In the field of regeneration two important and recurrent questions await for a satisfactory and “definitive” answer: 1) are there regeneration-specific genes that could be missed in species with poor regenerative capabilities? and, 2) up to what extend regeneration is a mere recapitulation of embryonic development? Most probably there will not be a common and unique answer for these questions that can be applied to all those classical models of regeneration.
In a recent paper from the laboratory of Elly Tanaka the authors have followed a high-throughput comparative transcriptomic approach to try to answer those questions in the axolotl limb regeneration paradigm (http://www.ncbi.nlm.nih.gov/pubmed/23658691). In this work the authors have carried out a comparative analysis of the transcriptional profiles of regenerating limbs, healing severe wounds and developing limb buds at different time points. Remarkably, the time course analysed has been impressively extensive with samples taken at 0, 3, 6, 9, 12, 24, 36, 54, 72, 120, 168, 288 and 528 hours after wounding or amputation.
Whereas most animals trigger a proper wound healing response after injury, only in few cases this wound healing is proceeded by the activation of a successful regeneration program. Therefore, by comparing the gene expression response to severe non-regenerative wounding (without amputation) to that triggered by amputation the authors sought first to identify common and distinct genes activated in those two contexts. One first remarkable result from these analyses is that they identified a molecular tripartite program that parallels the three phases of limb regeneration previously described based on morphological and cellular observations. Those phases comprise, basically: wound healing, blastema formation and growth of the new limb (with the establishment of a limb development program). This last phase resembles very much the normal growth of the limb bud during embryonic development. At the molecular level, the authors identified three distinct phases: 1) an initial phase up to 12 hours in which the genetic program activated after amputation resembles very much the activated program triggered by non-regenerative wounding; 2) from 24 up to 72 hours the amputation gene profile starts to diverge from the wound samples and, therefore, 24 hours represent the time point at which the regeneration-specific gene program is first discernible from the wound-healing program. And finally, 3) the amputated samples from 120 to 528 hours cluster more closely with the developing limb bud than to their corresponding wound samples. Thus, the 120-528 hours limb blastema would establish a limb development-like program.
A second interesting observation is that after amputation and non-regenerative wounding the behaviour of G1/S-phase genes and G2/M genes follow a similar dynamics in both context. Thus, the expression of G1/S genes rises to an initial peak at 72 hours whereas G2/M-associated genes peak at 120 hours. As this response is similar after amputation and wounding it seems reasonable to consider that this early proliferative response is associated to tissue injury. However, the samples coming from amputation display a second phase of expression of genes associated to proliferation that correlates to the phase in which the blastema is being formed. Thus, whereas after wounding there is just an initial proliferative response a second one appears to be specific for regeneration. As the authors point out these two waves of proliferation, one associated to injury and the second associated to regeneration and blastema formation resembles very much to what it has been previously described in freshwater planarians (http://www.ncbi.nlm.nih.gov/pubmed/20599901).
Next, the authors identified a set of 194 “regeneration-specific” genes, many of which fall into the GO categories, at the level of cellular function, of: 1) cellular stress, 2) chromatin associated factors, 3) epithelial function and differentiation, and 4) limb development module. Whole-mount in situ hybridizations validate the expression of many of those genes in the wound epidermis and the mesenchyme of the blastema. Finally, they wanted to identify those genes that would be present in the regenerative blastema but not in the limb bud. The reason for that is that although the growth of the blastema (once it is formed) into a new limb resembles very much the development of a limb, the initial phases of the blastema formation are quite different from the differentiation of the embryonic limb bud. Through comparative analyses they identified 20 genes that are expressed at low levels in the limb bud and are upregulated specifically after amputation. Many of those genes fall into the GO category of epidermal development and differentiation, which may highlight important functional differences between the epidermis of the blastema and the limb bud.
In summary the authors have identified here a set of regeneration-specific genes. Further analyses will determine the function of those genes during the phase of blastema formation in limb regeneration and may provide novel insights into this amazing process, not only in amphibians but also in other regeneration models.