Regeneration of the Urodele Limb

David L Stocum, Indiana University‐Purdue University, Indianapolis, Indiana, USA
Karen Crawford, Saint Mary's College of Maryland, Saint Mary's City, Maryland, USA

Published online: April 2015

DOI: 10.1002/9780470015902.a0001100.pub4

Abstract

Urodele limbs regenerate via a blastema of mesenchymal cells derived from muscle, connective tissue and nerve sheath cells in the vicinity of the amputation surface by a process of histolysis, dedifferentiation and release of stem cells. Blastema cells replicate their DNA, arrest in G2 and accumulate under an apical epidermal cap (AEC). G2 arrest is broken by factor(s) expressed by the AEC that bind to receptors on the blastema cell surface. Expression of these factors is dependent on factor(s) provided by axons reinnervating the AEC. The blastema cells proliferate and are patterned into new limb structures by suites of signalling factors such as retinoic acid, sonic hedgehog and Wnt, in addition to homeobox transcription factors that endow the cells with positional identities expressed as axial gradients on the cell surface. The degree to which the early blastema is determined is controversial, with some evidence arguing for developmental plasticity and other evidence arguing that the blastema is self‐organising.

Key Concepts

  • Urodeles are unique in their ability to form a regeneration blastema in response to amputation.
  • The cells of the regeneration blastema are derived from resident stem cells and/or by the reprogramming (dedifferentiation) of differentiated cells.
  • Blastema cells derived from the different limb tissues redifferentiate in a lineage‐specific manner, but blastema cells derived from fibroblasts can also transdifferentiate into cartilage and tendon.
  • The apical epidermal cap (AEC) of the wound epidermis is induced by regenerating nerve axons to produce a mitogen(s) that drives the proliferation of blastema cells.
  • Patterning of the blastema involves region‐specific contributions of limb cells, lineage‐specific redifferentiation of blastema cells, interactions between blastema cells of different positions to eliminate discontinuities and sorting out of blastema cells with different cell surface adhesion.
  • Cell interactions are mediated during axial patterning of the blastema by several signalling molecules and homeobox transcription factors.
  • How the axial pattern and limb type of the early blastema is determined, whether by induction, self‐organisation or both, remains controversial.

Keywords: blastema origin and formation; cell cycling; determination; patterning; retinoic acid; Hox genes

Figure 1. Four stages of regeneration in a larval urodele forelimb amputated through the distal radius (R) and ulna (U). (a): stage of accumulation blastema, 4 days post‐amputation. The arrow points to the forming AEC. Beneath the AEC lies the accumulation of undifferentiated mesenchymal cells, which are arrested in G2 of the cell cycle. Nerves are beginning to penetrate the AEC at this stage. (b): stage of medium bud, 1 week post‐amputation. G2 arrest has been broken and the mitosis has greatly enlarged the blastema. The arrow points to a gland cell (Leydig cell) in the AEC, which is now thoroughly innervated. The AEC is expressing AGP at this stage, which is mitogenic for the subjacent mesenchymal cells. (c): Late bud stage, 9 days post‐amputation. The asterisks indicate cartilage condensations that will form the carpals and first two digits. The AEC is diminishing in thickness, but gland cells are still prominent. (d): Four fingerbud stage, 12 days post‐amputation. U = ulna; C = differentiating carpals. Arrow points to regenerating muscle.
Figure 2. Diagram of affinophoresis assay. Conical blastemas (triangle) derived from the wrist (W) or elbow (E) of the axolotl forelimb were grafted to the blastema‐stump junction (green arrow) on the dorsal side of a hindlimb regenerating from the mid‐femur (F), and allowed to develop. The three segments of the hindlimb are depicted as rectangles. F, femur; T/f, tibia/fibula; Fo, foot; A, ankle joint and K, knee joint. The outline of the host hindlimb blastema at the start of the experiment is shown by the black arc. Dorsal is to the top, posterior towards the reader. As the hindlimb blastema grew and formed the segments of the hindlimb distal to the amputation plane, untreated elbow and wrist blastemas moved distally along the proximodistal axis to their corresponding levels of knee and ankle, respectively, and developed according to their level of origin. Retinoic acid treatment proximalised the positional identity of the elbow and wrist blastemas. Proximalisation abolished the distal sorting behaviour of the grafted blastemas, which remained at the level of grafting and developed shoulder girdle, humerus, radius/ulna and hand.
close

References

Adams DS, Masi A and Levin M (2007) H+ pump‐dependent changes in membrane voltage are an early mechanism necessary and sufficient to induce Xenopus tail regeneration. Development 134: 1323–1335.

