DEVELOPMENT AND STEM CELLS RESEARCH REPORT 513

Development 140, 513-518 (2013) doi:10.1242/dev.081752© 2013. Published by The Company of Biologists Ltd

INTRODUCTIONLimb development and regeneration involve the coordinated growthand patterning of several tissues, including bone, soft connectivetissue, muscle, epidermis, peripheral nerve and blood vessels. Duringlimb development, lateral plate mesoderm-derived cells can formrecognisable limb segments in the absence of muscle formation andit has been proposed that they are a dominant cell type directing limbpatterning (Christ et al., 1977; Grim and Wachtler, 1991; Kardon etal., 2002; Kardon et al., 2003; Hasson et al., 2010; Mathew et al.,2011). Molecular factors that influence the formation of the threedistinct limb segments (upper arm, lower arm and hand), such asMEIS and HOX genes, are expressed in multiple limb progenitor celltypes, including cells derived from lateral plate mesoderm, as wellas myogenic cells derived from somitic mesoderm (Yamamoto et al.,1998; Hashimoto et al., 1999; Mercader et al., 1999; Cooper et al.,2011; Roselló-Díez et al., 2011). These factors probably exert theirpatterning influence via expression in the lateral plate mesodermderivatives. Their expression in myogenic cells has been linked tomyogenic differentiation, although an influence on muscle patterninghas not been excluded (Knoepfler et al., 1999; Yamamoto andKuroiwa, 2003; Heidt et al., 2007).

Salamander limb regeneration provides another context in whichto study specification of proximo-distal limb identity. Upon limbamputation anywhere along the limb axis, only the missing portionof the limb distal to the amputation plane regenerates. Furthermore,when a hand blastema was transplanted to an upper arm stump, anormal limb regenerated. Examination of melanocytes or labelled

cartilage nuclei indicated that the hand blastema cells contributedonly to the hand, and the lower arm was formed from the upperarm stump cells through a phenomenon called intercalation(Stocum, 1975; Maden, 1980; Pescitelli and Stocum, 1980). Thisand other transplantation experiments showed that, as a whole, alimb blastema is autonomously specified to form the limbstructures distal to its site of origin (for review, see Nacu andTanaka, 2011). This is termed the rule of distal transformation.

The limb blastema is also composed of lineage-restrictedprogenitors (Kragl et al., 2009). Therefore, it is important to knowwhich cells obey the rule of distal transformation and thusdetermine the proximo-distal outcome of regeneration, a crucialaspect of regenerating a properly patterned limb. Kragl andcolleagues (Kragl et al., 2009) showed that upper limb blastemacells show heterogeneity in the nuclear expression of transcriptionfactors associated with limb patterning. Cartilage-derived andmuscle-derived blastema cells expressed MEIS, HOXA9 andHOXA13, as found for limb development, but Schwann cells didnot. Interestingly, the cellular behaviour of cartilage-derived cellsversus Schwann cells correlated with expression of these factors:cartilage-derived cells obeyed the rule of distal transformation,whereas Schwann cells did not. In addition to understanding whichcells obey the rule of distal transformation, it is also important todetermine the molecular mechanisms implemented in patterningduring limb regeneration, and to what extent they are common tothose used during development.

Here, we investigate the role of connective tissue (CT) cells,which derive from lateral plate mesoderm, and muscle cells inpatterning during limb regeneration by asking whether they obeythe rule of distal transformation. We find that CT-derived blastemacells display nuclear MEIS in upper arm, but not lower arm or handblastemas. At a cellular level, CT-derived blastema cells obey therule of distal transformation in the intercalation assay. By contrast,nuclear MEIS signal is not restricted to upper arm blastemas inmyogenic blastema cells. At a cellular level, myogenic cells break

1Center for Regenerative Therapies Dresden, Fetscherstraße 105, 01307, Dresden,Germany. 2Max Planck Institute of Molecular Cell Biology and Genetics,Pfotenhauerstraße 108, 01307, Dresden, Germany.

