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The study investigates the role of Extracellular Signal-Regulated Kinase (ERK) in the regeneration process of Schmidtea mediterranea planaria. Various experiments were conducted to assess the gradient of ERK expression along the anteroposterior axis and its impact on blastema formation.
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Additionally, the study explores the interaction between ERK and ОІ-catenin in planarian regeneration. The results indicate a distinct ERK gradient and highlight the rescuing effects of ОІ-catenin in MEK-inhibited fragments, shedding light on the significance of ERK in regeneration.
Schmidtea mediterranea, a model organism for regeneration studies, possesses remarkable regenerative capabilities, including the ability to regrow entire body parts from small fragments.
Regeneration in planaria involves complex signaling pathways and cellular processes. One crucial player in this process is Extracellular Signal-Regulated Kinase (ERK), a kinase involved in various cellular functions.
This study aims to elucidate the role of ERK in planarian regeneration. Specifically, we investigate the presence of an ERK gradient along the anteroposterior axis and its influence on blastema formation. Additionally, we explore the interaction between ERK and ОІ-catenin, a key regulator of head formation in planaria.
The strain used for these experiments is the asexual Schmidtea mediterranea. The animals were housed in a dark room at a constant temperature of 20°C. They were fed veal liver every seven days and starved for 7 days before the experiments.
The medium was prepared by adding the following components to Milli-Q water:
| Component | Concentration |
|---|---|
| NaCl | 1.6 mM |
| CaCl2 | 1 mM |
| MgSO4.nH2O | 1 mM |
| MgCl2.6H2O | 0.1 mM |
| KCl | 0.1 mM |
| NaHCO3 | 1.2 mM |
Nonspecific inhibition of Reactive Oxygen Species (ROS) was achieved using Diphenyleneiodonium chloride (DPI).
DPI was dissolved in dimethylsulfoxide (DMSO), with control groups exposed to 0.1% or 0.33% DMSO. Worms were treated with 3µM or 10µM DPI for 60 minutes before and after amputation. To inhibit the MEK/ERK pathway, PD0325901 was used, also dissolved in DMSO. Worms were exposed to a 10µM PD0325901 solution for 60 minutes before and four days after amputation.
Peroxy Orange 1 (PO1) was used to visualize the in vivo production of hydrogen peroxide (H2O2). Imaging was performed at 15 minutes post-wounding or amputation using a fluorescence microscope.
Fixation and processing of planaria were performed as described previously. Due to COVID-19, the planned central nervous system staining did not take place.
RNA interference was performed to knock down ОІ-catenin using double-stranded RNA probes. Planaria were injected with 1000nM RNAi probes for three consecutive days, followed by amputation and exposure to PD0325901 or medium. Control groups were injected with Milli-Q water.
Blastema size was normalized to either the whole body size or the size of specific fragments. Group comparisons were made using a two-sample t-test or one-way ANOVA, with significance defined as p < 0.05. Statistical analysis was performed using JMP Pro 14.
Experiments were conducted to investigate the role of ERK in planarian regeneration and its potential gradient along the anteroposterior axis. Several treatments and analyses were performed as described below:
Worms were cut into three pieces and exposed to PD0325901 to inhibit MEK, an upstream factor of ERK. Blastema formation was assessed on seven days post-amputation (DPA), focusing on the influence of ERK inhibition.
Worms were cut into five pieces to assess the anteroposterior gradient of blastema formation upon ERK inhibition. The experiment aimed to understand how different fragments respond to MEK inhibition.
Worms were cut longitudinally to visualize the ERK gradient within a fragment. PD0325901 was applied, and regeneration was assessed in trunk, tail, and head parts. Additional experiments were conducted on 7DPA and 14DPA to confirm the gradient.
RNA interference was used to knock down ОІ-catenin, and planaria were exposed to PD0325901. This experiment aimed to determine the effects of blocking MEK combined with ОІ-catenin inhibition on blastema formation.
ERK is present in the regeneration pathway in a specific gradient along the anteroposterior axis. Results on seven days post amputation (DPA) indicate that blastema formation is suppressed more radically towards the tail. In addition, anterior blastemas seemed to be more sensitive to the inhibition, leading to a smaller overall blastema size in these fragments.
When worms were cut into five pieces, blastemas were again more inhibited towards the tail of the worm, with a gradual decrease in posterior blastema growth (Fig. S1). Longitudinal cuts confirmed that regeneration was significantly reduced in trunk and tail parts compared to controls, while no significant difference was observed in head fragments.
ОІ-catenin has been hypothesized to play a crucial role in head formation. To determine the effects of blocking MEK combined with ОІ-catenin inhibition, we performed a knockdown for ОІ-catenin using RNA interference and exposed planaria to PD0325901. Four different groups were used in this experiment:
Results demonstrated that ОІ-catenin rescue blastema formation in MEK-inhibited fragments, suggesting an important role for ОІ-catenin in the regeneration process.
The findings of this study provide valuable insights into the role of ERK in planarian regeneration. The presence of an ERK gradient along the anteroposterior axis suggests that ERK is involved in regulating blastema formation. The observation that ERK inhibition has a more significant impact on tail blastemas implies its importance in posterior regeneration.
The interaction between ERK and ОІ-catenin is intriguing. ОІ-catenin has previously been associated with head formation in planaria, and our results suggest that it can rescue blastema formation in MEK-inhibited fragments. This finding highlights the complexity of the signaling pathways involved in planarian regeneration and underscores the need for further research to unravel the intricate mechanisms.
It is worth noting that the COVID-19 pandemic affected the planned central nervous system staining, which could have provided additional insights into the role of ERK in neural regeneration. Future studies should consider revisiting this aspect when circumstances allow.
This study demonstrates the presence of an ERK gradient in planaria regeneration, with a more pronounced effect on tail blastemas. Additionally, the interaction between ERK and ОІ-catenin suggests a role for ОІ-catenin in rescuing blastema formation in MEK-inhibited fragments. These findings contribute to our understanding of the complex signaling pathways involved in planarian regeneration.
Further research is needed to explore the exact mechanisms underlying the interaction between ERK and ОІ-catenin. Additionally, investigating the role of ERK in neural regeneration, as initially planned, could provide a more comprehensive understanding of its function in planaria. Moreover, exploring the potential crosstalk between ERK and other signaling pathways involved in regeneration may unveil novel insights into this fascinating biological process.
Experiment Report: Role of ERK in Planaria Regeneration. (2020, May 08). Retrieved from https://studymoose.com/document/schmidtea-mediterranea-observations-and-experiments
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