Bryant SV, French V and Bryant PJ (1981) Distal regeneration and symmetry. Science 212: 993–1002.

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.

Crawford K and Stocum DL (1988a) Retinoic acid coordinately proximalizes regenerate pattern and blastema differential affinity in axolotl limbs. Development 102: 687–698.

Crawford K and Stocum DL (1988b) Retinoic acid proximalizes level‐specific properties responsible for intercalary regeneration in axolotl limbs. Development 104: 703–712.

De Both NJ (1970) The developmental potencies of the regeneration blastema of the axolotl limb. Wilhelm Roux's Archiv Entwickungsmechanik der Organism 165: 242–276.

Endo T, Yokoyama H, Tamura K and Hiroyuki I (1997) Shh expression in developing and regenerating limb buds of Xenopus laevis. Developmental Dynamics 209: 227–232.

Faber J (1960) An experimental analysis of regional organization in the regenerating forelimb of the axolotl (Ambystoma mexicanum). Archives of Biology 71: 1–67.

Fei J‐F, Schuez M, Tazaki A, et al. (2014) CRISPR‐mediated genomic deletion of Sox2 in the axolotl shows a requirement in spinal cord neural stem cell amplification during tail regeneration. Stem Cell Reports 3: 444–459.

Flowers GP, Timberlake AT, Mclean KC, et al. (2014) Highly efficient targeted mutagenesis in axolotl sing Cas9 RNA‐guided nuclease. Development 141: 1–7.

Gardiner DM, Blumberg B, Komine Y and Bryant SV (1995) Regulation of HoxA expression in developing and regenerating axolotls. Development 121: 1731–1741.

Gardiner DM and Bryant SV (2007) Homeobox‐containing genes in limb regeneration. In: Papageorgiou S, (ed). HOX Gene Expression, pp. 102–100. Austin, TX: Landes Bioscience.

Graza‐Garcia A, Harris R, Esposito D, Gates PB and Driscoll PC (2009) Solution structure and phylogenetics of Prod1, a member of the three‐finger protein superfamily implicated in salamander limb regeneration. PLoS One 4 (9): e7123.

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

Han M‐J, An J‐Y and Kim W‐S (2001) Expression patterns of Fgf‐8 during development and limb regeneration of the axolotl. Developmental Dynamics 220: 40–48.

Holder N (1989) Organization of connective tissue patterns by dermal fibroblasts in the regenerating axolotl limb. Development 105: 585–593.

Imokawa Y and Yoshizato K (1997) Expression of sonic hedgehog gene in regenerating newt limb blastemas recapitulates that in developing limb buds. Proceedings of the National Academy of Sciences of the United States of America 94: 9159–9164.

Iten LE and Bryant SV (1975) The interaction between the blastema and stump in the establishment of the anterior‐posterior and proximal‐distal organization of the limb regenerate. Developmental Biology 44: 119–147.

Jenkins LS, Duerstock BS and Borgens RB (1996) Reduction of the current of injury leaving the amputation inhibits limb regeneration in the red spotted newt. Developmental Biology 178: 251–262.

Ju BG and Kim WS (1998) Upregulation of cathepsin D expression in the dedifferentiating salamander limb regenerates and enhancement of its expression by retinoic acid. Wound Repair and Regeneration 6: 349–357.

Kim WS and Stocum DL (1986) Retinoic acid modifies positional memory n the anteroposterior axis of regenerating axolotl limbs. Developmental Biology 114: 170–179.

