GSK3 Inhibition During Annelid Head Regeneration Results in a Hyper‐Proliferative Blastema and Impaired Differentiation

A major goal of regeneration biology is to uncover the molecular mechanisms that allow a wide range of animals to replace body parts through regeneration. Glycogen synthase kinase-3 (GSK3) is known to have significant regulatory influence on developmental pathways that are integral to successful regeneration in several animal groups. Annelids include some of the most highly regenerative animals known and, although GSK3 has been studied during regeneration in several species, its major roles remain unclear. Here, we characterize the effects of 1-azakenpaullone (AZP), a known GSK3 inhibitor and Wnt signaling agonist, during anterior and posterior regeneration in the highly regenerative oligochaete annelid Pristina leidyi using morphological markers, cell proliferation assays, and RT-qPCR of stem cell and differentiation associated genes. Although posterior regeneration was only minorly affected by AZP treatment, anterior regeneration was strongly impaired, producing a persistent "hyper-blastema" that fails to differentiate anterior-specific structures through at least 13 days, more than twice the normal time period of complete regeneration. At time points when regeneration should be complete, this hyper-blastema still exhibits high levels of cell proliferation and stem cell-associated gene expression, resembling an early blastema. These results suggest that GSK3 inhibition suppresses anterior-specific differentiation pathways during later phases of regeneration, consistent with a conserved role for GSK3 in anterior development. The results also support GSK3's conserved role in promoting neural differentiation. More broadly, our study implicates GSK3 in opposing the blastema's pro-regenerative signals in annelids, broadening understanding of the molecular mechanisms underlying animal regeneration.