A spatiotemporal blueprint of the planarian redox status: from drivers to physiological responses

The process of animal regeneration is characterized by the total recovery of lost or damaged tissues, organs, or entire body parts. Regenerative success, defined as a correct restoration, integration, and orientation of new tissues, requires a strict coordination of cell cycle dynamics and corresponding events. To date, researchers often only focus on the transcriptional changes during the different stages of regeneration, but upstream signals remain poorly understood. It is crucial to further explore upstream regulating events such as redox-related processes and map their dynamics in regenerating and non-regenerating organisms. In this PhD, I used the planarian Schmidtea mediterranea as a model organism because of its unique regeneration capacity. This organism is able to regenerate almost any missing tissue in only 7 days. Reactive oxygen species (ROS) are crucial messenger molecules during planarian regeneration and are kept in balance by antioxidative mechanisms. To date, researchers often only focus on the pro-oxidative part of the redox balance. It is crucial to further explore redox- related processes as a whole, to gain insights into the interplay between ROS and their antioxidant counterparts. The goal of my PhD is to determine the first steps towards a spatiotemporal blueprint of the planarians redox state, from drivers to physiological responses. In the first part of this study (Chapter I), I focused on how to monitor fast-changing redox parameters. I developed an inexpensive, easy, and reproducible method for live imaging in planarians. Immobilization in low melting point agarose allows to image in vivo time lapses without the need for anesthetics. The method is designed to allow functional and physical interference with the specimen during live imaging, in addition to being able to easily recover the organism after the imaging procedure. I further optimized the protocol for real-time in vivo imaging of general ROS production, as well as superoxide, and hydrogen peroxide production. In Chapter II, I performed an in-depth characterization of both the pro- and antioxidative part of the planarian redox balance during regeneration and homeostasis. Interfering with either side of the redox balance resulted in regenerative defects that were more pronounced in anterior blastemas compared to posterior blastemas. Adult planarians showed a high redox activity predominantly in the intestine and epidermis, which both play an important role in defense against external stressors. In the next part (Chapter III), I demonstrated how this amputation- and wound-induced ROS/H2O2 production triggers downstream signaling events such as the EGFR-MAPK-ERK signaling pathway during planarian regeneration. ROS, either by treatment with exogenous H2O2 or photomodulation therapy, have the potential to rescue regeneration in MEK-inhibited tails. I demonstrated that ROS and/or H2O2 activate the MAPK/ERK pathway during the early stages of regeneration, and that the EGFR pathway mediates ROS production and MAPK/ERK activation during planarian regeneration. In Chapter IV, I demonstrated how the ROS-mediated MAPK/ERK pathway is involved in pattern establishment. H2O2 production was higher and more prolonged in anterior blastemas compared to posterior blastemas. In addition, differences in H2O2 kinetics were detected depending on the position of the blastema along the A-P axis. Corresponding results were demonstrated on the level of MAPK/ERK activation, showing strong position-dependent effects of MEK inhibition. Knocking down β-catenin, responsible for posterior regeneration, rescued MEK inhibition-induced regenerative defects. These data suggest the presence of a ROS-controlled ERK gradient, presumably inversely related to the β-catenin gradient, in order to establish a correct antero-posterior axis. In the last chapter, Chapter V, I explored how the endogenous fluorophore riboflavin can be used as a redox marker in planarians. I visualized the presence of riboflavins and/or riboflavin-like molecules within a complex, yet undescribed system. The system is located peripherally in the head area, has a nerve-like morphology, and responds to various chemical and physical stimuli. It probably plays a role as a peripheral sensory organ. Strong adverse effects on planarian cognitive function and regeneration were observed after interfering with the riboflavin availability or related mitochondrial functioning, indicating their importance in planarians. In summary, the data presented in this PhD show the importance of the dynamic redox system in different physiological events in the planarian Schmidtea mediterranea, and the role of the ROS-mediated MAPK/ERK pathway in regeneration initiation and patterning. The thesis shows the value of monitoring dynamic processes such as ROS activity or the use of endogenous fluorophores, via real-time in vivo imaging. The spatiotemporal blueprint of the planarian redox system is far from fully mapped. However, this thesis is a beginning, as even the longest way starts with the first step.

Jaar van publicatie:2022

Toegankelijkheid:Embargoed