Regeneration of Vertebrate Appendages


Some vertebrates can faithfully replace complex parts of their body, a feature that is more commonly observed in invertebrates. They can perform this remarkable task because of their capacity to recruit progenitor cells and activate developmental programmes that largely parallel those used during embryogenesis. Tailed amphibians (urodeles), fish and deer provide the most striking examples of regeneration of appendages in adult animals. Frogs (anurans) can regenerate their limbs only at larval stages and can provide valuable models for studying loss of regenerative capability in the same species. Although birds and mammals cannot regenerate their limbs, digit tips display notable regenerative capability. The most striking example of mammalian appendage replacement is antler regeneration. Information on molecular landmarks of mature cell reprogramming in response to amputation is rapidly building up together with insights into the complex regulatory machinery controlling subsequent proliferation, differentiation and patterning of regenerating appendages.

Key Concepts:

  • Appendage regeneration can occur in some lower vertebrates, fish and amphibians.

  • Wound healing modality and appropriate inflammatory responses are crucial for regeneration.

  • Regeneration of vertebrate appendages occurs via formation of a growth zone, the blastema.

  • Blastema formation in adult lower vertebrates involves a reversal of the differentiated state.

  • Resident progenitor cells can contribute to regeneration.

  • Blastemal cells in regenerating limbs largely maintain their original identity.

  • The presence of a specialized wound epidermis is essential for regeneration throughout the process.

  • The presence of the nerve is crucial for the initial stages of limb and fin regeneration and also for mammalian digit tip regeneration.

  • Limb regeneration becomes nerve dependent after the developing limb is innervated.

  • Redeployment of certain developmental mechanisms underlies patterning in regenerating appendages.

  • Although, by and large, the same key signalling pathways are involved in regenerative responses across species, existence of speciesā€specific molecules may provide some rationale for differences in adult appendage regenerative capability.

Keywords: antler; amphibia; blastema; fin; fish; limb; mouse; regeneration

Figure 1.

Schematic representation of a limb regeneration blastema, a mound of undifferentiated progenitor cells covered by a specialised wound epithelium, the apical epithelial cap (AEC). The blastema gives rise to the amputated part of the appendage; after a rapid proliferation phase its differentiation progresses in a proximal to distal direction as indicated. Abbreviations: B, bone; M, muscle and N, nerve.

Figure 2.

Successive stages of regeneration and their approximate time course after limb amputation in the adult newt, N. viridescens. An appropriate inflammatory response is crucial for wound healing and underlies limb regeneration. Following wound healing, blastemal cells accumulate at the tip of the stump by a process of dedifferentiation, and they start to proliferate under the influence of the nerve and the thickened wound epithelium within 1 week after amputation. A clear blastema is usually visible by 2 weeks. Between 2 and 3 weeks after amputation, proliferation becomes nerve independent and differentiation begins. Some of the signalling pathways important at different stages of limb regeneration are indicated (red triangles) as well as one of the pathways regulated by retinoic acid unique to urodele limb regeneration. Abbreviations: d, day after amputation and w, weeks after amputation.

Figure 3.

Schematic representation of a normal pectoral fin skeleton (a) and of lepidotrichia in longitudinal (b) and cross‐section (c). (d) Schematic representation of ray stump and blastema. (e) Micrographs of cross‐ and longitudinal sections of normal fin and regenerating fins 1 and 5 days after amputation. Abbreviations: B, bone and bv, blood vessel.

Figure 4.

Schematic summary of interactions between different signalling pathways believed to regulate growth, differentiation and patterning in regenerating fins.



Akimenko MA, Mari‐Beffa M, Becerra J and Geraudie J (2003) Old questions, new tools, and some answers to the mystery of fin regeneration. Developmental Dynamics 226: 190–201.

Blum N and Begemann G (2012) Retinoic acid signaling controls the formation, proliferation and survival of the blastema during adult zebrafish fin regeneration. Development 139: 107–116.

Borsy A, Podani J, Steger V et al. (2009) Identifying novel genes involved in both deer physiological and human pathological osteoporosis. Molecular Genetics and Genomics 281: 301–313.

Brockes JP and Kumar A (2002) Plasticity and reprogramming of differentiated cells in amphibian regeneration. Nature Reviews Molecular Cell Biology 3: 566–574.

Cameron JA, Hilgers AR and Hinterberger TJ (1986) Evidence that reserve cells are a source of regenerated adult newt muscle in vitro. Nature 321: 607–610.

Christen B, Robles V, Raya M, Paramonov I and Izpisua Belmonte JC (2010) Regeneration and reprogramming compared. BMC Biology 8: 5.

