to use an optogenetic
Results approach to prevent developmental defects, correct ion
Absence ofH+efflux induces developmentaland regenerative imbalance, and initiate the restoration of a complex appendage,
defects the tail.
During Xenopus development, inhibition of H+-V-ATPase
function, molecularly or chemically, leads to both embryonic The light-activatedprotonpump,Arch, isactive inXenopus
and regenerative defects (Fig. 1). Injection of mRNA encoding cells
YCHE78,adominantnegativeH+-V-ATPasesubunit,attheone The Archaerhodopsin-3 (Arch) protein is a light-gated proton
cell stage results in craniofacial malformations including small pump (Chow et al., 2010) that is active when expressed in
branchial arches, and irregular eye, ear and olfactory bulb neurons. To determine whether Arch is functional in Xenopus
structure,(Fig. 1A–D).Treatmentwithconcanamycin,aspecific embryonic cells, we first examined Arch expression. After
Optogenetic regeneration 308
injection of the Arch mRNA into early embryos, a linked
fluorescent tag (GFP or Tomato) was imaged. Both
epifluorescence and spinning disk confocal microscopy
consistently detected strong Arch protein on cell surfaces from
approximately the 32-cell (Fig. 2A) through tadpole stages
(Fig. 2B,C), indicating that it is expressed as predicted.
Toverifythelight-dependentactivityofArchinXenopuscells,
we measured resting membrane voltage and intracellular pH in
vivo. Impaling early embryonic cells showed the expected
hyperpolarization of Arch-expressing light-stimulated cells
(Arch+Light) relative to Arch-expressing, unstimulated cells
maintained in the dark (Arch+Dark; 24067 mV versus
22268 mV; n512, t53.541, P50.002) (Fig. 2D). To establish
whether Arch-Tomato could pump H+ ions out of cells, we
measuredintracellularpHunderconditionswhenArchwaseither
inactive or active. BCECF is a fluorescent pH reporter whose
excitationwavelengthsdonotstronglystimulateArch(Fig. 2E).
UsingBCECF,wefoundthatthedifferencebetweenpHofcells
expressingArch-Tomatoandthosethatarenotexpressingitwas
0.360.2 pHunitsonaverage(Fig. 2F,G,H).UsingSNARF-5F,a
fluorescentpHreporterwhoseexcitationwavelengthsmaximally
stimulate Arch and for which the irradiance of the illumination
sourceusedwashigherthanthoseusedforBCECF,wefoundan
averagedifferenceof0.760.3 pHunits(Fig. 2I,J,K).Thesedata
confirmthatArchisactivatedbylighttopumpH+ionsoutofthe
minimally differentiated cells in the regeneration bud. Together,
n
the V measurements and the pH results establish that Arch
e mem
p functions in a light-dependent manner as a proton pump to
O
increase polarization in Xenopus cells in vivo.
y
g
o Light-inducedArchactivity rescuesdevelopmental defects
ol
Bi OurexperimentalevidencesuggeststhatArchactivityshouldbeable
tosubstituteforthefunctionofH+-V-ATPase,theendogenousproton
pump, during development. To test whether light-illuminated Arch Fig.2. ArchisexpressedandcanbeactivatedinXenopuscells.
couldinfactactasagainoffunctiontooltorescuedeficienciesinH+- (A–C)ExpressionofArch-GFPatthestagesindicated.mRNAforArch-GFPwas
fluxregulation,wefirstexaminedtheabilityofactiveArchtoprevent injectedattheonecellstageandlastsforatleastthe8daysrequiredforthelongest
embryonic defects caused by molecular inhibition of H+-flux. As experiments.(A)ViewofanimalpoleofaStage7embryo,thestageatwhich
electrophysiologywasperformed.Arch-GFPexpressionisespeciallyvisibleatcell–
shown in Fig.1, expression of YCHE78 blocks endogenous H+-V- cellboundaries(e.g.whitecircle)wherethecellshavelesspigment.(B)Arch-GFPis
ATPase function resulting in patterning and craniofacial stillpresentinthemembranesofhexagonally-packedectodermalcellsatstage30,
abnormalities(Vandenbergetal.,2011).Consistentwithrestoration wellafterthedevelopmentalstagesthatarerelevantforcraniofacialdevelopment.