*Authors for correspondence (eugeniu.nacu@crt-dresden.de; elly.tanaka@crt-dresden.de)

Accepted 23 November 2012

SUMMARYDuring salamander limb regeneration, only the structures distal to the amputation plane are regenerated, a property known as therule of distal transformation. Multiple cell types are involved in limb regeneration; therefore, determining which cell typesparticipate in distal transformation is important for understanding how the proximo-distal outcome of regeneration is achieved.We show that connective tissue-derived blastema cells obey the rule of distal transformation. They also have nuclear MEIS, whichcan act as an upper arm identity regulator, only upon upper arm amputation. By contrast, myogenic cells do not obey the rule ofdistal transformation and display nuclear MEIS upon amputation at any proximo-distal level. These results indicate that connectivetissue cells, but not myogenic cells, are involved in establishing the proximo-distal outcome of regeneration and are likely to guidemuscle patterning. Moreover, we show that, similarly to limb development, muscle patterning in regeneration is influenced by b-catenin signalling.

KEY WORDS: Limb, Regeneration, Proximo-distal, Muscle, Connective tissue, MEIS

Connective tissue cells, but not muscle cells, are involved inestablishing the proximo-distal outcome of limbregeneration in the axolotlEugen Nacu1,2,*, Mareen Glausch1, Huy Quang Le1, Febriyani Fiain Rochel Damanik1, Maritta Schuez1, Dunja Knapp1,2, Shahryar Khattak1,2, Tobias Richter2 and Elly M. Tanaka1,2,*

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the rule of distal transformation. These data indicate that, similarlyto limb development, lateral plate mesoderm-derived cells probablyplay a dominant role in patterning of distal structures, whereasmyogenic cells follow the patterning cues of lateral platemesoderm-derived cells. We highlight an additional parallel to limbdevelopment by showing that b-catenin activity influences musclepatterning.

MATERIALS AND METHODSLabelling of cell typesAmbystoma mexicanum (axolotl) expressing GFP in connective tissue cells(GFP-CT) or muscle cells (GFP-M) were generated by embryonictransplantation as previously described (Kragl et al., 2009; Nacu et al.,2009) (supplementary material Fig. S1).

TransgenicsSceI-CarAct:GFP plasmid (kind gift of Hajime Ogino, Nara Institute ofScience and Technology, Japan) was injected into axolotl eggs aspreviously described (Khattak et al., 2009).

Adult surgeryAll animal procedures were carried out in accordance with the laws andregulations of the State of Saxony. Animals were anesthetised in 0.03%benzocaine (Sigma) prior to surgery. Intercalation assays were performedas previously described (Maden, 1980). Blastemas, 7 or 10 days postamputation (dpa), were transplanted on animals 2.5-3.0 cm and 3.0-4.0 cmin length (snout to cloaca), respectively. Blastemas were transplanted ontohosts amputated at mid-upper arm or shoulder. Animals were covered bybenzocaine-soaked tissues for 1 hour and afterwards transferred into tapwater. Only animals that regenerated distinct upper arm, lower arm andhand were analysed.

Sample collection and immunohistochemistrySamples were fixed in MEMFA, embedded in Tissue-tek (O.C.T. compound,Sakura) or 7.5% gelatine (Sigma), sectioned at 10 µm thickness and stainedwith antibodies (supplementary material Table S1) as previously described(Mchedlishvili et al., 2007; Mchedlishvili et al., 2012).

