RESEARCH ARTICLE

Ablation Studies on the Developing Inner EarReveal a Propensity for Mirror DuplicationsErik H. Waldman,† Aldo Castillo, and Andres Collazo*

The inner ear develops from a simple ectodermal thickening known as the otic placode. Classicembryological manipulations rotating the prospective placode tissue found that the anteroposterior axiswas determined before the dorsoventral axis. A small percentage of such rotations also resulted in theformation of mirror duplicated ears, or enantiomorphs. We demonstrate a different embryologicalmanipulation in the frog Xenopus: the physical removal or ablation of either the anterior or posterior halfof the placode, which results in an even higher percentage of mirror image ears. Removal of the posteriorhalf results in mirror anterior duplications, whereas removal of the anterior half results in mirror posteriorduplications. In contrast, complete extirpation results in more variable phenotypes but never mirrorduplications. By the time the otocyst separates from the surface ectoderm, complete extirpation results inno regeneration. To test for a dosage response, differing amounts of the placode or otocyst were ablated.Removal of one third of the placode resulted in normal ears, whereas two-thirds ablations resulted inabnormal ears, including mirror duplications. Recent studies in zebrafish have demonstrated a role for thehedgehog (Hh) signaling pathway in anteroposterior patterning of the developing ear. We have usedoverexpression of Hedgehog interacting protein (Hip) to block Hh signaling and find that this strategyresulted in mirror duplications of anterior structures, consistent with the results in zebrafish.Developmental Dynamics 236:1237–1248, 2007. © 2007 Wiley-Liss, Inc.

Key words: enantiomorph; Xenopus; regeneration; inner ear; hedgehog; placode; otocyst; mirror image; hair cell;organogenesis

Accepted 28 February 2007

INTRODUCTION

The inner ear is a highly asymmetricalsensory structure with clearly definedanteroposterior, dorsoventral, and me-diolateral axes. Its sensory organs,which contain the mechanosensory haircells, are stereotypically located withinthe inner ear, which is important fortheir normal function in balance andhearing (Hudspeth, 1989). The numberof sensory organs contained in the innerear varies between vertebrates, but all

have at least six: five for the vestibularsystem plus one for the auditory system(Fritzsch et al., 2002). Approximately acentury of descriptive and experimentalembryological studies have provided abasic understanding of the developmentof the inner ear from the otic placode, anectodermal thickening adjacent to thedeveloping hindbrain (Fritzsch et al.,1998; Baker and Bronner-Fraser, 2001;Rinkwitz et al., 2001; Fekete and Wu,2002).

To test when during developmentthe three main axes (anteroposterior,dorsoventral, and mediolateral) of theinner ear were determined, embryo-logical manipulations involving therotation of the otic placode were done(Yntema, 1955). Most of these studieswere conducted in several differentspecies of amphibians, but more re-cent work in avian embryos came tothe same conclusion, that is, that theanteroposterior axis is determined be-

Leslie and Susan Gonda (Goldschmied) Department of Cell and Molecular Biology, House Ear Institute, Los Angeles, CaliforniaGrant sponsor: NIH NIDCD; Grant number: RO1 DC004061; Grant sponsor: the Deafness Research Foundation and Quota International.†Dr. Waldman’s present address is Department of Otolaryngology & Communication Disorders, Children’s Hospital Boston, 300 LongwoodAvenue, LO367, Boston, MA 02115.*Correspondence to: Andres Collazo, Leslie and Susan Gonda (Goldschmied) Department of Cell and Molecular Biology,House Ear Institute, Los Angeles, CA 90057. E-mail: acollazo@hei.org

DOI 10.1002/dvdy.21144Published online 29 March 2007 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 236:1237–1248, 2007

© 2007 Wiley-Liss, Inc.

fore the dorsoventral axis (Wu et al.,1998). Interestingly, in a small per-centage of cases in the amphibian ro-tation studies, mirror duplicated earswere generated (Harrison, 1936).These embryological manipulationswere the only known method of gener-ating such duplications until recently.

A zebrafish study identified thatmembers of the Hedgehog (Hh) signal-ing pathway are involved in antero-posterior patterning of the developinginner ear (Hammond et al., 2003).This finding contrasts with the role ofHh signaling in mouse inner ear de-velopment, where Sonic hedgehog(Shh) plays a role in dorsoventral pat-terning by promoting ventral identityand restricting Wnt genes to the dor-sal region (Riccomagno et al., 2002,2005; Ohyama et al., 2006). In chick-ens, Shh has also been shown to beimportant for ventral patterning ofthe otocyst (Bok et al., 2005). Hh (asecreted and lipid-modified signalingprotein) binds to its receptor patched,which releases inhibition of anothertransmembrane protein, smoothened,transducing the signal by means ofthe Gli transcription factors (Ingham,1998; McMahon et al., 2003; Pana-kova et al., 2005; Fuccillo et al., 2006).Two zebrafish mutants known to elim-inate Hh signaling were found to havepartial duplications of both anteriorinner ear structures and gene expres-sion in the posterior half, as well asreduction of posterior gene expres-sion. One of these mutations is in theHh signal transducer Smoothened andis one of the strongest Hh signalingmutants so far isolated in zebrafish(Hammond et al., 2003). These effectswere phenocopied by overexpressionof the patched1 mRNA, which also re-duces Hh signaling. Augmenting Hhsignaling by such manipulations asoverexpressing Shh generated the re-verse phenotype, duplicating bothposterior structures and gene expres-sion in the anterior half of the innerear. Of the three hedgehog genesknown in zebrafish, only Shh andTiggy-winkle hedgehog were involved.

