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A strict coordination between pro- and antioxidative molecules is needed for normal animal physiology, although their exact function and dynamics during regeneration and development remains largely unknown. Via in vivo imaging, we were able to locate and discriminate between reactive oxygen species (ROS) in real-time during different physiological stages of the highly regenerative planarian Schmidtea mediterranea. All ROS signals were strong enough to overcome the detected autofluorescence. Combined with an in situ characterisation and quantification of the transcription of several antioxidant genes, our data showed that the planarian gut and epidermis have a well-equipped redox system. Pharmacological inhibition or RNA interference of either side of the redox balance resulted in alterations in the regeneration process, characterised by decreased blastema sizes and delayed neurodevelopment, thereby affecting tails more than heads. Focusing on glutathione, a central component in the redox balance, we found that it is highly present in planarians and that a significant reduction in glutathione content led to regenerative failure with tissue lesions, characterised by underlying stem cell alterations. This exploratory study indicates that ROS and antioxidants are tightly intertwined and should be studied as a whole to fully comprehend the function of the redox balance in animal physiology. [ABSTRACT FROM AUTHOR]