ofH+effluxbylight-activatedArch,raisingembryosexpressingboth (C)Arch-GFPisstillstronglyexpressedatstage47,therefractorystage,during
which,ifthetailiscut,itdoesnotgrowbackduetoendogenousinhibition.Theblack
YCHE78 and Arch under light prevented those developmental
arborizedcellsaremelanophores,thelongeralignedcellsaremusclecells,andthe
defects,aswellaslethality,ascomparedtosiblingembryosraisedin hexagonallyarrayedcellsareepidermis.(D)Restingpotential(V )ofimpaled
mem
thedark,(n5149,Z53.423,P,0.001).Similarly,edemasinducedby cellsinembryosatstage7.Arch-tomatowasinjectedat1cell,thenhalfofthese
concanamycin treatment at the tadpole stage (Fig.1F) were also embryosandhalfoftheuninjected(NT5notreatment)controlswereplacedunder
bluelightwhiletheotherhalveswerekeptinthedarkuntilstage7.TheV of
suppressed by concurrent Arch activation via light treatment. Thus mem
untreatedembryoswasabout22962mVandwasunaffectedbylight.Arch-
Arch activity functionally replaced the normal H+ efflux during injectedembryoskeptinthedarkweredepolarizedwhileArch-injectedembryos
development and suppressed embryonic defects and physiologic keptinthelightwerehyperpolarized.(E)ExplanationofpHmeasuringexperimental
imbalance. protocol.TwodyeswereusedtocomparepHofArch-tomatoexpressingcellswith
pHofcellsshowingnoexpression.BCECFisexcitedby450and500nmlight,
whichdoesnotactivateArchverystrongly,whileSNARF-5Fisexcitedby540nm,
Light-inducedArchactivity initiatesregeneration whichmaximallyactivatesArch.ThepredictionisthatthepHsmeasuredbyBCECF
HavingdemonstratedthatArchfunctionsinnon-excitableembryonic willdifferbylessthanthepHsmeasuredbySNARFbecauseArchshouldbe
tissuestosuppressdefectscausedbymolecularinhibitionofH+-V- maximallyactivatedunderthelatterconditions,thuscausingpHtoincreaseinArch
ATPase, we sought to test the therapeutic potential of using this expressingcells.(F–K)ResultsofpHmeasurementsusingtwodyes.(F,I)
Brightfieldimagesshowingpositionsofregenerationbuds.(G,J)ImagesofArch-
approach to induce vertebrate regenerative repair. During the
tomatofluorescenceusedtochooseregionsofinterest(ROIs)definedbyhighand
refractory period, tadpoles exhibit human-like age-dependent lowtomatofluorescence.(H,K)ROIsfromGandJweretransferredtotheratio
regenerative ability: tail regeneration is endogenously blocked imagesgeneratedbythedyes(seeMaterialsandMethods).Aspredicted,
duringonephaseoftadpoledevelopment,justasdigitregeneration measurementsmadeunderlowArch-activationconditions(BCECF,F–H)show
onlya0.360.2pHunitdifferencebetweenArch-tomatoexpressingandnon-
isendogenouslyblockedinchildrenovertheageofseven(Douglas,
expressingcells.Incontrast,underArch-activatingconditions(SNARF-5F,I–K),the
1972; Illingworth, 1974; Illingworth and Barker, 1980). To test
pHdifferenceisapproximatelydoubledto0.760.3pHunits.Anterioristotheleftin
whetherArchactivitycouldreversethisnaturalinhibition,weasked allimagesexceptA.Scalebars5100mm.
Optogenetic regeneration 309
whether light-activated Arch H+ pump activity can initiate Table S1). Moreover, in contrast to previous results showing
regenerationduringtherefractoryperiod. thatconstitutiveH+-effluxbothbeforeandafteramputationcould
Arch-Tomato mRNA was injected into earlyembryos and the lead to refractory regeneration, here we initiated hyperpolariza-
embryos were allowed to develop in darkness. During the tion only after amputation, a more relevant model for clinical
refractory period, tadpole tails were amputated and immediately applications, and for only 2 days. Importantly, after 2 days of
placedeitherunderlightorinthedarkfor2daysandthenfurther light exposure, Arch activity did not cause any side effects such
grown in the dark for 5 additional days. We found that the age- as tissue abnormalities.
specific refractory inhibition of regeneration was reversed in To better characterize the role of Arch activity in the light-
light-stimulatedArch-expressingtadpolesbutnotinunstimulated induced refractory regeneration, we examined the spatial
Arch-expressing tadpoles, with light approximately doubling distribution of Arch expression in regenerated and non-
regenerative ability (n516 groups representing 428 tadpoles regenerated tails. Concentrated Arch expression (as evidenced
total, t53.709, P50.002) (Fig. 3A; supplementary material bythepresenceofTomatofluorescence)wasseenatthedistaltip
of all of the successful tail regenerates after light stimulation
(Fig. 3B,C). Non-regenerated tail stumps, in contrast, showed
randomizedregionsofArchexpression.Similarly,nocorrelation
betweenArchlocalizationandregenerativesuccesswasobserved
in tails of tadpoles expressing Arch but grown in the dark
(Fig. 3D).
