Role of Wnt/Beta-catenin Signaling Pathway in Post-amputation Cephalic Regeneration in the Annelid Worm Pristina leidyi

Regeneration is a developmental process that enables organisms to reconstruct lost body parts. During this process cell-to-cell signaling pathways play an important role in activating or deactivating cellular mechanisms required for regeneration, such as cell proliferation, formation of an undifferentiated blastema, and cell re-differentiation. The Wnt/β-catenin signaling pathway is well known for its involvement on cell fate specification during embryonic development in many species. We studied the role played by Wnt/ β-catenin signaling in regeneration of cephalic segments in the annelid worm Pristina leidyi. We propose that during regeneration in P. leidyi, activation of Wnt/β-catenin signaling promotes blastema formation and cell proliferation and inhibits cell re-differentiation. To test this hypothesis, we performed in situ m­RNA hybridization of P. leidyi β-catenin and found that expression of this gene is strongly up-regulated after amputation in the regeneration blastema. We then treated amputees with small-molecule inhibitor drugs targeting different components of Wnt/β-catenin pathway and measured the size and morphology of regenerates. We found that 2-20 uM 1-Azakenpaullone (AZK), a pathway activator, causes an increase in blastema size, and an inhibition in differentiation of internal structures, but no increase in cell proliferation as measured by bromodideoxyuridine incorporation. A different pathway activator, 0.25 uM Wnt Agonist, caused reductions in regenerate size and number of segments. These results support a role for Wnt/ β-catenin signaling on blastema formation and re-differentiation during cephalic regeneration in annelids, and will be followed up by analyzing changes on expression of β-catenin and stem-cell markers vasa and nanos after drug treatment.