Cytoskeletal Regulation of Neoblast Dynamics during Homeostasis and Regeneration in Schmidtea mediterranea

The planarian flatworm Schmidtea mediterranea is a powerful model for studying adult stem cell biology because its exceptional regenerative capacity depends on neoblasts, a heterogeneous population of proliferating cells able to sustain tissue homeostasis and replace missing structures after injury. Previous studies have established that neoblast proliferation, migration, differentiation and spatial organization are tightly coordinated during regeneration, while cytoskeletal dynamics and specific gene regulatory programs contribute to the control of these processes. Building on this framework, this work aimed to examine how neoblast behavior changes when key cellular mechanisms are experimentally perturbed and to connect these responses with stem cell persistence and regenerative competence. The first approach focused on microtubule disruption using colchicine and nocodazole. Since microtubules are essential for mitosis, intracellular organization, and cell morphology, these treatments were used to test how interference with the cytoskeleton affects neoblast proliferation, distribution and recovery. The observed responses were consistent with the known anti-mitotic effects of these compounds, while also extending previous observations by showing changes in piwi-1 and H3P-positive cell distribution, wound-associated responses and the morphology of Piwi1- positive cellular protrusions. A second approach investigated the behavior of selected FACS-isolated neoblast populations after transplantation into lethally irradiated planarians. This strategy was designed to explore differences in the ability of isolated cell fractions to survive, persist, and maintain stem cell-associated features within a depleted host environment, thereby providing additional information on the functional heterogeneity of neoblast populations. Finally, an RNA interference screen was performed on genes enriched in neoblasts to assess their possible contribution to stem cell maintenance and regeneration. The resulting phenotypes supported the importance of several candidate genes already associated with cellular homeostasis, while also identifying targets that warrant further investigation. Together, these complementary approaches investigated neoblast biology from cellular, functional and molecular perspectives during regeneration, perturbation and recovery. Overall, this work confirms established principles of neoblast biology and expands them by integrating cytoskeletal perturbation, transplantation, and functional gene screening within a single experimental framework.