graded expression of emx-2 in the adult newt limb and its corresponding regeneration blastema

Article No. mb981782 J. Mol. Biol. (1998) 279, 501±511

Graded Expression of Emx-2 in the Adult Newt Limband its Corresponding Regeneration Blastema

Michel Beauchemin1, Katia Del Rio-Tsonis2, Panagiotis A. Tsonis2

Monique Tremblay1 and Pierre Savard1*

1Neuroscience Research UnitUniversity Hospital ResearchCenter of QueÂbec, CHULPavilion, 2705 Laurier blvSainte-Foy, Canada G1V 4G22Department of BiologyThe University of Dayton300 College Park, DaytonOH 45469-2320, USA

Abbreviations used: RA, retinopolymerase chain reaction and racDNA ends; DMSO, dimethylsulf

0022±2836/98/230501±11 $25.00/0

Amputation of a newt limb causes stump cells to organize the reforma-tion of the missing structures. The phenomenon is remarkably precise inthat the regeneration is perfect. During the ®rst few days followingamputation, the tissue proximal to the plane of amputation gives rise tothe blastema, an area of growth composed of mesenchymal cells coveredby a single epithelium. The blastema possesses a morphogenetic potentialcharacteristic of the structures that have been amputated. Looking forcontrol genes putatively involved in regeneration, we cloned the newtversion of the mouse and human Emx-2. Its expression is restricted to theskin of the regeneration territories and is graded along the proximal-dis-tal axis of both forelimb and hindlimb, with higher levels in distalregions. The regeneration blastema also show this proximal-distal gradedlevel of expression with distal blastemas (mid-radius and ulna) showinghigher levels of expression when compared to blastemas of more proxi-mal origin (mid-humerus). Finally, retinoic acid proximalizes both thelevel of Emx-2 expression and the positional memory of the blastemasuggesting Emx-2 may participate in pattern formation by specifyingpositional information.

# 1998 Academic Press Limited

Keywords: Emx; positional information; regeneration; urodele; newt*Corresponding author

Introduction

Urodela amphibians, such as the newtNotophthalmus viridescens can replace amputatedportions of limb and tail by the process of epi-morphic regeneration (for a review, see Wallace,1981; Tsonis, 1996). Immediately after amputation,epidermal cells of the stump edge migrate rapidlyto cover the wound and form the apical ectodermalcap. Then the underlying tissue becomes dis-organized and dedifferentiates to a commonmesenchymatous-like cell type. These cells acquirea high proliferation rate, which, in conjunctionwith the dedifferentiation process, leads to aconical-shaped accumulation of multipotent cells,called the blastema. During subsequent days, theblastema grows rapidly and progressively differen-tiates from its proximal region to undergo morpho-genesis, leading to the perfect replacement of themissing structures.

ic acid; PCR-RACE,pid ampli®cation ofoxide.

A few regions of the newt body, referred to asthe regeneration territories including the limbs,the tail, the snout, and the dorsal ®n, are capableof epimorphic regeneration (GuyeÂnot et al., 1948;Kiortsis, 1953). Other body parts that do notexpress epimorphic regeneration capabilities, suchas the ¯ank, are known as neutral territories.Some intrinsic properties of the cells in a regener-ation territory enable their reversal of differen-tiation, their re-entry in cell cycle, and theirparticipation in the formation of a blastema(Ferretti & Brockes, 1991; Lo et al., 1993; Tanakaet al., 1997; Brockes, 1997), as though they havethe morphogenetic capabilities of embryonic cellspresent in morphogenetic ®elds. Indeed, theregeneration territories of the adult newt andsome embryonic ®elds have similar morphogen-etic capabilities (Muneoka & Bryant, 1984;Muneoka & Sassoon, 1992). For example, bothtissues allow the regulative development of apredictable body structure. It would not be sur-prising to ®nd in both tissues the expression ofgenes particular to morphogenetic ®elds.

The blastema is an autonomous system thatalways gives rise to the structure of its origin,

# 1998 Academic Press Limited

502 Regulation of Emx-2 in Regeneration

suggesting that blastema cells possess a memoryof the type of appendage to form (reviewed byStocum, 1984). This was demonstrated by thetransplantation of limb blastema onto X-ray irra-diated tail, where the blastema regrew a limb-likeappendage. As predicted, the converse exper-iment of transplanting a tail blastema onto anX-ray irradiated limb led to the growth of a tail-like appendage. Moreover, amputation of a limbat any level along the proximal-distal axis willalways produce a faithful regeneration of themissing parts only. Amputation at the level ofthe wrist induces the reformation of a paw,whereas amputation at the level of the elbowinduces the reformation of a forearm and a paw.Therefore, the regeneration blastema also pos-sesses a memory of its position along the appen-dage (reviewed by Stocum, 1984).

Retinoic acid (RA) and its derivatives are theonly chemicals known to affect the determinationof blastema cells; among its effects, it proximalizesthe proximal-distal, memory of a limb blastema.Thus, instead of forming a paw, a distal blastematreated with RA will regenerate a more proximalstructure (humerus, radius, ulna) in a dose-depen-dent fashion (Niazi & Saxena, 1978; Maden, 1982).The action of RA depends on nuclear receptors(Brockes, 1996; Gann et al., 1996; Pecorino et al.,1996a,b), which bind to the regulatory region oftarget genes, to modulate their expression. Theeffects of RA on homeobox gene expression duringdevelopment could account for the effects of RAon pattern formation (IzpisuÂa-Belmonte et al., 1991;Kessek & Gruss, 1991; Nohno et al., 1991; Kessel,1992; Marshall et al., 1993).

Homeobox-containing genes code for transcrip-tion factors that are involved in the determinationof the different structures of the body (reviewed byMcGinnis & Krumlauf, 1992). Many are expressedduring limb development, with expression patternssuggesting their involvement in the spatial pattern-ing of the anterior-posterior (DolleÂ et al., 1989;Oliver et al., 1989; IzpisuÂa-Belmonte et al., 1991,1992; Ros et al., 1992; Peterson et al., 1994), theproximal-distal (Yokouchi et al., 1991; Peterson

SmaI; Ss, SspI; St, StuI. (B) Southern blot analysis of newt ggested DNA (10 mg); lane 2, EcoRI digested DNA (25 mg); lamarkers are the fragments of l-DNA digested with HindIII.

et al., 1994), and the dorsal-ventral axes (Davieset al., 1991; Peterson et al., 1994). The homeoboxgenes Emx-2 and Emx-1 are vertebrate homologs ofa Drosophila head gap gene ems (Simeone et al.,1992a,b). Homozygous Emx-2 mutant mice displaydefects in development of the dorsal telencephalon(Yoshida et al., 1997) and mice die due to failure ofurogenital system development (Miyamoto et al.,1997). Emx-2 is also expressed in the epidermis ofthe developing mouse limbs (Simeone et al., 1992a)and its function in limb development is not knownyet.

