Our identification of nAG as a ligand for the PD determinant Prod 1 has underlined that patterning and cell division are linked at the molecular level. We envisage that PD identity is manifested by the quantitative gradation of Prod 1 (6
), and that nAG has no role in specifying that identity but acts through Prod 1 to promote cell division. Blastemal growth is stimulated in experimental confrontations of cells differing in positional identity, for example in PD intercalation where a wrist level blastema is grafted onto a shoulder stump (31
), and this is always dependent on the presence of the nerve. In a recent study of supernumerary limb formation in the axolotl, the deflection of the brachial nerve into a skin wound provided a growth stimulus to form an ectopic blastema or ‘bump’; such bumps only progress to form limbs if a piece of skin is grafted from the contralateral skin to the wound site so as to provide dermal fibroblasts of disparate identity (14
). It is interesting that dermal fibroblasts express Prod 1 and this expression is upregulated by retinoic acid (6
Two prior studies on Anterior Gradient proteins are relevant to the present results. First, the human AG2 protein was used as bait in a yeast two hybrid assay and found to complex with a GPI-anchored protein called C4.4 which is associated with metastasis (24
). This protein has two Ly6-type domains which are related in sequence to urokinase-type plasminogen activator receptor (uPAR) (34
). The degree of relatedness between the three dimensional structures of Prod 1 and C4.4 domains is not yet resolved, but taken together these results suggest that functional interactions between AG proteins and this class of small Cys-rich protein domains may be conserved.
In the second study it was found that overexpression of the XAG2 protein in early cleavage stage Xenopus
embryos could induce formation of an ectopic cement gland which expressed XAG2 (22
). We find that expression of nAG induces formation of nAG positive glands in the denervated newt wound epidermis. After amputation, nAG appears first in the Schwann cells of the distal nerve sheath and then in glands in the wound epidermis. If axonal regeneration is prevented by denervation, neither the Schwann cell nor the glandular expression is detected. Our results suggest that nAG is released by the distal sheath and induces the formation of glands in the wound epidermis. It appears that the secreted nAG acts directly on the limb blastemal cells. It is unclear how the regenerating axons act on the sheath cells, although the membrane form of neuregulin is a candidate in view of its importance for such interactions (35
). The nerve dependence of regeneration offers a distinction between limb development and regeneration, since the outgrowth of the limb bud is not dependent on its innervation (36
). Nonetheless the ingrowth of the nerve is critical for establishing the nerve dependence, as shown in elegant transplantation experiments on axolotl larvae (37
). The identification of nAG offers a new opportunity to study the mechanisms underlying this switch.
Nerve dependence of regeneration is conserved in phylogeny. It has been studied in regeneration of the fish fin, the taste barbel in catfish, the arms of crinoid and asteroid species in echinoderms, and the body axis in annelids (28
). In most vertebrate appendages the density of innervation is lower than in salamanders and Singer suggested that this is a primary determinant for the loss of regenerative ability, for example in mammals (9
). This hypothesis now seems unlikely as there are other variables apparently curtailing regeneration (1
). It is striking that the expression of a single protein can rescue limb regeneration in an adult animal (), and this finding underlines that the blastema is an autonomous unit of organisation for which there is no obvious mammalian counterpart. We have suggested that one approach for regenerative medicine would be to understand the specification of the blastema at a level of detail that would allow it to be engineered in mammals (1
). The local delivery of permissive regulators like nAG could then evoke formation of the appropriate structures without the need for subsequent intervention.