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Keywords:

  • blastema;
  • cell cycle;
  • c-Jun N-terminal kinase;
  • planarian;
  • regeneration;
  • stem cell

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The robust regenerative abilities of planarians absolutely depend on a unique population of pluripotent stem cells called neoblasts, which are the only mitotic somatic cells in adult planarians and are responsible for blastema formation after amputation. Little is known about the molecular mechanisms that drive blastema formation during planarian regeneration. Here we found that treatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 blocked the entry of neoblasts into the M-phase of the cell cycle, while allowing neoblasts to successfully enter S-phase in the planarian Dugesia japonica. The rapid and efficient blockage of neoblast mitosis by treatment with the JNK inhibitor provided a method to assess whether temporally regulated cell cycle activation drives blastema formation during planarian regeneration. In the early phase of blastema formation, activated JNK was detected prominently in a mitotic region (the “postblastema”) proximal to the blastema region. Furthermore, we demonstrated that undifferentiated mitotic neoblasts in the postblastema showed highly activated JNK at the single cell level. JNK inhibition by treatment with SP600125 during this period caused a severe defect of blastema formation, which accorded with a drastic decrease of mitotic neoblasts in regenerating animals. By contrast, these animals still retained many undifferentiated neoblasts near the amputation stump. These findings suggest that JNK signaling plays a crucial role in feeding into the blastema neoblasts for differentiation by regulating the G2/M transition in the cell cycle during planarian regeneration.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Regeneration ability enables animals to replace damaged cells after disease and injury. The “blastema” is a typical indicator structure of regeneration ability that is formed at the amputation stump and acts as a major source of cells for replenishing regenerating tissues. For the sake of completion of successful tissue regeneration, spatio-temporal regulation of mitotic activity in regenerative cells plays a crucial role in maintaining a certain pool of blastema cells.

Planarians show high regenerative ability and provide a good opportunity to elucidate the molecular mechanism underlying stem cell dynamics during regeneration (Agata & Umesono 2008; Umesono & Agata 2009; Shibata et al. 2010). The robust regenerative abilities of planarians absolutely depend on a unique population of pluripotent stem cells called neoblasts (Baguñà 1981; Baguñàet al. 1989; Agata & Watanabe 1999; Newmark & Sánchez Alvarado 2000), which are the only mitotic somatic cells in adult planarians (Fig. 1A–E). Neoblasts are morphologically defined as undifferentiated cells with an euchromatic nucleus (Hayashi et al. 2006; Higuchi et al. 2007) and specifically express a set of “stemness” genes including piwi family genes (Reddien et al. 2005b; Rossi et al. 2006; Yoshida-Kashikawa et al. 2007; Eisenhoffer et al. 2008; Rouhana et al. 2010). X-ray irradiation specifically eliminates neoblasts, and not differentiated cells, and thus results in complete lack of blastema formation during regeneration (Wolff & Dubois 1948), clearly indicating that neoblasts are the definitive source of blastema formation. Neoblasts also allow planarians to maintain the complexity of their body and tissue architecture by continuous cell replacement during normal physiological cell turnover (Pellettieri & Sánchez Alvarado 2007). Recently, by applying the method that we developed single-cell gene profiling of neoblasts using fluorescent activated cell sorting (FACS), we definitely identified a certain population of neoblasts in the S–M phase of the cell cycle during homeostasis (Hayashi et al. 2010).

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Figure 1.  Proliferating and mitotic neoblasts are dispersed throughout almost the entire body during homeostasis. (A) Anti-DjPiwiA (magenta) staining as a reliable neoblast-specific marker. (B) The expression pattern of mcm2 gene as a marker for proliferating neoblasts. (C) Anti-pH3 (green) staining as a marker for mitotic neoblasts. (D) Double staining with anti-pH3 (green) and anti-DjPiwiA (magenta). (E) Higher-magnification views of double staining with anti-pH3 (green) and anti-DjPiwiA (magenta). Anti-pH3(+) neoblasts are a subpopulation of anti-DjPiwiA(+) neoblasts (arrowheads). (F) The early steps of the planarian regeneration process. After amputation, first, planarians show wound healing. Then neoblasts in the amputated planarians form non-proliferative blastema region (pink) and a proliferative postblastema region (green) close to the blastema (Sálo & Baguñà 1984). Scale bars: (A–C) 500 μm, (D) 250 μm, (E) 15 μm.

