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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

In situ hybridizations of anterior (smed-notum and smed-sfrp-1) and posterior (smed-wnt-1 and smed-fz-4) markers at different time points during regeneration after DPI exposure. (a) The expression of anterior markers smed-notum and smed-sfrp-1 in regenerating head, trunk, and tail fragments at different time points during regeneration. Posterior expression of smed-notum was observed after DPI exposure (3 μM) at 1 DPA in both head (n = 3/5) and trunk fragments (n = 3/6). At 7 DPA, an increased expression of both smed-notum and smed-sfrp-1 was observed in anterior blastemas of the DPI-exposed trunk and tail fragments in comparison to the control animals. Differences in gene expression are indicated with red arrow heads. Scale bar: 500 μm. (b) The expression of posterior markers smed-wnt-1 and smed-fz-4 in regenerating head, trunk, and tail fragments at different time points during regeneration. Increased expressions of smed-wnt-1 and smed-fz-4 were observed at 7 DPA in DPI-exposed trunk and tail fragments (3 μM) in comparison to control animals. Differences in gene expression are indicated with red arrow heads. Scale bar: 500 μm.
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fig9: In situ hybridizations of anterior (smed-notum and smed-sfrp-1) and posterior (smed-wnt-1 and smed-fz-4) markers at different time points during regeneration after DPI exposure. (a) The expression of anterior markers smed-notum and smed-sfrp-1 in regenerating head, trunk, and tail fragments at different time points during regeneration. Posterior expression of smed-notum was observed after DPI exposure (3 μM) at 1 DPA in both head (n = 3/5) and trunk fragments (n = 3/6). At 7 DPA, an increased expression of both smed-notum and smed-sfrp-1 was observed in anterior blastemas of the DPI-exposed trunk and tail fragments in comparison to the control animals. Differences in gene expression are indicated with red arrow heads. Scale bar: 500 μm. (b) The expression of posterior markers smed-wnt-1 and smed-fz-4 in regenerating head, trunk, and tail fragments at different time points during regeneration. Increased expressions of smed-wnt-1 and smed-fz-4 were observed at 7 DPA in DPI-exposed trunk and tail fragments (3 μM) in comparison to control animals. Differences in gene expression are indicated with red arrow heads. Scale bar: 500 μm.

Mentions: Based on the diminished cephalic ganglia, posterior expression of neuronal cells, and seemingly increased expression of the anterior marker smed-ndl-4, we investigated the effect of an inhibited ROS production on polarization and patterning. In situ hybridizations with the polarity determinants smed-notum [48, 77] and smed-wnt-1 [47, 78, 79] were performed at different time points during regeneration to further characterize the effects of DPI on polarity. During normal regeneration smed-notum follows a distinct pattern of expression. Within 12 HPA, smed-notum is expressed at the anterior wound site in a number of distinct cells. By 1 DPA, the number of smed-notum-expressing cells decreases and at approximately 3 DPA these smed-notum-positive cells gather at the tip of the anterior blastema. In the final phases, between 5 and 8 DPA, the expression of smed-notum becomes restricted to a small number of cells located at the tip of the head, along the midline and in the two cephalic ganglia. After DPI exposure, smed-notum was normally expressed at the anterior wound sites at the 1 DPA and 3 DPA (Figure 9(a)). At 7 days post amputation smed-notum expression seemed to be upregulated in the anterior blastemas after DPI exposure in comparison with the control animals (Figure 9(a)). The accumulation of smed-notum-expressing cells in DPI-exposed trunk and tail fragments in comparison with the control animals might be a result of the reduced blastema and mispatterned brain, similar to the expression of smed-th, smed-tbh, and smed-ndl-4 (Figure 8). Interestingly, smed-notum-expressing cells were also present in some posterior blastemas after DPI exposure at 1 DPA. This defect was no longer visible at 3 DPA or 7 DPA. The ectopic expression of smed-notum clearly indicates that ROS signaling is required to obtain a correct posterior identity, which is confirmed by the presence of anteriorly situated neuronal cells (dopaminergic smed-th-expressing cells and mechanosensory smed-cintillo-expressing cells, Figure 8) in the posterior blastema of DPI-exposed trunk parts. These data correlate with the decreased wnt/β-catenin signaling observed by Love and colleagues in DPI- and APO-exposed Xenopus, which also indicates defects in the establishment of posterior identity after inhibition of ROS production [7]. These possible defects on posterior identity were supported by the ectopic expression of smed-sfrp-1 in DPI-exposed fragments at 7 DPA (Figure 9(a)). smed-sfrp-1, an anterior marker related to the Wnt signaling pathway, is expressed at the anterior wound site within 3 HPA during normal regeneration and a strong cluster of smed-sfrp-1-expressing cells (similar to intact organisms) is observed starting 1 DPA [80]. An increase of smed-sfrp-1 expression was observed in both blastemas in the DPI-exposed animals (Figure 9(a)). In addition to the data obtained by the in situ hybridizations, the gene expression data show that at 4 HPA both smed-sfrp-1 and smed-pbx were significantly downregulated in DPI-exposed head fragments (pbx, p = 0.016; sfrp-1, p = 0.063, Figure 3, supplementary Table 1) [80, 81].