Christensen RN, Weinstein M and Tassava RA (2002) Expression of fibroblast growth factors 4, 8, and 10 in limbs, flanks, and blastemas of Ambystoma. Developmental Dynamics 223: 193–203.

Corcoran JP and Ferretti P (1999) RA regulation of keratin expression and myogenesis suggests different ways of regenerating muscle in adult amphibian limbs. Journal of Cell Science 112: 1385–1394.

Gardiner DM and Bryant SV (1996) Molecular mechanisms in the control of limb regeneration: the role of homeobox genes. International Journal of Developmental Biology 40: 797–805.

Gardiner DM and Bryant SV (1998) The tetrapod limb. In: Ferretti P and Géraudie J (eds) Cellular and Molecular Basis of Regeneration: From Invertebrates to Humans, p. 187–205. Chichester, UK: John Wiley and Sons, Ltd.

Geraudie J and Ferretti P (1997) Correlation between RA‐induced apoptosis and patterning defects in regenerating fins and limbs. International Journal of Developmental Biology 41: 529–532.

Geraudie J and Ferretti P (1998) Gene expression during amphibian limb regeneration. International Review of Cytology 180: 1–50.

Giampaoli S, Bucci S, Ragghianti M, Mancino G and Ferretti P (2003) Expression of FGF‐2 in the limb of two Salamandridae correlates with their regenerative capability. Proceedings of the Royal Society B: Biological Sciences 270: 2197–2205.

Godwin JW, Pinto AR and Rosenthal NA (2013) Macrophages are required for adult salamander limb regeneration. Proceedings of the National Academy of Sciences of the USA 110: 9415–9420.

Goss JM (1969) Horns and antlers. In: Priciples of Regeneration, p. 223–254. New York: Academic Press.

Grotek B, Wehner D and Weidinger G (2013) Notch signaling coordinates cellular proliferation with differentiation during zebrafish fin regeneration. Development 140: 1412–1423.

Han M, Yang X, Farrington JE and Muneoka K (2003) Digit regeneration is regulated by Msx1 and BMP4 in fetal mice. Development 130: 5123–5132.

Holman EC, Campbell LJ, Hines J and Crews CM (2012) Microarray analysis of microRNA expression during axolotl limb regeneration. PLoS One 7: e41804.

Hu W, Meng X, Lu T et al. (2013) MicroRNA1 inhibits the proliferation of Chinese sika deer derived cartilage cells by binding to the 3′‐untranslated region of IGF1. Molecular Medicine Reports 8(2): 523–528.

Ivanova AS, Tereshina MB, Ermakova GV, Belousov VV and Zaraisky AG (2013) Agr genes, missing in amniotes, are involved in the body appendages regeneration in frog tadpoles. Scientific Reports 3: 1279.

Jhamb D, Rao N, Milner DJ et al. (2011) Network based transcription factor analysis of regenerating axolotl limbs. BMC Bioinformatics 12: 80.

Kierdorf U, Li C and Price JS (2009) Improbable appendages: deer antler renewal as a unique case of mammalian regeneration. Seminars in Cell and Developmental Biology 20: 535–542.

King MW, Neff AW and Mescher AL (2012) The developing Xenopus limb as a model for studies on the balance between inflammation and regeneration. Anatomical Record (Hoboken) 295: 1552–1561.

Knapp D, Schulz H, Rascon CA et al. (2013) Comparative transcriptional profiling of the axolotl limb identifies a tripartite regeneration‐specific gene program. PLoS One 8: e61352.

Knopf F, Hammond C, Chekuru A et al. (2011) Bone regenerates via dedifferentiation of osteoblasts in the zebrafish fin Developmental Cell 20: 713–724.

Kragl M, Knapp D, Nacu E et al. (2009) Cells keep a memory of their tissue origin during axolotl limb regeneration. Nature 460: 60–65.

Kumar A and Brockes JP (2012) Nerve dependence in tissue, organ, and appendage regeneration. Trends in Neurosciences 35(11): 691–699.

Kumar A, Godwin JW, Gates PB, Garza‐Garcia AA and Brockes JP (2007) Molecular basis for the nerve dependence of limb regeneration in an adult vertebrate. Science 318: 772–777.

Kumar A, Velloso CP, Imokawa Y and Brockes JP (2000) Plasticity of retrovirus‐labelled myotubes in the newt limb regeneration blastema. Developmental Biology 218: 125–136.

Kumar A, Velloso CP, Imokawa Y and Brockes JP (2004) The regenerative plasticity of isolated urodele myofibers and its dependence on MSX1. PLoS Biology 2: E218.

