Simulation of adult limb regeneration with lizard tail spinal cord implants reveals distinct roles of radial glia and microglia populations.

Lizards are the closest relatives of humans able to suppress fibrosis and regrow multiple tissue lineages following appendage regeneration. As amniotes capable of tail, but not limb regrowth, lizards are also distinguished as the only vertebrate group that include both regenerative and non-regenerative appendages in the same animal. Lizard tail stumps naturally form blastemas - heterogenous collections of fibroblasts, adult stem cells, and immune cells that suppress scar formation and potentiate new tissue growth. Conversely, amputated lizard limbs form scars similar to those observed in human patients. Lizard blastema formation is dependent upon tail spinal cord tissue, which contains distinct populations of radial glia and microglia. Using the parthenogenetic lizard Lepidodactylus lugubris as a platform for tail-to-limb spinal cord implantations, we developed an ectopic blastema model toward defining the roles of radial glial and microglia in appendage regeneration. Removal of either population inhibits fibroblast proliferation and blastema formation, but only microglia depletion leads to enhanced fibrosis. Similarly, effects of radial glia, but not microglia, depletion on fibroblast proliferation are reversed via Hedgehog agonism. Taken together, these results indicate that lizard limbs contain all the necessary cell types and biological responses necessary for blastema formation but lack the proliferative and anti-fibrotic signals provided by tail spinal cord radial glia and microglia, respectively. Radial glia contribute Hedgehog signals that cause fibroblast proliferation but do not affect fibrosis. Conversely, microglia enhance fibroblast sensitivity to Hedgehog signaling and inhibit differentiation into fibrocytes. In summary, this study demonstrates blastema stimulation in amputated limbs of adult amniotes with application of lizard spinal cord cells and holds promise as a blueprint for limiting painful scarring and supporting new tissue growth following amputation injuries in human patients.