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Reactive Oxygen Species in Planarian Regeneration: An Upstream Necessity for Correct Patterning and Brain Formation.

Pirotte N, Stevens AS, Fraguas S, Plusquin M, Van Roten A, Van Belleghem F, Paesen R, Ameloot M, Cebrià F, Artois T, Smeets K - Oxid Med Cell Longev (2015)

Bottom Line: Inhibition of ROS production by diphenyleneiodonium (DPI) or apocynin (APO) causes regeneration defaults at both the anterior and posterior wound sites, resulting in reduced regeneration sites (blastemas) and improper tissue homeostasis.ROS signaling is necessary for early differentiation and inhibition of the ROS burst results in defects on the regeneration of the nervous system and on the patterning process.Our results indicate that ROS are key players in neuroregeneration through interference with the differentiation and patterning processes.

View Article: PubMed Central - PubMed

Affiliation: Centre for Environmental Sciences, Hasselt University, Agoralaan Building D, 3590 Diepenbeek, Belgium.

ABSTRACT
Recent research highlighted the impact of ROS as upstream regulators of tissue regeneration. We investigated their role and targeted processes during the regeneration of different body structures using the planarian Schmidtea mediterranea, an organism capable of regenerating its entire body, including its brain. The amputation of head and tail compartments induces a ROS burst at the wound site independently of the orientation. Inhibition of ROS production by diphenyleneiodonium (DPI) or apocynin (APO) causes regeneration defaults at both the anterior and posterior wound sites, resulting in reduced regeneration sites (blastemas) and improper tissue homeostasis. ROS signaling is necessary for early differentiation and inhibition of the ROS burst results in defects on the regeneration of the nervous system and on the patterning process. Stem cell proliferation was not affected, as indicated by histone H3-P immunostaining, fluorescence-activated cell sorting (FACS), in situ hybridization of smedwi-1, and transcript levels of proliferation-related genes. We showed for the first time that ROS modulate both anterior and posterior regeneration in a context where regeneration is not limited to certain body structures. Our results indicate that ROS are key players in neuroregeneration through interference with the differentiation and patterning processes.

No MeSH data available.


Related in: MedlinePlus

Relative gene expression levels of genes of interest; representing different classes (antioxidative, polarization-related, differentiation-related, proliferation-related, neuronal, and apoptosis-related genes). (a) Gene expression levels of DPI-exposed head fragments relative to the control group (0.01% DMSO) 4 hours post amputation (HPA). (b) Gene expression levels of DPI-exposed head fragments relative to the control group (0.01% DMSO) 72 hours post amputation (HPA). The values indicated in the graphs are the mean ± SEM of minimum 4 biological replicates. There was no effect of DMSO exposure on the expression levels of the measured genes. Significant effects (as compared to the corresponding 0.01% DMSO-exposed control worms): ∗p < 0.1, ∗∗p < 0.05. p values were obtained via the Kruskal-Wallis test.
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fig3: Relative gene expression levels of genes of interest; representing different classes (antioxidative, polarization-related, differentiation-related, proliferation-related, neuronal, and apoptosis-related genes). (a) Gene expression levels of DPI-exposed head fragments relative to the control group (0.01% DMSO) 4 hours post amputation (HPA). (b) Gene expression levels of DPI-exposed head fragments relative to the control group (0.01% DMSO) 72 hours post amputation (HPA). The values indicated in the graphs are the mean ± SEM of minimum 4 biological replicates. There was no effect of DMSO exposure on the expression levels of the measured genes. Significant effects (as compared to the corresponding 0.01% DMSO-exposed control worms): ∗p < 0.1, ∗∗p < 0.05. p values were obtained via the Kruskal-Wallis test.

Mentions: Since homologue NOX or DUOX enzymes have not yet been identified in this species, we used two types of ROS inhibitors to test our hypothesis. Diphenyleneiodonium chloride (DPI, Sigma Aldrich, D2926) is a nonspecific flavoprotein inhibitor which interferes with many different electron transporters [3, 39]. Apocynin (APO, 4′-hydroxy-3′-methoxy-acetophenone, Sigma Aldrich, A10809) inhibits the NOX enzymes, acting on the translocation of the cytoplasmic subunits of the enzymes [39, 40]. As such, a maximum reduction of ROS levels during regeneration is ascertained to explore the effects of impaired ROS signaling. After an initial range-finding experiment, we chose to expose the animals to 2 or 3 μM DPI, depending on the type of experiment and time points of interest or to 400 μM APO. Animals were incubated for 1 hour prior to amputation or staining and exposed during regeneration. Experiments were performed on regenerating head, trunk, and tail fragments, unless described otherwise (Figures 1–3). DPI and APO solutions were prepared in 0.01% dimethylsulfoxide (DMSO, Sigma Aldrich, 471267) and a DMSO control group was added to each experiment to investigate possible effects of DMSO exposure. DMSO is a solvent which is regularly used to dissolve hydrophobic compounds. However, in higher concentrations, DMSO is known to influence cell proliferation and have neurotoxic characteristics in S. mediterranea [38]. Therefore, we used the lowest concentration of DMSO possible to dissolve both DPI and APO and always added a DMSO-exposed control group.


