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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

Phenotypical characterization during DPI and APO exposure. (a) The effect of DPI (3 μM) and APO (400 μM) exposure on regenerating head, trunk, and tail fragments after 7 days of regeneration. Animals kept in cultivation medium or a solution of 0.01% DMSO regenerated normally (n = 10), while worms exposed to DPI failed to form proper blastemas and photoreceptors (heads: 3/3, trunks: 6/7, tails: 6/6), with a survival rate of 3/10 for the head fragments, 7/10 for trunk fragments, and 6/10 for tail fragments after 7 days of DPI exposure. Additional experiments showed that DPI exposure also diminished blastema formation in a concentration-dependent manner. APO exposure induced similar regeneration defaults, including reduced blastema formation and degeneration of the photoreceptors (3/4 head fragments) or lack of regeneration of the photoreceptors (trunks: 5/5, tails: 5/5), with one head fragment lethality at 7 days post amputation. Scale bar: 500 μm. (b) The effect of DPI and APO exposure on intact organisms. Head regression was observed in all animals exposed to DPI (8/8). APO exposure resulted in the development of lesions, specifically in the anterior region (8/8). Worms were exposed to both inhibitors for 7 days. b′: close-up of an APO-exposed animal. b′′: close-up of a DPI-exposed animal in an early phase of head regression. Scale bar: 1 mm. (c) Presentation of the relative blastema sizes during DPI or APO exposure in comparison to the control animals in regenerating head, trunk, and tail fragments at 7 DPA. The average relative blastema size for the trunk fragments was obtained using the relative sizes of both the anterior and posterior blastemas. (n = 10 in both control groups, n = 3 DPI-exposed head fragments, n = 7 DPI-exposed trunk-fragments, n = 6 DPI-exposed tail fragments, and n = 5 APO-exposed animals). ∗p < 0.1; ∗∗∗p < 0.01. p values were obtained via one-way ANOVA analysis. Asterisks show the level of significance. If significant differences were observed between the exposed group and just one of the control groups, a connective line is added between the bars. If the differences are significant in comparison with both control groups, the asterisks are placed above the bar of the exposed group.
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fig2: Phenotypical characterization during DPI and APO exposure. (a) The effect of DPI (3 μM) and APO (400 μM) exposure on regenerating head, trunk, and tail fragments after 7 days of regeneration. Animals kept in cultivation medium or a solution of 0.01% DMSO regenerated normally (n = 10), while worms exposed to DPI failed to form proper blastemas and photoreceptors (heads: 3/3, trunks: 6/7, tails: 6/6), with a survival rate of 3/10 for the head fragments, 7/10 for trunk fragments, and 6/10 for tail fragments after 7 days of DPI exposure. Additional experiments showed that DPI exposure also diminished blastema formation in a concentration-dependent manner. APO exposure induced similar regeneration defaults, including reduced blastema formation and degeneration of the photoreceptors (3/4 head fragments) or lack of regeneration of the photoreceptors (trunks: 5/5, tails: 5/5), with one head fragment lethality at 7 days post amputation. Scale bar: 500 μm. (b) The effect of DPI and APO exposure on intact organisms. Head regression was observed in all animals exposed to DPI (8/8). APO exposure resulted in the development of lesions, specifically in the anterior region (8/8). Worms were exposed to both inhibitors for 7 days. b′: close-up of an APO-exposed animal. b′′: close-up of a DPI-exposed animal in an early phase of head regression. Scale bar: 1 mm. (c) Presentation of the relative blastema sizes during DPI or APO exposure in comparison to the control animals in regenerating head, trunk, and tail fragments at 7 DPA. The average relative blastema size for the trunk fragments was obtained using the relative sizes of both the anterior and posterior blastemas. (n = 10 in both control groups, n = 3 DPI-exposed head fragments, n = 7 DPI-exposed trunk-fragments, n = 6 DPI-exposed tail fragments, and n = 5 APO-exposed animals). ∗p < 0.1; ∗∗∗p < 0.01. p values were obtained via one-way ANOVA analysis. Asterisks show the level of significance. If significant differences were observed between the exposed group and just one of the control groups, a connective line is added between the bars. If the differences are significant in comparison with both control groups, the asterisks are placed above the bar of the exposed group.

Mentions: The amputation-induced ROS burst is crucial for successful regeneration (Figure 2). Both ROS inhibitors DPI and APO noticeably reduced fluorescent signaling of ROS at all the different wound sites (Figures 1(a)–1(c), supplementary Figure 1(A)) causing improper regeneration of all fragments, similar to the observed tail regeneration defaults in Xenopus and zebrafish studies [6, 7]. Although no differences in ROS production were observed between anterior and posterior wound site, ROS inhibition was most effective in the head fragments (supplementary Figure 1(A)). This correlates with the observed differences in vulnerability between the different body fragments. Head fragments were more susceptible (especially to DPI exposure) in comparison to trunks and tails resulting in higher mortality rates and more significant effects on the regeneration capacity (Figure 2). In the regenerating head fragments, we investigated the effects of the DPI exposure on the redox balance via gene expression analyses and noticed that not only prooxidant levels, but also antioxidant levels were affected as shown by the upregulation of the antioxidative enzyme CuZnSOD at 72 HPA (hours post amputation, p = 0.016, Figure 3, supplementary Table 1).


