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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

Effects of DPI exposure on neoblast differentiation. (a) Smed-wi1 expression at 3 days post amputation (3 DPA) in head, trunk, and tail fragments. Exposure to DPI (3 μM) did not affect the expression (pattern) of the smedwi-1 gene (n ≥ 4). Scale bar: 1 mm. (b) Smedwi-1 expression at 7 days post amputation (7 DPA) in head, trunk, and tail fragments. Exposure to DPI (3 μM) did not affect the expression (pattern) of the smedwi-1 gene (n ≥ 5). Scale bar: 1 mm. (c) An increase of SMEDWI-1 positive cells was observed at the wound sites of trunk and tail fragments after exposure to DPI (3 μM) in comparison to the control fragments at 7 days post amputation (7 DPA) (n ≥ 5). Scale bar: 500 μm. (d) An increase in cell density was noticeable at the wound site of the DPI-exposed animals after 7 days of regeneration (7 DPA) in comparison to the control animals (n ≥ 5). Scale bar: 500 μm. (e) A decrease of early neoblast progeny, marked by the expression of smed-NB21.11e, was observed in DPI-exposed (3 μM) trunk and tail fragments (n ≥ 5). Scale bar: 500 μm. (f) Quantification of the number of smed-NB.21.11e-positive cells. The number of cells was counted in the prepharyngeal area of the regenerating trunk and tail fragments. A significant decrease was observed in the DPI-exposed trunk and tail fragments in comparison to the trunks and tails of the control groups. ∗∗∗p < 0.01. p values were obtained via one-way ANOVA analysis.
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fig6: Effects of DPI exposure on neoblast differentiation. (a) Smed-wi1 expression at 3 days post amputation (3 DPA) in head, trunk, and tail fragments. Exposure to DPI (3 μM) did not affect the expression (pattern) of the smedwi-1 gene (n ≥ 4). Scale bar: 1 mm. (b) Smedwi-1 expression at 7 days post amputation (7 DPA) in head, trunk, and tail fragments. Exposure to DPI (3 μM) did not affect the expression (pattern) of the smedwi-1 gene (n ≥ 5). Scale bar: 1 mm. (c) An increase of SMEDWI-1 positive cells was observed at the wound sites of trunk and tail fragments after exposure to DPI (3 μM) in comparison to the control fragments at 7 days post amputation (7 DPA) (n ≥ 5). Scale bar: 500 μm. (d) An increase in cell density was noticeable at the wound site of the DPI-exposed animals after 7 days of regeneration (7 DPA) in comparison to the control animals (n ≥ 5). Scale bar: 500 μm. (e) A decrease of early neoblast progeny, marked by the expression of smed-NB21.11e, was observed in DPI-exposed (3 μM) trunk and tail fragments (n ≥ 5). Scale bar: 500 μm. (f) Quantification of the number of smed-NB.21.11e-positive cells. The number of cells was counted in the prepharyngeal area of the regenerating trunk and tail fragments. A significant decrease was observed in the DPI-exposed trunk and tail fragments in comparison to the trunks and tails of the control groups. ∗∗∗p < 0.01. p values were obtained via one-way ANOVA analysis.