On the basis of limb development and limbregeneration possibly sharing some genetic con-trols, we looked for the expression of homeoboxcontaining genes in the newt regeneration terri-tories and their corresponding blastemas. Here, wereport the isolation of Emx-2. We have found thatits primary structure is strongly conserved amongvertebrates. Emx-2 expression was analyzed indifferent regions of the body and along the proxi-mal-distal axis of the limb corresponding blastema,either treated or not treated with RA. The resultsare discussed in relation to the putative role ofEmx-2 in the speci®cation of the proximal-distalmemory of the blastema.

Results

Cloning

A redundant oligonucleotide probe (U-box),speci®c for the third helix region of homeodo-mains, was used to screen a newt tail cDNAlibrary (Beauchemin & Savard, 1992). The p027clone (Figure 1(A)) included 56 of the 60 aminoacids of a homeodomain. An additional cDNAlibrary screening with p027 did not reveal othercDNA of the same family. The cloning wasextended in the 50 region of the homeodomain byPCR-RACE (Frohman et al., 1988; Frohman, 1990;Jain et al., 1992). Clones TA � 54, TA � 159, andTA � 217 showed 50-extensions of 54, 159, and217 bp, respectively (Figure 1(A)).

A Southern blot of newt genomic DNA washybridized with p027. HindIII digested sample

Figure 1. Schematic representa-tion of Emx-2 cDNA clones andSouthern blot analysis of newtgenomic DNA. (A) the cDNA clonep027 was isolated from a newt tailcDNA library. The clones TA � 54,TA � 159, and TA � 217 wereobtained by PCR-RACE ampli®ca-tion of newt limb cDNA; theyextend p027 by 54, 159, and217 bp, respectively. Open boxesrepresent coding regions whereashatched boxes represent the home-odomain. B, BamHI; P, PvuII; Sm,

enomic DNA with p027 cDNA as a probe. Lane 1, undi-ne 3, HindIII digested DNA (25 mg). The molecular mass

Regulation of Emx-2 in Regeneration 503

revealed a single band whereas EcoRI digestedsample revealed two bands (Figure 1(B)). Theintensity of the bands in the EcoRI digest areapproximately 50% the level of the HindIII band,suggesting that an EcoRI restriction site is locatedin the genomic DNA harboring the p027sequences. Since there is no EcoRI restriction site inp027, we believe Emx-2 is harboring an intron.Consistent with this interpretation, a splicing site isknown to be present in the homeodomain of thehuman and mouse versions of the same gene(Simeone et al., 1992a). We conclude that Emx-2 isa single-copy gene.

Computerized data bank searches revealedthat the newt Emx-2 homeodomain shares ahigh degree of homology with the correspond-ing region in the Drosophila ems (Dalton et al.,1989). Moreover, 131 of the 135 amino acids ofthe coding region are identical to an equivalentregion in the mouse and human EMX-2(Simeone et al., 1992a; Figure 2). We concludedthese sequences are versions of a strongly con-served gene in vertebrates. The similaritiesbetween newt Emx-2 and other vertebratehomologs include a domain located upstreamfrom the homeodomain that is a divergentversion of the conserved homeopentapeptideIle/Phe-Tyr-Pro-Trp-Met present in several ver-tebrate homeobox genes belonging to the HOXclusters (Scott et al., 1989), the two residuesArg-Lys immediately upstream of the homeodo-main, and the three acidic residues Glu-Glu-Gluimmediately downstream of the homeodomain.

Figure 2. Nucleotide sequence of the newt Emx- cDNA anboxed. In the coding region, 131 amino acids are identicalthese are labeled with stars. Three conserved peptide motifs

Expression in the adult newt appendages

Emx-2 expression in different body regions ofthe newt was analyzed on Northern blots(Figure 3). A single rare transcript of approxi-mately 2.5 kb was revealed in RNA samples oftail, hindlimb, forelimb, and their correspondingblastema, whereas there was no signal in the¯ank. Emx-2 was also found to be expressed inthe adult kidney (as for many other homeoboxcontaining genes), whereas it was not in liverand spleen (data not shown).

Several normalizing genes were used for thecomplementary information they provide on RNAintegrity and origin (muscle or skin). A Xenopuscytokeratin probe revealed three messenger RNAs(3.5, 3.2, and 2.8 kb) that are speci®cally expressedin the epidermis (Mathisen & Miller, 1987; Ferrettiet al., 1991). The intensity of the signal (Figure 3)demonstrated that the proportion of RNA comingfrom the epidermis is quite similar in samples oftail, hindlimb, ¯ank, and forelimb, whereas it isapproximately two times higher in blastemasamples. Xenopus actin probe (Mohun et al., 1984)revealed a 1.4 kb and a 1.8 kb transcript (low strin-gency hybridization) corresponding to muscular(a-sarcomeric) and cytoplasmic (b-cytoplasmic)actin, respectively. Analysis of the intensity of thesignal (Figure 3) showed that RNA samples fromdifferent body structures, like the limb and tail,contain different ratios of muscular-to-cytoplasmicactin whereas blastema tissue only expresses cyto-plasmic actin. Therefore, actin is a probe of choice

d its conceptual translation product. The homeodomain isto the human Emx-2, and four amino acids are different;(see the test) are underlined.

Figure 3. Body distribution of Emx-2 in the adult newt.A Northern blot loaded with 5 mg of poly(A)�RNA(absorbance at 260 nm) of various samples was seriallyhybridized with Emx-2, cytokeratin, and actin probes.T, tail; BT, tail blastema; HL, hindlimb; Fk, ¯ank, FL,forelimb, BL, forelimb blastema. The numbers at right ofeach panel indicate the apparent length of thetranscripts.


to normalize RNA samples prepared from differentharvests of blastemas.