For MEIS/PAX7 double staining, the slides were first incubated withanti-MEIS, then with Fab goat anti-mouse, and finally with Alexa647-conjugated Fab rabbit anti-goat. Slides were then blocked with 20% goatserum in PBS and then incubated with anti-PAX7, followed by Cy3-conjugated donkey anti-rabbit.

b-Catenin electroporationUpper arm blastemas (6 dpa) were injected with 2.5 mg/ml pCS2+ plasmidexpressing a constitutively active form of b-catenin (kind gift of GilbertWeidinger, Universität Ulm, Germany) or 2.5 mg/ml CAGGS:GFP ascontrol. CAGGS:mCherry was added to each mix (final concentration 0.25mg/ml). Limbs were electroporated at 300 V/cm, 50 ms pulse length, 5pulses on a BTX-ECM-830 electroporator.

Whole-mount staining of electroporated limbsFixed limbs were stained with mouse anti-MHC antibody, then Cy3-coupled Fab goat anti-mouse and cleared as previously described (Ertürket al., 2012).

Imaging and image processingWhole-limb images were acquired on Olympus SZX-16. Whole-mountstainings and limb sections were imaged on Zeiss AxioImagerZ.2LSM780, Zeiss AxioObserverZ.1 or Olympus-BX61VS. Images wereprocessed with Fiji software for better visualisation and Volocity software(PerkinElmer) for 3D reconstruction.

RESULTS AND DISCUSSIONConnective tissue cells obey the rule of distaltransformationIn this study, we were interested in elucidating whether myogenicand connective tissue (CT)-derived blastema cells, both expressing

MEIS and HOXA13, obey the rule of distal transformation, whichis linked to the proximo-distal outcome of regeneration. Indevelopment, lateral plate mesoderm-derived cells are sufficient forforming a properly patterned limb (Christ et al., 1977; Grim andWachtler, 1991). Therefore, we postulated that during regenerationCT cells, which are derived from lateral plate mesoderm, obey therule of distal transformation.

To test this hypothesis, we first investigated whether MEIS,which can act as an upper arm identity regulator (Capdevila et al.,1999; Mercader et al., 1999; Mercader et al., 2005), is present innuclei of CT cells only upon upper arm amputation. We generated,by embryonic transplantation, animals in which the vast majorityof CT cells constitutively express GFP (GFP-CT) (supplementarymaterial Fig. S1). In 10 dpa upper arm blastemas of GFP-CT limbs,we identified that 37-62% of GFP+ cells have nuclear MEIS, incontrast to 0.6% of GFP+ cells having nuclear MEIS+ in 10 dpalower arm blastemas (Fig. 1A-F,M-Q; supplementary materialTable S2). These results suggested that connective tissue cellscould indeed obey the rule of distal transformation duringregeneration.

To address the question at a cellular level, we set up theintercalation assay as previously described: a wrist blastema wastransplanted onto an upper arm stump (Fig. 2A,C,E). If cells fromthe grafted blastema end up more proximally of their origin, thosecells break the rule of distal transformation. When wrist blastemasfrom GFP-CT animals were transplanted onto upper arm stumps,the vast majority of GFP+ cells homed to the hand (Fig. 2C,D;supplementary material Fig. S2). In control experiments of upperarm GFP-CT blastema transplantations, GFP+ cells were found inupper arm, lower arm and hand (Fig. 2E,F; Table 1). These resultsshow that CT-derived blastema cells obey the rule of distaltransformation.

Myogenic cells break the rule of distaltransformationNext, we investigated whether myogenic blastema cells displaynuclear MEIS signal only upon upper arm amputation by usinganimals in which myogenic somitic mesoderm derivativesconstitutively express GFP (GFP-M) (supplementary material Fig.S1). We found that, in contrast to CT cells, myogenic blastemacells have nuclear MEIS after amputation through any limbsegment: mid-upper arm (Fig. 1R-V), mid-lower arm (Fig. 1G-L),and metacarpals (supplementary material Fig. S3). Specifically, 51-80% of GFP+ myogenic blastema cells have nuclear MEIS in 10dpa upper arm blastemas, and 23-86% in 10 dpa lower armblastemas (supplementary material Table S2). Because myogenicblastema cells express PAX7 (supplementary material Table S2)(Kragl et al., 2009), we explored whether PAX7+ myogenicprogenitors have nuclear MEIS. Double staining of MEIS andPAX7 revealed that 36.8-91.4% of PAX7+ cells in the blastema areMEIS+ (supplementary material Fig. S4, Table S3). These resultssuggested that during regeneration, myogenic blastema cells havea different proximo-distal determination system from connectivetissues.

