Salamanders are fascinating vertebrate organisms because they routinely regenerate complex tissues. Within only a few weeks of losing a piece of limb to a hungry predator or scalpel-wielding scientist, a salamander perfectly reforms the missing structure. In the early history of salamander regeneration research, scientists innovated elegant experimental designs to probe the anatomical basis of regeneration [1
]. More recently and in parallel with the discovery of conserved regulatory genes and developmental pathways among metazoans, scientists have focused attention on candidate molecules and signaling pathways whose functions were deduced first from studies of model organisms. In particular, much research has been devoted to understanding aspects of limb regeneration associated with wound healing that recapitulate limb development; this strategy has yielded many useful insights and molecular probes [2
]. Although it is clear that key regulatory molecules play important roles in the development of all organisms, it is not clear that a framework for understanding regeneration can be constructed using a generic and limited molecular toolkit. There is a need to go beyond candidate molecules and use unbiased approaches to characterize the molecular complexity underlying salamander regeneration.
Recent research resource development for the Mexican axolotl now allows classic regeneration experiments to be re-examined with powerful and unbiased genomic approaches. One particularly elegant experiment performed almost 200 years ago showed that salamander limb regeneration requires the presence of peripheral nerves. Todd [15
] found in 1823 that limb regeneration does not occur if the sciatic nerve of the hindlimb is severed shortly before or immediately after a more distal limb amputation. Subsequent research showed that the brachial nerves entering the forelimb are required to promote limb outgrowth and patterning of a new limb [16
]. Within a few days of limb amputation, cells proximal to the amputation plane dedifferentiate and accumulate to form a blastema. Blastema cells proliferate and progressively differentiate during regeneration to give rise to all mesodermal structures of a typical vertebrate limb [18
]. Upon amputation, nerve fibers in the vicinity of the amputation plane extend into the blastema and play a supportive role in cell proliferation [11
]. Transection of the spinal nerves that enter the limb in the axolotl leads to a decrease in cycling cells and there is resorption of distal tissues of the amputated limb. Histological and cell proliferation analyses suggest that early cellular events are similar between denervated and innervated limbs, but denervated limbs are incapable of blastema formation because they do not support significant cell proliferation and outgrowth [20
Although limb regeneration is a complex developmental process, nerve dependency and other aspects of regeneration have often been conceptualized as having a simple molecular basis, involving relatively few regulatory factors. For example, Ferretti and Brockes [26
] hypothesized that in the absence of nerves, Schwann cells produce an inhibitory factor that prevents blastema cell proliferation. This mechanism is supported by experimental results although the hypothetical factor has not been identified [27
]. Alternative mechanisms for nerve dependency have also been proposed for several factors with growth promoting effects [29
]. A recent study identified newt anterior gradient protein (nAG) as a blastema cell growth-promoting factor in vitro
, whose over-expression was sufficient to rescue regeneration of denervated and amputated limbs in vivo
]. Most recently, nerve-dependent expression of the transcription factor sp9
has been identified as an early marker of dedifferentiation of the wound epithelium and the initiation of limb regeneration [11
]. While considerable progress has been made in investigating the functions of candidate regulatory factors and signaling pathways, a broader systems-level perspective is needed to understand why multiple aspects of limb regeneration are dependent upon the presence of a nerve.
Genomic tools are now available that allow global characterization of the regeneration process in salamanders. Expressed sequence tag (EST) information has facilitated the development of an Ambystoma
salamander Affymetrix microarray platform [35
]. This platform and a high-throughput 454 cDNA sequencing approach were used in this study to compare transcript abundance among uninjured limbs, regenerating limbs, and limbs denervated at the time of amputation. The results show that innervated (NR) and denervated (DL) limbs exhibited similar (but not identical) gene expression patterns at five days post amputation (dpa) but then diverged as a blastema formed under the influence of nerves. The results are discussed within the context of previous studies of nerve dependency, highlighting specific genes and biological processes that are associated with blastema formation and outgrowth, and more generally, the Mexican axolotl's unparalleled ability to regenerate limbs.