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.

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

Kumar A, Nevill G, Brockes JP and Forge A (2010) A comparative study of gland cells implicated in the nerve dependence of salamander limb regeneration. Journal of Anatomy 217: 16–25.

Kumar A, Delgado JP, Gates PB, et al. (2011) The aneurogenic limb identifies developmental cell interactions underlying vertebrate limb regeneration. Proceedings of the National Academy of Sciences of the United States of America 108: 13588–13593.

Lheureux E (1975) Regeneration des membres irradies de Pleurodeles waltlii Micah (Urodele). Influence des qualites et orientations des greffons non irradies. Wilhem Roux's Archive of Developmental Biology 176: 303–327.

Ludolph D, Cameron JA and Stocum DL (1990) The effect of retinoic acid on positional memory n the dorsoventral axis of regenerating axolotl limbs. Developmental Biology 140: 41–52.

Maden M (1983) A test of the predictions of the boundary model regarding supernumerary limb structure. Journal of Embryology and Experimental Morphology 76: 147–155.

Maden M (1998) Retinoids as endogenous components of the regenerating limb and tail. Wound Repair and Regeneration 6: 358–365.

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.

Matsuda H, Yokoyama H, Endo T, et al. (2001) An epidermal signal regulates Lmx-1 expression and dorsal‐ventral pattern duringxenoplus limb regeneration. Developmental Biology 229: 351–362.

McCusker CD and Gardiner DM (2013) Positional information is reprogrammed in blastema cells of the regenerating limb of the axolotl (Ambystoma mexicanum). PLoS One 8: e77064.

Meinhardt H (1983) A boundary model for pattern formation in vertebrate limbs. Journal of Embryology and Experimental Morphology 76: 115–137.

Mercader N, Tanaka E, Torres M (2005) Proximodistal identity during vertibrate limb regeneration is regulated by Meis homeodomain proteins. Development 132: 4131–4142.

Mescher AL (1992) Trophic activity of regenerating peripheral nerve. Comments on Developmental Neurobiology 1: 373–390.

Mittenthal JE (1981) The rule of normal neighbors: a hypothesis for morphogenetic pattern regulation. Developmental Biology 88: 15–26.

Monaghan J, Epp L, Putta S, et al. (2009) Microarray and sequence analysis of transcription during nerve‐dependent limb regeneration. BMC Biology 7: 1.

Monaghan J, Athippozhy A, Seifert A, et al. (2012) Gene expression patterns specific to the regenerating limb of the Mexican axolotl. Biology Open 1: 937–948.

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

Muneoka K, Fox WF and Bryant SV (1986) Cellular contribution from dermis and cartilage to the regenerating limb blastema in axolotls. Developmental Biology 116: 256–260.

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.

Nardi JB and Stocum DL (1983) Surface properties of regenerating limb cells: evidence for gradatin along the proximodistal axis. Differentiation 25: 27–31.

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

Rao N, Song F, Jhamb D, et al. (2014) Proteomic analysis of fibroblastema formation in regenerating hindlimbs of Cenopus laevis froglets and comparison to axolotl. BMC Developmental Biology 14: 32.

Roensch K, Tazaki A, Chara O and Tanaka EM (2014) Progressive specification rather than intercalation of segments during limb regeneration. Science 342: 1375–1379.

Sandoval‐Guzman T, Wan GH, Khattak S, et al. (2014) Fundamental differences in dedifferentiation and stem cell recruitment during skeletal muscle regeneration in two salamander species. Cell Stem Cell 14: 1–14.

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.

Singer M (1952) The influence of the nerve in regeneration of the amphibian extremity. Quarterly Review of Biology 27: 169–200.

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.

Stocum DL (1968) The urodele limb regeneration blastema: a self‐organizing system: II. Morphogenesis and differentiation of autografted whole and fractional blastemas. Developmental Biology 18: 457–480.

Stocum DL (1980) Autonomous development of reciprocally exchanged regeneration blastemas of normal forelimbs and symmetrical hindlimbs. Journal of Experimental Zoology 212: 361–371.

