As we discussed in Chapter 15, neural activity with target cells is required for synapse maturation and neuronal survival. Thus, it was initially postulated that neural activity might be the necessary stimulus for limb regeneration. However, experiments have shown that neural conductance (action potentials and release of acetylcholine) is not required to promote salamander limb regeneration (Sidman and Singer 1951; Drachman and Singer 1971; Stocum 2011). If neural activity is not required, then what are the nerve axons providing to the limb blastema? These neurons are believed to release factors necessary for the proliferation of the blastema cells (Singer and Caston 1972; Mescher and Tassava 1975). There have been many candidates for such a nerve-derived blastema mitogen, but probably the best candidate is newt anterior gradient protein (nAG). This protein can cause blastema cells to proliferate in culture, and it permits normal regeneration in salamander limbs that have been denervated (Figure 1; Kumar et al. 2007a). If activated nAG genes are electroporated into the dedifferentiating tissues of amputated limbs that have been denervated, the limbs are able to regenerate. If nAG is not administered, the limbs remain stumps. Moreover, nAG is only minimally expressed in normal limbs, but it is induced in the Schwann cells that surround the regenerating axons within 5 days of amputation.
Further support for the stimulatory role of nAG comes from the study of aneurogenic limbs. In this experiment, two embryonic salamanders were joined together by parabiosis, ensuring similar conditions and survival of both. In one of the salamanders, the neural tube was removed. Both salamanders survived this procedure, and all limbs grew successfully, but the limbs of the salamander lacking a neural tube were aneurogenic, completely devoid of any neural innervation. Based on the previous findings that denervated limbs are unable to regenerate, one would hypothesize that these aneurogenic limbs would likewise be unable to regenerate following amputation. Strikingly, these aneurogenic limbs did regenerate. When researchers compared the expression of nAG of normal limbs with that of both denervated limbs and these aneurogenic limbs, they discovered that the aneurogenic limbs had a uniquely high level of nAG expression in the epidermis (Figure 2A; Kumar et al. 2011). Moreover, upon limb amputation, nAG increased first in the nerve sheath of normal limbs, but was present throughout the blastema in the aneurogenic limbs (Figure 2B). These results suggest that nAG alone is the primary mitogen responsible for nerve-dependent regeneration. The receptor for nAG, Prod1, has since been discovered and found to be expressed in a proximal-to-distal gradient in the salamander limb (Morais et al. 2002; Kumar et al. 2007a and b). This ligand-receptor relationship seems to be conserved across regeneration-competent salamander species (Geng et al. 2015).
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