Mentions: During regeneration, stem cells not only need to proliferate, but also need to differentiate to regrow the missing tissues. Various redox-modulated signaling pathways, such as the MAPK cascades, regulate this differentiation process [66]. Since stem cell proliferation was not affected after inhibition of the ROS production, the effect of DPI exposure on differentiation was investigated. Both the expression of smedwi-1 and the presence of the SMEDWI-1 protein were studied in control and DPI-exposed animals. During normal regeneration, smedwi-1-expressing neoblasts migrate to the wound site where they will differentiate to form the blastema. As the neoblasts differentiate, they no longer express smedwi-1; however, the protein will remain present in the early neoblast progeny and will eventually degrade in the differentiated cells [66, 67]. Therefore, smedwi-1 expression is observed throughout the mesenchyme of the animal and this gene is not highly expressed within the blastema. After DPI exposure, smedwi-1 expression was not affected at an early (3 DPA, Figure 6(a)) or a later (7 DPA, Figure 6(b)) time point of regeneration. Smedwi-1 was expressed in similar quantities and patterns in all the regenerating organisms. These data confirm that the defects on blastema formation do not result from depletion of the stem cell population. When the SMEDWI-1 protein was observed, the SMEDWI-1 positive cells were normally distributed within the anterior blastema of the control trunk and tail fragments, indicating that the blastemas consist of early neoblast progeny and differentiated cells. However, in DPI-exposed animals, these SMEDWI-1-positive cells were more abundant in comparison with the control animals and accumulated at the wound sites (Figure 6(c)), suggesting that those neoblasts in the DPI-exposed animals fail to fully differentiate. This increased cell density was also observed in the blastemas of DPI-exposed organisms with the DAPI staining, which visualizes the nuclei of the cells (Figure 6(d)). These data suggest that the SMEDWI-1-positive cells are unable to proceed to a differentiated state which confirms that the phenotypical defects observed after exposure to the ROS inhibitor do not result from any effects on neoblast proliferation or survival but rather result from an effect on stem cell dynamics. To clarify the effects of DPI exposure on early neoblast differentiation, the number of cells expressing smed-NB21.11e, a marker for early neoblast postmitotic progeny [68], was determined in the prepharyngeal area at 3 DPA using fluorescent in situ hybridization. The number of early neoblast progeny cells significantly decreased in both DPI-exposed trunk and tail parts in comparison with the control animals (27-28% less smed-NB21.11e-positive cells in DPI-exposed trunk and tail fragments in comparison with control (p = 0.0014) and DMSO-exposed animals (p = 0.0008), Figures 6(e) and 6(f)) confirming that ROS signaling is necessary for proper early neoblast differentiation rather than neoblast proliferation or maintenance. Gene expression analyses reinforce this hypothesis since expression levels of smedwi-2 were significantly downregulated at 4 HPA (p = 0.056) and upregulated at 72 HPA (p = 0.016) in DPI-exposed regenerating head fragments. SMEDWI-2 is an enzyme which is needed for the production of neoblast progeny capable of replacing aged differentiated cells during homeostasis and missing tissues during regeneration [67]. Smedwi-2(RNAi) planarians display defects comparable to the DPI- and APO-exposed phenotypes, including regression of the tissue anterior to the photoreceptors and incapability of regeneration [67]. Also, smedwi-2(RNAi) did not affect the normal proliferative wounding response of the neoblasts, since stem cells were able to proliferate and migrate to the wound site [67].


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)