To follow muscle cells in the intercalation assay, we transplantedwrist blastemas from germline CarAct:GFP transgenic animals,which express GFP in muscle fibres, onto upper arm stumps(Mohun et al., 1986). Upon regeneration, GFP+ muscle fibres wereobserved in the upper arm, lower arm and hand, indicating thatmyogenic blastema cells break the rule of distal transformation(Fig. 2A,B; Table 1). GFP+ muscle fibres were observed in allmuscles of the upper arm. The humeroantebrachialis and

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anconaeus humeralis medialis, which originate in the humerus(Walthall and Ashley-Ross, 2006), had the highest abundance ofGFP+ muscle fibres (supplementary material Fig. S5, Tables S4,S5).

Muscle fibres form by fusion of precursor cells; thus, theobserved proximalisation could be the result of fusion of labelledmyogenic blastema cells in the grafted blastema with pre-existingmuscle fibres in the stump. To account for this, we examinedwhether satellite cells (mononucleate myogenic cells) were alsoproximalised. Upper arms of regenerates that were generated bytransplanting CAGGS:GFP wrist blastemas (in which all cells

express GFP) onto upper arm stumps, were analysed with anantibody against PAX7, which marks satellite cells. PAX7+GFP+cells were present in the upper arms of the regenerates (seven outof seven limbs), indicating that myogenic blastema cells gave riseto proximalised satellite cells in the intercalation assay (Table 1;supplementary material Fig. S5, Table S5). These results show thatmyogenic cells break the rule of distal transformation.

Our results indicate that connective tissue cells, but not myogeniccells, guide the proximo-distal outcome of regeneration. In thisregard, patterning in regeneration might recapitulate development,during which limb bud cells of lateral plate mesoderm origin direct

515RESEARCH REPORTCells in distal transformation

Fig. 1. Connective tissue (CT)-derivedblastema cells have nuclear MEIS onlyupon upper arm amputation, whereasmyogenic blastema cells have nuclearMEIS upon upper and lower armamputation in axolotl. (A-F) In 10 dpalower arm blastemas, CT-derived cells(green) have no nuclear MEIS (red). (B)Enlargement of the boxed area in Ashowing that MEIS+ cells are surroundedby GFP+ cells, but do not colocalise. (C-F)Enlargements of the boxed area in B;yellow arrowheads indicate MEIS+GFP–cells. (G-L) In 10 dpa lower arm blastemas,myogenic cells (green) have nuclear MEIS(red). H is an enlargement of the boxedarea in G. (I-L) Enlargements of the boxedarea in H; white arrowheads indicateMEIS+GFP+ cells. (M-V) In 10 dpa upperarm blastemas, CT-derived cells (M-Q,green) and myogenic cells (R-V, green)show nuclear MEIS (red). (N-Q)Enlargements of the boxed area in M. (S-V)Enlargements of the boxed area in R.White arrowheads indicate MEIS+GFP+cells. (W) Percentage of myogenic or CTcells that are MEIS+ in 10 dpa lower armblastemas. Number of limbs counted:dermis, n=5; muscle, n=5 (supplementarymaterial Table S2). (X) Percentage ofmyogenic or CT-derived cells that areMEIS+ in 10 dpa upper arm blastemas.Number of limbs counted: dermis, n=3;muscle, n=2. Scale bars: in A,G,M,R, 1mm; in B,H, 200 m; in C-F,I-L, 50 m, inN-Q,S-V, 100 m.