We discovered another embryologi-cal manipulation that results in theformation of mirror duplicated innerears. In these experiments, either theanterior or posterior half of the pla-code is physically removed and theembryos are left to develop. Signifi-

cant percentages resulted in dupli-cated inner ears, with two anterior ortwo posterior halves fused as if mirrorimages. These percentages were muchhigher than was ever seen with theamphibian rotation studies (Yntema,1955) or even the zebrafish Hh signal-ing pathway mutants (Hammond etal., 2003). Labeled grafting experi-ments confirmed that the regeneratedtissue was from the remaining placodeand not surrounding tissues. Com-plete extirpation experiments addressthe following question: given the re-moval of the placode or otocyst what isthe potential of the surrounding tis-sues to generate a normal inner ear?The results of completely removingthe placode or early otocyst revealthat the inner ear is much less regen-erative than other placodes such asthe olfactory (Stout and Graziadei,1980) and somewhat contradict ear-lier ablation studies in salamander(Kaan, 1926). Lastly, to investigatethe potential involvement of thehedgehog signaling pathway on an-teroposterior inner ear development,experiments overexpressing Hedge-hog interacting protein (Hip) were un-dertaken. Hip has been demonstratedto be a potent inhibitor of Hh signal-ing (Koebernick et al., 2003; Cornesseet al., 2005). Hip overexpression re-sulted in mirror anterior duplications.By showing that Hh signaling is in-volved in the anteroposterior pattern-ing of the inner ear, these results sug-gest that Xenopus is more likezebrafish than mouse and chickens.They also suggest that, during thecourse of evolution, the role of Hh sig-naling in inner ear patterningchanged from an inducer of posterioridentity to an inducer of ventral iden-tity in amniotes.

RESULTS

Complete Ablations SuggestThat the RegenerativeAbility of the Inner EarQuickly Declines With Age

To test the ability of surrounding tis-sues to regenerate the inner ear, theplacode or otocyst was completely re-moved leaving the surrounding tissueintact. The contralateral side servedas a nonoperated control. Previousstudies in salamanders found that the

inner ear could still develop after ab-lation of the prospective otic placode,even when an area larger than the oticplacode was ablated (Kaan, 1926).Our complete extirpations in Xenopusembryos were done at stages rangingfrom 21 to 30 (Nieuwkoop and Faber,1967), which represent three catego-ries of inner ear development: earlyplacode (21–23), late placode (24–27),and otocyst (28–30; Fig. 1; Schlosserand Northcutt, 2000).

The morphology of inner ears re-sulting from complete ablation couldbe placed into four categories: (1) com-plete regeneration, (2) formation ofabnormal otocysts without sensorycells, (3) formation of abnormal oto-cysts with sensory cells, and (4) com-plete absence of regeneration (Fig. 2;Table 1). The stage the ears werescored, typically 48, was a tadpolestage where the semicircular canalsare present and five of the eventualeight sensory organs have developed(Kil and Collazo, 2001; Bever et al.,2003). The ear was scored as com-pletely regenerated if it appearedidentical to nonmanipulated controlears. The abnormal inner ears in theother two categories were oftensmaller in size and did not have fullyformed semicircular canals. The ab-normal otocysts with sensory cellstypically had fewer sensory organsthan control ears, but we could notdetermine whether these findings re-sulted from fusions or absence of sen-sory organs. These sensory organswere often in ectopic positions and ofvariable sizes, so identification ofthese sensory organs as cristae ormacula was not always possible. Thecomplete absence of regenerationcould be judged by blood vessels thatnormally ended at the lateral edge ofthe inner ear continuing to the hind-brain and by the apposition of anteriorand posterior lateral line ganglia thatare normally separated by the innerear (Fig. 2D,D�).

The ability of surrounding tissues toregenerate any type of inner earquickly drops from placode to otocyststages (Fig. 1). Complete regenerationof the inner ear was only seen at earlyplacode stages (13%, Table 1). Remov-ing the otic structure at placode stagestypically resulted in some form of re-generation, although the ears weremostly abnormal. Xenopus embryos

1238 WALDMAN ET AL.

fail to regenerate ears if the extirpa-tion occurs during the otocyst stagesof development (48/49 showed no re-generation). The inner ear, unlikeother placodally derived sensorystructures, such as the olfactory pla-code (Stout and Graziadei, 1980), hasless regenerative potential, eventhough the olfactory and otic placodedifferentiate at similar stages (Fig. 1;Nieuwkoop and Faber, 1967; Schlosserand Northcutt, 2000).

The most common category of innerear seen at early and late placodestages was otocysts without sensoryorgans (Table 1). Of the four catego-ries of ears resulting from completeablation, no regeneration was the sec-ond most common at late placodestages and tied with abnormal oto-cysts with sensory organs at early pla-code stages. The lack of regenerationapparent at otocyst stages, but alsoseen at placode stages, suggests thatany regenerated tissue in partial ab-lation would likely be derived from theremaining placode and not surround-ing tissues. In summary, completelynormal inner ears are only seen atearly placode stages, but even at thisstage, no regeneration is more com-mon, and by otocyst stages, completeablation results in no regeneration.

Half Ablations Result in aHigh Percentage of MirrorDuplicated Inner Ears

Previous otic placode rotation experi-ments revealed a small percentage ofembryos with an interesting pheno-type: mirror duplicated (enantiomor-phic) inner ears (Harrison, 1936; Yn-tema, 1955). These findings wereeither mirror anterior (with two setsof anterior and lateral cristae andutricular maculae but no posteriorcristae) or mirror posterior (with twosets of posterior cristae, papilla am-phibiorum, and lagenar maculae butno anterior and lateral cristae orutricular maculae). In the course ofour ablation experiments, we discov-ered another embryological manipula-tion that results in mirror-duplicatedears at even higher frequencies thanseen in previous studies.