Reactive Oxygen Species in Planarian Regeneration: An Upstream Necessity for Correct Patterning and Brain Formation.

Pirotte N, Stevens AS, Fraguas S, Plusquin M, Van Roten A, Van Belleghem F, Paesen R, Ameloot M, Cebrià F, Artois T, Smeets K - Oxid Med Cell Longev (2015)

Relative gene expression levels of genes of interest; representing different classes (antioxidative, polarization-related, differentiation-related, proliferation-related, neuronal, and apoptosis-related genes). (a) Gene expression levels of DPI-exposed head fragments relative to the control group (0.01% DMSO) 4 hours post amputation (HPA). (b) Gene expression levels of DPI-exposed head fragments relative to the control group (0.01% DMSO) 72 hours post amputation (HPA). The values indicated in the graphs are the mean ± SEM of minimum 4 biological replicates. There was no effect of DMSO exposure on the expression levels of the measured genes. Significant effects (as compared to the corresponding 0.01% DMSO-exposed control worms): ∗p < 0.1, ∗∗p < 0.05. p values were obtained via the Kruskal-Wallis test.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4477255&req=5

fig3: Relative gene expression levels of genes of interest; representing different classes (antioxidative, polarization-related, differentiation-related, proliferation-related, neuronal, and apoptosis-related genes). (a) Gene expression levels of DPI-exposed head fragments relative to the control group (0.01% DMSO) 4 hours post amputation (HPA). (b) Gene expression levels of DPI-exposed head fragments relative to the control group (0.01% DMSO) 72 hours post amputation (HPA). The values indicated in the graphs are the mean ± SEM of minimum 4 biological replicates. There was no effect of DMSO exposure on the expression levels of the measured genes. Significant effects (as compared to the corresponding 0.01% DMSO-exposed control worms): ∗p < 0.1, ∗∗p < 0.05. p values were obtained via the Kruskal-Wallis test.
Mentions: Since homologue NOX or DUOX enzymes have not yet been identified in this species, we used two types of ROS inhibitors to test our hypothesis. Diphenyleneiodonium chloride (DPI, Sigma Aldrich, D2926) is a nonspecific flavoprotein inhibitor which interferes with many different electron transporters [3, 39]. Apocynin (APO, 4′-hydroxy-3′-methoxy-acetophenone, Sigma Aldrich, A10809) inhibits the NOX enzymes, acting on the translocation of the cytoplasmic subunits of the enzymes [39, 40]. As such, a maximum reduction of ROS levels during regeneration is ascertained to explore the effects of impaired ROS signaling. After an initial range-finding experiment, we chose to expose the animals to 2 or 3 μM DPI, depending on the type of experiment and time points of interest or to 400 μM APO. Animals were incubated for 1 hour prior to amputation or staining and exposed during regeneration. Experiments were performed on regenerating head, trunk, and tail fragments, unless described otherwise (Figures 1–3). DPI and APO solutions were prepared in 0.01% dimethylsulfoxide (DMSO, Sigma Aldrich, 471267) and a DMSO control group was added to each experiment to investigate possible effects of DMSO exposure. DMSO is a solvent which is regularly used to dissolve hydrophobic compounds. However, in higher concentrations, DMSO is known to influence cell proliferation and have neurotoxic characteristics in S. mediterranea [38]. Therefore, we used the lowest concentration of DMSO possible to dissolve both DPI and APO and always added a DMSO-exposed control group.

Bottom Line: Inhibition of ROS production by diphenyleneiodonium (DPI) or apocynin (APO) causes regeneration defaults at both the anterior and posterior wound sites, resulting in reduced regeneration sites (blastemas) and improper tissue homeostasis.ROS signaling is necessary for early differentiation and inhibition of the ROS burst results in defects on the regeneration of the nervous system and on the patterning process.Our results indicate that ROS are key players in neuroregeneration through interference with the differentiation and patterning processes.

View Article: PubMed Central - PubMed

Affiliation: Centre for Environmental Sciences, Hasselt University, Agoralaan Building D, 3590 Diepenbeek, Belgium.

ABSTRACT
Recent research highlighted the impact of ROS as upstream regulators of tissue regeneration. We investigated their role and targeted processes during the regeneration of different body structures using the planarian Schmidtea mediterranea, an organism capable of regenerating its entire body, including its brain. The amputation of head and tail compartments induces a ROS burst at the wound site independently of the orientation. Inhibition of ROS production by diphenyleneiodonium (DPI) or apocynin (APO) causes regeneration defaults at both the anterior and posterior wound sites, resulting in reduced regeneration sites (blastemas) and improper tissue homeostasis. ROS signaling is necessary for early differentiation and inhibition of the ROS burst results in defects on the regeneration of the nervous system and on the patterning process. Stem cell proliferation was not affected, as indicated by histone H3-P immunostaining, fluorescence-activated cell sorting (FACS), in situ hybridization of smedwi-1, and transcript levels of proliferation-related genes. We showed for the first time that ROS modulate both anterior and posterior regeneration in a context where regeneration is not limited to certain body structures. Our results indicate that ROS are key players in neuroregeneration through interference with the differentiation and patterning processes.

No MeSH data available.


Related in: MedlinePlus