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the migration and patterning of naive myogenic progenitors (seeIntroduction). Next, we were interested in uncovering the moleculesthat might play a role in patterning muscle cells.

b-Catenin signalling is involved in musclepatterning during regenerationIn limb development, TCF4 of the WNT/b-catenin pathway isexpressed in early muscle connective fibroblasts and plays a crucialrole in dictating the placement of limb muscle masses (Kardon etal., 2003; Mathew et al., 2011). Kardon and colleagues (Kardon etal., 2003) showed that expression of constitutively active b-cateninin chicken limb buds gives rise to ectopic muscle formation. We

were therefore interested to determine whether b-catenin signallingaffects muscle patterning in axolotl limb regeneration.

We overexpressed constitutively active b-catenin (Yost et al.,1996) in axolotl blastemas using electroporation. Five out of 11limbs electroporated with b-catenin showed ectopic muscleformation in the regenerated upper arm, such as on the dorsal sideof the limb close to the triceps (Fig. 3; supplementary material Fig.S6). Control GFP-electroporated regenerates showed defects inmusculature, as occurs normally during regeneration (Diogo et al.,2013), but we did not observe in any of the ten samples ectopicmuscle formation similar to that seen in b-catenin-electroporatedlimbs.

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Fig. 2. Connective tissue cells, but not myogeniccells, obey the rule of distal transformation inaxolotl limb regeneration. (A) Schema of wristblastema transplantation from an animal thatexpresses GFP in muscle fibres (green) onto a non-transgenic upper arm (UA) stump. (B) Experimentalresult of A: upon regeneration, GFP-muscle fibres(green) are found in the UA, lower arm (LA) and hand.(C,D) Upon wrist blastema transplantation from a GFP-CT animal, in which connective tissue cells expressGFP, onto a non-transgenic UA stump, GFP+connective tissue cells are found only in the hand ofthe regenerate. (E,F) UA blastema transplantationfrom a GFP-CT animal onto a non-transgenic UAstump results in GFP-connective tissues cells in UA, LAand hand of the regenerate. Dashed lines in B,D,Foutline the limb. Scale bars: 2 mm.

Table 1. Muscle fibres and satellite cells, but not connective tissue cells, are found more proximal of their origin when a wristblastema is transplanted onto an upper arm stump

Number of regenerates with GFP+ cells in the limb segments

Cell type followed Donor blastema origin* Total transplantations (n) Upper arm Lower arm Hand

GFP+ muscle fibres‡ Wrist 12 11 12 12Upper arm 10 10 10 10

GFP+ satellite cells§ Wrist 7 7 n.a. n.a.Upper arm 5 5 n.a. n.a.

GFP+ connective tissue cells¶ Wrist 8 1 1 8Upper arm 6 3 6 6

*The donor blastemas were grafted onto non-transgenic upper arm stumps.‡Muscle fibres were followed by transplanting CarAct:GFP blastemas.§Satellite cells were followed by transplanting CAGGS:GFP blastemas.¶Connective tissue cells were followed by transplanting GFP-CT blastemas.n.a., origin of satellite cells in the lower arm or hand was not analysed. D

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ConclusionsWe show through intercalation assays that myogenic cells break therule of distal transformation, whereas connective tissue cells obeyit. These results suggest that, similarly to development, theconnective tissue cells guide muscle patterning during regeneration.Therefore, it will be important in the future to study connectivetissue cells in order to understand how the proximo-distal outcomeof regeneration is established.

We show that myogenic blastema cells break the rule of distaltransformation and display nuclear MEIS, which can functionallyproximalise blastema cells (Mercader et al., 2005). We also foundmyogenic cells expressing HOXA11 and HOXA13 in 10 dpa lowerarm blastemas (data not shown). It is possible that MEIS, HOXA11and HOXA13 play a role in patterning of myogenic cells duringregeneration; however, their expression is regulated by signals thatmyogenic cells receive from connective tissue cells. For example,in chicken, HOXA10 predisposes, in an autonomous way, themyogenic progenitors in the somite to a limb fate (Alvares et al.,2003). In Drosophila, the identity of muscle cells in different larvalsegments is believed to be the result of integration of extrinsic cueswith autonomous expression of HOX homologues (Greig andAkam, 1993; Roy and Vijayraghavan, 1997).