In situ analysis in regenerating limb revealedEmx-2 was mainly expressed in the newt limb skinand the wound epithelium of the regenerate(Figure 4). The restricted expression of Emx-2 inthe skin fraction was con®rmed on Northern blot(Figure 5). Newt hindlimbs, forelimbs, and theircorresponding blastemas were mechanically dis-sected to separate skin and muscle (intact limbs),or mesenchyme and epidermis (regeneration blas-temas); the tissues were frozen and kept in liquidnitrogen before poly(A)�RNA extraction. The skinfraction of adult hindlimb contained epidermis,dermis, and connective cells, whereas the musclefraction contained almost exclusively muscularcells. On the other hand, the blastema is composedof a cone of mesenchymal cells and a layer ofcovering epidermis; the separation of these tissuesfrom each other is dif®cult and can only result inenriched fractions with more or less contaminationof the other tissue fraction.

A Northern blot, loaded with samples ofpoly(A)�RNA of the different tissue fractions,was serially hybridized with cytokeratin, dlx-1(NvHBox-4), Hox C6 (NvHBox-1), actin, andEmx-2 probes (Figure 5). As expected, the cytoker-atin signal was enriched in the epidermis fraction,whereas it was almost not detected in themesenchymal fraction of the regeneration blaste-ma; the low intensity signal may represent thelevel of epidermal contamination of the mesench-ymal sample or the mesenchymal expression of ablastema speci®c cytokeratin (Ferretti et al., 1991).dlx-1 is a distal less like (dll-like) homeobox-con-

taining gene that is expressed ubiquitously inthe skin fraction of the adult newt limbs(Beauchemin & Savard, 1992). Its pattern ofexpression in different tissue fractions of the limband its corresponding blastema is identical tocytokeratin (Figure 5). The newt Hox C6 (Savardet al., 1988; Savard & Tremblay, 1995) expresseda 1.8 kb transcript in the muscle and skin frac-tions of the adult newt limb and in the epidermisand mesenchymal fractions of blastemas(Figure 5). The signals revealed with the actinprobe show the enrichment of the muscular actintranscript in the muscle fraction, and the cyto-plasmic one in the skin fraction of the limb(Figure 5). In the blastema, the cells are not yetdifferentiated, and the level of cytoplasmic actinis representative of all the cells in the blastema,including its epidermis.

Finally, the expression of Emx-2 was similar inlimb skin of ventral or dorsal origin when usingthe cytokeratin signal to correct for differences inload (data not shown).

Graded expression along the proximal-distal axis

We looked for a feature of Emx-2 expression thatwould be particular in pattern formation. Emx-2mRNA was measured in different regions alongthe proximal-distal axis of the limb and in blaste-mas originating from different levels of amputa-tion. Such blastemas regenerate different amountsof structure and show qualitative or quantitativedifferences, such as cellular adhesiveness, homeo-box gene expression, and differential regulationof RA-activated gene expression (Crawford &Stocum, 1988; Savard et al., 1988; Brown &Brockes, 1991; Ferretti et al., 1991; Brockes, 1991;Simon & Tabin, 1993). Poly(A)�RNA was extractedfrom different parts of the forelimb and hindlimb(as depicted in Figure 6(A)) and analyzed onNorthern blots.

Hybridization with Emx-2 revealed a 2.5 kbtranscript that gradually increases in intensity inthe more distal regions of both forelimb andhindlimb (Figure 6(B)). Distal blastema (mid-radius and ulna level) also shows a strongaccumulation of Emx-2 when compared withproximal blastema (mid-humerus level). The blotwas normalized to ascertain that the variations ofEmx-2 expression were related to position alongthe limb, and not to variations in the proportionof expressing tissue in various limb parts. Emx-2signal in distal blastemas is approximately seven-fold higher than proximal blastemas (actin as anormalizer). The same ratio is seen in proximaland distal portions of the adult limb (dlx-1 andcytokeratin 3.5 kb as normalizers). Emx-2expression increases gradually along the proxi-mal-distal axis of both forelimb and hindlimb asit is shown by in situ hybridization (Figure 4).The proximal-distal gradient in the intact limb isseen in Figure 4C by looking at the epidermis

Figure 4. In situ hybridization ofEmx-2 in the adult newt limb andits regeneration blastema, Cross-section of a newt forelimb threeweeks after amputation; the planeof amputation is labeled with adotted line (a-a). The intact tissue ifon the left side (proximal) of theline whereas the blastema tissue ison the right side (distal). (A) Bright®eld section. (B) Autoradiograms ofthe same section. (C) Two timesmagni®cation of a selected portionof the posterior epidermis (regionincluded between the arrowheads),to better show the proximo-distalgradient in the epidermis of theintact limb; the gradient is seen atthe epidermis layer between mar-kers m and a (left to the a-a line).


layer between markers m and a (left to thea-a line). Moreover, up-regulation of Emx-2expression in the wound epidermis of the blaste-ma is seen by comparing the signal left and rightof the a-a line.

We found similar amounts of dlx-1 messengerin the different proximal-distal regions of bothforelimb and hindlimb (Figure 6(B)) as previouslyreported (Beauchemin & Savard, 1992). The con-stant level of dlx-1 expression along the limbindicates that the variations of Emx-2 expressiondid not result from variations in the amount ofskin tissue present in each sample. The cytokeratinprobe revealed three transcripts whose expressionlevels are differentially regulated according toposition along the limb (Figure 6(B)). Althoughthe amount of 3.5 kb transcript is constant indifferent regions of the limb, its seems to be up-regulated in distal blastema (when comparedwith proximal blastema). The 3.2 kb transcript is

detectable only in the more distal regions of thelimb, a pattern that is similar to the cytokeratingene reported by Ferretti et al., 1991). Finally, the2.8 kb transcript level decreases gradually fromthe proximal to the distal region of the limb, agradient of expression that is complementary toEmx-2. The actin probe was useful to verify RNAintegrity of blastemal samples, as there is noterminal differentiation in the tissue.

Emx-2 and retinoic acidinduced proximalization

RA has a profound in¯uence on blastema devel-opment when it is injected into a newt after ampu-tation (Niazi & Saxena, 1978; Maden, 1982). Distalblastemas (wrist level) of RA treated animalsbehave like proximal blastemas. Thus, RA isthought to somehow respecify the proximal-distalpositional memory of the blastema. The relation-

Figure 5. Tissue distribution of Emx-2 in the hindlimbof adult newt. A Northern blot loaded with 5 mg ofpoly(A)�RNA (absorbance at 260 nm) of varioussamples was serially hybridized with cytokeratin, dlx-1,Hox C6, Actin, and Emx-2. Abbreviations; Fk, ¯ank; HLt,total hindlimb; HLs, skin of the hindlimb; HLm, muscleof the hindlimb; Blt, total limb blastema, BLe, epidermisof the blastema; BLm, mesenchyme of the blastema. Thenumbers of right of each panel indicate the apparentlength of the transcripts. The lowest band in the Emx-2panel is speci®c to Emx-2, whereas the highest is aresidual dlx-1 signal that did not wash off completelybefore rehybridization.