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During regeneration, neoblasts proliferate in response to wounding stimuli (Wenemoser & Reddien 2010). After effective wound healing, dorso-ventral (DV) interaction induces the expression of Dugesia japonica noggin-like gene A (DjnlgA) in X-ray-insensitive differentiated cells at the stump, and this expression serves as an early indicator of the onset of regeneration prior to blastema formation (Ogawa et al. 2002), leading to blastema formation and differentiation (Kato et al. 1999). Interestingly, it has been reported that mitotic activity of neoblasts associated with blastema formation is found at tissues positioned closest to the blastema, the so-called “postblastema” region (Sálo & Baguñà 1984; Fig. 1F), whereas no mitosis is found within the blastema itself despite the steadily increasing number of blastema cells during regeneration (Sálo & Baguñà 1984; Eisenhoffer et al. 2008; Wenemoser & Reddien 2010). These observations highlight a crucial role of the “postblastema” region as a major source of blastema cells. However, it still remains unknown how cell cycle progression of neoblasts is regulated in the postblastema and how it is related to blastema formation during regeneration.

Mitogen-activated protein kinase (MAPK) signaling pathways are evolutionarily conserved kinase cascades that regulate diverse cellular functions, including cell proliferation, differentiation, migration, and response to stress (Nishida & Gotoh 1993; Chang & Karin 2001). The three major subgroups of kinases in the MAPK family are p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK). Interestingly, recent studies provide evidence directly linking JNK and ERK activation to the entry of the cell cycle into M-phase in vertebrates (Wang et al. 2007; Gutierrez et al. 2010).

In this study, we demonstrated that treatment with the JNK inhibitor SP600125 blocked the entry of neoblasts into M-phase of the cell cycle in the planarian Dugesia japonica. Furthermore, both the spatio-temporal profile of JNK activation and the SP600125-induced phenotype fulfilled the criteria for concluding that JNK activation plays a role in the “postblastema” early in the process of blastema formation. As far as we know, this is the first report regarding a potential role of JNK activity in regulating the entry into M-phase in the cell cycle in invertebrates.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Animals

A clonal strain of the planarian Dugesia japonica derived from the Iruma River in Gifu prefecture, Japan, which was maintained in autoclaved tap water at 22–24°C, was used in this study. Planarians 6–8 mm in length were starved for at least 1 week before experiments.

Treatment with chemical inhibitors

The JNK inhibitors SP600125 (Sigma-Aldrich) and AS601245 (Calbiochem), and the MAPK/ERK kinase (MEK) inhibitor U0126 (Cell Signaling Technology) were dissolved in dimethylsulfoxide (DMSO) and each was used at a final concentration of 25 μmol/L. Amputated planarians were allowed to regenerate in tap water supplemented with each inhibitor either immediately after amputation (0 h) or from 12 h after amputation until the indicated period of regeneration for each experiment.

X-ray irradiation

X-ray irradiation was performed as described by Yoshida-Kashikawa et al. (2007). Five days after irradiation, planarians were amputated and allowed to undergo regeneration.

BrdU incorporation and detection

Bromo-2’-deoxyuridine (BrdU) (Sigma-Aldrich) labeling was performed by injection basically as described previously (Newmark & Sánchez Alvarado 2000). Twelve hours after injection, samples were fixed and treated with 2 N hydrochloric acid (HCl) for 30 min at room temperature. After washing with phosphate-buffered saline (PBS) containing 0.1% Triton X-100, samples were treated overnight at 4°C with 1/25 diluted Anti-BrdU (Becton, Dickinson). Then BrdU signals were detected using a tyramide signal amplification (TSA) Labeling kit No. 2 (Molecular Probes).

Whole-mount in situ hybridization

Animals were treated with 2% HCl in 5/8 Holtfreter’s solution for 5 min at 4°C and fixed in 5/8 Holtfreter’s solution containing 4% paraformaldehyde and 5% methanol for <2 h at 4°C. Hybridization and color detection of digoxigenin (DIG)-labeled RNA probes were carried out as previously described by Umesono et al. (1997). A TSA Labeling kit No. 2 or No. 15 (Molecular Probes) was used for detection of fluorescent color (Yoshida-Kashikawa et al. 2007).

Reverse transcription and quantitative RT-PCR analysis

The reverse transcription reaction was carried out with total RNA from 10 intact planarians using a QuantiTect Transcription kit (Qiagen). Reverse transcription–polymerase chain reaction (RT–PCR) was performed as previously reported (Yazawa et al. 2009).