Alternatively, MEIS and HOX genes might play a role notrelated to positional identity in myogenic cells. MEIS, in complexwith PBX, binds DNA cooperatively with MYOD and marks genesfor activation by MYOD (Knoepfler et al., 1999; Heidt et al.,2007). Also, HOXA11 and HOXA13 have been shown to repressMYOD and maintain the myogenic progenitors in a proliferativestate during limb development (Yamamoto and Kuroiwa, 2003).Therefore, it is possible that MEIS, HOXA11 and HOXA13 are notinvolved in patterning, but have a role only in proliferation anddifferentiation of myogenic cells during regeneration. Todistinguish between these hypotheses, it will be important toknockdown MEIS and assess the effect on muscle cellproximalisation in regeneration.

We further show that, similarly to development, activation ofthe b-catenin pathway influences muscle patterning, indicatingthat molecules involved in muscle patterning are likely to beconserved between regeneration and development. In addition toTCF4/b-catenin, TBX4 and TBX5 non-autonomously influencelimb muscle patterning during mouse limb development (Hassonet al., 2010). Brand-Saberi and colleagues (Brand-Saberi et al.,1996) previously suggested that N-cadherin might participate inmyoblast path finding. It will be interesting to investigatewhether these molecules play a role in muscle patterning duringregeneration also.

AcknowledgementsWe thank Heino Andreas, Sabine Mögel and Beate Gruhl for axolotl care. Weacknowledge Hajime Ogino for CarAct:GFP transgenesis construct and GilbertWeidinger for the b-catenin construct. We thank Walter Bonacci and ShirleyGalbiati for advice on the manuscript. We apologise for the limitation inpossibility to extensively discuss and cite the literature on patterning duringlimb development and regeneration.

FundingThis research was funded by grants from the German Research Foundation(DFG) [DFG TA 274/3-1, DFG TA 274/3-2, DFG TA 274/3-2]; the VolkswagenFoundation [I/85018]; and funds from Max Planck Society and the Center forRegenerative Therapies Dresden [to E.M.T.].

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.081752/-/DC1

ReferencesAlvares, L. E., Schubert, F. R., Thorpe, C., Mootoosamy, R. C., Cheng, L.,

Parkyn, G., Lumsden, A. and Dietrich, S. (2003). Intrinsic, Hox-dependentcues determine the fate of skeletal muscle precursors. Dev. Cell 5, 379-390.

Brand-Saberi, B., Gamel, A. J., Krenn, V., Müller, T. S., Wilting, J. and Christ,B. (1996). N-cadherin is involved in myoblast migration and muscledifferentiation in the avian limb bud. Dev. Biol. 178, 160-173.

Capdevila, J., Tsukui, T., Rodríquez Esteban, C., Zappavigna, V. and IzpisúaBelmonte, J. C. (1999). Control of vertebrate limb outgrowth by the proximal factor Meis2 and distal antagonism of BMPs by Gremlin. Mol. Cell 4,839-849.

Christ, B., Jacob, H. J. and Jacob, M. (1977). Experimental analysis of the originof the wing musculature in avian embryos. Anat. Embryol. 150, 171-186.

Cooper, K. L., Hu, J. K.-H., ten Berge, D., Fernandez-Teran, M., Ros, M. A.and Tabin, C. J. (2011). Initiation of proximal-distal patterning in the vertebratelimb by signals and growth. Science 332, 1083-1086.