We ablated either the anterior orposterior half of the otic placode atlate placode to early otocyst stages(24–27; Table 2). Unlike complete ab-lations in which the inner ear oftennever regenerated or formed vesicleswithout sensory organs, these half ab-lations all (71/71) had sensory organsin the otocyst and many of these weremirror duplicated inner ears. The re-sulting ears could be placed in three

categories, perfect mirror duplica-tions, normal, and an intermediatecategory with either partial duplica-tions of certain structures, but not all,or noticeably smaller but normal-look-ing ears (Fig. 3). Anterior mirror du-plications contained a symmetricalinner ear with two anterior halvesfused about the anteroposterior axis,whereas the presumed posterior mir-ror duplications seemed to contain asymmetrical inner ear with two poste-rior halves fused about the anteropos-terior axis (Fig. 4). The identity of thecristae in what we call mirror poste-rior duplications could instead repre-sent two lateral cristae, two anteriorcristae, or an anterior and posteriorcrista. We think these ears have twoposterior cristae because the resultingcristae have hair cells in the horizon-tal arrangement typical of anteriorand posterior cristae rather than thedenser and more vertically arrangedhair cells in lateral crista. Also theseears are missing the utricle and itsotoliths, which typically lie just belowthe anterior cristae. The intermediatecategory (called “other” in Fig. 3 and“other otocysts with sensory” in Table2) for the half ablations mainly con-sisted of ears with partial but not com-plete duplications. For anterior halfablations, this included ears wherethe cristae were duplicated but not themacula with its otolith or vice versa,whereas for posterior half ablations,this included ears missing the endog-enous utricle or either the lateral oranterior crista. The smaller normal-looking ears that were also part of thiscategory were seen less often than thepartial duplications discussed, espe-cially in posterior half ablations.

Anterior half ablations leaving theposterior half resulted in mirror dupli-cated posterior halves in 31% of cases.If we include those with partial butnot perfect duplications, the percent-age rises to almost 60%. Posterior halfablations, leaving the anterior half,resulted in mirror duplicated anteriorhalves in 42% of cases. Includingthose with partial but not completeduplications increases the percentageto almost 80%. The remaining ears foreither half ablation looked completelynormal, suggesting some ability to re-generate the normal pattern remainsin the leftover half. The mirror dupli-cated ears after anterior half ablation

Fig. 1. Results of complete ablations. Inner ear placodes or otocysts were ablated at stagesrepresenting three different categories encompassing several Nieuwkoop and Faber (1967) stages.The Y-axis lists the percentage of embryos (out of 142, see Table 1) that had the observedphenotype. The four categories are discussed in the text. Previously published results showing thepercentage of complete regeneration seen after prospective olfactory tissues were ablated (nasalregeneration) are included for comparison (Stout and Graziadei, 1980).

INNER EAR MIRROR DUPLICATIONS 1239

were usually smaller than those afterposterior half ablation (Fig. 4), sug-gesting that there is more potentialfor growth in the anterior half of theotic placode.

To determine the source of the re-generated tissue, we transplantedrhodamine-labeled half placodes intoembryos in which the endogenous pla-code had been removed. The resultingregenerated inner ears consisted en-tirely of labeled tissue, demonstratingthat the surrounding tissues do notcontribute to the regenerated ear (Fig.5). The example shown resulted in anormal inner ear; other examples hadpartial mirror duplications (data notshown). The rhodamine fluorescencewas concentrated in the thicker tis-

sues of the inner ear, such as the cris-tae with their densely packed haircells and supporting cells (Fig. 5D).The otic ganglion neurons were alsolabeled.

Results of Other PartialAblations

To see if there was a dose responsebased on the amount of tissue left, weremoved either one third or two thirdsof the placode (Table 2). When onethird of the anterior or posterior endwas removed (n � 10; stages 24 to 27),the resulting ears were all normal.When two thirds of the placode wasremoved (n � 20; stages 24 to 27), theresulting ears were more variable, but

complete regeneration was neverseen, similar to what we observed af-ter full ablation at these stages (Table1). Unlike after complete ablation,two-thirds ablations resulted in a goodnumber of mirror duplications (30%)that were consistent with the side be-ing ablated as seen after anterior orposterior half ablations. It was alsothe only manipulation besides com-plete ablation that resulted in otocystswithout any sensory organs. The ab-normal otocysts with sensory organsseen after two-thirds ablations (cate-gory “Other otocysts with sensory” inTable 2; “Otocyst with sensory” in Ta-ble 1) were similar to those seen aftercomplete ablation, with fewer orlarger sensory organs in ectopic loca-

Fig. 2. Examples of the phenotypes seen after complete ablation. A,A�: Complete regeneration (stage 21 ablation). B,B�: Otocyst without sensoryorgans (stage 25 ablation). C,C�: Otocyst with sensory organs (stage 24 ablation). D,D�: No regeneration (stage 21 ablation). A–D: Brightfield viewedat stages 47–48. A�–D�: Corresponding fluorescent image. L, left side ablated; R, right side control. Dorsal views, anterior to top. Tadpoles were imagedalive through an epifluorescent compound microscope using a low light level camera. Gray brackets indicate the length of inner ear. mb, midbrain; hb,hindbrain; sc, spinal cord; as, anterior semicircular canal (scc); ls, lateral scc; ps, posterior scc; uo, utricular otolith; so, saccular otolith; suo, saccular&/or utricular otolith; allg, anterior lateral line ganglion � facial; pllg, posterior lateral line ganglion; ac, anterior crista; lc, lateral crista; pc, posteriorcrista; um, utricular macula; sm, saccular macula; uso, unidentified sensory organ; ki, kidney autofluorescence; A, anterior; D, dorsal. Scale bar �750 �m.