The PCR primers used were as follows:

DjpiwiA forward, 5′-CGAATCCGGGAACTGTCGTAG-3′;

DjpiwiA reverse, 5′-GGAGCCATAGGTGAAATCTCATTTG-3′;

Djpcna forward, 5′-ACCTATCGTGTCACTGTCTTTGACCGAAAA-3′;

Djpcna reverse, 5′-TTCATCATCTTCGATTTTCGGAGCCAGATA-3′;

DjMCM2 forward, 5′-CGCTGTTGGACAAGGTCAGAAGAATGAACA-3′;

DjMCM2 reverse, 5′-CCAGAAACACAAATCTACATCTTCCAAAGG-3′;

DjMCM3 forward, 5′-GAGTCAGTTCCAAATCATCGATTATATCCT-3′;

DjMCM3 reverse, 5′-TTCAAGGATGTCCTGAAGAAGACGAACAAG-3′.

Whole-mount immunostaining

Planarians were treated with 2% HCl in 5/8 Holtfreter’s solution for 6 min at room temperature and washed three times with 5/8 Holtfreter’s solution at room temperature. Then they were fixed in 5/8 Holtfreter’s solution containing 4% paraformaldehyde, 5% methanol and PhosSTOP phosphatase inhibitor (Roche) for 3 h at 4°C. To decrease background signals, fixed planarians were treated with hybridization buffer for whole-mount in situ hybridization overnight at 55°C. They were then blocked with 10% goat serum in TPBS for 1 h at 4°C, and incubated with 1/1000 diluted mouse anti-DjPiwiA (Yoshida-Kashikawa et al. 2007) or 1/200 diluted rabbit anti-phosphorylated histone H3 (Upstate Biotechnology) or 1/500 diluted rabbit anti-diphosphorylated JNK (Promega) overnight at 4°C. The samples were washed with TPBS for 30 min four times. Signals were detected with 1/500 diluted Alexa Fluor 488- or 594-conjugated goat anti-rabbit or mouse IgG (Invitrogen) in 10% goat serum in TPBS for 3 h at room temperature in the dark. A TSA Labeling kit No. 2 (Molecular Probes) was used for signal amplification.

Western blotting

Sixty fragments including blastemas at 1 day of regeneration were dissolved in sample buffer (0.01 mol L−1 Tris–HCl, 2% sodium dodecyl sulfate [SDS], 6% 2-mercaptethanol, 10% glycerol) and boiled for 5 min. The samples were then subjected to gel electrophoresis and the gel was subjected to Western blotting. Blocking One-P (Nacalai Tesque) was used for the membrane blocking. Western blotting was performed using 1/500 diluted rabbit anti-phosphorylated JNK or 1/5000 diluted mouse anti-α-tubulin monoclonal antibody DM 1A (Sigma) as the primary antibody, and a 1/5000 dilution of each appropriate secondary antibody conjugated with horseradish peroxidase. Signal detection was performed using SuperSignal West Dura Extended Duration Substrate (Pierce).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The JNK inhibitor SP600125 prevents the entry of neoblasts into M-phase of the cell cycle

We examined the effects of a JNK inhibitor (SP600125), and a MEK inhibitor (U0126) that causes ERK inactivation, on the cell cycle progression of neoblasts during homeostasis. Intact planarians were kept in tap water supplemented with various concentrations of these inhibitors for a limited time period (12 h). The expression of DjpiwiA mRNA was used as a reliable marker of undifferentiated neoblasts (Yoshida-Kashikawa et al. 2007; Hayashi et al. 2010). Proliferating cell nuclear antigen (Djpcna) and minichromosome maintenance 2 and 3 (DjMCM2, 3) were used as marker genes for S-phase neoblasts (Salvetti et al. 2000; Orii et al. 2005; Hayashi et al. 2010). Incorporation of BrdU was used as an indicator of successful entry of neoblasts into the S-phase of the cell cycle (Newmark & Sánchez Alvarado 2000). An antibody against phosphohistone H3 (pH3) was used as a reliable marker for M-phase neoblasts (Hendzel et al. 1997; Newmark & Sánchez Alvarado 2000).