Diogo, R., Nacu, E. and Tanaka, E. M. (2013). Is salamander limb regenerationperfect? Anatomical and morphogenetic analysis of forelimb muscleregeneration in GFP-transgenic axolotls as a basis for regenerative,developmental and evolutionary studies. J. Anat. (in press).

Ertürk, A., Mauch, C. P., Hellal, F., Förstner, F., Keck, T., Becker, K., Jährling,N., Steffens, H., Richter, M., Hübener, M. et al. (2012). Three-dimensionalimaging of the unsectioned adult spinal cord to assess axon regeneration andglial responses after injury. Nat. Med. 18, 166-171.

Greig, S. and Akam, M. (1993). Homeotic genes autonomously specify oneaspect of pattern in the Drosophila mesoderm. Nature 362, 630-632.

Grim, M. and Wachtler, F. (1991). Muscle morphogenesis in the absence ofmyogenic cells. Anat. Embryol. 183, 67-70.

Hashimoto, K., Yokouchi, Y., Yamamoto, M. and Kuroiwa, A. (1999). Distinctsignaling molecules control Hoxa-11 and Hoxa-13 expression in the muscleprecursor and mesenchyme of the chick limb bud. Development 126, 2771-2783.

Hasson, P., DeLaurier, A., Bennett, M., Grigorieva, E., Naiche, L. A.,Papaioannou, V. E., Mohun, T. J. and Logan, M. P. O. (2010). Tbx4 and tbx5acting in connective tissue are required for limb muscle and tendon patterning.Dev. Cell 18, 148-156.

517RESEARCH REPORTCells in distal transformation

Fig. 3. Overexpression of constitutively active b-catenin induces ectopic muscle formation inaxolotl limb generation. (A,B) Maximum intensityprojection (MIP) from z-stacks through ventral (A) anddorsal (B) halves of a control electroporated limb. (C,D)MIP from z-stacks through ventral (C) and dorsal (D)halves of two example limbs electroporated with anexpression plasmid for constitutively active b-catenin.Red arrowheads point to ectopic muscles. Musclesnormally present in the upper arm are: triceps branchiigroup, green; coracobrachialis longus (CBL), blue;humeroantebrachialis (HAB), yellow; supracoracoideus(SC), purple. Scale bars: 1 mm.

DEVELO

PMENT

518

Heidt, A. B., Rojas, A., Harris, I. S. and Black, B. L. (2007). Determinants ofmyogenic specificity within MyoD are required for noncanonical E box binding.Mol. Cell. Biol. 27, 5910-5920.

Kardon, G., Campbell, J. K. and Tabin, C. J. (2002). Local extrinsic signalsdetermine muscle and endothelial cell fate and patterning in the vertebrate limb.Dev. Cell 3, 533-545.

Kardon, G., Harfe, B. D. and Tabin, C. J. (2003). A Tcf4-positive mesodermalpopulation provides a prepattern for vertebrate limb muscle patterning. Dev. Cell5, 937-944.

Khattak, S., Richter, T. and Tanaka, E. M. (2009). Generation of transgenicaxolotls (Ambystoma mexicanum). Cold Spring Harb. Protoc. 2009,pdb.prot5264.

Knoepfler, P. S., Bergstrom, D. A., Uetsuki, T., Dac-Korytko, I., Sun, Y. H.,Wright, W. E., Tapscott, S. J. and Kamps, M. P. (1999). A conserved motif N-terminal to the DNA-binding domains of myogenic bHLH transcription factorsmediates cooperative DNA binding with pbx-Meis1/Prep1. Nucleic Acids Res. 27,3752-3761.

Kragl, M., Knapp, D., Nacu, E., Khattak, S., Maden, M., Epperlein, H. H. andTanaka, E. M. (2009). Cells keep a memory of their tissue origin during axolotllimb regeneration. Nature 460, 60-65.

Maden, M. (1980). Intercalary regeneration in the amphibian limb and the rule ofdistal transformation. J. Embryol. Exp. Morphol. 56, 201-209.