TABLE 1. Complete Ablations

Stage at ablationNoregeneration

Otocystswithoutsensory

Otocysts withsensory

Completeregeneration Totals

Early placode (21-23) 11 23.9% 18 39.1% 11 23.9% 6 13.0% 46Late placode (24-27) 16 34.0% 21 44.7% 10 21.3% 0 0.0% 47Otocyst (28�) 48 98.0% 0 0.0% 1 2.0% 0 0.0% 49Totals 75 39 22 6 142

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tions (Fig. 2). Although the smallernormal otocysts seen in this categoryafter half ablation were not seen, twoembryos did have partial duplicationsthat were similar to what we de-scribed above in the half ablations.

These results suggest that the ante-rior or posterior-most half (maybeeven third) has the information neces-sary to replicate a mirror image ear.To test if there was something specialabout the middle portion of the pla-code, we removed the middle third ofthe placode at stages 24 to 25 (Table2). At least half of these looked en-tirely normal (four of seven), with theremainder having smaller ears. In onecase, two separate small ears wereformed. This finding was likely due tonot pushing the two pieces togetherafter removal of the middle third. Thesmaller ears had reduced or fused oto-liths, missing canals, and fewer haircells. All these results suggest thatthe placode is capable of regulating toform a normally patterned inner ear

after a loss of only a third or even halfof its tissue, but not if two thirds aremissing.

Blocking Hedgehog SignalingResults in Mirror AnteriorDuplications

In zebrafish, Hh signaling is requiredfor posterior identity; when it is inhib-ited, anterior structures are dupli-cated posteriorly (Hammond et al.,2003). Hip is a vertebrate-specific in-hibitor of Hh signaling that was dis-covered in mice (Chuang and McMa-hon, 1999; Koebernick et al., 2003).We blocked Hh signaling by overex-pressing the mouse Hip, which hasbeen shown to act the same as theendogenous Xenopus hip (Cornesse etal., 2005), in half of the embryo, leav-ing the contralateral side as a control.The inner ears of the injected side hadanterior mirror duplications in 30 to35% of the embryos (Table 3; Fig. 6).The three examples shown include

two where just the anterior and lat-eral cristae were duplicated and an-other in which both cristae and theutricular macula were duplicated. Theear on the noninjected side was al-ways normal. The smaller eye pheno-type previously described after Hip in-jection (Cornesse et al., 2005) wasseen in 50 to 100% of the embryosinjected (Table 3).

The extra sensory organs seen afterHip injection and half ablation do notresult from the splitting of endoge-nous sensory organs but from the in-duction of new, extra hair cells. Wecounted the number of hair cells inendogenous and ectopic cristae in theposterior half of mirror image and con-trol injected inner ears. The mediannumber of hair cells normally found inthe anterior and posterior cristae ofnormal tadpoles at stage 48 is thesame, approximately 12. The numberof hair cells in the ectopic “anterior”crista was not significantly differentfrom the number found in posteriorcrista of control ears injected with agreen fluorescent protein (GFP)mRNA (11.0 SD � 5.3; n � 5 vs. 11.7SD � 0.8; n � 15, respectively; P �0.63). If we add the hair cells in theectopic “lateral” crista, the mean num-ber of hair cells in the duplicated halfincreases to 23.8 (SD � 6.7; n � 5),which is significantly more than thetotal found in the single posteriorcrista of GFP mRNA injected controls(P � 0.001). The number of hair cellsin the endogenous anterior crista ofears with posterior mirror duplica-tions was not different from that ofcontrol embryos or the uninjected sideof the same embryo. Our results sug-gest that the role of Hh signaling in

Fig. 3. Results of anterior (posterior left) or posterior (anterior left) half ablations. The resultinginner ears were placed in three categories: Mirror, Normal, and Other (an intermediate category).These three categories are explained in the text. Percentages are from values in Table 2.

TABLE 2. Partial Ablations

Otocystswithoutsensory

Other otocystswith sensorya

Mirrorduplications

Completeregeneration Totals

Anterior half ablations 0 0.0% 10 28.6% 11 31.4% 14 40.0% 35Posterior half ablations 0 0.0% 14 38.9% 15 41.7% 7 19.4% 36Ant/Post 1/3 ablations 0 0.0% 0 0.0% 0 0.0% 10 100.0% 10Ant/Post 2/3 ablations 4 20.0% 10 50.0% 6 30.0% 0 0.0% 20Middle 1/3 ablations 0 0.0% 3 42.9% 0 0.0% 4 57.1% 7Totals 4 37 32 35 108

aFor the half ablations, this category consisted of ears with partial but not complete mirror duplications or normal-looking ears thatwere significantly smaller than the control side.

INNER EAR MIRROR DUPLICATIONS 1241

anteroposterior patterning of Xenopusinner ears is similar to that in ze-brafish.

DISCUSSION

Complete Ablation

Ablations studies have historicallybeen important for understandingwhat a given cell or tissue forms orinduces and for testing the ability ofsurrounding cells or tissues to recon-stitute lost structures (DuShane,1935, 1938; Scherson et al., 1993). Pre-vious ablation studies in salamanders(Kaan, 1926) found that most ears ab-lated at early and late placode stagescould regenerate at least some otic tis-sue (76% and 72%, respectively). Wefound less regeneration after completeablation then Kaan found and most ofour experiments were done at compa-rable stages of inner ear development.Complete regeneration of normal earswas only seen when the experimentswere done at early placode stages(13%), and by late placode stages, allresulting inner ears were abnormal,as were most resulting from early pla-code ablation (Fig. 1). Kaan foundthat, at early placode stages most ofthe regenerated ears were completelynormal (9 of 14), even when an areagreater than that of the otic placodewas ablated. By late placode stages,she did find that most of the ears wereabnormal, especially at the later oticcup stages. Most of the abnormal earsresulting from our complete ablationsat early and late placode stages wereotocysts with little or no invaginationof the semicircular canals and lackedany hair cells that could be detectedby our in vivo labeling. The otoliths,which are at least in part formed bythe support cells of the sensory organ(Thalmann et al., 2001; although inzebrafish lacking support cells, verysmall otoliths can form; Haddon et al.,1999), were also often missing. Theseresults led us to conclude that theseabnormal inner ears lack sensory or-gans. The formation of empty otocystsdevoid of sensory organs suggests thatthe formation of the nonsensory por-tion of the otocyst (the membranouslabyrinth) may be uncoupled fromthat of the sensory organs. This find-ing is consistent with recent studiesthat separate the roles of genes in-volved in otic placode specification

from those involved in later pattern-ing (Groves and Bronner-Fraser,2000; Saint-Germain et al., 2004; Kilet al., 2005).