Quantitative RT–PCR analysis showed that the expression levels of DjpiwiA were largely unchanged during the time of treatment with these inhibitors (Fig. 2A), suggesting that a constant number of neoblasts could survive in these inhibitor-treated animals. Moreover, treatment with these inhibitors did not affect the expression levels of all three S-phase marker genes (Fig. 2A). Consistent with these results, many neoblasts could incorporate BrdU successfully in the presence of the inhibitors (Fig. 2B–D). These observations suggest that neoblasts successfully entered S-phase.

image

Figure 2.  Treatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 prevents the entry of neoblasts into M-phase of the cell cycle. (A) Relative expression levels of the neoblast-specific marker genes determined by quantitative reverse transcription–polymerase chain reaction (RT–PCR) in intact animals at 12 h after SP600125 (bsl00001) and U0126 (inline image) treatment. Control (bsl00000). (B–D) Successful BrdU incorporation (yellow) in neoblasts in these inhibitor-treated animals. (E) Quantification of pH3(+) neoblasts in SP600125-treated animals (cells in the boxed area, n = 5). SP600125 treatment significantly reduced the number of anti-pH3(+) neoblasts (< 0.01). (F–H) Double staining with DjpiwiA gene probe (magenta) and an anti-pH3 antibody (green) in these inhibitor-treated animals. SP600125, but not U0126, severely blocked the expression of pH3 in neoblasts. Vertical short white lines indicate the midline of intact planarians. Anterior is to the top. Scale bars: 100 μm.

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In contrast, treatment with 25 μmol/L SP600125 caused the loss of almost all of the pH3(+) M-phase neoblasts (Fig. 2E–G). We confirmed that another JNK inhibitor (AS601245) produced the same result (data not shown). This strong effect was specific for the JNK inhibitors: treatment with 25 μmol/L U0126 allowed the expression of pH3 in neoblasts (Fig. 2H). These observations suggest that JNK inhibition prevents the transition of neoblasts from S- to M-phase in the cell cycle in planarians.

SP600125 treatment immediately after amputation blocked wound healing

We next examined the effect of treatment with SP600125 on planarian regeneration. When planarians were allowed to regenerate in tap water supplemented with 25 μmol/L SP600125 immediately after amputation (Fig. 3), they showed normal muscle contraction at the stump (data not shown), but failed to close the wound surface with epidermal cells (100% of animals, n = 15; Fig. 3A, B). This phenotype was induced only in the presence of the inhibitor during the process of wound healing (Fig. S1A). Treatment with AS601245 produced the same defect (100% of animals, n = 15; data not shown), suggesting that JNK activity may be required for effective wound healing in planarians. The SP600125-induced failure of wound healing severely inhibited the expression of DjnlgA at the stump (100% of animals, n = 10; Fig. 3C, C′, D, D′), indicating the complete lack of any detectable regeneration-specific response. This example in planarians provides clear evidence that effective wound healing is strictly required for the subsequent regeneration-specific response after injury, which accords with the notion that DV interaction occurring during wound closure plays a crucial role in evoking the onset of planarian regeneration (Kato et al. 1999).

image

Figure 3.  Effect of SP600125 on wound healing in planarians. (A) When the right side of the planarian body was partially amputated (i.e., cut through from the edge to about the midline), the wound site regenerated normally within 24 h after amputation. (B) Treatment with 25 μmol/L SP600125 immediately after partial amputation caused the wound site to remain open at 24 h of regeneration. Arrowheads indicate the site of partial amputation. (C and D) DjnlgA expression at 24 h of regeneration. Treatment with 25 μmol/L SP600125 caused the wound site to remain open and resulted in the failure of the DjnlgA induction at the stump. (C′ and D′) Higher-magnification views of the bracketed regions in panels C and D. (E) Sample sizes of panels A–D. Anterior is to the top. Scale bars: 500 μm.

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Application of SP600125 for limited time periods during planarian regeneration blocked blastema formation

To avoid wound healing defects resulting from treatment with SP600125, planarians were allowed to perform successful wound healing for 12 h after amputation, and then they were treated with 25 μmol/L SP600125 and challenged to complete regeneration (Fig. 4). We confirmed that these animals expressed DjnlgA normally at the stump as a result of successful wound healing (Fig. 4A–C). However, they failed to regenerate either head or tail structures independently of the inhibitor’s action in wound healing (100% of animals, n = 10; Fig. 4D, E). When the inhibitor-treated animals were put in tap water without the inhibitor from 24 h of regeneration, they showed a certain degree of restoration of the ability to regenerate (Fig. S1B), indicating that the effect of the inhibitor treatment was reversible. These regeneration defects caused by SP600125 were quite similar to those caused by loss of neoblasts as a result of X-ray irradiation (Fig. 4F, F′). Indeed, SP600125-treated animals contained almost no pH3(+) neoblasts (data not shown), but they still retained a large number of neoblasts (100% of animals, n = 10; Fig. 4D′, E′), as indicated by anti-DjPiwiA antibody staining (Yoshida-Kashikawa et al. 2007). These observations suggest that SP600125 treatment did not cause a drastic decrease of neoblasts due to impairment of their ability of self-renewal during the assay period.