Figure 6. Expression of Emx-2 along the proximal-distal axis of forelimbs and hindlimbs of adult newt.(A) Schematic representation of adult newt legs showingthe code used to identify the different regions of fore-limbs (F1 to F4), hindlimbs (H1 to H3) and forelimbblastema (PB, proximal blastema; DB, distal blastema).(B) Poly(A)�RNA samples of various regions of bothforelimb and hindlimb were loaded on Northern blotsthat were serially hybridized with Emx-2, dlx-1, cytokera-tin, and actin probes. The numbers at the right of eachpanel indicate the apparent length of the transcripts.


ship between proximal-distal axial position andthe level of expression of Emx-2 prompted us toinvestigate the effect of RA on Emx-2 expression.Therefore, we used RA to proximalize the pos-itional memory of a forelimb distal blastema anddetermine whether the level of Emx-2 expression isreset to a value characteristic of a more proximalblastema.

Newts were amputated at the wrist level andinjected either with RA or dimethylsulfoxide(DMSO) ®ve days after amputation. Blastemaswere harvested seven days later, when they havereached the early bud stage. Emx-2 expression inRA treated distal blastema was compared withthat of DMSO treated distal and proximal blaste-mas (Figure 7). Expression along the proximal-dis-tal axis of the hindlimb was also compared. In thehindlimbs of DMSO treated newts, expression ofEmx-2 is graded with higher values in more distalregions (like untreated newt Figure 6(B)). Follow-ing RA treatment, the graded expression of Emx-2persists, but the general level of expression in allfractions is decreased by at least 50%. The sameeffect of RA was obtained with forelimb blastemasof different origins along the proximal-distal axis.Thus, the level of Emx-2 expression is higher in

distal blastema compared with proximal ones, andRA treatment on distal blastema reset the distallevel of expression to a more proximal level. Thenormalizing probe used in comparing treated anduntreated blastemas was the cytoplasmic actin,because it appeared unaffected by RA treatment,and the signal correlates well with the ethidiumbromide staining of the gel.

Discussion

Gene

We cloned a newt homeobox-containing cDNA(Emx-2) related to the Drosophila ems gene (Daltonet al., 1989). Emx-2 is a single copy gene thatencodes a 2.5 kb transcript from which we report615 bp that include an open reading frame of 135amino acids, starting 26 amino acids upstream ofthe homeodomain and ®nishing in the 30-untrans-lated portion of the cDNA. The coding region is97% identical (131 of 135) to the human EMX-2(Simeone et al., 1992a). The degree of homology ishigh enough to conclude these are the newt andhuman version of the same gene. The high level ofconservation of the protein in the phyla suggeststhat it plays similar functions in development andregeneration.

Figure 7. Effect of RA-treatment on Emx-2 expression inadult newt limbs. (A) Poly(A)�RNA samples of varioushindlimb regions and of proximal and distal forelimbblastemas of newts, which were treated or not with RA,were loaded on Northern blots that were seriallyhybridized with Emx-2, cytokeratin and actin probes.(B) Schematic representation of the ratio Emx-2/cyto-plasmic actin (transcript of 1.8 kb). H1 to H3 representdifferent hindlimb regions (see Figure 6); BLP � DMSO,proximal forelimb blastema of newts treated withDMSO; BLD � RA, distal forelimb blastema of newtstreated with RA; BLD � DMSO, distal forelimb blastemaof newts treated with DMSO. The results show that RAdecreases the overall Emx-2 expression in the adult limband its regeneration blastema.


Pattern of expression

Emx-2 is mainly expressed in the epidermal com-ponents of the regeneration blastema. Itsexpression in the mouse limb bud is also restrictedto the epidermis (Simeone et al., 1992a). Therefore,the homology between the newt and the vertebrateversion of Emx-2 extends to their domain ofexpression and this argues in favor of the prop-osition that limb development and limb regener-ation make use of the same genes to direct patternformation.

An Emx-2-related transcript was revealed in themuscular fraction of the limbs by reducing thestringency of hybridization (data not shown),suggesting that there is more than a single ems-likehomeobox gene in the newt. This observation is inaccordance with the existence of two ems-likehomeobox genes (Emx-1 and Emx-2) in human andmouse (Simeone et al., 1992a,b); both genes share a

very homologous DNA-binding domain and differin their amino and carboxy-terminal ends.

Most homeobox containing genes studied todate in both insects and vertebrate are transcribedin developing tissue and show restrictedexpression, if any, in the adult. This is part of thegeneral evidence that argues for their instructiverole in development. The maintenance ofexpression of Emx-2 in adult limb and tail suggeststhe skin of these appendages may have some prop-erties particular to embryonic tissue. The regener-ation territories of the adult newt are believed tocorrespond to the embryonic ®elds that are at theorigin of the formation of the body structure(Muneoka & Bryant, 1984). Therefore, sustainedexpression of genes of development, like Emx-2, inthe adult newt may be an interesting possibility forexplaining a predisposition for regeneration. Forexample, Emx-2 could provide the epidermis of theadult newt with developmental properties, such asthe ability to form the apical ectodermal cap.Emx-2 could also provide positional informationalong the proximal-distal axis of the limb, to regu-late the regeneration only of the missing structures.

Emx-2 is expressed in the limbs and tail of thenewt and not in the ¯ank; the restricted expressionto regeneration territories suggests Emx-2 may berelated to the regeneration potential. Analysis of itsexpression at the cellular level may permit delinea-tion of the precise frontiers of the regeneration ter-ritories. It would also be interesting to investigate,in vertebrates that lose their regeneration potentialduring development (Dent, 1962), the time courseof loss of Emx-2 expression with the loss of regen-erative ability, and possibly even to maintainexpression in the adult limb through appropriatemanipulations (Brockes, 1992; Lourenssen &Carlone, 1993; Schilthuis et al., 1993; Burns et al.,1994; Pecorino et al., 1994). Emx-2 expression inchick limb mutants, like limbless (no AER), diplop-dia-5, and talpid2 (which have a thickened AER),could also be helpful in understanding the functionof the gene during limb development.

