IDENTIFYING THE CELLULAR AND MOLECULAR DYNAMICS INVOLVED IN BLASTEMA FORMATION IN THE ANNELID PRISTINA LEIDYI

Abstract

Annelids (segmented worms) include some of the most remarkable regenerators of the animal kingdom. Like many other animals, annelids regenerate by forming a blastema, a mass of proliferative undifferentiated cells at the wound from which new structures develop. However, how their blastema is initially formed is still poorly understood. Other animals with extensive regenerative properties such as flatworms employ migrating pluripotent stem cells to carry out regeneration, leading some to hypothesize the existence of a similar mechanism in annelids. To better understand where and when blastemal cells arise in annelids, I performed a fine-scale time series of cellular and molecular assays after head amputation in the freshwater oligochaete Pristina leidyi. I demonstrate that differentiated cells from multiple tissues near the wound begin expressing stem cell markers de novo within hours of amputation, and later start to undergo cell divisions in association with nerve outgrowth. Furthermore, proliferation studies did not support a pluripotent stem cell model. Together, these findings strongly support a model for annelid regeneration in which tissues adjacent to the wound contribute cells to the regeneration blastema in a lineage-specific manner. I then further investigated the origin of the mesodermal tissue in the blastema, which has been hypothesized to at least partly originate from migrating cells along the lateral body wall. I described a population of cells that, after injury, migrate along a lateral neuromuscular cord that expresses multiple wnt genes. These cells appear to contribute to the mesoderm of the blastema. I propose that these cells originate from a quiescent mesoderm-specific stem cell population. Finally, I investigated the role of cell signaling in anterior regeneration and found that overactivation of wnt signaling via pharmacological GSK3 inhibition after decapitation causes hyperproliferation and impairs differentiation in the regenerated head. This supports a conserved role of wnt signaling inhibition for properly differentiating anterior tissues. Overall, these findings support a model for annelid regeneration involving nerve-dependent dedifferentiation and lineage-restricted contributions, along with additional input from tissue-specific quiescent progenitors, strikingly similar to vertebrate limb regeneration.