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)

Phenotypical characterization during DPI and APO exposure. (a) The effect of DPI (3 μM) and APO (400 μM) exposure on regenerating head, trunk, and tail fragments after 7 days of regeneration. Animals kept in cultivation medium or a solution of 0.01% DMSO regenerated normally (n = 10), while worms exposed to DPI failed to form proper blastemas and photoreceptors (heads: 3/3, trunks: 6/7, tails: 6/6), with a survival rate of 3/10 for the head fragments, 7/10 for trunk fragments, and 6/10 for tail fragments after 7 days of DPI exposure. Additional experiments showed that DPI exposure also diminished blastema formation in a concentration-dependent manner. APO exposure induced similar regeneration defaults, including reduced blastema formation and degeneration of the photoreceptors (3/4 head fragments) or lack of regeneration of the photoreceptors (trunks: 5/5, tails: 5/5), with one head fragment lethality at 7 days post amputation. Scale bar: 500 μm. (b) The effect of DPI and APO exposure on intact organisms. Head regression was observed in all animals exposed to DPI (8/8). APO exposure resulted in the development of lesions, specifically in the anterior region (8/8). Worms were exposed to both inhibitors for 7 days. b′: close-up of an APO-exposed animal. b′′: close-up of a DPI-exposed animal in an early phase of head regression. Scale bar: 1 mm. (c) Presentation of the relative blastema sizes during DPI or APO exposure in comparison to the control animals in regenerating head, trunk, and tail fragments at 7 DPA. The average relative blastema size for the trunk fragments was obtained using the relative sizes of both the anterior and posterior blastemas. (n = 10 in both control groups, n = 3 DPI-exposed head fragments, n = 7 DPI-exposed trunk-fragments, n = 6 DPI-exposed tail fragments, and n = 5 APO-exposed animals). ∗p < 0.1; ∗∗∗p < 0.01. p values were obtained via one-way ANOVA analysis. Asterisks show the level of significance. If significant differences were observed between the exposed group and just one of the control groups, a connective line is added between the bars. If the differences are significant in comparison with both control groups, the asterisks are placed above the bar of the exposed group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: Phenotypical characterization during DPI and APO exposure. (a) The effect of DPI (3 μM) and APO (400 μM) exposure on regenerating head, trunk, and tail fragments after 7 days of regeneration. Animals kept in cultivation medium or a solution of 0.01% DMSO regenerated normally (n = 10), while worms exposed to DPI failed to form proper blastemas and photoreceptors (heads: 3/3, trunks: 6/7, tails: 6/6), with a survival rate of 3/10 for the head fragments, 7/10 for trunk fragments, and 6/10 for tail fragments after 7 days of DPI exposure. Additional experiments showed that DPI exposure also diminished blastema formation in a concentration-dependent manner. APO exposure induced similar regeneration defaults, including reduced blastema formation and degeneration of the photoreceptors (3/4 head fragments) or lack of regeneration of the photoreceptors (trunks: 5/5, tails: 5/5), with one head fragment lethality at 7 days post amputation. Scale bar: 500 μm. (b) The effect of DPI and APO exposure on intact organisms. Head regression was observed in all animals exposed to DPI (8/8). APO exposure resulted in the development of lesions, specifically in the anterior region (8/8). Worms were exposed to both inhibitors for 7 days. b′: close-up of an APO-exposed animal. b′′: close-up of a DPI-exposed animal in an early phase of head regression. Scale bar: 1 mm. (c) Presentation of the relative blastema sizes during DPI or APO exposure in comparison to the control animals in regenerating head, trunk, and tail fragments at 7 DPA. The average relative blastema size for the trunk fragments was obtained using the relative sizes of both the anterior and posterior blastemas. (n = 10 in both control groups, n = 3 DPI-exposed head fragments, n = 7 DPI-exposed trunk-fragments, n = 6 DPI-exposed tail fragments, and n = 5 APO-exposed animals). ∗p < 0.1; ∗∗∗p < 0.01. p values were obtained via one-way ANOVA analysis. Asterisks show the level of significance. If significant differences were observed between the exposed group and just one of the control groups, a connective line is added between the bars. If the differences are significant in comparison with both control groups, the asterisks are placed above the bar of the exposed group.
Mentions: The amputation-induced ROS burst is crucial for successful regeneration (Figure 2). Both ROS inhibitors DPI and APO noticeably reduced fluorescent signaling of ROS at all the different wound sites (Figures 1(a)–1(c), supplementary Figure 1(A)) causing improper regeneration of all fragments, similar to the observed tail regeneration defaults in Xenopus and zebrafish studies [6, 7]. Although no differences in ROS production were observed between anterior and posterior wound site, ROS inhibition was most effective in the head fragments (supplementary Figure 1(A)). This correlates with the observed differences in vulnerability between the different body fragments. Head fragments were more susceptible (especially to DPI exposure) in comparison to trunks and tails resulting in higher mortality rates and more significant effects on the regeneration capacity (Figure 2). In the regenerating head fragments, we investigated the effects of the DPI exposure on the redox balance via gene expression analyses and noticed that not only prooxidant levels, but also antioxidant levels were affected as shown by the upregulation of the antioxidative enzyme CuZnSOD at 72 HPA (hours post amputation, p = 0.016, Figure 3, supplementary Table 1).

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