Emx-2 and positional information

It is interesting to relate the graded expressionseen in both the forelimb and the hindlimb to theconcept of positional information introduced byLewis Wolpert (Wolpert, 1969, 1989). One particu-larity of this model is that positional information intwo different systems could be set with the samemechanism even though the developmental out-come of each system (dependent on cell history) isdifferent. For example, Crawford & Stocum (1988)obtained evidence that position-speci®c properties(such as cellular adhesiveness) of the blastema arehomologous (or identical) in forelimb and hind-limb of the axololt. The graded expression of Emx-2in both hindlimb and forelimb may be anotherexample of such a mechanism.

Other homeobox containing genes are expressedin the appendages of the adult newt: two distal-


less-like transcripts (NvHBox-4 and NvHBox-5) arefound constitutively and ubiquitously in newt skin(Beauchemin & Savard, 1992); FH-1 is also foundin the skin, but only in the forelimbs and not thehindlimb (Tabin, 1989); Hox C6, Hox A11, Hox A2,Hox A3, Hox-C10 and Msx-1 are present in themesenchymal and skin fractions of limb, tail, andtheir corresponding blastemas (Savard et al., 1988;Beauchemin & Savard, 1993; Simon & Tabin, 1993;Beauchemin et al., 1994; Crews et al., 1995). Twoother homeobox genes (Hox D10 and Hox D11) areexpressed only in the mesenchymal fraction of theregeneration blastema (Brown & Brockes, 1991;Simon & Tabin, 1993). It is interesting that HoxA11, Hox C6, Hox D10, and Hox D11 show a higherlevel of expression in more proximal regions of thelimb, whereas Emx-2 shows an opposite gradient,meaning that the epidermal and mesenchymalcompartments of the limb blastema are expressingdifferent combinations of homeobox containinggenes, depending on their location in the limb.This, we suspect, could be related to the identi®-cation of cell position in the appendage. Homeoboxcontaining genes are suspected to play importantfunctions in the patterning of vertebrate limbs, andcraniofacial structures by providing an orderedmolecular system of positional values (IzpisuÂa-Belmonte et al., 1991; Kessel & Gruss, 1991; Nohnoet al., 1991; Kessel, 1992; Marshall et al., 1993).Homeobox containing gene expression in the adultnewt limb, and its regeneration blastema, suggeststhat the combination of genes used during limbdevelopment may still be active or reactivatedduring regeneration in the adult newt. Because thecovering epidermis appears to provide only a per-missive in¯uence on blastema development(Stocum & Dearlove, 1972; Tassava & Mescher,1975), it is unlikely that a homeobox combinationfound in the epidermis would be instructive to theregeneration blastema. In turn, the identity of theunderlying mesenchyme might induce the over-lying epidermis to acquire expression of speci®chomeobox genes just as it can induce speci®c ecto-dermal derived structures (Hamburgh, 1970;Saunders, 1980). Therefore, signaling frommesenchyme to epidermis may induce ectodermalphenotypes that are important in positional speci®-cation.

The hypothesis that Emx-2 level of expression isrelated to cell position in the limb, and thus partici-pates in blastema determination, can be certi®edby changing the blastema determination and ana-lyzing whether Emx-2 expression is respeci®ed to avalue characteristic of the new blastemal fate. Ithas been possible to proximalize both the blaste-mal fate and the level of expression of Emx-2 usingRA treatment. A similar type of proximal regu-lation in RA treated blastema was observed withHox D10 and Hox A13 (Simon & Tabin, 1993;Gardiner et al., 1995). Knowing the important roleof homeobox-containing genes in the determi-nation of positional information during develop-ment, it is conceivable that homeobox-containing

genes would act by specifying position along thelimb to ensure that only portions distal to theamputation plan are regenerated. Indeed, it wasproposed that the wound epidermis whichmigrates over the amputation surface serves as thedistal boundary during regeneration (Maden, 1977;Repesh & Oberbriller, 1978; Stocum, 1984).

RA receptors (RARs) are strong candidates to bethe initial targets in altering positional memory ofthe blastema. The results of Pecorino et al. (1996a)support this hypothesis since the activation of theRAR d2 isoform speci®cally mediates cell proxima-lization. On the other hand, the four isoforms ofRAR analyzed to date do not shown markeddifferences in expression either along the proximal-distal axis of intact limbs or between proximal anddistal blastemas (GigueÁre et al., 1989; Ragsdale et al.,1989, 1992a,b; Ragsdale & Brockes, 1991; Hill et al.,1993).

Therefore, we do not exclude the participation ofsecondary factors that would interact differentlywith the RAR pathways. The expression of tran-scription factors, like homeobox-containing genes,could be candidate for such a mechanism.

Materials and Methods

Animals and treatment

Adult N. viridescens were purchased from C. D.Sullivan Co. Inc. (Nashville, Tennessee). Anesthesia andsurgical procedures were as reported (Savard et al.,1988). Blastemas were harvested at the mid-bud stage.When needed, blastema epidermis was mechanically dis-sected of the mesenchyme in a calcium and magnesiumfree PBS buffer (20 minutes of incubation). Proximaliza-tion of distal blastemas was done ®ve days postamputa-tion with a single intraperitoneal injection of 20 ml of asolution of DMSO-RA (15 mg/ml). Control animalswere injected with DMSO. The blastemas were collectedseven days after treatment.

Cloning, PCR-RACE, and sequencing

The p027 clone was isolated by screening a newtcDNA library with a redundant oligonucleotide recog-nizing a highly conserved region of the homeoboxsequence. Screening was as described (Beauchemin &Savard, 1992). cDNA extensions in the 50 region of thegene were cloned by PCR-RACE (Frohman et al., 1988;Frohman, 1990; Jain et al., 1992). Brie¯y, 1 mg of forelimbpoly(A)�RNA was oligo(dT) primed to synthesizecDNA; the mix was subsequently ®ltrated on a Centri-con 100 spun-column, and poly(A)-tailed with 17 unitsof T4 deoxynucleotidyl transferase (Pharmacia) for 15minutes at 37�C, in 200 ml of DNA-tailing buffer (0.1 Mpotassium-cacodylate (pH 7.2), 2 mM cobalt chloride,0.2 mM dithiothreitol). A ®rst series (30 rounds) of PCRampli®cation was done with 10 ml of the previousreaction and 150 ng of both oligonucleotides poly(T)(50-AAGGATCCGTCGACAT-CGATAATACGAC(T)17-30),and PUY-40 (50-CTATTCGCCACCTGTTGA-30; position341 to 358 of the reported sequence in Figure 2) in 50 ml;of PCR reaction buffer (67 mM Tris (pH 8.8), 6.7 mMmagnesium chloride, 170 mg/ml bovine serum albumin,17 mM ammonium sulfate, 1.5 mM deoxynucleotide