Effects of DPI exposure on neoblast differentiation. (a) Smed-wi1 expression at 3 days post amputation (3 DPA) in head, trunk, and tail fragments. Exposure to DPI (3 μM) did not affect the expression (pattern) of the smedwi-1 gene (n ≥ 4). Scale bar: 1 mm. (b) Smedwi-1 expression at 7 days post amputation (7 DPA) in head, trunk, and tail fragments. Exposure to DPI (3 μM) did not affect the expression (pattern) of the smedwi-1 gene (n ≥ 5). Scale bar: 1 mm. (c) An increase of SMEDWI-1 positive cells was observed at the wound sites of trunk and tail fragments after exposure to DPI (3 μM) in comparison to the control fragments at 7 days post amputation (7 DPA) (n ≥ 5). Scale bar: 500 μm. (d) An increase in cell density was noticeable at the wound site of the DPI-exposed animals after 7 days of regeneration (7 DPA) in comparison to the control animals (n ≥ 5). Scale bar: 500 μm. (e) A decrease of early neoblast progeny, marked by the expression of smed-NB21.11e, was observed in DPI-exposed (3 μM) trunk and tail fragments (n ≥ 5). Scale bar: 500 μm. (f) Quantification of the number of smed-NB.21.11e-positive cells. The number of cells was counted in the prepharyngeal area of the regenerating trunk and tail fragments. A significant decrease was observed in the DPI-exposed trunk and tail fragments in comparison to the trunks and tails of the control groups. ∗∗∗p < 0.01. p values were obtained via one-way ANOVA analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig6: Effects of DPI exposure on neoblast differentiation. (a) Smed-wi1 expression at 3 days post amputation (3 DPA) in head, trunk, and tail fragments. Exposure to DPI (3 μM) did not affect the expression (pattern) of the smedwi-1 gene (n ≥ 4). Scale bar: 1 mm. (b) Smedwi-1 expression at 7 days post amputation (7 DPA) in head, trunk, and tail fragments. Exposure to DPI (3 μM) did not affect the expression (pattern) of the smedwi-1 gene (n ≥ 5). Scale bar: 1 mm. (c) An increase of SMEDWI-1 positive cells was observed at the wound sites of trunk and tail fragments after exposure to DPI (3 μM) in comparison to the control fragments at 7 days post amputation (7 DPA) (n ≥ 5). Scale bar: 500 μm. (d) An increase in cell density was noticeable at the wound site of the DPI-exposed animals after 7 days of regeneration (7 DPA) in comparison to the control animals (n ≥ 5). Scale bar: 500 μm. (e) A decrease of early neoblast progeny, marked by the expression of smed-NB21.11e, was observed in DPI-exposed (3 μM) trunk and tail fragments (n ≥ 5). Scale bar: 500 μm. (f) Quantification of the number of smed-NB.21.11e-positive cells. The number of cells was counted in the prepharyngeal area of the regenerating trunk and tail fragments. A significant decrease was observed in the DPI-exposed trunk and tail fragments in comparison to the trunks and tails of the control groups. ∗∗∗p < 0.01. p values were obtained via one-way ANOVA analysis.
Mentions: During regeneration, stem cells not only need to proliferate, but also need to differentiate to regrow the missing tissues. Various redox-modulated signaling pathways, such as the MAPK cascades, regulate this differentiation process [66]. Since stem cell proliferation was not affected after inhibition of the ROS production, the effect of DPI exposure on differentiation was investigated. Both the expression of smedwi-1 and the presence of the SMEDWI-1 protein were studied in control and DPI-exposed animals. During normal regeneration, smedwi-1-expressing neoblasts migrate to the wound site where they will differentiate to form the blastema. As the neoblasts differentiate, they no longer express smedwi-1; however, the protein will remain present in the early neoblast progeny and will eventually degrade in the differentiated cells [66, 67]. Therefore, smedwi-1 expression is observed throughout the mesenchyme of the animal and this gene is not highly expressed within the blastema. After DPI exposure, smedwi-1 expression was not affected at an early (3 DPA, Figure 6(a)) or a later (7 DPA, Figure 6(b)) time point of regeneration. Smedwi-1 was expressed in similar quantities and patterns in all the regenerating organisms. These data confirm that the defects on blastema formation do not result from depletion of the stem cell population. When the SMEDWI-1 protein was observed, the SMEDWI-1 positive cells were normally distributed within the anterior blastema of the control trunk and tail fragments, indicating that the blastemas consist of early neoblast progeny and differentiated cells. However, in DPI-exposed animals, these SMEDWI-1-positive cells were more abundant in comparison with the control animals and accumulated at the wound sites (Figure 6(c)), suggesting that those neoblasts in the DPI-exposed animals fail to fully differentiate. This increased cell density was also observed in the blastemas of DPI-exposed organisms with the DAPI staining, which visualizes the nuclei of the cells (Figure 6(d)). These data suggest that the SMEDWI-1-positive cells are unable to proceed to a differentiated state which confirms that the phenotypical defects observed after exposure to the ROS inhibitor do not result from any effects on neoblast proliferation or survival but rather result from an effect on stem cell dynamics. To clarify the effects of DPI exposure on early neoblast differentiation, the number of cells expressing smed-NB21.11e, a marker for early neoblast postmitotic progeny [68], was determined in the prepharyngeal area at 3 DPA using fluorescent in situ hybridization. The number of early neoblast progeny cells significantly decreased in both DPI-exposed trunk and tail parts in comparison with the control animals (27-28% less smed-NB21.11e-positive cells in DPI-exposed trunk and tail fragments in comparison with control (p = 0.0014) and DMSO-exposed animals (p = 0.0008), Figures 6(e) and 6(f)) confirming that ROS signaling is necessary for proper early neoblast differentiation rather than neoblast proliferation or maintenance. Gene expression analyses reinforce this hypothesis since expression levels of smedwi-2 were significantly downregulated at 4 HPA (p = 0.056) and upregulated at 72 HPA (p = 0.016) in DPI-exposed regenerating head fragments. SMEDWI-2 is an enzyme which is needed for the production of neoblast progeny capable of replacing aged differentiated cells during homeostasis and missing tissues during regeneration [67]. Smedwi-2(RNAi) planarians display defects comparable to the DPI- and APO-exposed phenotypes, including regression of the tissue anterior to the photoreceptors and incapability of regeneration [67]. Also, smedwi-2(RNAi) did not affect the normal proliferative wounding response of the neoblasts, since stem cells were able to proliferate and migrate to the wound site [67].

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