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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

Visualization of ROS levels using carboxy-H2DCFDA, 30 minutes post amputation. For each condition a representative image of the entire animal is shown followed by close-ups of both the anterior and posterior wound sites as bright field (upper) and fluorescence (lower) images. (a) ROS levels in regenerating head parts. ROS were produced at the amputation site in control animals (10 out of 13 (10/13) heads displayed fluorescence at the wound site), while ROS levels were visibly diminished in DPI (4/6 displayed diminished fluorescence) and APO-exposed organisms (2/3 displayed diminished fluorescence). (b) ROS levels in regenerating trunks. Amputation-induced ROS were produced at both the anterior and posterior wound site of the control trunk fragment (10/13 trunks displayed fluorescence at the anterior wound sites and 11/13 trunks displayed fluorescence at the posterior wound sites). During DPI and APO exposure, ROS levels were visibly reduced at both amputation sites (DPI: 5/5 displayed diminished fluorescence at the anterior wound sites and 4/5 displayed diminished fluorescence at the posterior wound sites; APO: 3/4 displayed diminished fluorescence at the anterior wound sites and 4/4 displayed diminished fluorescence at the posterior wound sites). A close-up of each wound site is pictured with first the anterior wound site and next the posterior wound site. (c) ROS levels in regenerating tails. ROS are produced at the anterior amputation site (8/10 tails displayed fluorescence at the wound site). DPI and APO exposure reduced ROS levels at the anterior wound site (DPI: 5/7 displayed diminished fluorescence; APO: 2/3 displayed diminished fluorescence). A close-up is shown of each anterior wound site. ROS production in control animals was studied in at least 10 individual fragments. Animals were exposed to 3 μM DPI (n ≥ 5) or 400 μM APO (n ≥ 3) administered in the cultivation medium. All animals were exposed for at least one hour before the staining procedure. Scale bars total image: 200 μm, scale bars close-ups: 400 μm.
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fig1: Visualization of ROS levels using carboxy-H2DCFDA, 30 minutes post amputation. For each condition a representative image of the entire animal is shown followed by close-ups of both the anterior and posterior wound sites as bright field (upper) and fluorescence (lower) images. (a) ROS levels in regenerating head parts. ROS were produced at the amputation site in control animals (10 out of 13 (10/13) heads displayed fluorescence at the wound site), while ROS levels were visibly diminished in DPI (4/6 displayed diminished fluorescence) and APO-exposed organisms (2/3 displayed diminished fluorescence). (b) ROS levels in regenerating trunks. Amputation-induced ROS were produced at both the anterior and posterior wound site of the control trunk fragment (10/13 trunks displayed fluorescence at the anterior wound sites and 11/13 trunks displayed fluorescence at the posterior wound sites). During DPI and APO exposure, ROS levels were visibly reduced at both amputation sites (DPI: 5/5 displayed diminished fluorescence at the anterior wound sites and 4/5 displayed diminished fluorescence at the posterior wound sites; APO: 3/4 displayed diminished fluorescence at the anterior wound sites and 4/4 displayed diminished fluorescence at the posterior wound sites). A close-up of each wound site is pictured with first the anterior wound site and next the posterior wound site. (c) ROS levels in regenerating tails. ROS are produced at the anterior amputation site (8/10 tails displayed fluorescence at the wound site). DPI and APO exposure reduced ROS levels at the anterior wound site (DPI: 5/7 displayed diminished fluorescence; APO: 2/3 displayed diminished fluorescence). A close-up is shown of each anterior wound site. ROS production in control animals was studied in at least 10 individual fragments. Animals were exposed to 3 μM DPI (n ≥ 5) or 400 μM APO (n ≥ 3) administered in the cultivation medium. All animals were exposed for at least one hour before the staining procedure. Scale bars total image: 200 μm, scale bars close-ups: 400 μm.

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)

Visualization of ROS levels using carboxy-H2DCFDA, 30 minutes post amputation. For each condition a representative image of the entire animal is shown followed by close-ups of both the anterior and posterior wound sites as bright field (upper) and fluorescence (lower) images. (a) ROS levels in regenerating head parts. ROS were produced at the amputation site in control animals (10 out of 13 (10/13) heads displayed fluorescence at the wound site), while ROS levels were visibly diminished in DPI (4/6 displayed diminished fluorescence) and APO-exposed organisms (2/3 displayed diminished fluorescence). (b) ROS levels in regenerating trunks. Amputation-induced ROS were produced at both the anterior and posterior wound site of the control trunk fragment (10/13 trunks displayed fluorescence at the anterior wound sites and 11/13 trunks displayed fluorescence at the posterior wound sites). During DPI and APO exposure, ROS levels were visibly reduced at both amputation sites (DPI: 5/5 displayed diminished fluorescence at the anterior wound sites and 4/5 displayed diminished fluorescence at the posterior wound sites; APO: 3/4 displayed diminished fluorescence at the anterior wound sites and 4/4 displayed diminished fluorescence at the posterior wound sites). A close-up of each wound site is pictured with first the anterior wound site and next the posterior wound site. (c) ROS levels in regenerating tails. ROS are produced at the anterior amputation site (8/10 tails displayed fluorescence at the wound site). DPI and APO exposure reduced ROS levels at the anterior wound site (DPI: 5/7 displayed diminished fluorescence; APO: 2/3 displayed diminished fluorescence). A close-up is shown of each anterior wound site. ROS production in control animals was studied in at least 10 individual fragments. Animals were exposed to 3 μM DPI (n ≥ 5) or 400 μM APO (n ≥ 3) administered in the cultivation medium. All animals were exposed for at least one hour before the staining procedure. Scale bars total image: 200 μm, scale bars close-ups: 400 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Visualization of ROS levels using carboxy-H2DCFDA, 30 minutes post amputation. For each condition a representative image of the entire animal is shown followed by close-ups of both the anterior and posterior wound sites as bright field (upper) and fluorescence (lower) images. (a) ROS levels in regenerating head parts. ROS were produced at the amputation site in control animals (10 out of 13 (10/13) heads displayed fluorescence at the wound site), while ROS levels were visibly diminished in DPI (4/6 displayed diminished fluorescence) and APO-exposed organisms (2/3 displayed diminished fluorescence). (b) ROS levels in regenerating trunks. Amputation-induced ROS were produced at both the anterior and posterior wound site of the control trunk fragment (10/13 trunks displayed fluorescence at the anterior wound sites and 11/13 trunks displayed fluorescence at the posterior wound sites). During DPI and APO exposure, ROS levels were visibly reduced at both amputation sites (DPI: 5/5 displayed diminished fluorescence at the anterior wound sites and 4/5 displayed diminished fluorescence at the posterior wound sites; APO: 3/4 displayed diminished fluorescence at the anterior wound sites and 4/4 displayed diminished fluorescence at the posterior wound sites). A close-up of each wound site is pictured with first the anterior wound site and next the posterior wound site. (c) ROS levels in regenerating tails. ROS are produced at the anterior amputation site (8/10 tails displayed fluorescence at the wound site). DPI and APO exposure reduced ROS levels at the anterior wound site (DPI: 5/7 displayed diminished fluorescence; APO: 2/3 displayed diminished fluorescence). A close-up is shown of each anterior wound site. ROS production in control animals was studied in at least 10 individual fragments. Animals were exposed to 3 μM DPI (n ≥ 5) or 400 μM APO (n ≥ 3) administered in the cultivation medium. All animals were exposed for at least one hour before the staining procedure. Scale bars total image: 200 μm, scale bars close-ups: 400 μm.
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