Mathew, S. J., Hansen, J. M., Merrell, A. J., Murphy, M. M., Lawson, J. A.,Hutcheson, D. A., Hansen, M. S., Angus-Hill, M. and Kardon, G. (2011).Connective tissue fibroblasts and Tcf4 regulate myogenesis. Development 138,371-384.

Mchedlishvili, L., Epperlein, H. H., Telzerow, A. and Tanaka, E. M. (2007). Aclonal analysis of neural progenitors during axolotl spinal cord regenerationreveals evidence for both spatially restricted and multipotent progenitors.Development 134, 2083-2093.

Mchedlishvili, L., Mazurov, V., Grassme, K. S., Goehler, K., Robl, B., Tazaki,A., Roensch, K., Duemmler, A. and Tanaka, E. M. (2012). Reconstitution ofthe central and peripheral nervous system during salamander tail regeneration.Proc. Natl. Acad. Sci. USA 109, E2258-E2266.

Mercader, N., Leonardo, E., Azpiazu, N., Serrano, A., Morata, G., Martínez,C. and Torres, M. (1999). Conserved regulation of proximodistal limb axisdevelopment by Meis1/Hth. Nature 402, 425-429.

Mercader, N., Tanaka, E. M. and Torres, M. (2005). Proximodistal identity duringvertebrate limb regeneration is regulated by Meis homeodomain proteins.Development 132, 4131-4142.

Mohun, T. J., Garrett, N. and Gurdon, J. B. (1986). Upstream sequencesrequired for tissue-specific activation of the cardiac actin gene in Xenopus laevisembryos. EMBO J. 5, 3185-3193.

Nacu, E. and Tanaka, E. M. (2011). Limb regeneration: a new development?Annu. Rev. Cell Dev. Biol. 27, 409-440.

Nacu, E., Knapp, D., Tanaka, E. M. and Epperlein, H. H. (2009). Axolotl(Ambystoma mexicanum) embryonic transplantation methods. Cold SpringHarb. Protoc. 2009, pdb.prot5265.

Pescitelli, M. J. and Stocum, D. L. (1980). The origin of skeletal structures during intercalary regeneration of larval Ambystoma limbs. Dev. Biol. 79, 255-275.

Roselló-Díez, A., Ros, M. A. and Torres, M. (2011). Diffusible signals, notautonomous mechanisms, determine the main proximodistal limb subdivision.Science 332, 1086-1088.

Roy, S. and Vijayraghavan, K. (1997). Homeotic genes and the regulation ofmyoblast migration, fusion, and fibre-specific gene expression during adultmyogenesis in Drosophila. Development 124, 3333-3341.

Stocum, D. (1975). Regulation after proximal or distal transposition of limbregeneration blastemas and determination of the proximal boundary of theregenerate. Dev. Biol. 45, 112-136.

Walthall, J. C. and Ashley-Ross, M. A. (2006). Postcranial myology of theCalifornia newt, Taricha torosa. Anat. Rec. A Discov. Mol. Cell. Evol. Biol. 288,46-57.

Yamamoto, M. and Kuroiwa, A. (2003). Hoxa-11 and Hoxa-13 are involved inrepression of MyoD during limb muscle development. Dev. Growth Differ. 45,485-498.

Yamamoto, M., Gotoh, Y., Tamura, K., Tanaka, M., Kawakami, A., Ide, H.and Kuroiwa, A. (1998). Coordinated expression of Hoxa-11 and Hoxa-13during limb muscle patterning. Development 125, 1325-1335.

Yost, C., Torres, M., Miller, J. R., Huang, E., Kimelman, D. and Moon, R. T.(1996). The axis-inducing activity, stability, and subcellular distribution of beta-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3. GenesDev. 10, 1443-1454.

RESEARCH REPORT Development 140 (3)

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