Forsuccessfulregenerationtooccur,propernervepatterningis
essential, so we examined whether Arch expression correlated
with the position of neurons. Surprisingly, expression of Arch-
Tomato in the spinal cord was not required for full regeneration
(Fig. 3E,F). Consistent with this observation, amputated tail
stumps that contained strong Arch-Tomato expression in the
neural tissue, but lacked strong distal expression, failed to
n regenerate after light activation (Fig. 3G,H). Our data establish
e that temporally inducing H+ efflux via Arch activation after
p
O refractory tail amputation is sufficient to initiate regeneration
y during this endogenous non-regenerative state. Furthermore,
g
Archactivityisnotrequiredintheneuralcellsoftheamputated
o
ol tail stump, but rather in a small distal population.
Bi
Fig.3. Arch-Tomatoactivitypromotesregenerativerepair.
(A)Comparisonofregenerationindices,ameasureofregenerationsuccess,of
thetailsofArch-Tomatoexpressingtadpolesamputatedattherefractorystage.
Significantlybetterregenerationwasfoundintadpolesthatwereexposedto
Arch-activatinglightfor48hoursfollowingamputationrelativetothosekept
inthedark(t-test).(B)ComparisonoflocalizationofArch-Tomatointadpoles
exposedtodarkversuslightfor48hourspost-amputation.UnderArch-
activatinglight,regenerationcorrelatedperfectlywiththepresenceofagroup
ofArch-Tomatoexpressingcellsattheverytipoftheregeneratingtail(seeC
below).Incontrast,amongthetadpoleskeptinthedark,regenerationsuccess
didnotcorrelateatallwithlocalizationofArch-tomato.Thedistributionsare
highlysignificantlydifferent(x2).(C)Arch-Tomatolocalizationina
regeneratedtail8daysafteramputationatthenon-regenerativestage47.Arch-
Tomatoexpressionisstillstrongintheuncutpartofthetailtotheleft,andina
smallclumpofcellsatthedistalmosttipoftheregeneratingtail(whitearrow).
Otherwise,asexpected,thereisverylowifanyexpressionofArch-tomatoin
theregenerate.Scalebar5100mm.(D)Arch-Tomatoexpressioninatailthat
didnotregenerate.Whilethereisexpressionallthewaytothedistaledgeofthe
amputationplane,expressionisrelativelyeven,thatis,thereisnogroupof
particularlybrightcells.ScalebarasinC.(E–H)Highermagnification
brightfieldandfluorescenceviewsoflight-stimulatedtailsandArch-Tomato
expression.YellowNsindicatethenotochords.WhiteSCsandsmall
arrowheadsindicatethespinalcord.Letterswerepositionedonthebrightfield
imagesthentransferredtothefluorescenceimages.Atthedistalends(tothe
right)largerarrowheadsindicatetheposteriorextentsofthenotochord(yellow)
andspinalcord(white).Scalebars525mm.(E,F)Brightfieldandfluorescence
imagesofaregeneratedtailshowingthebrightcellstobefurtherdistal,and
largelyventral,tothespinalcord.Thebrightcellsareindicatedbyawhite
arrowinF.(G,H)ArefractorytailthatdidnotregenerateshowingArch-tomato
expressingneuraltissueextendingtothedistaltipofthenon-regeneratingbud.
Forallimages,anterioristotheleftanddorsalisup.
