Regeneration is the ability of an organism to replace lost or damaged structures without scar formation. This extraordinary capacity is possessed by only a few animals among which the planarian species Schmidtea mediterranea. The most upstream identified regulator of regeneration are reactive oxygen species (ROS). To characterize ROS, we have visualized one specific ROS-form being hydrogen peroxide (H202) at both regenerative- and healing wounds. ROS subsequently activates the MAPK/ERK pathway leading to regeneration. To demonstrate the interaction of different factors in this pathway and their gradients, we cut the worms in various ways and inhibited MEK/ERK with PD0325901.
Analysis indicated that there is impaired regeneration from head to tail. By combining MEK/ERK inhibition with ОІ-catenin knockdown, we showed a rescue mechanism of the inhibited phenotype. Although further observations are needed, it appears that: hydrogen peroxide plays a role in regeneration possibly by activating the MAPK/ERK pathway, other factors might take over the role of ERK in regeneration after MEK/ERK inhibition and when we combine different inhibitions, a partial normalization of regeneration occurs.
Inroduction
Regeneration
Planarians are becoming a widely used model in scientific research. This is mainly due to their extraordinary regenerative capacities. Regeneration is the process by which animals can replace lost or damaged structures without scar formation (1). The regenerative ability of planarians is the result of the presence of neoblasts (pluripotent adult somatic stem cells). These cells make up approximately 30% of all adult cells in their body and can migrate and differentiate into all cell types (2, 3).
Active neoblast proliferation is essential in blastema formation and regeneration (4, 5). A blastema is an unpigmented regeneration bud at the wound location that eventually grows and replaces the missing tissue (6). Besides planaria, there are other species that can regenerate missing or damaged body parts. Some invertebrates like Hydra and even some vertebrates like Xenopus, , and zebrafish (7-10). Mammals such as humans usually have limited regenerative abilities. They can repair some damaged tissues, but cannot replace whole body parts (11). All of these model organisms are essential for understanding the complex regeneration mechanisms, but we will focus on the master of them all: Schmidtea mediterranea.
Schmidtea mediterranea
When S. mediterranea. is cut into pieces, each piece develops into a fully functional worm. In approximately seven days after amputation, this species can regenerate almost any part of their body, including a fully functional brain (2, 11, 12). Additionally, the fact that much is known about their genome, makes S. mediterranea an interesting animal model to study regeneration (13). The main advantage, however, is the possibility to study this master regenerator in vivo. It enables the researchers to extensively explore the regeneration process and the involved stem cells (14). There is more to the regeneration process than one would think. It is a strictly controlled complex of pathways and networks that collaborate to recreate a fully functional organism. The most upstream identified regulator in this process has been found to be reactive oxygen species (ROS) (15).
Reactive oxygen species in regeneration
ROS are oxygen-derived chemicals that are considered to have important roles in development and regeneration (12, 16). They regulate cell signaling, proliferation, migration, cell death, immune signaling, etc (16, 17). Nonetheless, there is also a negative side to them called oxidative stress. When in disbalance with antioxidants (in favor of ROS), ROS can cause damage to proteins, lipids, membranes, nucleic acids, and organelles and consequently lie at the heart of many pathologies (12, 18, 19). In planaria, Pirotte et al. demonstrated that ROS are produced after amputation. They elucidated that ROS signaling is essential for neoblast differentiation and nervous system regeneration, but not for neoblast proliferation (12).
The importance of ROS in regeneration has not only been proven in planaria but also in other species. Love et al. showed that ROS are required for successful Xenopus tadpole tail regeneration (15) and Sehring et al. demonstrated the same for zebrafish fin regeneration (20). There is no question about the importance of ROS, but this research would like to focus on a specific type of ROS called hydrogen peroxide (H2O2). H2O2 is the preferred reactive oxygen species as it is able to cross cell membranes via aquaporins and has been shown to activate the extracellular signal-related protein kinase phosphorylation cascade (pERK) in planarian regeneration (21, 22). The process of regeneration, however, is more than just ROS activating one specific signaling pathway, but consists of a complex orchestra of interactions between numerous pathways and molecules.
Regeneration pathways
Some of the main signaling pathways in planarian regeneration are the MAPK/ERK and the Wnt/ОІ-catenin pathway. Both are necessary for proper regeneration, but the link between them has never been completely elaborated before. The Wnt/ОІ-catenin pathway is important to instruct anteroposterior polarity during homeostasis but is also upregulated at the wound site after injury. Wnt activates ОІ-catenin, which is mainly present posteriorly and gradually decreases towards the anterior (11, 23). The ERK gradient has been hypothesized to be inversely related to Wnt/ОІ-catenin (24, 25). This MAPK/ERK pathway is, among others, activated by ROS. When ROS bind to the EGFR3-receptor (endothelial growth factor 3), MAPK/ERK kinase (MEK) is phosphorylated and consequently activated.
The phosphorylation cascade goes on activating ERK and eventually the early growth response 4 (egr4) transcription factor, which leads to a successful regenerative outcome. (26, 27). Even though it is known that these pathways play important roles in regeneration, the interaction between them and the gradients of their components remains mostly unexplored. Pietak et al. did find that ОІ-catenin inhibits ERK anteriorly (28). Here, we will try to demonstrate the interrelation between the different factors of these pathways and their gradients. Furthermore, we will investigate the presence of hydrogen peroxide in the worms and how it affects components of the previously mentioned mechanisms. Our hypothesis states that amputation induced ROS production activates the MEK/ERK signaling pathway via a gradient along the anteroposterior axis and is linked with the existing ОІ-catenin gradient.
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