Nerve independent limb induction in axolotls

Abstract

Urodele amphibians can regenerate their limbs. During limb regeneration, dermal fibroblasts are transformed into undifferentiated cells called blastema cells. These dermis-blastema cells show multipotency. Such so-called endogenous reprogramming of cell differentiation is one of the main targets of amphibian limb regeneration studies. It is well recognized that nerve presence controls the initiation of limb regeneration. Accordingly, nerve factors have been sought in amphibian limb regeneration. To investigate it, a relatively new study system called the accessory limb model (ALM) was developed. Using ALM, two signaling cascades (Fgf and Gdf5 signaling) came under focus. In the present study, Growth and differentiation factor-5 (Gdf5) application to wounded skin initiated limb regeneration responses and resulted in induction of a blastema-like structure in the absence of a nerve. However, the Gdf5-induced structure showed defects as a regeneration blastema, such as absence of detectable Prrx1 expression by in situ hybridization. The defects could be remedied by additional Fibroblasts growth factor (Fgf) inputs. These two inputs (Gdf5 and Fgfs) were sufficient to substitute for the nerve functions in the induction of limb regeneration. Indeed, Fgf2, Fgf8, and Gdf5 applications with the contralateral skin graft resulted in limb formation without nerve supply. Furthermore, acquisition of cartilage differentiation potential of dermal fibroblasts was tested in an in vivo and in vitro combination assay. Dermal fibroblasts cultured with Gdf5 were difficult to participate in cartilage formation when the cultured cells were grafted into cartilage forming region. In contrast, dermal fibroblasts cultured with Fgf2 and Fgf8 became easier to participate into cartilage formation in the same procedure. These results contribute to our understanding of molecular mechanisms of the early phase of amphibian limb regeneration.

A brief history of the study of nerve dependent regeneration

“…A 2013 study by Makanae et al showed that Gdf5 and FGFs are together capable of inducing blastema growth and limb formation in this model even in the absence of a deviated nerve. 61 Even more recently, Satoh et al have shown that FGF8 and Bmp7 are expressed at the ends of peripheral nerves, and knockdown of these factors inhibits blastema formation in regenerating limbs. 72 These findings reinforce the validity of the AL model as an analog to the amputated limb while demonstrating the necessity of FGFs and BMPs for nerve dependent limb regeneration.…”

Regulation of Regeneration by Heparan Sulfate Proteoglycans in the Extracellular Matrix

“…The recent discovery of the involvement of FGF (fibroblast growth factor) and BMP (bone morphogenetic protein) signaling [24–26] are encouraging in terms of being able to tease apart the specifics of how this early step in regeneration is controlled. Although an ectopic blastema can be induced to form in response to signaling from the deviated nerve and wound epithelium, it lacks the positional information to make a new limb since the cells recruited from around the wound site all share similar positional codes.…”

The Axolotl Limb Regeneration Model as a Discovery Tool for Engineering the Stem Cell Niche

“…This later step is dependent on signals from the nerve that interact with the newly formed wound epithelium to induce formation of a signaling center that recruits mesenchymal cells from the adjacent connective tissue (see [5,21,22]. This step that is dependent on nerve-associated signals in vivo can be regulated by the therapeutic delivery of a defined cocktail of growth factors [23-25]. In so doing, a deviated nerve is no longer required, and the downstream pathways (next steps in the regeneration cascade) leading to blastema formation can be targeted directly.…”

“…human recombinant growth factors induce ectopic blastema formation in the axolotl [23,25]). The lack of a regenerative response in humans may be a consequence of a failure to produce the necessary signals at the right time or place, but it is unlikely that there is a problem in the underlying pathways themselves.…”