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Molecular Basis for the Nerve Dependence of Limb Regeneration in an Adult Vertebrate

Anoop Kumar et al.

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Unlike human, salamanders can regrow an amputated limb. This regeneration occurs only if there is simultaneous regeneration of the severed nerves. Although we usually think of the nervous system as carrying information between nerve cells and their sensory and motor targets, nerves are also essential for tissue regeneration. When a salamander limb is amputated at any position, from the shoulder to the fingertips, the stump forms a blastema, a mound of stem cells from which regeneration begins. The nerve supply cut by the amputation also regenerates, and this regrowth is required for the proliferation of the blastemal cells. If the nerves are cut at the base of the limb, deeper than the regenerative tissue, the limb stump is permanently denervated, the axons cannot regenerate, and limb regeneration fails.

The nerve dependence of limb regeneration was discovered in 1823 by Tweedy John Todd, an English physician, and analyzed in the 1940s and 1950s by Marcus Singer. Nerves are required for many different sorts of regeneration in both vertebrates and invertebrates (1), perhaps to ensure that the regenerated tissue receives adequate innervation. Singer showed that either sensory or motor nerves could support regeneration and that neither conduction of the nerve impulse nor neurotransmitter release was required. The molecular basis for communication between nerves and regenerating tissue has been unclear, although various growth factors have been proposed as mediators. Our paper now identifies a protein that can rescue the denervated blastema and induce regeneration of the limb.

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Regeneration of salamander limbs requires concomitant regeneration of the severed nerves. A single protein, nAG, can substitute for the regenerating nerve cells.

Credit: C. Bickel/Science

The limb blastema gives rise to structures precisely distal to its site of origin (that is, a blastema at the wrist gives rise to a hand, not an upper arm). The stem cells of the blastema can thus be specified according to what structures are being formed, not just what cell types are needed. Analysis of this property led to the previous identification of Prod 1, a small protein anchored to the surface of blastemal cells and expressed in a gradient along the limb (more Prod 1 is found proximally and less at distal points). Prod 1, a member of a protein superfamily called Ly6, determines the proper identity of the regenerating tissues. To search for possible ligands for Prod 1, we conducted a screen in yeast cells and identified the n(ewt)AG protein. This candidate ligand, nAG, is a member of a large conserved family, the anterior gradient proteins, each of which contains a fold named after the protein thioredoxin and is secreted from cells.

With the help of antibodies reactive to two regions of nAG, we identified where in the regen-erating newt limb the protein is expressed. After amputation, the severed axons retract within the stump and then grow back along the nerve sheath (see the figure, A and B). The Schwann cells that make up the nerve sheath in this region express nAG (B and C) as the first blastemal cells divide (B). Later, nAG appears within gland cells in the specialized wound epidermis at the end of the limb (C). This epidermis is critical for sustaining later cell proliferation in the blastema. The appearance of nAG in both the Schwann cells and the wound epidermis is abrogated by denervation (D), showing that expression in both locations depends on axons. In isolated cells cultured from the blastema, nAG promotes proliferation (F). nAG can be artificially expressed in the denervated stump by injecting plasmid DNA that encodes nAG and passing an appropriate current across the limb, a procedure called electroporation that encourages cells to take up external DNA (D). In denervated blastema so treated, nAG is expressed widely and is secreted, and the otherwise missing glands in the wound epidermis are re-established (E). These results suggest that nAG that is normally released from the Schwann cells during regeneration induces the glands (B, C). Most striking, the artificial expression of nAG rescues the denervated blastema so that the limb regenerates through the entire missing proximal-distal axis and forms digits (G). Therefore, the requirement for the presence of nerve cells to achieve normal, regenerated limb shape and structure can be met by expression of a single protein–nAG.

A geneticist might prefer to see the function of nAG tested by leaving the nerve intact and knocking out the expression of the nAG gene in the relevant cells after amputation. This manipulation is not possible on the salamander at present, but the rescue experiment we report may hold promise for future efforts to promote limb regeneration in mammals (2).

Summary References

  1. B. M. Carlson, Principles of Regenerative Biology (Elsevier, London, 2007).
  2. A. Parson, J. Life Sci., p. 60 (September 2007).

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