By otocyst stages, we found a com-plete lack of regeneration after abla-tion. This finding is a dramatic de-crease in regenerative potential fromlate placode stages. Kaan did not doany ablations at these stages, but theonly examples where she saw no re-generation were her latest otic cup ab-lations (Kaan, 1926). That even atearly and late placode stages we foundno regeneration to be the second mostcommon result further demonstratesthe lack of regenerative potential ofsurrounding tissues. This finding wassurprising given that other placodesin Xenopus such as the olfactory arehighly regenerative at comparablestages of development (Stout and Gra-ziadei, 1980). Of interest, the otic pla-code also differs from the lens placode

in that ear induction appears to beginlater and end earlier than lens induc-tion (Gallagher et al., 1996).

Two concerns arising from any ab-lation experiment are being certainthat the tissue was completely re-moved and making sure that only thetissue of interest and not surroundingtissues are removed. Placodes and oto-cysts are relatively easy to distinguishand remove. That combined with ourmolecular marker, X-dll3, allowed usto be certain that we could reliablyremove the whole inner ear, and justthis tissue, for our total extirpationexperiments (Fig. 7). Although someneural crests cells that adhere to theotocyst may also be removed, theywould be few in number. Earlier abla-tion studies in amphibians removedthe otic placode as well as tissuearound and beyond the area of theplacode (Kaan, 1926). Because of thelack of regeneration in our complete

Fig. 4. Mirror image duplicated inner ears. Dorsal views. A,A�: Normal unoperated ear. B,B�:Mirror anterior duplications after the posterior half was removed. C,C�: Mirror posterior duplicationsafter the anterior half was removed. A–C�: Fluorescent (A–C) and brightfield (A�–C�) images at stage48. All panels are to scale except for C, which was magnified to better visualize the hair cells. Thefigures are montages of multiple planes of focus. Tadpoles were imaged alive through an epifluo-rescent compound microscope using a low light level camera. ls, lateral semicircular canal (scc); ps,posterior scc; uo, utricular otolith; so, saccular otolith; ac, anterior crista; lc, lateral crista; pc,posterior crista; um, utricular macula; sm, saccular macula; A, anterior; L, lateral. Scale bars� 100 �m.

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extirpation experiments, it was fairlystraightforward to be certain that wewere just eliminating the inner earand not surrounding tissues. Tadpoleswith missing inner ears had all theother tissues expected around the earsuch as the lateral line ganglia, bloodvessels, branchial arches, and normalappearing hindbrain, suggesting that

these tissues were not removed, as weconfirmed visually when doing the ex-tirpations. When removals were donebefore placode formation the embryoslost many structures such as the neu-ral crest-derived branchial arches ofthat side (data not shown). This find-ing meant that we could not do abla-tions earlier than placode stages, so

we could not determine whether, atearlier time points, the placode couldregenerate more completely as mightbe extrapolated back from Figure 1.The fate map in chicken reveals thatthe prospective otic placode cells areintermixed with neural crest, hind-brain, and other placodes, so our re-sults before placode formation are notsurprising (Streit, 2002).

Mirror Duplications

Why do anteroposterior half ablationsresult in mirror image duplications?The simplest explanation might bethat, in the case of posterior half ab-lations, the tissue that would respondto Hh signaling is being eliminatedand the resulting regenerated ear onlyreceives anterior identity signals. Foranterior half ablations, the reversewould be true, although the identitiesof the molecules involved in anteriorpatterning are not known. These re-sults have implications for how manysignals are necessary to pattern theinner ear and suggest that at least twosignals are required for anteroposte-rior patterning. A one-signal modelwould require a gradient of activityand should result in normal, ifsmaller, ears. That mirror image du-plications can still form, even after 2/3ablations, suggests that prospectivepatterning centers reside in or adja-cent to the anterior and posterior-most tips of the otic placode. Thesepatterning signals could be intrinsicand/or extrinsic to the inner ear. Forexample, the Hh signal for inner earpatterning in zebrafish, chicken, andmouse likely arises from adjacent tis-sues (Hammond et al., 2003; Bok etal., 2005; Riccomagno et al., 2005).

Although anterior versus posteriorsignals can explain why the regener-ated half has the same identity as theoriginal half, it less clear why the du-plicated inner ear structures are inmirror image to those in the originalhalf. One possibility is that, duringregeneration, a central anteroposte-rior boundary could result in a thirdsignal that subsequently polarizeseach half. Another alternative is thatthe endogenous and reconstituted sig-nals each produce lateral inhibitionsthat cause them to be distally located,resulting in a mirror image pattern.In the latter case, the anteroposterior

Fig. 5. Results after transplanting fluorescent rhodamine-labeled half placode where endogenousplacode was completely removed. The resulting inner ear was normal and consisted entirely ofrhodamine-labeled cells. A–D: Brightfield (A,C) and fluorescent images (B,D) at stage 48. Transplantdone at stage 28. C,D: Higher magnification images of the left ear shown in A,B. Tadpole slightlytilted so the left ear saccular otolith is clearly visible while the one in the right ear is under thehindbrain. C: Montage of multiple planes of focus. L, left side transplanted; R, right side unoperatedcontrol. Dorsal views, anterior to top. The tadpole was imaged alive through an epifluorescentcompound microscope using a low light level camera. hb, hindbrain; ls, lateral semicircular canal(scc); ps, posterior scc; uo, utricular otolith; so, saccular otolith; ac, anterior crista; lc, lateral crista;pc, posterior crista. Scale bars � 200 �m. A and B are at same magnification; C and D are at samemagnification.