Stocum DL (2011) The role of peripheral nerves in limb regeneration. European Journal of Neuroscience 34: 908–916.

Stocum DL and Dearlove GE (1972) Epidermal‐mesodermal interaction during morphogenesis of the limb regeneration blastema in larval salamanders. Journal of Experimental Zoology 181: 49–62.

Stocum DL and Melton DA (1977) Self‐organizational capacity of distally transplanted limb regeneration blastemas in larval salamanders. Journal of Experimental Zoology 201: 451–472.

Takahashi K, Tanabe K, Ohnuki M, et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131: 861–872.

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

Tanaka EM, Gann AAF, Gates PB and Brockes JB (1997) Newt myotubes reenter the cell cycle by phosphorylation of the retinoblastoma protein. Journal of Cell Biology 136: 155–165.

Tseng A‐S, Beane WS, Lemire JM, et al. (2010) Induction of vertebrate regeneration by a transient sodium current. Journal of Neuroscience 30: 13193–13200.

Tsonis PA, English D and Mescher AL (1991) Increased content of inositol phosphates in amputated limbs of axolotl larvae and the effect of beryllium. Journal of Experimental Zoology 259: 252–258.

Vethamany‐Globus S, Globus M and Tomlinson B (1978) Neural and hormonal stimulation of DNA and protein synthesis in cultured regeneration blastema in the newt Notophthalmus viridescens. Developmental Biology 65: 183–192.

Vinarsky V, Atkinson DL, Stevenson T, Keating MT and Odelberg SJ (2005) Normal newt limb regeneration requires matrix metalloproteinase function. Developmental Biology 279: 86–98.

Wada N, Kimura I, Tanaka H, Ide H and Nohno T (1998) Glycosylphosphatidylinositol‐anchored cell surface proteins regulate position‐specific cell affinity in the limb bud. Developmental Biology 202: 244–252.

Yajima H, Yonei‐Tamura S, Watanabe N, Tamura K and Ide H (1999) Role of N‐cadherin in the sorting out of mesenchymal cells and in the positional identity along the proximodistal axis of the chick limb bud. Developmental Dynamics 216: 274–284.

Yu J, Vodyanik MA, Smuga‐Otto K, et al. (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318: 1917–1920.

Yun MH, Gates PB and Brockes JP (2013) Regulation of p53 is critical for vertebrate limb regeneration. Proceedings of the National Academy of Sciences of the United States of America 110: 17392–17397.

Further Reading

Brockes JP (1997) Amphibian limb regeneration: rebuilding a complex structure. Science 276: 81–87.

Brockes JP and Kumar A (2008) Comparative aspects of animal regeneration. Annual Review of Cell and Developmental Biology 24: 525–549.

Carlson BM (2007) Principles of Regenerative Biology379 pp.. San Diego, CA: Elsevier/Academic Press.

Gardiner DM, Carlson MRJ and Roy S (1999) Towards a functional analysis of limb regeneration. Seminars in Cell and Developmental Biology 10: 385–394.

Mescher AL (1996) The cellular basis of limb regeneration in urodeles. International Journal of Developmental Biology 40: 785–796.

Stocum DL (1984) The urodele limb regeneration blastema: determination and organization of the morphogenetic field. Differentiation 27: 13–28.

Stocum DL (1996) A conceptual framework for analyzing axial patterning in regenerating urodele limbs. International Journal of Developmental Biology 40: 773–783.

Stocum DL and Cameron JA (2011) Looking proximally and distally: 100 years of limb regeneration. Developmental Dynamics 240: 943–968.

Stocum DL (2012) Regenerative Biology and Medicine, 2nd edn., p. 465 San Diego, CA: Elsevier/Academic Press.

Related Sub-Topics:

Genes, Molecules and Cellular Processes | Regeneration: Vertebrates
Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

Article Title: Regeneration of the Urodele Limb
 
How to Cite close
Stocum, David L, and Crawford, Karen(Apr 2015) Regeneration of the Urodele Limb. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0001100.pub4]