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ABSTRACT
Prohibitins (PHBs) are a family of evolutionary conserved proteins that play a key role in multiple cellular processes including cell cycle regulation, cell surface signalling, senescence, apoptosis, regulation of sister chromatid cohesion and mitochondrial function.
Moreover, it has been hypothesized by proteomic analysis an in vitro correlation between PHB expression and the maintenance of undifferentiated and proliferating hepatic stem-like (HSL) cells. Up to now, no data are available about PHB’s in vivo role in adult stem cell biology.
Recently, it has been demonstrated that the planarian PHB2 homologous (DjPHB2) is expressed in a population of adult pluripotent stem cells, the neoblasts. The presence of neoblasts makes planarians a sound model system for in vivo investigations of adult stem cell biology. Indeed, neoblasts are the only proliferating cells of planarians and they are able to differentiate into all cell type. This stem cell population is involved in both homeostasis any regenerative process, during which neoblasts actively proliferate to create a blastema from which lost body parts will be rebuilt.
5 Aim of this work was to investigate in vivo the function of DjPHB2. Firstly, the complete DjPHB2 gene sequence was obtained using the 3’-5’ RACE technique. Then, DjPHB2 function was analyzed in both intact and regenerating planarians using RNAi.
The results showed that DjPHB2 silencing induced the death in both intact and regenerating planarians. By analyzing the mitotic index and using immunohistochemistry, in situ hybridization experiments and electron microscopy, we demonstrated a significant reduction in the neoblast number in intact planarians. In particular, neoblasts scattered through out the parenchyma as well as those accumulated in dorsal lateral clusters were reduced in numbers. Neoblasts were always detected in clusters accumulated along the body midline.
When DjPHB2 RNAi planarians were amputated (first regeneration), animals were able to regenerate. However, a reduction in the neoblast number was observed in these injected animals similarly to what observed in intact animals: scattered neoblasts were progressively reduced and, then, they only were restricted in clusters accumulated along the body midline.
Finally, DjPHB2 RNAi planarians at the second regeneration showed regenerative defects: head fragments were not able to produce a blastema, while tail fragments showed a delay in regeneration and morphogenetic defects with respect to the controls. Although in RNAi animals a significative reduction in the neoblast number occurred, these cells were always present at the body midline, also in animals lacking a blastema.
In conclusion, the findings obtained demonstrate that DjPHB2 is a neoblast molecular marker. The RNAi experiments suggest that DjPHB2 is involved in the maintenance of both neoblasts scattered through the parenchyma and
6 accumulated in lateral clusters. However, it is not involved in the maintenance of the neoblasts accumulated along the body midline. Indeed, this population of cells is always present in DjPHB2 RNAi animals.
Taking into account that in situ hybridization experiments indicate that DjPHB2 is expressed in all neoblasts, also in those located along the midline, a
hypothesis is that DjPHB2 plays different roles in different cell populations.
However, it is not possible to completely rule out the presence of a DjMCM2- and DjPiwi-1-positive neoblast sub-population, located along the midline, which does not express DjPHB2.
