Reactive Oxygen Species have been implicated in multiple cellular processes. A disturbed redox balance has been associated to cancer and neurodegenerative diseases. On the other hand, redox signalling may also positively modulate some important processes such as immune response, neurological functioning and wound healing. Recently, some papers have shown that ROS play an important role as early triggers of the regenerative response in vertebrates such as Xenopus and zebrafish. In them, ROS seems to play a positive action on cell proliferation, apoptosis and/or the activation of signalling pathways such as Wnt and JNK that ultimately lead to proper blastema formation and regeneration. Now, a recent paper from the laboratory of Karen Smeets reports on the role of ROS in freshwater planarians, which are capable of regenerating a whole animal from a tiny piece of their bodies (http://www.hindawi.com/journals/omcl/2015/392476/abs/).
Firs the authors used carboxy-H2DCFDA to visualize ROS levels during regeneration. Thirty minutes after amputation ROS were detected at the wound regions. Treatment with two types of inhibitors: DPI (a non-specific flavoprotein inhibitor) and APO (an inhibitor of NOX-like enzymes) blocked the induction of ROS at the wound region after amputation. This drug-mediated inhibition of ROS resulted in severe problems of regeneration visualized by the formation of very small blastemas. In contrast, treatment with BSO (an inhibitor glutathione synthesis) and oligomycin A (an inhibitor of ATP synthase) that promote ROS production did not cause any regeneration defect, despite causing an overproduction of ROS at the wound region. In intact, non-regenerating animals treatment with DPI or APO resulted in head regression and lesions ultimately leading to animal death.
DPI treatments at different times before or after amputation indicated that ROS inhibition did not affect wound closure or early stages of regeneration. In contrast to other regeneration models in which ROS inhibition decreases cell proliferation, this does not seem to be the case in planarians. In planarians, regeneration depends on the presence of totipotent stem cells, called neoblasts. No differences in mitotic rates were seen during the first 3 days of regeneration and in intact animals in DPI-treated animals compared to controls. Also, FACS analyses showed no differences in the stem cell population. Next, the authors investigated whether ROS inhibition could be affecting cell differentiation. By using an antibody against SMEDWI-1, a piwi homolog, specifically expressed in neoblasts the authors found that after DPI treatment SMEDWI-1 positive cells accumulated within the blastema compared to controls. In a normal situation the number of SMEDWI-1 positive cells within the blastema decreases over time as those cells fully differentiate into the different cell types. Therefore, an accumulation of SMEDWI-1 within the blastema is interpreted as neoblasts having problems in differentiating. In the case of ROS inhibition this was further supported by the fact that when using markers of early neoblast progeny, those were also significantly reduced. Overall, these results suggested that ROS inhibition might be affecting normal cell differentiation.
Next and to further analyse the possible function of ROS on cell differentiation the authors turned into the central nervous system (CNS). ROS inhibition resulted in a significant reduction of the brain size and in the number of specific neuronal populations as well as defects in the normal pattern of the brain. Remarkably, in some cases the authors found an ectopic expression in posterior blastemas of anterior neural markers. For this reason, the authors studied the polarity determinants Smed-notum and Smed-wnt1 to see whether polarity was affected after ROS inhibition. In anterior blastemas the expression of Smed-notum was not affected during the early stages of regeneration in which polarity is re-established. Interestingly, in few animals Smed-notum was expressed in posterior blastemas by day 1, an expression that was not observed in controls. These results suggest that ROS could be important for posterior identity, although further experiments would be necessary to better understand the role of ROS on the re-establishment of axial identity during planarian regeneration.
In summary, this paper describes the effects that ROS inhibition has on planarians, a model for whole body regeneration. Future experiments should help to clarify the exact role of ROS controlling cell differentiation and patterning in these animals.