Optogenetic regeneration 310
pHchangealone isnot sufficientto restoreregeneration (Praetoriusetal.,2000;Sabirovetal.,1999).Incubationofthese
SinceArchfunctionmodulatesboth,V andpH,wesoughtto embryosinculturemediumcontaininghigh[Na+]thatfacilitates
mem
determine which of these physiological properties was causal in H+-efflux through this antiport should, like Arch activity, raise
initiatingregeneration.Previousdataalreadyshowedthatcellular internal pH but unlike Arch activity, should not affect V . In
mem
hyperpolarization(usingachloridetransporter,independently of the tail regeneration rescue assays performed during the non-
proton flux) is sufficient to induce regeneration (Tseng and regenerativerefractoryperiod,comparisonsofNHE3-expressing
Levin, 2012). Thus, we asked whether pH change alone would tadpolesinhigh[Na+]mediumtothoseinnormalmediumorto
improve regenerative response. To alter pH without changing uninjected controls in either high [Na+] or normal medium gave
V , embryos were injected with mRNA encoding no evidence of improved regenerative ability (total n5255,
mem
constitutively active NHE3, an electroneutral Na+/H+ exchanger Kruskal–Wallis plusDunn’s Q, P.0.5 for allcomparisons). We
interprettheseresultstobeinconsistentwithamodelinwhichan
increase in pH is sufficient to induce refractory regeneration.
Together with the evidence that shows V change alone is
mem
sufficient to rescue refractory regeneration (Tseng and Levin,
2012), we conclude that it is principally the effect of light-
stimulated Arch on V , not on pH, that initiates regeneration.
mem
Stimulation of Arch-dependentH+-efflux restores regeneration
bytriggering endogenousdownstream geneticmechanisms
Because Arch activity restored refractory regeneration, we
examined the potential downstream mechanisms by which this
process was carried out. Notch1 and Msx1 are two well-
characterizedbiochemicalsignalingcomponentsthatarerequired
to drive regenerative outgrowth and patterning (Beck et al.,
2003).WeaskedwhetherArchpromotedtailregrowthbyacting
through these known downstream regenerative pathways. In situ
n
e hybridization revealed that light stimulation of Arch induced
p upregulation of both Notch1 and Msx1 (Coffman et al., 1990;
O
Feledy et al., 1999) (Fig. 4A,B as compared to Fig. 4C,D). We
y
g alsoexaminedtheeffectofArchstimulationoncellproliferation
o and nerve patterning, two processes that are required for
ol
Bi regeneration. Light-dependent Arch activation more than
doubled the number of mitotic cells in the tail regeneration bud
region (n522, t55.273, P,0.001) (Fig. 4E) and promoted
proper nerve patterning (compare Fig. 4F,G to Fig. 4H,I).
Together, these results show that Arch-induced H+ efflux
initiates regenerative tail growth through endogenous
mechanisms.
Discussion
In this study, we asked whether an optogenetic strategy could
successfully initiate regeneration of a complex vertebrate
structure in vivo. Previous work from our laboratory has
demonstrated that a specific early proton efflux generated by
Fig.4. Endogenousregenerativepathwaysfollowarchactivationbylight.
(A–D)Whole-mountinsituhybridizationsforNotch1andMsx1,components
ofregenerationsignalingpathways.(A,B)Expressionintheregenerationbuds
ofArch-tomatoinjectedembryosmaintainedinthedark(whitearrows).(C,D)
Expressioninthe3dayspost-amputation(dpa)regenerationbudsofArch-
Tomatoinjectedembryosmaintainedinthelightfor48hoursafteramputation
(redarrows).Expressionismuchhigherinthelight-stimulatedtails.
(E)Comparisonofthenumberofcellspositiveforphospho-histoneH3(H3P,a
markerofmitoticactivity)intheregenerationbudsofArch-tomatoexpressing
tailskeptinthedarkversusthelight.Thereweresignificantlymoreinthelight-
stimulatedtails(t-test).(F–I)DICandfluorescenceimagesofa-tubulin(a
neuronalmarker)ofArch-Tomatoexpressingtailsat3dpa.Tadpoleswerekept
inthedarkversusthelightfor48hpa.(F,G)Non-regeneratingtailskeptinthe
darkshowthetypicalarrangementofneuronsthathavestoppedgrowingor
growninacurvetowardsthemidline.(H,I)Regeneratingtailskeptinthelight
showthenormalarrangementofneuronsinaregeneratingtailthatisgrowing
paralleltotheAPaxisofthetailandextendingintothegrowingtail,although
notreachingallthewaytothedistaltip.Inallimages,anterioristotheleftand
dorsalisup.Scalebars525mm.
Optogenetic regeneration 311
trigger normal biochemical signaling pathways downstream of
bioelectric triggers.