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Figure 4.  Effect of SP600125 on blastema formation. Planarians were allowed to undergo effective wound healing for 12 h after amputation, and then the animals were treated with 25 μmol/L SP600125. (A–C) DjnlgA expression in differentiated cells at 24 h of regeneration. The expression of DjnlgA, an early indicator of the onset of regeneration, was not affected by the inhibitor treatment (B) or by selective elimination of neoblasts induced by X-ray irradiation (C). (D–F) Dorsal live images of regenerates at 3 days of regeneration. SP600125 caused severe regeneration defects. Brackets indicate the regenerating head and tail. (D′–F′) Distribution pattern of anti-DjPiwiA(+) neoblasts (magenta). Hoechst staining indicates nuclei (blue). (G) Sample sizes of panels A–F′. Anterior is to the top. Scale bars: 500 μm.

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In summary, we applied SP600125 for limited time periods during planarian regeneration, and thereby obtained evidence that SP600125 treatment affected both wound healing and blastema formation.

JNK is highly activated in the postblastema early during the process of blastema formation

To further assess the requirement for JNK signaling for blastema formation, we examined the spatio-temporal patterns of JNK activation associated with the blastema formation by using an antibody against phosphorylated JNK (pJNK, an indicator of JNK activity). Judging from our findings in the JNK inhibitor experiments described above, active blastema formation should start at least from 12 h after amputation. Consistent with this idea, we were first able to detect the accumulation of neoblasts at the stump around 12 h after amputation (data not shown). These neoblasts increased in number and formed a blastema structure, which could be clearly recognized as a mass of DjPiwiA(+) cells at the stump at 24 h after amputation (Fig. 5A). The pJNK signal was also detected at about the same time near the stump (Fig. 5A). This signal was severely decreased by X-ray irradiation (Fig. 5B), suggesting that JNK activation occurs in a population of neoblast-derived cells. Interestingly, the pJNK signal was found in tissues positioned closest to the blastema, the so-called “postblastema” region, as indicated by the presence of many pH3(+) mitotic neoblasts in this area (Fig. 5C, D). Similar neoblast dynamics were commonly observed in both the anterior and posterior blastemas (data not shown).

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Figure 5.  c-Jun N-terminal kinase (JNK) activation early during the process of the blastema formation. (A and B) Double staining with anti-DjPiwiA (magenta) and anti-phosphorylated JNK (pJNK) (green) at 24 h of regeneration. A collar of pJNK signal was detected in the region adjacent to the anti-DjPiwiA(+) blastema region. This collar was severely reduced by selective elimination of neoblasts induced by X-ray irradiation. (C) Double staining with anti-pJNK (green) and anti-pH3 (white) at 24 h of regeneration. Many anti-pH3(+) cells (strong dotted signals) were observed in the anti-pJNK(+) region. Anti-pH3 staining produced high background signal in the epidermis at the stump. (D) Schematic drawing of a merged view. Anterior is to the top. Scale bars: (A and B) 500 μm, (C) 250 μm.

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Finally, we examined pJNK expression in the postblastema at the single cell level. We confirmed that pH3(+) neoblasts had condensed chromosomes (Fig. 6A, B), as assayed by Hoechst staining, demonstrating that pH3(+) neoblasts were actually undergoing the process of mitosis. By performing triple staining with Hoechst, DjpiwiA probe and anti-pJNK, we found that DjpiwiA mRNA(+) undifferentiated neoblasts with condensed chromosomes in the postblastema showed highly activated JNK (100% of cells, n = 11; Fig. 6C, D). Furthermore, anti-pJNK staining showed a centrosome-like pattern in dividing neoblasts (Fig. 6D) in a pattern similar to that in human HeLa cells (MacCorkle-Chosnek et al. 2001). These observations supported our idea that JNK activity is required by mitotic neoblasts.