Laforest L, Brown CW, Poleo G et al. (1998) Involvement of the sonic hedgehog, patched 1 and bmp2 genes in patterning of the zebrafish dermal fin rays. Development 125: 4175–4184.

Lee Y, Hami D, De Val S et al. (2009) Maintenance of blastemal proliferation by functionally diverse epidermis in regenerating zebrafish fins. Developmental Biology 331: 270–280.

Lehoczky JA, Robert B and Tabin CJ (2011) Mouse digit tip regeneration is mediated by fate‐restricted progenitor cells. Proceedings of the National Academy of Sciences of the USA 108: 20609–20614.

Levesque M, Gatien S, Finnson K et al. (2007) Transforming growth factor: beta signaling is essential for limb regeneration in axolotls. PLoS One 2: e1227.

Li C, Harper A, Puddick J, Wang W and McMahon C (2012a) Proteomes and signalling pathways of antler stem cells. PLoS One 7: e30026.

Li C, Stanton JA, Robertson TM et al. (2007) Nerve growth factor mRNA expression in the regenerating antler tip of red deer (Cervus elaphus). PLoS One 2: e148.

Li C, Yang F and Sheppard A (2009) Adult stem cells and mammalian epimorphic regeneration‐insights from studying annual renewal of deer antlers. Current Stem Cell Research and Therapy 4: 237–251.

Li L, Yan B, Shi YQ, Zhang WQ and Wen ZL (2012b) Live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration. Journal of Biological Chemistry 287: 25353–25360.

Lin G, Chen Y and Slack JM (2013) Imparting regenerative capacity to limbs by progenitor cell transplantation. Developmental Cell 24: 41–51.

Loof S, Straube WL, Drechsel D, Tanaka EM and Simon A (2007) Plasticity of mammalian myotubes upon stimulation with a thrombin‐activated serum factor. Cell Cycle 6: 1096–1101.

Love NR, Chen Y, Ishibashi S et al. (2013) Amputation‐induced reactive oxygen species are required for successful Xenopus tadpole tail regeneration. Nature Cell Biology 15: 222–228.

Maden M (1997) Retinoic acid and its receptors in limb regeneration. Seminars in Cell and Developmental Biology 8: 445–453.

Makanae A and Satoh A (2012) Early regulation of axolotl limb regeneration. Anatomical Record (Hoboken) 295: 1566–1574.

Maki N, Suetsugu‐Maki R, Tarui H et al. (2009) Expression of stem cell pluripotency factors during regeneration in newts. Developmental Dynamics 238: 1613–1616.

Monaghan JR and Maden M (2012) Visualization of retinoic acid signaling in transgenic axolotls during limb development and regeneration. Developmental Biology 368: 63–75.

Morrison JI, Loof S, He P and Simon A (2006) Salamander limb regeneration involves the activation of a multipotent skeletal muscle satellite cell population. Journal of Cell Biology 172: 433–440.

Mount JG, Muzylak M, Allen S et al. (2006) Evidence that the canonical Wnt signalling pathway regulates deer antler regeneration. Developmental Dynamics 235: 1390–1399.

Nacu E, Glausch M, Le HQ et al. (2013) Connective tissue cells, but not muscle cells, are involved in establishing the proximo‐distal outcome of limb regeneration in the axolotl. Development 140: 513–518.

Neff AW, King MW and Mescher AL (2011) Dedifferentiation and the role of sall4 in reprogramming and patterning during amphibian limb regeneration. Developmental Dynamics 240: 979–989.

Odelberg SJ, Kollhoff A and Keating MT (2000) Dedifferentiation of mammalian myotubes induced by msx1. Cell 103: 1099–1109.

Ohgo S, Itoh A, Suzuki M et al. (2010) Analysis of hoxa11 and hoxa13 expression during patternless limb regeneration in Xenopus. Developmental Biology 338: 148–157.

Ozpolat DB, Zapata M, Daniel Fruge J et al. (2012) Regeneration of the elbow joint In the developing chick embryo recapitulates development. Developmental Biology 372: 229–238.

Pita‐Thomas W, Fernandez‐Martos C, Yunta M et al. (2010) Gene expression of axon growth promoting factors in the deer antler. PLoS One 5: e15706.

Quint E, Smith A, Avaron F et al. (2002) Bone patterning is altered in the regenerating zebrafish caudal fin after ectopic expression of sonic hedgehog and bmp2b or exposure to cyclopamine. Proceedings of the National Academy of Sciences of the USA 99: 8713–8718.