Prior to measuring V , Arch-expressing embryos were
mem
incubated under the light or in the dark, but then were kept at
ambientlightforupto15 minutespriortobeingmeasured.Thus,
theaveragerelativehyperpolarizationof22 mVcausedbylight-
stimulationofArchwasstableforatleastthatlong,orevenmore
pronounced before the time spent at ambient conditions. This
outcome highlights a critical difference between the cells under
investigation here and the previously reported kinetics of Arch
activation,wheretheassayforactivityisinhibitionorstimulation
of an action potential lasting milliseconds. Specifically, the
changeinV thatisinitiatedbyArchstimulationissustained
mem
for a much longer time.
Light activation of Arch induces a H+-flux. Consistent with
this function, our results showed that light-activated Arch alters
both V and pH in Xenopus cells. Changes in V correlate
mem mem
with tadpole regenerative ability whereas a role for pH in
regeneration has not been documented. To address this question
we changed V and pH individually, to see whether either
mem
alone was sufficient for inducing regeneration. We recently
showed that altering V by the use of an exogenous chloride
mem
channel is sufficient to regulate regenerative ability in tadpoles
(TsengandLevin,2012).Here,wereportthatexpressionofNHE
(asodium-protonexchangerthatproducesthepHgradientbyan
electroneutral process), which tested the role of pH without a
n
e concomitant Vmem change, induced no improvement of
p Fig.5. SummaryoftheeffectsoflightactivationofArch-tomatoon regeneration. Combined, these results suggest that it is unlikely
O
embryoswithblockedH+-V-ATPase.(A)Embryosexpressingadominant- that pH is a critical part of the mechanism for the initiation of
gy negativeH+-V-ATPasesubunitdevelopseverecraniofacialdefects. regeneration and implicate the voltage change induced by Arch
o (B)Illuminatingco-expressedArch-tomatofromstage9tostage26 as the key causal factor.
ol significantlyreducesthenumberofcraniofacialabnormalities.(C)Embryos
Bi exposedtothehighlyspecificH+-V-ATPaseinhibitorconcanamycinfor Wealsodiscoveredthatage-dependentregenerationrescueby
24hoursafteramputationatstage40developstereotypicalswellingsthendie, light-stimulated H+-pumping only occurs in tails with small
consistentwithaneffectonosmoticbalanceregulation.(D)IlluminatingArch- populations of Arch-expressing cells at the distal tip of the tail;
tomatoduringexposuretoconcanamycinpreventsswellingandsubsequent
furthermore,thereisnocorrelationbetweenthepositionofthese
mortality.(E)Tadpoletailsamputatedatstage47normallydonotregenerate.
cells and the positions of neural tissue. Correct nerve patterning
(F)IlluminatingArch-tomatofor48hoursafteramputationsignificantly
increasesthenumberofregeneratingtailsifthereisasmallgroupofArch- has been shown to be necessary for full regeneration; however,
expressingcellsatthedistalmosttipofthetail.Scalebars51mm. thepresenceofArch-expressingcellsindistal,non-neuronalcells
of regenerates is more highly correlated with stimulation of
the V-ATPase H+ pump at the amputation site is required to regeneration,aswellaswithnormalgrowthoftheneuronsalong
initiate Xenopus tail regeneration, while inactivation of V- the anterior–posterior axis. As H+-V-ATPase is not expressed in
ATPase function in amputated tail stumps leads to regenerative thespinalcordampullaeaftertailamputation,thesedatasuggest
failure.Asproof-of-concept,wedemonstratethatthelight-gated that the repolarization of regeneration bud cells through H+-
H+ pump, Archaerhodopsin, can be expressed in non-excitable efflux is likely acting non-cell autonomously to drive
embryonic and tail cells of Xenopus laevis and it hyperpolarizes regeneration of neural tissue and then the tail. The
thosecellswhenexposedtolight(Fig. 2).Furthermore,weshow development of tissue-specific tools in Xenopus will enable us
that light-activated Arch activity restores regeneration in the to further address this question in the future.