triphosphate, and 10% DMSO) containing 2.5 units TaqDNA polymerase (Pharmacia). The PCR programfollowed these conditions: a hot start (95�C for sevenminutes, Taq addition, 75�C for two minutes, 45�C fortwo minutes, and 72�C for 40 minutes), and 30 regularcycles (94�C for 45 seconds, 50�C for 25 seconds, 72�Cfor three minutes with an extension cycle of ®veseconds). A second series (30 rounds) of PCR ampli®ca-tion was done with 1 ml of the ®rst ampli®cation and150 ng of both oligonucleotides Ri (50-GACATCGATAA-TACGAC-30; poly(T)-complementary sequence) andPUY-41 (50-GTGCCCTTCTTCTTCTGC-30; position 315 to336 of the reported sequence in Figure 2). The ®nalproducts of ampli®cation were cloned in the PCR-II vec-tor (TA-cloning kit, In Vitrogen Corporation). Positiveclones were detected by colony hybridization using anEmx-2 speci®c oligonucleotide located in the homeobox.Nucleotide sequence determination was performed bythe method of Sanger, using the kits and methodsprovided by United States Biochemical Corporation andPharmacia.

RNA isolation, Northern blots andin situ hybridization

RNA was extracted as described by Chomczynski &Sacchi (1987). Poly(A)�RNA was puri®ed on anoligo(dT)-cellulose column following the procedureprovided by the supplier (Boehringer Mannheim). About5 mg of poly(A)�RNA (evaluated with the optical densityat 260 nm) was run onto an agarose-formaldehyde gel(Maniatis et al., 1982) and transferred onto Genescreenmembranes according to the supplier (New EnglandNuclear). The hybridization procedure and normalizingprobes were as previously described (Beauchemin &Savard, 1992). Autoradiograms were scanned with animage analyzer RAS-1000 (Amersham) to evaluate bandintensity.

For in situ hybridization, regenerating limbs were fro-zen in Optimal Cutting Temperature compound (OCT),sectioned and processed as described (Del Rio-Tsoniset al., 1995). Brie¯y, sections were ®xed in 4% parafor-maldehyde, rinsed in PBS, treated with triethanolamine,washed with 2� sodium citrate buffer (SSC), and dehy-drated by ethanol series (50% to 100%). Slides wereallowed to air dry and were then incubated overnightat 55�C with either the sense or antisense 35S-labelledEmx-2 probes. The next day, the slides were rinsed in 2�SSC, then washed in a solution containing 50% forma-mide, 1� SSC, 0.1% b-mercaptoethanol at 52�C followedby RNase treatment, and ®nally washed with 0.1� SSCand 0.1% b-mercaptoethanol. Sections were dehydratedand air dried so that they could be dipped in photo-graphic emulsion and exposed for two weeks. Slideswere counterstained with either hematoxylin-eosin orvan Gieson's stain.

Acknowledgments

We thank Ms Jean Daly for helping in the preparationof the manuscript. M.B. had a studentship from theGeorges PheÂnix Foundation and P.S. had a scholarshipfrom the Medical Research Council of Canada (MRC)and the ``Fonds de la Recherche en SanteÂ du QueÂbec.This work was funded by the MRC (MT-10664).

References

Beauchemin, M. & Savard, P. (1992). Two Distal-lessrelated homeobox-containing genes expressed inregeneration blastemas of the newt. Dev. Biol. 154,55±65.

Beauchemin, M. & Savard, P. (1993). Expression of ®vehomeobox genes in the adult newt appendages andregeneration blastemas. In Limb Development andRegeneration (Fallon, J. F., Goetinck, P. F., Kelly,R. O. & Stocum, D. L., eds), pp. 41±50, Wiley-Liss,Inc., USA.

Beauchemin, M., Noiseux, N., Tremblay, M. & Savard,P. (1994). Expression of Hox A11 in the limb andthe regeneration blastema of adult newt. Int. J. Dev.Biol. 38, 641±649.

Brockes, J. P. (1992). Introduction of a retinoid reportergene in urodele limb blastema. Proc. Natl Acad. Sci.USA, 89, 11386±11390.

Brockes, J. P. (1996). Retinoid signalling and retinoidreceptors in amphibian limb regeneration. Biochem.Soc. Symp. 62, 137±142.

Brockes, J. P. (1997). Amphibian limb regeneration:rebuilding a complex structure. Science, 276, 81±87.

Brown, R. & Brockes, J. P. (1991). Identi®cation andexpression of a regeneration-speci®c homeoboxgene in the newt limb blastema. Development, 111,489±496.

Burns, J. C., Matsubara, T., Lozinski, G., Yee, J.-K.,Friedman, T., Washabaugh, C. H. & Tsonis, P. A.(1994). Pantropic retroviral vector-mediated genetransfer, integration, and expression in culturednewt limb cells. Dev. Biol. 165, 285±289.

Chomczynski, P. & Sacchi, N. (1987). Single-step methodof RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162,156±159.

Crawford, K. & Stocum, D. L. (1988). Retinoic acidcoordinately proximalizes regenerate pattern andblastema differential af®nity in axolotl limbs. Devel-opment, 102, 687±698.

Crews, L., Gates, P. B., Brown, R., Joliot, A., Foley, C.,Brockes, J. P. & Gann, A. A. (1995). Expression andactivity of the newt Msx-1 gene in relation to limbregeneration. Proc. Roy. Soc. Lond. 259, 161±171.

Dalton, D., Chadwick, R. & McGinnis, W. (1989).Expression and embryonic function of emptyspaces: a Drosophila homeobox gene with twopatterning functions on the anterior-posterior axisof the embryo. Genes Dev. 3, 1940±1956.

Davies, C. A., Holmyard, D. P., Millen, K. J. & Joyner,A. L. (1991). Examining pattern formation inmouse, chicken and frog embryos with an En-speci®c antiserum. Development, 111, 287±298.

Del, Rio-Tsonis K., Washabaugh, C. H. & Tsonis, P. A.(1995). Expression of pax-6 during urodele eyedevelopment and lens regeneration. Proc. Natl Acad.Sci. USA, 92, 5092±5096.