Rao N, Jhamb D, Milner DJ et al. (2009) Proteomic analysis of blastema formation in regenerating axolotl limbs. BMC Biology 7: 83.

Rinkevich Y, Lindau P, Ueno H, Longaker MT and Weissman IL (2011) Germ‐layer and lineage‐restricted stem/progenitors regenerate the mouse digit tip. Nature 476: 409–413.

Rodrigues AM, Christen B, Marti M and Izpisua Belmonte JC (2012) Skeletal muscle regeneration in Xenopus tadpoles and zebrafish larvae. BMC Developmental Biology 12: 9.

Rolf HJ, Niebert S, Niebert M et al. (2013) Intercellular transport of Oct4 in mammalian cells: a basic principle to expand a stem cell niche? PLoS One 7: e32287.

Santosh N, Windsor LJ, Mahmoudi BS et al. (2011) Matrix metalloproteinase expression during blastema formation in regeneration‐competent versus regeneration‐deficient amphibian limbs. Developmental Dynamics 240: 1127–1141.

Satoh A, Graham GM, Bryant SV and Gardiner DM (2008) Neurotrophic regulation of epidermal dedifferentiation during wound healing and limb regeneration in the axolotl (Ambystoma mexicanum). Developmental Biology 319: 321–335.

Satoh A, Makanae A, Hirata A and Satou Y (2011) Blastema induction in aneurogenic state and Prrx‐1 regulation by MMPs and FGFs in Ambystoma mexicanum limb regeneration. Developmental Biology 355: 263–274.

Savard P, Gates PB and Brockes JP (1988) Position dependent expression of a homeobox gene transcript in relation to amphibian limb regeneration. EMBO Journal 7: 4275–4282.

Shaikh N, Gates PB and Brockes JP (2011) The Meis homeoprotein regulates the axolotl Prod 1 promoter during limb regeneration. Gene 484: 69–74.

da Silva SM, Gates PB and Brockes JP (2002) The newt ortholog of CD59 is implicated in proximodistal identity during amphibian limb regeneration. Developmental Cell 3: 547–555.

Singer M (1974) Neurotrophic control of limb regeneration in the newt. Annals of the New York Academy of Sciences 228: 308–322.

Singh BN, Doyle MJ, Weaver CV, Koyano‐Nakagawa N and Garry DJ (2012a) Hedgehog and Wnt coordinate signaling in myogenic progenitors and regulate limb regeneration. Developmental Biology 371: 23–34.

Singh SP, Holdway JE and Poss KD (2012b) Regeneration of amputated zebrafish fin rays from de novo osteoblasts. Developmental Cell 22: 879–886.

Sousa S, Afonso N, Bensimon‐Brito A et al. (2011) Differentiated skeletal cells contribute to blastema formation during zebrafish fin regeneration. Development 138: 3897–3905.

Stewart R, Rascon CA, Tian S et al. (2013) Comparative RNA‐seq analysis in the unsequenced axolotl: the oncogene burst highlights early gene expression in the blastema. PLoS Computational Biology 9: e1002936.

Stewart S and Stankunas K (2012) Limited dedifferentiation provides replacement tissue during zebrafish fin regeneration. Developmental Biology 365: 339–349.

Stocum D (1985) Role of the skin in urodele limb regeneration. In: Sicard RE (ed.) Regulation of Vertebrate Limb Regeneration, p. 32–53. New York: Oxford University Press.

Stocum DL (1991) Retinoic acid and limb regeneration. Seminars in Developmental Biology 2: 199–210.

Takeo M, Chou WC, Sun Q et al. (2013) Wnt activation in nail epithelium couples nail growth to digit regeneration. Nature 499(7457): 228–232.

Tanaka EM, Drechsel DN and Brockes JP (1999) Thrombin regulates S‐phase re‐entry by cultured newt myotubes. Current Biology 9: 792–799.

Taylor AJ and Beck CW (2012) Histone deacetylases are required for amphibian tail and limb regeneration but not development. Mechanisms of Development 129(9–2): 208–218.

Thatcher EJ, Paydar I, Anderson KK and Patton JG (2008) Regulation of zebrafish fin regeneration by microRNAs. Proceedings of the National Academy of Sciences of the USA 105: 18384–18389.

Tu S and Johnson SL (2011) Fate restriction in the growing and regenerating zebrafish fin. Developmental Cell 20: 725–732.

White JA, Boffa MB, Jones B and Petkovich M (1994) A zebrafish retinoic acid receptor expressed in the regenerating caudal fin. Development 120: 1861–1872.