absence of endogenous H+ efflux (Fig. 3). Exploiting the Thedatarevealedseveralimportantnewaspectsofbioelectric
temporal control of Arch afforded by its light-sensitivity, we control of regeneration. First, the light stimulation was stopped
establish that the duration of H+-flux needed to start the precisely at 48 hours post-injury; reliable wash-out of
regeneration pathway need not be long term, and, importantly pharmacological inhibitors or inactivation of misexpressed
forfuturebiomedicaluse,thatH+-effluxtherapycanbeginafter genes cannot be done in this system, and these data
amputation. Consistent withthe knownrole of voltage-mediated demonstrated that hyperpolarization during the first 2 days is
earlystepsinregeneration,light-activatedArchsignalsupstream sufficienttoinducethe entirecomplex,highlycoordinated,self-
of canonical pathways required for tail regrowth (Fig. 4). limiting7-daycascadeoftailrebuilding.Second,itwasseenthat
Importantly, it overcomes the chemical and molecular function of Arch in the spinal cord was not required for
inhibition of endogenous H+-flux and, notably, the regeneration (Fig. 3C,D). Given the known importance of nerve
physiological inhibition of regeneration such as is found in supplyforregeneration(Gaeteetal.,2012;Thornton,1970),itis
humans.Toourknowledge,thesearethefirstdatatodemonstrate important for the development of biomedical strategies to note
thatanoptogeneticapproachisfeasibleandusefulindeveloping that bioelectric stimulation of other tissues can induce a strong
and regenerative systems in vivo, and that it can successfully repairprogram.Finally,weshowedthatoptogeneticinductionof
Optogenetic regeneration 312
regenerative repair activates known transcriptional cascades asimilartemperature,butunderalightblockingcover.After2days,tadpoleswere
transferredtofresh0.16MMRat22˚C,thenscored5dayslater.Priortodirect
(Fig. 4) and can thus potentially be used to regulate these
measurementofmembranevoltage(V )byimpalingwithamicroelectrode,the
pathways in multiple contexts. lightregimewasalteredto5secondsmoenmthen10secondsofftoinsurethatArch
In summary, we utilized the temporal activation of an ion wasactiveandcouldbelight-stimulatedbeforecellsweretoosmall.
current via a light-gated pump to initiate coordinated system-
level changes to prevent craniofacial malformations or restore pHmeasurements
Tails of Arch-Tomato expressing stage 47 tadpoles were amputated. Four days
age-dependent loss of regenerative ability (Fig. 5). Instead of
later,afterincubationinthelight,tadpoleswereincubatedin5mM29,79-Bis-(2-
controllinganimalbehaviororspikingintheCNSormuscle,our Carboxyethyl)-5-(and6)-Carboxyfluorescein,AcetoxymethylEster(BCECF-AM)
studyindicatesthatitispossibletoaffectfundamentalalteration or Semi-Naphthyl Rhodofluor-5 Acetoxymethyl Ester (SNARF-5F) for 2hours.
in body structures, without adversely affecting overall Excess dye was washed out, then the embryos were lightly anesthetized with
0.15%MS222andimaged.MeasurementsweremadeusinganOlympusBX-61
development, by manipulation of bioelectrical signals. This
withepifluorescenceoptics;illuminationwasfromaLumen200metalhalidelamp.
finding has important implications since ion transport is well- ToimageBCECF,adualexcitationdye,filterswereEX450/20,D460,EM535/
knownforbeingessentialnotonlyforexcitablecellbehavior,but 30(theisobesticpoint)andEX500/20,D515,EM535/30.ToimageSNARF-5F,
adualemissiondye,filterswereEX540/25,D565andD610,andEM580/25(the
also for the body’s anatomical homeostasis. Furthermore, the
isoemissive point) or EM 640/25. Metamorph software was used to perform
regulationofionfluxhasincreasinglybeenidentifiedasacritical darkfieldandflatfieldcorrectionsandtocreateratioimages,500/450forBCECF
player in diseases including cancer. Moreover, there have and 640/580 for SNARF-5F. Images were transferred to PhotoshopTM with no
recently been concerns raised about the safety of proton pump changestopixelintensitiesandthehistogramfunctionwasusedtocalculatemeans
andstandarddeviationsofpixelintensitiesinregionsofinterest(ROIs)chosenon
inhibitors taken during pregnancy and their links to resulting
images of Arch-tomato fluorescence then transferred to the ratio images.
birth defects (Gill et al., 2009; Pasternak and Hviid, 2010). Our Conversion from differences in intensity to differences in pH were made using
application of optogenetics to the control of complex conversiondatafromtheliterature(Morleyetal.,1996;Sasakietal.,1992).