Dent, J. N. (1962). Limb regeneration in larvae andmetamorphosing individuals of the South Africanclawed toad. J. Morphol. 110, 61±78.

DolleÂ, P., IzpisuÂa-Belmonte, J.-C., Falkenstein, H.,Renucci, A. & Duboule, D. (1989). Coordinateexpression of the murine Hox-5 complex homeo-box-containing genes during limb pattern for-mation. Nature, 342, 767±772.

Ferretti, P. & Brockes, J. P. (1991). Cell origin and iden-tity in limb regeneration and development. GLIA, 4,214±224.


Ferretti, P., Brockes, J. P. & Brown, R. (1991). A newttype II keratin restricted to normal and regeneratinglimbs and tails is responsive to retinoic acid. Devel-opment, 111, 497±507.

Frohman, M. A. (1990). RACE: Rapid ampli®cation ofcDNA ends. In PCR Protocols (Innis, M. A., Gelfand,D. H., Snincky, J. J. & White, T. J., eds), pp. 28±38,Academic Press, San Diego, CA.

Frohman, M. A., Dush, M. K. & Martin, G. R. (1988).Rapid production of full-length cDNAs from raretranscripts: ampli®cation using a single gene-speci®c oligonucleotide primer. Proc. Natl Acad. Sci.USA, 85, 8998±9002.

Gann, A. A. F., Gates, P. B., Stark, D. & Brockes, J. P.(1996). Receptor isoform speci®city in a cellularresponse to retinoic acid. Proc. Roy. Soc. London Ser.B, 263, 729±734.

Gardiner, D. M., Blumberg, B., Komine, Y. & Bryant,S. V. (1995). Regulation of HoxA expression indeveloping and regenerating and axolotl limbs.Development, 121, 1731±1741.

GigueÁre, V., Ong, E. S., Evans, R. M. & Tabin, C. J.(1989). Spatial and temporal expression of the reti-noic acid receptor in the regenerating amphibianlimb. Nature, 337, 566±569.

GuyeÂnot, E., Dinichert-Favarger, J. & Galland, M. (1948).L'exploration du territoire de la patte anteÂrieure duTriton. Rev. Suisse Zool. 55, 1±120.

Hamburgh, M. (1970). Theories of Differentiation, Elsevier,New York.

Hill, D. S., Ragsdale, C. W., Jr & Brockes, J. P. (1993).Isoform-speci®c immunological detection of newtretinoic acid receptor d1 in normal and regeneratinglimbs. Development, 117, 937±945.

IzpisuÂa-Belmonte, J.-C., Tickle, C., DolleÂ, P., Wolpert, L.& Duboule, D. (1991). Expression of the homeoboxHox-4 genes and the speci®cation of position inchick wing development. Nature, 350, 585±589.

IzpisuÂa-Belmonte, J.-C., Ede, D. A., Tickle, C. &Duboule, D. (1992). The mis-expression of posteriorHox-4 genes in talpid (ta3) mutant wings correlateswith the absence of anteroposterior polarity. Devel-opment, 114, 959±963.

Jain, R., Gomer, R. H. & Murtagh, J. J. (1992). Increasingspeci®city from the PCR-RACE technique. Biotechni-ques, 12, 58±59.

Kessel, M. (1992). Respeci®cation of vertebral identitiesby retinoic acid. Development, 115, 487±501.

Kessel, M. & Gruss, P. (1991). Homeotic transformationsof murine vertebrae and concomitant alteration ofHox codes induced by retinoic acid. Cell, 67, 89±104.

Kiortsis, V. (1953). PotentialiteÂs du territoire patte chezle Triton. Rev. Suisse Zool. 60, 301±410.

Lo, D. C., Allen, F. & Brockes, J. P. (1993). Reversal ofmuscle differentiation during urodele limb regener-ation. Proc. Natl Acad. Sci. USA, 90, 7230±7234.

Lourenssen, S. & Carlone, R. (1993). Transfection ofadult newt limb blastema cells in vivo: increasedef®ciency with direct injection of plasmid DNA. InLimb Development and Regeneration (Fallon, J. F.,Goetinck, P. F., Kelly, R. O. & Stocum, D. L., eds),pp. 193±201, Wiley-Liss, Inc., USA.

Maden, M. (1977). The regeneration of positional infor-mation in the amphibian limb. J. Theoret. Biol. 69,735±753.

Maden, M. (1982). Vitamin A and pattern formation inthe regenerating limb. Nature, 295, 672±675.

Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982). In Mol-ecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY.

Marshall, H., Nonchev, S., Sham, M. H., Muchamore, I.,Lumsden, A. & Krumlauf, R. (1993). Retinoic acidalters hindbrain Hox code and induces transform-ation of rhombomeres 2/3 into a 4/5 identity.Nature, 360, 737±741.

Mathisen, P. M. & Miller, L. (1987). Thyroid hormoneinduction of keratin genes: a two-step activation ofgene expression during development. Genes Dev. 1,1107±1117.

McGinnis, W. & Krumlauf, R. (1992). Homeobox genesand axial patterning. Cell 68, 283±302.

Miyamoto, N., Yoshida, M., Kuratani, S., Matsuo, I. &Aizawa, S. (1997). Defects of urogenital develop-ment in mice lacking Emx2. Development, 124,1653±1664.

Mohun, T. J., Brennan, S., Dathan, N., Fairman, S. &Gurdon, J. B. (1984). Cell-type-speci®c activation ofactin genes in the early amphibian embryo. Nature,311, 716±721.

Muneoka, K. & Bryant, S. V. (1984). Cellular contri-bution to supernumerary limbs resulting from theinteraction between developing and regeneratingtissues in the axololt. Dev. Biol. 105, 179±187.

Muneoka, K. & Sassoon, D. (1992). Molecular aspects ofregeneration in developing vertebrate limbs. Dev.Biol. 152, 37±49.

Niazi, A. A. & Saxena, S. (1978). Abnormal hindlimbregeneration in tadpoles of the toad, Bufo ander-soni, exposed to excess vitamin A. Folia Biol.(Krakow), 26, 3±11.

Nohno, T., Noji, S., Koyama, E., Ohyama, K., Myokai,F., Kuroiwa, A., Saito, T. & Taniguchi, S. (1991).Involvement of the Chox-4 chicken homeoboxgenes in determination of anteroposterior axialpolarity during limb development. Cell, 64, 1197±1205.

