Sub-Lethal 5-Fluorouracil Dose Challenges Planarian Stem Cells Promoting Transcriptional Profile Changes in the Pluripotent Sigma-Class Neoblasts

Abstract

Under physiological conditions, the complex planarian neoblast system is a composite of hierarchically organized stem cell sub-populations with sigma-class neoblasts, including clonogenic neoblasts, endowed with larger self-renewal and differentiation capabilities, thus generating all the other sub-populations and dominating the regenerative process. This complex system responds to differentiated tissue demands, ensuring a continuous cell turnover in a way to replace aged specialized cells and maintain tissue functionality. Potency of the neoblast system can be appreciated under challenging conditions in which these stem cells are massively depleted and the few remaining repopulate the entire body, ensuring animal resilience. These challenging conditions offer the possibility to deepen the relationships among different neoblast sub-populations, allowing to expose uncanonical properties that are negligible under physiological conditions. In this paper, we employ short, sub-lethal 5-fluorouracil treatment to specifically affect proliferating cells passing through the S phase and demonstrate that S-phase slowdown triggers a shift in the transcriptional profile of sigma neoblasts, which reduces the expression of their hallmark sox-P1. Later, some cells reactivate sox-P1 expression, suggesting that some neoblasts in the earlier steps of commitment could modulate their expression profile, reacquiring a wider differentiative potential.

Cell quiescence in planarian stem cells, interplay between p53 and nutritional stimuli

“…Indeed, planarians change their body size depending on the nutritional status: feeding induces ASC proliferation that results in an increase in cell number and in animal growth, while following starvation, the number of cells decreases and body size shrinks leading to degrowth [52,53], suggesting that energy metabolism is a central cue for proliferation control, thus linking the control of cell quiescence to its original evolutionary origin in unicellular organisms. DMC cells are not significantly activated during the regeneration process [18,29], neither their poor proliferative activity seems to play an essential role in the repopulation process following ASC partial depletion [36]. On the contrary, here we demonstrate that these cells respond to nutritional stimuli, not as a direct consequence of intestinal filling but belatedly, suggesting the intermediation of some still unknown molecular signal.…”

“…Five days after irradiation some DjPiwiA -positive cells are still detectable in planarian body region unrelated to the DMC (figure 1 d ). Strikingly, DCM cells are resistant to the treatment with high doses of the genotoxicant 5-fluorouracyl (5FU) (5FU CT6000) [35,36] that specifically affect cells during their transit through the S phase of the cell cycle [37], thus suggesting that DMC cells are someway arrested or cycle at a very slow rate (figure 1 d ). Another scenario in which DMC cells show higher resistance with respect to dispersed neoblasts is following the silencing of the prohibitin 2 homologue DjPhb2 [38].…”

“…Accordingly, mitoses are almost undetectable (figure 3 c ). However, some rare dividing cells are detectable among DMC cells in challenging conditions after 5FU treatment [35,36], thus allowing to exclude that they are all definitely committed post-mitotic progenies and suggesting that some specific stimuli might trigger, at least for some of them, proliferative activity. Also in the case of DjPhb2 silencing, a dramatic drop in S phase (figure 4 a , b ) and mitosis (figure 4 c ) is detectable, together with a significant decrease in G2 cells, while G1 cells increase proportionally (figure 4 b ).…”

Microtubule-associated protein 1B is implicated in stem cell commitment and nervous system regeneration in planarians

“…At the end of the cycle, in the case of asymmetric division, a newly produced neoblast could reenter the cell cycle and specialize towards a different cell fate suggesting that a specialized neoblast could reacquire pluripotency [ 5 ]. Additional evidence in support of such plasticity have been recently provided [ 6 , 7 ].…”

“…DNA templates for DjMCM2 , Djgata4/5/6 , DjCoe , DjNB-21 . 11 and DjEgr-1 were prepared by RT-PCR as described in Salvetti and coworkers [ 23 ], Gambino and coworkers [ 22 ], Gambino and coworkers [ 24 ], Cassella and coworkers [ 20 ] and Gambino and coworkers [ 7 ], respectively. DjMap1B template was obtained using the following primers:…”