developmental and regenerative bioelectrical events, and the
Electrophysiology
regulation of known transcriptional cascades by brief optical
V wasmeasureddirectlyusingaWarnerInstrumentsOocyteClampAmplifier
stimulation of cells demonstrates the enormous potential for mem
model OC-725C with oocyte pathclamp model 7251.I (Harvard Apparatus
expanding the use of light-controlled ion flux beyond regulating Company, Hampden, CT). Borosilicate microelectrodes (O.D. 1.0mm, I.D.
action potentials in nerve and muscle tissues. Indeed, there is 0.50mm)werepulledonaSutterInstrumentsFlamingBrownP-97micropipette
puller (P5500; heat5257; pull560; vel5100; time5200) and the tips were
tremendous potential to address questions about ion-flux based
n brokenbyhand.Electrodeswerefilledwith2MKClandtheembryoswerebathed
e disease, such as cancer, and especially for new approaches in in0.16MMR.EmbryosthathadbeeninjectedwithArchmRNAattheonecell
p regenerative medicine. stage,thenkeptunderlightorinthedark,weremovedtotherigandkeptunder
O ambientlightforupto15minutespriorto,andthenduring,measurement.
y
g Materials and Methods
o Xenopushusbandryandexperimentation Scoringofregenerationefficiency
ol Thisstudywascarriedoutinstrictaccordancewiththerecommendationsinthe Regenerativeabilitywasquantifiedbyexaminingtheamputatedtails7daysafter
Bi GuidefortheCareandUseofLaboratoryAnimalsoftheNationalInstitutesof amputation.Ascoringsystemwasusedtodividethetadpolesintofourcategories:
no,bad,good,andfullregeneration,asdescribedpreviously(Adamsetal.,2007).
Health.TheprotocolwasapprovedbyTuftsUniversity’sInstitutionalAnimalCare
Regenerative ability was then summarized as a score called the Regeneration
andUseCommittee(TuftsIACUCprotocol#M2011-70).Embryoswerecollected
Index,with0indicatingnoregenerationofanyindividualand300indicatingfull
and maintained as described previously (Sive et al., 2000) and grown in 0.16
regeneration of all animals. YCHE78’s effect on development was scored by
ModifiedMarc’sRingers(MMR).StagingwasaccordingtoNieuwkoopandFaber
dividing embryos into three categories: normal patterning and face; heterotaxia
(Nieuwkoop and Faber, 1967). Tail amputation was performed at stage 47 (a
(abnormal left–right positioning of the gut, gall bladder or heart loop) and/or
refractorystage)forphysiologicalinhibition,asdescribedpreviously(Adamset
craniofacialabnormalities;ordead.
al.,2007;Tsengetal.,2010).Formolecularstudies,Xenopusembryoswerefixed
overnight in MEMFA (Sive et al., 2000). In situ hybridization was carried out
accordingtostandardprotocols(Harland,1991)withprobestoNotch1(Coffman Microscopy
etal.,1990),andMsx1(Feledyetal.,1999).Antibodiesusedwereanti-acetylated Fluorescence microscopy was performed on an Olympus BX-61 compound
a-tubulin(Sigma#T6793),andanti-phospo-histone-3(H3P;Upstate#05-598)at microscope using, for Arch-GFP EX470/20, D485, EM517/23, and for Arch-
1:1000dilution. tomatoEX545/20,D565,EM595/50.Forlowermagnification/resolutionimages,a
NikonSMZ-1500withepifluorescenceopticsandsimilarfiltersetswasused.
mRNAinjection
Arch-GFPorArch-Tomatowasinjectedat60610pg;thisdosewasdeterminedto Statistics
haveactivitywithoutcausinglethality.BecauseoftheverylowsalinityofMMR, Alltestsonoptogeneticconstructscomparedthelightversusdarktreatments.The
othercandidatehyperpolarizingoptogeneticreagentswerefoundtobeunsuitable effectsoflightanddarkintheabsenceofArch(noArchcontrols)werecompared
forthissystem;additionally,nooptogeneticreagentthatcandepolarizeembryonic separatelyandshowednodifferences(supplementarymaterialTableS1).T-tests
orectodermalXenopuscellswerefoundinourtestingofdozensofoptogenetic were used to compare the results of electrophysiological measurements,
protein constructs. Injection of 461ng of YCHE78 mRNA at the one cell physiological inhibition experiments, and numbers of H3P-positive cells. A
(fertilized egg) stage caused both left–right patterning defects and craniofacial Mann–Whitney U test was used to compare the distributions of wild-type,
abnormalities.Formolecularinhibitionrescue,60610pgofArch-TomatomRNA abnormal,anddeadtadpolesresultingfromYCHE78injection.ForArchactivity,
wasco-injected with the YCHE78 mRNA (Adams etal., 2007). Forthe NHE3 thenumberofH3P-positivecells,andArch+YCHE,samplesizeswerethenumber
experiments, because intracellular [Na+] in blastomeres is higher than [Na+] of ofindividualtadpoles.Tocompareregenerationindices(regenerationability)after
MMR(21mMversus9.9mM),0.16MMRwassupplementedwith42mMNa- refractory amputation, the sample sizes were the number of dishes, which
gluconatetofacilitateactivityoftheNHE3antiporter. collectively represented 428 tadpoles. The effects of NHE by [Na+]ext were
comparedusingaKruskal–WallistestfollowedbyDunn’sQtoidentifypairwise
significantdifferences.
