Regeneration: Growth Factors in Limb Regeneration

Abstract

The remarkable phenomenon of limb regeneration in vertebrates is limited to some, but not all, amphibians. Despite this, it is widely believed that the ancestor of all vertebrates was capable of limb regeneration, suggesting that this ability could be reawakened in humans. Research into limb regeneration has benefited from the use of two complementary model systems. The urodele amphibians (newts and salamanders) can regenerate perfect limbs even as adults, initiating a programme of wound healing, blastema formation and redevelopment. Urodeles can be used to investigate how common wound‐healing processes can subsequently activate a regeneration programme. Anuran amphibians (frogs and toads), on the other hand, progressively lose the ability to regenerate as they progress through their life cycle, and can be used in gain of function experiments to test potential regeneration factors. Using both of these models, growth factor signalling pathways have been identified as being involved in regulating all stages of the limb regeneration process.

Key Concepts

  • Regeneration of the limb is perfect in urodeles (newts and salamanders), declines with age in frogs and is absent in mammals.
  • Urodele regeneration is divided into three distinct phases, wound healing, blastema formation and redevelopment.
  • Mechanisms unique to regeneration are likely to be involved in the transition from phase I to phase II.
  • Growth factors are important in regulating all three phases of limb regeneration.
  • Amphibians are good models for studying the role of growth factors in limb regeneration, as both loss and gain of function experiments can be performed.

Keywords: regeneration; growth factors; blastema; axolotl, Xenopus

Figure 1. Regeneration of the urodele limb. (a) Skeletal preparation of a larval (approx. stage 54) axolotl, blue is stained cartilage and red is ossified bone. Forelimb on the right shows complete pattern of the skeletal elements, the stylopod or upper/proximal limb (humerus), zeugopod or middle limb (radius and ulna) and the autopod or distal limb (the wrist and hand, including carpals, metacarpals and phalanges). The left limb is undergoing regeneration after being amputated at the distal humerus by one of its siblings and has formed a blastema. The limb will regenerate perfectly in a few weeks. (b) Illustration of the three phases of limb regeneration in urodeles. Phase I, wound healing, phase II, blastema formation and phase III redifferentiation. Blastemal cells are formed from Schwann cells, satellite cells, dermal fibroblasts, chondrocytes (cartilage cells) and, in at least some cases, mononuclear myocytes (muscle cells derived from the muscle fibres). WE, wound epidermis; AEC, apical epithelial cap. Arrows in B show direction of epithelial cell migration to form the WE.
Figure 2. The ontogenetic decline in anuran hindlimbs. Summary of Dent's experiments using (Dent, ) where hindlimbs were operated at the level of the distal femur (dashed line) at stages 51 to 59 (left column, stage in bold black) and scored at metamorphic climax (right column, average number of digits in grey).
Figure 3. The froglet forelimb can be used to test factors for their ability to enhance regeneration. In these diagrams, only the skeletal structures are illustrated. (a) an uncut froglet forelimb has clear anterior–posterior pattern (digits II to V) and proximodistal pattern (from proximal to distal: humerus, radioulna, carpals, metacarpals and phalanges). (b) Amputation through the mid radioulna of a newly metamorphosed froglet results in the formation of a hypomorphic limb spike containing only cartilage, fully formed after 42 days. (c) Addition of a BMP containing bead to the stump can induce the formation of segmented cartilage, perhaps the forerunner of a joint (Satoh ., ). (d) Adding a hedgehog agonist to the stump results in the formation of branched, more complex cartilage (Yakushiji ., ). In (e) and (f), the stumps have received a patch of cells from a transgenic stage 53 (regeneration competent, see table) hindlimb, which express an activated beta catenin. These cells are therefore actively signalling through the canonical wnt pathway. The froglets used were derived from athymic tadpoles (thymus destroyed by cauterisation) in order to prevent rejection of the graft cells. (e) Stumps have received a cell patch, as well as shh and fgf10 applied on beads. Branching of the cartilages and the formation of disctinct segments were observed, as well as a degree of ossification and muscle cell regeneration (not shown). (f) Stumps that were treated as in (e) but also received thymosin B4 developed ossified structures resembling digits, joints, metacarpal and carpal‐like structures, indicating the partial recovery of anterior–posterior and proximodistal patterning (Lin ., ).
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Further Reading

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Yin VP and Poss KD (2008) New regulators of vertebrate appendage regeneration. Current Opinion in Genetics and Development 18: 381–386.

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Beck, Caroline W(Apr 2015) Regeneration: Growth Factors in Limb Regeneration. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0001104.pub2]