image

Figure 6.  Undifferentiated mitotic neoblasts in the postblastema showed highly activated c-Jun N-terminal kinase (JNK). (A and B) Double staining with anti-pH3 (green) and Hoechst (blue). Anti-pH3(+) neoblasts had condensed chromosomes (broken circles), showing that pH3(+) neoblasts were undergoing phases of mitosis such as metaphase in (A) and anaphase in (B) based on their condensed chromosomes. (C and D) Triple staining with Hoechst (blue), DjpiwiA probe (magenta) and anti-phosphorylated JNK (pJNK) (green). (C) Strong pJNK signal was detected only in a metaphase neoblast (broken circle), but not in other DjpiwiA mRNA(+) undifferentiated neoblasts (stars) in the postblastema. (D) Strong pJNK signal was detected in an anaphase neoblast (broken circle), but not in another neoblast (star). The strong punctate signals (arrowheads) shown by anti-pJNK staining in (D) were likely to show a centrosomal pattern. Mitotic neoblasts in (C) and (D) showed widespread expression of DjpiwiA mRNA, presumably due to breakdown of the nuclear envelope, whereas neoblasts not undergoing mitosis showed a tight localization of DjpiwiA mRNA in the cytoplasm (stars). Five postblastema regions (1698 cells in total) were checked by Hoechst staining and 11 cells entering M-phase of the cell cycle were thereby identified, all of which were DjpiwiA mRNA(+) and pJNK(+) neoblasts (100% of cells, n = 11). Scale bar: 20 μm.

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SP600125 treatment prevented JNK activation in the postblastema and resulted in a drastic decrease of blastema cells early during the process of blastema formation

We performed more detailed analysis of the effect of the JNK inhibitor SP600125 on the process of blastema formation. The pJNK signal was significantly reduced by treatment with SP600125 starting at 12 h post-amputation (Fig. 7A, B). We confirmed by Western blotting that the pJNK signal was indeed reduced by treatment with SP600125 (Fig. 7C). Under these conditions, inhibitor-treated animals possessed very few DjPiwiA(+) blastema cells at 24 h of regeneration compared with control animals (Fig. 7D, E). This defect was associated with a lack of mitotic cells in the presumptive postblastema region (Fig. 7D, E).

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Figure 7.  Effects of SP600125 on phosphorylated c-Jun N-terminal kinase (pJNK) and neoblast dynamics involved in blastema formation. (A and B) Anti-pJNK staining at 24 h of regeneration. JNK activation was sensitive to treatment with SP600125. (C) Western blotting analysis indicated that treatment with 25 μmol/L SP600125 markedly reduced the pJNK signal. For this analysis, protein extracts were prepared from fragments including the anterior and posterior blastema at 24 h after amputation. (D and E) Triple staining with Hoechst for nuclei (cyan), anti-DjPiwiA (magenta) and anti-pH3 (green) at 24 h of regeneration. Treatment with SP600125 starting from 12 h after amputation dramatically decreased the number of anti-DjPiwiA(+) cells in the presumptive blastema region (arrowheads), which accorded with the severely reduced number of anti-pH3(+) cells in the presumptive postblastema region (brackets). Nuclear staining confirmed a lack of accumulation of blastema cells at the stump in the inhibitor-treated animals. Anterior is to the top. Scale bars: (A and B) 500 μm, (D and E) 250 μm.

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We next examined the effect of the JNK inhibitor on undifferentiated neoblasts in the postblastema region. In control animals around 24 h of regeneration, we found that DjPiwiA(+) blastema cells showed a drastic decrease of the expression level of DjpiwiA mRNA (Fig. 8A). This finding revealed a clear difference between blastema cells and undifferentiated neoblasts in the postblastema depending on the DjpiwiA mRNA expression level. In contrast to the severe defect of blastema formation, we found many DjpiwiA mRNA(+) undifferentiated neoblasts in the presumptive postblastema region of the inhibitor-treated animals (Fig. 8A, B). As we described above, the expression of DjnlgA occurred normally at the stump in the inhibitor-treated animals (Fig. 4B), indicating that a certain regeneration program that promoted blastema formation and differentiation was actually in progress after the completion of effective wound healing. However, the inhibitor-treated animals had no expression of nou-darake (ndk), a marker gene for the brain primordium in planarians (Cebriàet al. 2002), indicating that DjpiwiA mRNA(+) undifferentiated neoblasts in the presumptive postblastema region failed to participate in blastema formation in the presence of the JNK inhibitor (Fig. 8C, D).

