It is well known that denervated amphibian limbs do not regenerate which has lead to the idea of limb regeneration being nerve-dependent. In fact, this nerve dependency for regeneration has been suggested not only for amphibian limbs but also for many other organs and structures in several regeneration models. However, and until now, very few neural factors have been shown to explain satisfactorily this nerve dependency. On the other side, the fact that it has been hypothesized in different models suggests that nerve dependency could be a common and specific feature for animal regeneration.
Now, a recent paper from the laboratory of Akira Satoh reports on the cooperative roles of Bmp and Fgf signalling pathways to induce limb formation in the absence of innervation in urodeles (http://www.ncbi.nlm.nih.gov/pubmed/25286122). The model they have used is the Accessory Limb Model (ALM). In this model, when a piece of skin is wounded and if, at the same time, a nerve is rerouted to the wound this wound can be induced to form a regenerative blastema, that does not grow into any patterned structure, and in fact it regresses over time. However, if together with this ectopic nerve supply a small piece of skin from the contralateral side of the wound is grafted into this wound, then the formation of an overall well-patterned ectopic limb is induced.
Previous studies have shown that when different Fgfs (fibroblast growth factors) are applied to skin wounds, an initial regenerative response with blastema formation can be induced, even in the absence of a nerve supply. However, an ectopic limb is not formed in these cases. So, Fgfs are not sufficient to induce a complete regenerative response. Here, the authors analysed the regeneration inductive function of Fgfs and Bmps in skin wounds. First, they found that Fgf2, Fgf8, Bmp2 and Bmp7 were expressed in dorsal root ganglion neurons and, therefore, can be considered as nerve factors. Then, the authors applied different combinations of these Fgfs and Bmps to see whether or not they could induce blastema and limb formation in skin wounds in the absence of nerve rerouting. The application of beads soaked either with Fgf2 and Fgf8 or Bmp7 alone was able to induce a blastema but not limb formation. However, when Bmp2+Fgf2+Fgf8 or Bmp7+Fgf2+Fgf8 were applied together, those induced blastemas were capable to keep growing and formed a patterned limb with digits, in most cases. Those induced blastemas showed normal expression of the blastema markers Prrx1 and Msx1 and the ectopic limbs, although missing some skeletal elements, showed an overall proper pattern with quite normal innervation.
The use of specific inhibitors of the Fgf or Bmp signalling pathways blocked limb formation in these skin wounds in which beads soaked with Bmp7+Fgf2+Fgf8 had been applied. Interestingly, in those cases, blastema formation was not inhibited indicating that single input of Fgf or Bmp signalling was sufficient to induce this blastema formation. However, simultaneous activation of both Fgf and Bmp pathways was necessary to induce limb formation. Next, the authors investigated whether Fgfs and Bmps were capable of inducing a blastema in denervated limbs. After denervation by removing the brachial plexus at the forelimb level and skin wounding and skin grafting, they applied beads with Bmp7+Fgf2+Fgf8. As a result they observed the formation of ectopic blastemas positive for Prrx1 and Msx2. Those blastemas, however, did not keep growing into a limb. The probable reason for that is that they were not innervated, suggesting that the later growing phase was dependent on axons attracted by the induced blastema cells.
Finally, the authors show that the use of Bmp2+Fgf2+Fgf8 or Bmp7+Fgf2+Fgf8 rescued also the regeneration ability in a denervated and amputated limb. Similarly to the rescue observed after skin wounding in denervated limbs, these factors induced blastema formation in denervated and amputated limbs, with a normal regenerative mitogenic response. Identical inductive effects of Bmps and Fgfs were seen when using the ALM model in the newt Pleurodeles waltl, indicating that these factors could work as general transformative agents from skin wound healing to limb formation in urodele amphibians.
Overall, this study further supports the idea of Fgfs and Bmps as neural factors that may explain, at least partly, nerve dependency of amphibian limb regeneration. In addition to an initial important role of those neural factors in blastema formation, the results obtained here also indicate that nerves may play important roles at later stages of limb formation. Thus, in denervated limbs Fgfs and Bmps induce blastema formation but these never grow into limbs as those blastemas do not get innervated. Further analyses those determine if these same Bmps and Fgfs or other nural factors are responsible of promoting blastema growth into a limb after axons are attracted from the stump region into the regenerative blastema.