TABLE 3. Hip mRNA Injections

Concentration 4 ng 5 ng 9 ng

Normal 2 20% 0 0% 1 5%Smaller eye 8 80% 15 100% 19 95%Mirror duplicationsa 3 30% 5 33% 7 35%Total 10 15 20

aAll embryos with mirror duplications also had the smaller eye phenotype on the sameside.

INNER EAR MIRROR DUPLICATIONS 1243

boundary is not functionally impor-tant. Whereas our middle third abla-tions might suggest that a central an-teroposterior boundary is not requiredfor normal development, we cannotrule out the possibility that, after themiddle third is removed, a new an-teroposterior boundary is being recon-

stituted by the remaining anteriorand posterior signals.

Our discussion up to this point hassuggested that regional identity andaxial polarity are the same, but thisdoes not have to be the case. The es-tablishment of polarity may be occur-ring first in the remaining placode and

only later is the identity of that tissuein terms of anterior or posterior struc-tures established. A key assumptionof most patterning models is that theregenerated inner ear is only derivedfrom the remaining placode and notsurrounding tissues as we confirmedfor our half ablations (Fig. 5). Thissuggests that, after an ablation, a“new” anterior or posterior edge is es-tablished along the cut portion of theremaining placode. It is necessary forthe partially ablated placode to main-tain or reestablish anterior or poste-rior polarity whether it is going to re-sult in a normal or mirror imagepattern. While the same molecules in-volved in anterior and posterior iden-tity may also be involved in axial po-larity, they could also be different.

The key manipulation for generat-ing mirror duplications appears to beremoval of half or more of the ear.Although two-thirds ablations canalso produce mirror duplications, it isin smaller numbers when partial du-plications are included. Also it is crit-ical that the half removed for mirrorduplications to occur has to be the an-terior or posterior and not the dorsalor ventral halves. When dorsal or ven-tral halves are ablated, no mirror du-plications occur and the abnormalphenotypes are quite different (Forri-stall and Collazo, unpublished data)but similar to the results of dorsaland ventral half ablations done insalamanders (Kaan, 1926).

Fate map studies in frog indicatethat cell mixing is occurring during

Fig. 6. Inhibiting Hedgehog (Hh) signaling results in mirror anterior duplications. Projection of multiple confocal sections. A–C: Dorsal view of right(A) or left (B,C) ears. Three examples are shown. ac, anterior crista; lc, lateral crista; pc, posterior crista; um, utricular macula; A, anterior. Grayarrowhead, background fluorescence, mostly outside of the inner ear. Scale bars � 50 �m.

Fig. 7. Examples of whole and partial ablations. A,B: After complete placode extirpation on oneside of an albino embryo (stage 25), it was fixed for whole-mount in situ hybridization. The probeused was to Xenopus homeobox gene Distal-less 3 (X-dll3). A: Right-side lateral view of theunoperated side showing the inner ear. B: Left-side lateral view showing that the inner ear wascompletely removed. C,D: Partial ablation where the posterior half was removed. Pigmentedsuperficial epidermis is peeled away to expose otocyst (edges indicated by arrows). Note howquickly it heals back in the few minutes between C and D. C: The unoperated otic placode at stage26 (surrounded by arrowheads). D: The posterior half has been removed: arrowheads point toanterior half, while the dotted line indicates half removed. pr, pharyngeal region; a, anterior; d,dorsal (same orientation in B,C,D); A and B are at same original magnification; C and D are at sameoriginal magnification.

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the stages we did our ablation, yet ourhalf ablation results are also consis-tent with a compartment model of in-ner ear development in that the re-maining half often could onlyregenerate tissues with its own iden-tity (Brigande et al., 2000; Kil andCollazo, 2001). These two results mayseem to contradict, but our ablationsand the fate map experiments addressdifferent questions that are not com-parable. The fate map experimentsare looking at normal development,while the ablations are challengingthe fate of the remaining tissues. Thetwo results can be reconciled for ex-ample if we assume that cells in theanterior half that would normally con-tribute to posterior structures insteadform anterior structures in the mirrorduplicated ears. Also, many of theears in the half ablations were nor-mal, suggesting that cells with poste-rior identity could reside in the ante-rior half. As a review of the inner earfate map literature suggests, geneswith complex and dynamic expressionpatterns may also be important forinner ear patterning during develop-ment, which would be consistent withthe extensive cell mixing observed (Kiland Collazo, 2002).

Hedgehog Inhibition Resultsin Mirror Duplications

Almost all the components of the Hhsignaling pathway are expressed in oraround the developing inner of Xeno-pus. In addition to Sonic Hh (Shh),Xenopus has Banded (Bhh) and Ce-phalic hedgehogs (Chh), homologuesof mammalian Indian and Deserthedgehogs, respectively, of which onlyBhh is expressed directly in the oto-cyst (Ekker et al., 1995). AlthoughShh is not expressed in the ear, it isexpressed in adjacent tissues. Also,the Hh receptor and transducers areexpressed in the developing inner earof Xenopus (Takabatake et al., 2000;Koebernick et al., 2001).

