Regeneration of the Urodele Limb


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 in vivo 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.


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Further Reading

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

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Stocum, David L, and Crawford, Karen(Apr 2015) Regeneration of the Urodele Limb. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0001100.pub4]