Whited JL, Tsai SL, Beier KT et al. (2013) Pseudotyped retroviruses for infecting axolotl in vivo and in vitro. Development 140: 1137–1146.

Wu Y, Wang K, Karapetyan A et al. (2013) Connective tissue fibroblast properties are position‐dependent during mouse digit tip regeneration. PLoS One 8: e54764.

Yakushiji N, Yokoyama H and Tamura K (2009) Repatterning in amphibian limb regeneration: a model for study of genetic and epigenetic control of organ regeneration. Seminars in Cell and Developmental Biology 20: 565–574.

Yin VP, Thomson JM, Thummel R et al. (2008) Fgf‐dependent depletion of microRNA‐133 promotes appendage regeneration in zebrafish. Genes and Development 22: 728–733.

Yoo SK, Freisinger CM, LeBert DC and Huttenlocher A (2012) Early redox, Src family kinase, and calcium signaling integrate wound responses and tissue regeneration in zebrafish. Journal of Cell Biology 199: 225–234.

Yu L, Han M, Yan M et al. (2010) BMP signaling induces digit regeneration in neonatal mice. Development 137: 551–559.

Yu L, Han M, Yan M, Lee J and Muneoka K (2012) BMP2 induces segment‐specific skeletal regeneration from digit and limb amputations by establishing a new endochondral ossification center. Developmental Biology 372: 263–273.

Zhang J, Jeradi S, Strahle U and Akimenko MA (2012) Laser ablation of the sonic hedgehog‐a‐expressing cells during fin regeneration affects ray branching morphogenesis. Developmental Biology 365: 424–433.

Zhu W, Pao GM, Satoh A et al. (2012) Activation of germline‐specific genes is required for limb regeneration in the Mexican axolotl. Developmental Biology 370: 42–51.

Further Reading

Brockes JP (1998) Progenitor cells for regeneration: origin by reversal of the differentiated state. In: Ferretti P and Géraudie J (eds) Cellular and Molecular Basis of Regeneration: From Invertebrates to Humans, pp 63–77. Chichester, UK: John Wiley and Sons, Ltd.

Carlson B (1998) Development and regeneration, with special emphasis on the amphibian limb. In: Ferretti P and Géraudie J (eds) Cellular and Molecular Basis of Regeneration: From Invertebrates to Humans, pp 45–61. Chichester, UK: John Wiley and Sons, Ltd.

Carlson B (2007) Principles of Regenerative Biology, p. 167–185. Amsterdam; London: Elsevier.

Dinsmore CE and Mescher AL (1998) The role of the nervous system in regeneration. In: Ferretti P and Géraudie J (eds) Cellular and Molecular Basis of Regeneration: From Invertebrates to Humans, pp 79–108. Chichester, UK: John Wiley and Sons, Ltd.

Ferretti P and Géraudie J (eds) (1998) Cellular and Molecular Basis of Regeneration: From Invertebrates to Humans. Chichester, UK: John Wiley and Sons, Ltd.

Géraudie J, Akimenko MA and Smith M (1998) The dermal skeleton. In: Ferretti P and Géraudie J (eds) Cellular and Molecular Basis of Regeneration: From Invertebrates to Humans, pp 167–185. Chichester, UK: John Wiley and Sons, Ltd.

Heber‐Katz H and Stocum DL (eds) (2013) New Perspectives in Regeneration. Current Topics in Microbiology and Immunology, vol. 367. Berlin, Heidelberg: Springer‐Verlag.

Sicard ER (ed.) (1985) Regulation of Vertebrate Limb Regeneration. New York: Oxford University Press.

Stocum D (2004) Amphibian regeneration and stem cells. Current Topics in Microbiology and Immunology 280: 1–70.

Stocum D (2006) Regenerative Biology and Medicine Canada: Academic Press.

Spallanzani L (1768) Prodomo di un opera da imprimersi sopra la riproduzioni animali. Modena: Giovanni Montanari. [Maty M (transl.) (1769) An Essay on Animal Reproduction. London: T Becket and De Hondt].

Tal TL, Franzosa JA and Tanguay RL (2010) Molecular signaling networks that choreograph epimorphic fin regeneration in zebrafish – a mini‐review. Gerontology 56: 231–240.

Tsonis PA (ed.) (1996) Limb regeneration. In: Barlow PW, Bard JBL, Green PB and Kirk DL (Series editors) Developmental and Cell Biology Series. New York: Cambridge University Press.

Wallace H (1981) Vertebrate Limb Regeneration. Chichester: Wiley.

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Ferretti, Patrizia(Oct 2013) Regeneration of Vertebrate Appendages. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0001099.pub3]