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Figure 8.  Effects of SP600125 on undifferentiated neoblasts in the postblastema. (A and B) Double staining with anti-DjPiwiA (magenta) and DjpiwiA gene probe (green) at 24 h of regeneration. A blastema can be recognized as a mass of anti-DjPiwiA(+) cells at the stump, in which DjpiwiA mRNA expression has decayed, indicative of these cells undergoing a transition from the undifferentiated state to a differentiated state. For this reason, the postblastema region (brackets) can be recognized as a region in which DjpiwiA mRNA was strongly expressed. Treatment with SP600125 severely blocked accumulation of anti-DjPiwiA(+) blastema cells at the stump but had little effect in the postblastema. (C and D) Expression of nou-darake (ndk) at 24 h of regeneration. The expression of ndk was detected in the brain primordium in control animals and was inhibited by treatment with SP600125. Anterior is to the top. Scale bars: 500 μm.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

JNK signaling is the best candidate signaling responsible for the postblastema function during planarian regeneration

A previous study reported that knockdown of various genes required for cell-cycle progression caused regeneration failure in the planarian Schmidtea mediterranea (Reddien et al. 2005a), providing evidence of a close linkage between cell-cycle activation of neoblasts and blastema formation. However, it has remained unclear whether temporally regulated cell cycle activation of neoblasts drives blastema formation during regeneration, because simple RNAi experiments caused severe defects in neoblast maintenance, making it hard to understand the temporal requirement for cell cycle activation during the course of regeneration. To address this question, we applied a chemical JNK inhibitor during various phases of planarian regeneration, and thereby obtained evidence that temporally regulated cell-cycle activation of neoblasts is likely to be indispensable for blastema formation in the planarian Dugesia japonica. Furthermore, the role of JNK signaling seemed to fulfill the criteria for that of the “postblastema” early in the process of blastema formation. On the basis of our findings, we propose that blastema cells originate from a population of mitotic neoblasts in which JNK signaling is highly activated in the postblastema, and then immediately enter a differentiating state, resulting in a clear distinction between the postblastema and blastema regions in planarians (Fig. 9A, B).

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Figure 9.  A proposed model for the role of c-Jun N-terminal kinase (JNK) signaling in blastema formation. (A) The normal process of blastema formation in planarians. JNK activation in the postblastema region appears to promote cell cycle progression of neoblasts, which is required for the generation of blastema cells (differentiating neoblast progeny) during regeneration. That is, undifferentiated neoblasts with highly active JNK signaling (tentatively called “regenerative neoblasts”) divide to give rise to one or two blastema cells. (B) When JNK activity was inhibited by treatment with SP600125, animals showed a lack of both mitotic cells in the presumptive postblastema region and blastema cells. This suggests that undifferentiated neoblasts seemed unlikely to differentiate directly without undergoing cell division. (C) Proposed explanation of the role of JNK activation in the cell cycle progression of neoblasts during homeostasis and regeneration. JNK signaling may be activated strongly in neoblasts in the postblastema by regeneration stimuli and may cause acceleration of the transition of neoblasts from S-phase to G2/M-phase in the cell cycle, contributing to the rapid generation of an initial cohort of blastema cells.

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In addition to the model we have proposed here, reduced directional motility of neoblasts toward the amputation stump could also account for the failure of blastema formation induced by SP600125 (Fig. 9A, B). It has been reported that directional cell migration is involved in both wound healing and blastema formation in planarians (Pascolini et al. 1984; Hori 1989, 1991), suggesting that JNK signaling may also regulate cytoskeletal machineries that drive cell migration between these distinct cellular contexts in common. We therefore speculate that the strong defect of blastema formation induced by SP600125 may result from a combination of firstly the efficient blockage of neoblast mitosis and secondly the reduced directional motility of blastema cells into their correct locations at the stump.

Potential role of JNK signaling in blastema formation

We demonstrated that JNK activity is required for the entry of neoblasts into M-phase in the cell cycle regardless of whether the animals are regenerating amputated animals or non-regenerating intact animals. Interestingly, we found that undifferentiated neoblasts undergoing the process of mitosis showed highly activated JNK in the postblastema region (Fig. 6C, D). Why was JNK activated preferentially in regenerative neoblasts and how was it related to blastema formation? We speculate that strong activation of JNK, which is presumably induced by regeneration stimuli, might cause acceleration of neoblast mitosis to generate blastema cells (Fig. 9C). This mechanism would ensure the differentiation of an initial cohort of blastema cells for a limited time period (from 12 to 24 h after amputation), which is likely to be required for the blastema function in the AP patterning that we have proposed (Agata et al. 2003, 2007).