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)

In situ hybridizations of anterior (smed-notum and smed-sfrp-1) and posterior (smed-wnt-1 and smed-fz-4) markers at different time points during regeneration after DPI exposure. (a) The expression of anterior markers smed-notum and smed-sfrp-1 in regenerating head, trunk, and tail fragments at different time points during regeneration. Posterior expression of smed-notum was observed after DPI exposure (3 μM) at 1 DPA in both head (n = 3/5) and trunk fragments (n = 3/6). At 7 DPA, an increased expression of both smed-notum and smed-sfrp-1 was observed in anterior blastemas of the DPI-exposed trunk and tail fragments in comparison to the control animals. Differences in gene expression are indicated with red arrow heads. Scale bar: 500 μm. (b) The expression of posterior markers smed-wnt-1 and smed-fz-4 in regenerating head, trunk, and tail fragments at different time points during regeneration. Increased expressions of smed-wnt-1 and smed-fz-4 were observed at 7 DPA in DPI-exposed trunk and tail fragments (3 μM) in comparison to control animals. Differences in gene expression are indicated with red arrow heads. Scale bar: 500 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig9: In situ hybridizations of anterior (smed-notum and smed-sfrp-1) and posterior (smed-wnt-1 and smed-fz-4) markers at different time points during regeneration after DPI exposure. (a) The expression of anterior markers smed-notum and smed-sfrp-1 in regenerating head, trunk, and tail fragments at different time points during regeneration. Posterior expression of smed-notum was observed after DPI exposure (3 μM) at 1 DPA in both head (n = 3/5) and trunk fragments (n = 3/6). At 7 DPA, an increased expression of both smed-notum and smed-sfrp-1 was observed in anterior blastemas of the DPI-exposed trunk and tail fragments in comparison to the control animals. Differences in gene expression are indicated with red arrow heads. Scale bar: 500 μm. (b) The expression of posterior markers smed-wnt-1 and smed-fz-4 in regenerating head, trunk, and tail fragments at different time points during regeneration. Increased expressions of smed-wnt-1 and smed-fz-4 were observed at 7 DPA in DPI-exposed trunk and tail fragments (3 μM) in comparison to control animals. Differences in gene expression are indicated with red arrow heads. Scale bar: 500 μm.
Mentions: Based on the diminished cephalic ganglia, posterior expression of neuronal cells, and seemingly increased expression of the anterior marker smed-ndl-4, we investigated the effect of an inhibited ROS production on polarization and patterning. In situ hybridizations with the polarity determinants smed-notum [48, 77] and smed-wnt-1 [47, 78, 79] were performed at different time points during regeneration to further characterize the effects of DPI on polarity. During normal regeneration smed-notum follows a distinct pattern of expression. Within 12 HPA, smed-notum is expressed at the anterior wound site in a number of distinct cells. By 1 DPA, the number of smed-notum-expressing cells decreases and at approximately 3 DPA these smed-notum-positive cells gather at the tip of the anterior blastema. In the final phases, between 5 and 8 DPA, the expression of smed-notum becomes restricted to a small number of cells located at the tip of the head, along the midline and in the two cephalic ganglia. After DPI exposure, smed-notum was normally expressed at the anterior wound sites at the 1 DPA and 3 DPA (Figure 9(a)). At 7 days post amputation smed-notum expression seemed to be upregulated in the anterior blastemas after DPI exposure in comparison with the control animals (Figure 9(a)). The accumulation of smed-notum-expressing cells in DPI-exposed trunk and tail fragments in comparison with the control animals might be a result of the reduced blastema and mispatterned brain, similar to the expression of smed-th, smed-tbh, and smed-ndl-4 (Figure 8). Interestingly, smed-notum-expressing cells were also present in some posterior blastemas after DPI exposure at 1 DPA. This defect was no longer visible at 3 DPA or 7 DPA. The ectopic expression of smed-notum clearly indicates that ROS signaling is required to obtain a correct posterior identity, which is confirmed by the presence of anteriorly situated neuronal cells (dopaminergic smed-th-expressing cells and mechanosensory smed-cintillo-expressing cells, Figure 8) in the posterior blastema of DPI-exposed trunk parts. These data correlate with the decreased wnt/β-catenin signaling observed by Love and colleagues in DPI- and APO-exposed Xenopus, which also indicates defects in the establishment of posterior identity after inhibition of ROS production [7]. These possible defects on posterior identity were supported by the ectopic expression of smed-sfrp-1 in DPI-exposed fragments at 7 DPA (Figure 9(a)). smed-sfrp-1, an anterior marker related to the Wnt signaling pathway, is expressed at the anterior wound site within 3 HPA during normal regeneration and a strong cluster of smed-sfrp-1-expressing cells (similar to intact organisms) is observed starting 1 DPA [80]. An increase of smed-sfrp-1 expression was observed in both blastemas in the DPI-exposed animals (Figure 9(a)). In addition to the data obtained by the in situ hybridizations, the gene expression data show that at 4 HPA both smed-sfrp-1 and smed-pbx were significantly downregulated in DPI-exposed head fragments (pbx, p = 0.016; sfrp-1, p = 0.063, Figure 3, supplementary Table 1) [80, 81].

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