Lighttreatment
LighttreatmentconsistedofplacingPetridishesoftail-amputatedtadpoleseither
(a)beneathanarrayofsixLEDs(471nm)atadistanceresultinginirradianceof Acknowledgements
0.560.1mW/mm2or(b)intoalight-tightboxcontainingtwofiberopticcables TheauthorsthankPunitaKoustubhanandAmberBrandforXenopus
connected to SugarCube LEDs (Boston Engineering, Waltham, MA) delivering care;EdBoydenforArch-GFPandadviceonoptogenetics;Ravshan
0.860.1mW/mm2of463nmlight.Topreventphototoxicityandheatingofthe
SabirovfortheNHE3plasmid;JoanLemireandClaireStevensonfor
mediumduringthe48hoursofexposure,disheswereplacedoncoolingstagesto
maintainthetemperaturebetween18and20˚Candlightswererepeatedlyturned cloningoftheplasmids;andTalShomratandBrookChernetforhelp
onfor500millisecondsthenofffor2seconds.Alldarkcontroldisheswerekeptat with electrophysiology. M.L. gratefully acknowledges support from
Optogenetic regeneration 313
NIH (GM078484, AR055993), NSF (DBI-1152279), and the Illingworth,C.M.(1974).Trappedfingersandamputatedfingertipsinchildren.J.
G. Harold and Leila Y. Mathers Charitable Foundation. This work Pediatr.Surg.9,853-858.
Illingworth, C. M. and Barker, A. T. (1980). Measurement of electrical currents
wasalsosupportedbytheTelemedicine andAdvanced Technology
emergingduringtheregenerationofamputatedfingertipsinchildren.Clin.Phys.
Research Center (TATRC) at the US Army Medical Research and Physiol.Meas.1,87.
Materiel Command (USAMRMC) through award W81XWH-10-2- Kno¨pfel,T.,Lin,M.Z.,Levskaya,A.,Tian,L.,Lin,J.Y.andBoyden,E.S.(2010).
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Levin,M.(2009).Bioelectricmechanismsinregeneration:uniqueaspectsandfuture
perspectives.Semin.CellDev.Biol.20,543-556.
Competing Interests Levin, M. (2012). Molecular bioelectricityin developmental biology:new tools and
The authors havenocompetinginterests todeclare. recentdiscoveries:controlofcellbehaviorandpatternformationbytransmembrane
potentialgradients.Bioessays34,205-217.
Morley, G. E., Taffet, S. M. and Delmar, M. (1996). Intramolecular interactions
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Optogenetic regeneration S1
Supplementary Material
DanySpencerAdamsetal.doi:10.1242/bio.20133665
Table S1. Summary of statistical analyses.
Valuefor Valuefor Valueof
Experiment Measurement N dark light Test statistic P
Archactivity V 12 2268 4067 t-test t53.541 0.002
mem
Arch+YCHE Craniofacialabnormalities 149 6309 4866 MWU Z53.423 ,0.001
Arch+stage47amputation Regenerativeability 16 45624 111658 t-test t53.709 0.002
Arch+stage47amputation NumberofH3P-positivecells 22 463 1264 t-test t55.237 ,0.001
NoArchcontrols
Notreatment V 6 2962 2962 t-test t50.346 0.426
mem
YCHE78 Craniofacialabnormalities 105 2665 2901 MWU Z520.67 .0.5
Stage47amputation Regenerativeability 14 69644 49630 t-test t51.464 0.167
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