In developmental contexts, stem cells usually switch from their pluripotent or multipotent state to a committed state via their active cell cycle progression. In vertebrates, it has been demonstrated that stem cells are the most developmentally plastic when they are passing through S phase in the cell cycle (McConnell & Kaznowski 1991; Fischer et al. 2007; Pop et al. 2010). In planarians, we found that blastema cells immediately enter their differentiating state (Fig. 8A, C), suggesting that JNK-dependent mitosis is indispensable for the generation of differentiating neoblast progeny during regeneration. This observation raises the possibility that JNK signaling might play a role in the coordination of cell cycle progression and differentiation during blastema formation. In accordance with this possibility, the role of JNK signaling has been demonstrated in the regeneration of Drosophila imaginal discs. In the imaginal disc, JNK is activated at the leading edges of healing tissue and is required for wound healing and subsequent regenerative cell proliferation (Bergantiños et al. 2010).

Interestingly, JNK activation in response to wounding stimuli is also involved in switching cellular identities of disc cells in a process known as transdetermination (Lee et al. 2005). In cells undergoing transdetermination, JNK activation downregulates Polycomb group (PcG) genes (Lee et al. 2005), which encode transcriptional repressors that regulate chromatin dynamics to maintain cellular identity during metazoan development (Ringrose & Paro 2004). In mammals, recent studies have highlighted the role of PcG genes in maintaining the pluripotency of embryonic stem cells by repressing a large cohort of developmental genes (Boyer et al. 2006). Although the role of PcG genes in neoblasts is still unknown, these observations encourage us to speculate that high levels of JNK activity might promote passage through S phase in the cell cycle and coordinate chromatin remodeling as well, which would contribute to the robust expression of differentiation-related genes in blastema cells. JNK seems likely to be highly active only in mitotic regenerative neoblasts (Fig. 6C, D), supporting our idea. We are looking forward to investigating the role of PcG genes in neoblasts and their functional relationship with JNK signaling in blastema formation and differentiation.

A recent study in zebrafish demonstrated that JNK activity is required for fin regeneration and cell-cycle activation of blastema cells (Ishida et al. 2010; Kawakami 2010), highlighting crucial roles of JNK signaling early during the regenerative response of vertebrates. However, we must point out a discrepancy between the definition of “blastema cells” between planarians and other regenerative animals. In planarians, blastema cells are definitely identified as postmitotic cells at the stump, which originate absolutely from undifferentiated neoblasts (Agata & Watanabe 1999; Agata et al. 2003). By contrast, the potential ambiguity of the origin and cellular state of blastema cells remains open to debate in other regeneration models (Stoick-Cooper et al. 2007). According to the classical definition, blastema cells are identified as proliferating cells observed at the stump in response to wounding stimuli. If the zebrafish blastema cells are really in an undifferentiated state and divide to give rise to differentiated daughter cells, we consider it likely that regenerative neoblasts possess a counterpart ability to that of the zebrafish blastema cells, raising the intriguing possibility that both of these animals may share a common JNK function in regeneration contexts. We must keep in mind that we may need to reconsider classical definitions and concepts of regeneration based on findings at the cellular and molecular levels (Agata et al. 2007).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Dr Eisuke Nishida (Department of Cell and Developmental Biology Graduate School of Biostudies, Kyoto University) for his kind gifts of anti-phosphorylated JNK antibodies. We thank Dr Labib Rouhana for helpful discussions and for critical reading of the manuscript. We also thank Dr Elizabeth Nakajima for critical reading of the manuscript, and all other laboratory members for their help and encouragement. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas to Y. U. (22124004), a Grant-in-Aid for Scientific Research on Innovative Areas to K. A. (22124001), a Grant-in-Aid for Creative Scientific Research to K. A. (17GS0318), the Global COE Program A06 of Kyoto University, the Naito Foundation, the Sasakawa Scientific Research Grant, and a JSPS Research Fellowship.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1. The effects of the c-Jun N-terminal kinase (JNK) inhibitor treatment were reversible.

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DGD_1254_sm_f1.pdf174KSupporting info item

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