Oliver, G., Sidell, N., Fiske, W., Heinzmann, C.,Mohandas, T., Sparkes, R. S. & De Robertis, E. M.(1989). Complementary homeo protein gradients indeveloping limb buds. Genes Dev. 3, 641±650.

Pecorino, L. T., Lo, D. C. & Brockes, J. P. (1994). Iso-form-speci®c induction of a retinoid-responsiveantigen after biolistic transfection of chimaeric reti-noic acid/thyroid hormone receptors into a regener-ating limb. Development, 120, 325±333.

Pecorino, L. T., Entwistle, A. & Brockes, J. P. (1996a).Activation of a single retinoic acid receptor isoformmediates proximodistal respeci®cation. Curr. Biol. 6,563±569.

Pecorino, L. T., Brockes, J. P. & Entwistle, A. (1996b).Semi-automated positional analysis using laserscanning microscopy of cells transfected in aregenerating newt limb. J. Histochem. Cytochem. 44,559±569.

Peterson, R. L., Papenbrock, T., Davda, M. M. &Awgulewitsch, A. (1994). The murine HoxC clustercontains ®ve neighboring AbdB-related Hox genesthat show unique spatially coordinated expressionin posterior embryonic subregions. Mech. Dev. 47,253±260.

Ragsdale, C. W. & Brockes, J. P. (1991). Retinoic recep-tors and vertebrate limb morphogenesis. In NuclearHormone Receptors (Parker, M. G., ed.), pp. 269±295,Academic Press, London.

Ragsdale, C. W., Petkovich, M., Gates, P. B., Chambon,P. & Brockes, J. P. (1989). Identi®cation of a novel


retinoic acid receptor in regenerative tissues of thenewt. Nature, 341, 654±657.

Ragsdale, C. W., Gates, P. B. & Brockes, J. P. (1992a).Identi®cation and expression pattern of a secondisoform of the newt alpha retinoic acid receptor.Nucl. Acid Res. 20, 5851.

Ragsdale, C. W., Gates, P. B., Hill, D. S. & Brockes, J. P.(1992b). Delta retinoic acid receptor isoform d1 isdistinguished by its exceptional N-terminalsequence and abundance in the limb regenerationblastema. Mech. Dev. 40, 99±112.

Repesh, L. A. & Oberbriller, J. L. (1978). Scanning elec-tron microscopy of epidermal cell migration inwound healing during limb regeneration in theadult newt, Notophthalmus viridescens. Am. J. Anat.151, 539±556.

Ros, M. A., Lyons, G., Kosher, R. A., Upholt, W. B.,Coelho, C. N. D. & Fallon, J. F. (1992). Apical ridgedependent and independent mesodermal domainsof GHox-7 and GHox-8 expression in chick limbbuds. Development, 116, 811±818.

Saunders, J. W., Jr (1980). Developmental Biology, Macmil-lan, New York.

Savard, P. & Tremblay, M. (1995). Differential regulationof Hox C6 in the appendages of adult urodeles andanurans. J. Mol. Biol. 249, 879±889.

Savard, P., Gates, P. B. & Brockes, J. P. (1988). Positiondependent expression of a homeobox gene tran-script in relation to amphibian limb regeneration.EMBO J. 7, 4275±4282.

Schilthuis, J. G., Gann, A. A. F. & Brockes, J. P. (1993).Chimeric retinoic acid-thyroid hormone receptorsimplicate RAR-a1 as mediating growth inhibitionby retinoic acid. EMBO J. 12, 3459±3466.

Scott, M. P., Tamkun, J. W. & Hartzell, G. W., III (1989).The structure and function of the homeodomain.Biochem. Biophys. Acta, 989, 25±48.

Simeone, A., Gulisano, M., Acampora, D., Stornaiuolo,A., Rambaldi, M. & Boncinelli, E. (1992a). Two ver-tebrate homeobox genes related to the Drosophilaempty spiracles gene are expressed in the embryo-nic cerebral cortex. EMBO J. 11, 2541±2550.

Simeone, A., Acampora, D., Gulisano, M., Stornaiuolo,A. & Boncinelli, E. (1992b). Nested expressiondomains of four homeobox genes in developing ros-tral brain. Nature, 358, 687±690.

Simon, H.-G. & Tabin, C. J. (1993). Analysis of Hox-4.5and Hox-3.6 expression during newt limb regener-ation: differential regulation of paralogous Hoxgenes suggest different roles for members of differ-ent Hox clusters. Development, 117, 1397±1407.

Stocum, D. L. (1984). The urodele limb regenerationblastema: determination and organization of themorphogenetic ®eld. Differentiation, 27, 13±28.

Stocum, D. L. & Dearlove, G. E. (1972). Epidermal-meso-dermal interaction during morphogenesis of thelimb regeneration blastemas in larval salamanders.J. Exp. Zool. 181, 49±61.

Tabin, C. J. (1989). Isolation of potential vertebrate limb-identity genes. Development, 105, 813±820.

Tanaka, E. M., Gann, A. A., Gates, P. B. & Brockes, J. P.(1997). Newt myotubes reenter the cell cycle byphosphorylation of the retinoblastoma protein.J. Cell. Biol. 136, 155±165.

Tassava, R. A. & Mescher, A. L. (1975). The roles ofinjury, nerves and the wound epidermis during theinitiation of amphibian limb regeneration. Differen-tiation, 4, 23±24.

Tsonis, P. A. (1996). Limb Regeneration, CambridgeUniversity Press, Cambridge, UK.

Wallace, H. (1981). Vertebrate Limb Regeneration, JohnWiley & Sons, Chichester.

Wolpert, L. (1969). Positional information and the spatialpattern of cellular differentiation. J. Theoret. Biol. 25,1±47.

Wolpert, L. (1989). Positional information revisited.Development, 109(suppl.), 3±12.

Yokouchi, Y., Sasaki, H. & Kuroiwa, A. (1991). Homeo-box gene expression correlated with the bifurcationof limb cartilage development. Nature, 353, 443±445.

Yoshida, M., Suda, Y., Matsuo, I., Miyamoto, N.,Takeda, N., Kuratani, S. & Aizawa, S. (1997). Emx1and Emx2 functions in development of dorsaltelencephalon. Development, 124, 101±111.

Edited by J. Karn

(Received 24 November 1997; received in revised form 9 March 1998; accepted 16 March 1998)

graded expression of emx-2 in the adult newt limb and its corresponding regeneration blastema

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