FGF, BMP, and RA signaling are sufficient for the induction of complete limb regeneration from non-regenerating wounds on Ambystoma mexicanum limbs

Some organisms, such as the Mexican axolotl, have the capacity to regenerate complicated biological structures throughout their lives. Which molecular pathways are sufficient to induce a complete endogenous regenerative response in injured tissue is an important question that remains unanswered. Using a gain-of-function regeneration assay, known as the Accessory Limb Model (ALM), we and others have begun to identify the molecular underpinnings of the three essential requirements for limb regeneration; wounding, neurotrophic signaling, and the induction of pattern from cells that retain positional memory. We have previously shown that treatment of Mexican axolotls with exogenous retinoic acid (RA) is sufficient to induce the formation of complete limb structures from blastemas that were generated by deviating a nerve bundle into an anterior-located wound site on the limb. Here we show that these ectopic structures are capable of regenerating and inducing new pattern to form when grafted into new anterior-located wounds. We additionally found that the expression of Alx4 decreases, and Shh expression increases in these anterior located blastemas, but not in the mature anterior tissues, supporting the hypothesis that RA treatment posteriorizes blastema tissue. Based on these and previous observations, we used the ALM assay to test the hypothesis that a complete regenerative response can be generated by treating anterior-located superficial limb wounds with a specific combination of growth factors at defined developmental stages. Our data shows that limb wounds that are first treated with a combination of FGF-2, FGF-8, and BMP-2, followed by RA treatment of the resultant mid-bud stage blastema, will result in the generation of limbs with complete proximal/distal and anterior/posterior limb axes. Thus, the minimal signaling requirements from the nerve and a positional disparity are achieved with the application of this specific combination of signaling molecules.