Our results inhibiting Hh signalingin Xenopus produced the same resultsseen in zebrafish: mirror anterior du-plications. Whereas Hh signaling inzebrafish plays an important role inanteroposterior patterning, in mouseand chicken, it appears to play a dif-ferent role. Loss of Hh signaling inmouse, as seen in the Shh knockout,

results in loss of ventral molecularmarkers and structures, such as thecochlea and cochleovestibular (otic)ganglion, whereas ectopic activationof Hh signaling dorsally results indown-regulation of dorsal markersand the turning on of some ventralmarkers (Riccomagno et al., 2002). Hhsignaling is also important for inhib-iting the expression of more dorsalgenes, such as Wnt, ventrally (Ricco-magno et al., 2005; Ohyama et al.,2006). Similar morphological defectsto those observed in mouse are seenafter loss of Shh activity in chickenembryos (Bok et al., 2005). The mouseand zebrafish studies are not strictlycomparable as the zebrafish study re-quired that two Hedgehogs, Shh andTiggy-winkle Hh (Twhh), be knockeddown before any mirror duplicationswere noted. The strongest duplica-tions were found to occur in the stron-gest inhibitor of Hh signaling in ze-brafish, the Smo mutant, whose innerear phenotype in mouse has not beendescribed because it does not survivelong enough (Zhang et al., 2001). Ourdata in Xenopus combined with thefindings in zebrafish suggest that,during the course of evolution, theremay have been a change in the role ofHh signaling in ear development frompromoting posterior identity to pro-moting ventral identity in amniotes.

Gain and loss of function experi-ments in Xenopus suggested that Hhsignaling was involved in otic placodeinduction (Koebernick et al., 2003).Either activation or inhibition of Hhsignaling produced the same results,ectopic otic vesicles. One reagent usedto inhibit Hh signaling, cyclopaminetreatment, did not produce ectopic oticvesicles but instead enlarged the en-dogenous otocyst. Later work from thesame group showed that the effects onotic induction may have been due tosome of the reagents used, specificallyHip, disrupting Fgf and Wnt signaling(Cornesse et al., 2005), two pathwaysknown to be involved in otic placodeinduction (Groves and Bronner-Fraser, 2000; Noramly and Grainger,2002). Therefore, we cannot rule outthat the mirror duplications we ob-served could be due to Hip inhibitingother cell signaling pathways such asFgf and Wnt.

What is interesting about the re-sults in zebrafish is that the mirror

image duplications are the results ofnot just creating anterior structuresin the posterior half (in the case ofanterior duplications) but also of in-hibiting posterior structures and geneexpression. This suggests that the cre-ation of mirror image duplications is atwo-step process first involving thesuppression of genes and structures inthat half and then the promotion ofgenes and structures with the newidentity. However, the percentage ofembryos with complete duplicationsafter Hh signaling was disrupted issmaller than the percentage seen af-ter our partial ablations. This findingwould suggest that, while Hh signal-ing is an important component of an-teroposterior patterning, other mole-cules are likely to be involved. Ourhalf ablations will provide a powerfulassay for determining other moleculespotentially involved in anteroposte-rior patterning by providing a readysource of material for further molecu-lar analyses.

EXPERIMENTALPROCEDURES

Experimental Manipulationof Otic Tissue

Xenopus laevis eggs were fertilized invitro and staged according to the nor-mal table of Nieuwkoop and Faber(Nieuwkoop and Faber, 1967). Theplacode is first visible morphologicallyas early as stage 21 (Nieuwkoop andFaber, 1967; Hausen and Riebesell,1991). The otic placode begins to in-vaginate at stages 22–23 and beginsto form a vesicle at stage 24 but theotic vesicle does not completely sepa-rate from the surface ectoderm untilstage 28 (Nieuwkoop and Faber, 1967;Schlosser and Northcutt, 2000).

Embryos were chemically dejelliedfor 10 min in a 2% cysteine-HCl solu-tion in rearing solution (20 mM In-stant Ocean: a commercial aquariumsalt) at pH 8.2. For stages beforehatching, the vitelline membraneswere removed with sharpened watch-maker forceps (Dumont #5). Livingembryos were anesthetized with tric-aine methanosulfonate (Finquel), inrearing solution, at concentrationsranging from 1:10,000 to 1:2,500. Tobetter visualize the otic placode(stages 21–27) and otocyst (stages 28–

INNER EAR MIRROR DUPLICATIONS 1245

31), the pigmented epidermis waspeeled away using quartz glass mi-cropipettes pulled into fine needles us-ing a P-2000 micropipette puller (Sut-ter Instrument Co.). Removing thisepidermis does not disturb ear devel-opment (Kil and Collazo, 2001); itheals quickly, and this most superfi-cial epidermis does not contribute anytissue to the inner ear at placode orotocyst stages (Hausen and Riebesell,1991; arrows in Fig. 7C,D). After ex-posure, the placode or otocyst was ab-lated in total, half (anterior or poste-rior), or thirds leaving either nothingor the unablated portion (Fig. 7). Im-mediately after surgery, each partialablation was scored poor, fair, good, orexcellent, and only those embryoswith good or excellent surgeries wereanalyzed further. These ablationswere done with quartz micropipettes(described above), an eyelash knife,and/or Dumont #5 forceps. A smallsubset of ablations were also accom-plished by aspiration with a mouthpipette (Sigma A-5177) attached to amicropipette broken to the appropri-ate tip diameter. Results were similarfor all methods of removal used. Pic-tures of the partial ablations weretaken on a Zeiss M2Bio dissecting mi-croscope using a color Zeiss Axiocamand the Axiovision 2.0 software.

To make certain that the otocystwas completely removed; we practicedextirpating the placode on one side ofalbino embryos and fixed them imme-diately after the operation for whole-mount in situ hybridization (protocolbelow; Fig. 7A,B). As a probe, we usedthe Xenopus homeobox gene Distal-less 3 (X-dll3; actually Dlx5; Stock etal., 1996), which is strongly and glo-bally expressed in the otic placode andotocyst (Papalopulu and Kintner,1993). We have found that X-dll3 isexpressed in prospective otic regionswell before placode formation (stage13, which is just after gastrulation),making it one of the earliest otic genesexpressed in Xenopus, although notearlier than the transcription factorsSox9 and Pax8 (Saint-Germain et al.,2004). These ablations were done un-til 100% of labeled embryos were com-pletely missing their inner ears on theoperated side, indicating that the ex-perimenter had achieved proficiency.Only when the experimenter demon-strated and believed that he or she

was proficient did we proceed to theactual experiments that were scored.

Transplants

To determine the tissue source of re-generated ears, grafting experimentsof labeled donor ears into unlabeledhosts were undertaken. To generatelabeled donors, fertilized eggs of Xeno-pus were injected with a fluorescentlabel, lysinated rhodamine dextran(LRD; Molecular Probes D-1817), at aconcentration of 50 to 100 mg/ml atthe one-cell stage. Rhodamine-labeledanterior or posterior half placodeswere transplanted into unlabeled ani-mals whose placode had been com-pletely removed. Animals were al-lowed to develop to Nieuwkoop andFaber stage 47–49 at which time anyregenerated form was analyzed as de-scribed below. Regenerated tissue wasscored for the presence or absence ofrhodamine labeling.

Labeling Hair Cells In Vivo

Labeling of hair cells was done as pre-viously described (Kil and Collazo,2001). Briefly, inner ears of stage47–49 tadpoles, or any structure thathad regenerated in its place, were in-jected with a solution of the styryl flu-orescent dye 4-Di-2-ASP or FM1-43(Molecular Probes) to visualize haircells (Meyers et al., 2003). This pro-cess was accomplished using quartzmicropipettes (described above) back-filled with the vital dye and a pico-injector, PLI-100 from Harvard Appa-ratus. At these stages, many of thesensory hair cells have differentiatedand can be labeled with the dye. Thefive sensory organs (anterior, lateral,and posterior cristae and maculae ofthe utricle and saccule) visible atthese stages and the two otoliths as-sociated with these maculae werecompared with the control side. Oto-liths were scored before injection, asthe injection process sometimesmoved the calcium carbonate crystalsof which they consist. Other sensoryorgans, like the amphibian papillaand the basal papilla, develop later(stage 50�; Nieuwkoop and Faber,1967; Quick and Serrano, 2005) and sowere not scored during our manipula-tions.

Imaging and Scoring ofManipulated Inner Ears

Living embryos were prepared for im-aging by being placed on a bed of 2%Bactoagar just after injection atstages 47–49. Tadpoles are transpar-ent at these stages, facilitating obser-vation. Labeled cells were visualizedon either a Zeiss Axiophot2 Mot epi-fluorescence microscope or Zeiss LSM410 Scanning Laser Confocal micro-scope. Long working distance air orwater objectives ranging up to a mag-nification of �40 were used. Datawere recorded digitally from a light-intensifying camera (HamamatsuSIT) using MetaMorph 3.5 (MolecularDevices) on the epifluorescence micro-scope. Any regenerated structure wasscored for the presence or absence ofsensory cells, otoliths, semicircular ca-nals, and the pattern these structuresformed. Ears on the unoperated sidewere always normal. Adobe Photo-shopTM (Versions 6.0 to CS) was usedto view and process images.

Whole-Mount In SituHybridization

The method of Knecht and workers(Harland, 1991; Knecht et al., 1995)was used to perform whole-mount insitu hybridization (ISH). Antisenseand sense probes were made to X-dll3.The sense probe served as a negativecontrol. Pictures of the ISH weretaken with a ProgRes 3012 digitalcolor CCD camera using the Roche Di-agnostic software running under Win-dows for Workgroup 3.11. Imageswere collected through either a ZeissSV11 dissecting microscope or ZeissAxioplan upright compound micro-scope.

Injection of Hip mRNA

Injection of mouse Hedgehog-interact-ing protein 1 (Hip) was used to blockHh signaling as has been previouslydescribed (Koebernick et al., 2003).Previous authors have found that Hipgives the same phenotype as the en-dogenous Xenopus hip (Cornesse etal., 2005). Hip mRNA was injectedwith the same apparatus describedabove for styryl dye injection into theear. One cell at the two-cell stage wasinjected with 4 to 9 ng of mRNA, re-

1246 WALDMAN ET AL.

sulting in half the animal being af-fected, leaving the other half as a con-trol. In initial experiments, a smallamount of fluorescent lysinated rho-damine dextran (10,000 molecularweight, Molecular Probes) or GFPmRNA was co-injected so the experi-mental side could be identified by itsfluorescence. The smaller eye pheno-type previously described after Hip in-jection in Xenopus (Cornesse et al.,2005), always correlated with the la-beled side, so we used this phenotypeto identify the affected half in somelater experiments. Embryos werescored by labeling the hair cells asdescribed above and counting thenumber of hair cells in the anteriorand posterior cristae. Statistical anal-yses were done using the paired stu-dent’s t-test in Microsoft Excel (Office2000) and the Web site http://stat-pages.org for unpaired t-tests.

ACKNOWLEDGMENTSWe thank Drs. Olivier Bricaud, DonnaFekete, Caryl Forristall, Andy Groves,and Sung-Hee Kil for stimulating dis-cussions and advice during our stud-ies. We also thank Olivier Bricaud,Andy Groves, and two anonymous re-viewers for helpful comments on ear-lier versions of this study. A.C. wasfunded by the NIH and NIDCD, andE.H.W. was supported by grants fromthe Deafness Research Foundationand Quota International. We alsothank Drs. Katja Koebernick andNancy Papalopulu for the mouse Hipand X-dll3 plasmids, respectively.

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