After bisection, Hydra polyps regenerate their head from the lower half thanks to a head-organizer activity that is rapidly established at the tip. Head regeneration is also highly plastic as both the wild-type and the epithelial Hydra (that lack the interstitial cell lineage) can regenerate their head. In the wild-type context, we previously showed that after mid-gastric bisection, a large subset of the interstitial cells undergo apoptosis, inducing compensatory proliferation of the surrounding progenitors. This asymmetric process is necessary and sufficient to launch head regeneration. The apoptotic cells transiently release Wnt3, which promotes the formation of a proliferative zone by activating the beta-catenin pathway in the adjacent cycling cells. However the injury-induced signaling that triggers apoptosis is unknown. We previously reported an asymmetric immediate activation of the mitogen-activated protein kinase/ribosomal S6 kinase/cAMP response element binding protein (MAPK/RSK/CREB) pathway in head-regenerating tips after mid-gastric bisection. We show here that pharmacological inhibition of the MAPK/ERK pathway or RNAi knockdown of the RSK, CREB, CREB binding protein (CBP) genes prevents apoptosis, compensatory proliferation and blocks head regeneration. As the activation of the MAPK pathway upon injury plays an essential role in regenerating bilaterian species, these results suggest that the MAPK-dependent activation of apoptosis-induced compensatory proliferation represents an evolutionary-conserved mechanism to launch a regenerative process.
The freshwater Hydra polyp is a classical model system to investigate the cellular and molecular basis of regeneration (Galliot & Schmid 2002; Steele 2002; Holstein et al. 2003; Bosch 2007). Hydra, which belongs to Cnidaria, exhibits a tube shape, with a mouth opening surrounded by tentacles at its apical pole, and a basal disc that secretes mucus at the basal one (Fig. 1A). The bilayered cellular organization relies on myoepithelial cells that constitute the endodermal and ectodermal layers, whereas the intermingled interstitial stem cells differentiate into neurons, mechano-sensory cells (nematocytes), gland cells and gametes (Fig. 1B). Therefore three distinct stem cell populations permanently self-renew and differentiate in the adult polyp, where cycling cells, i.e. stem cells and progenitors are found along the gastric region, whereas the extremities (tentacles, basal disc) are made of terminally differentiated cells that are continuously renewed as they get sloughed off. This highly dynamic maintenance of the Hydra polyp likely explains its amazing plasticity in homeostatic conditions as well as its possibilities for adult developmental processes (Galliot & Ghila 2010). Indeed adult animals can stand weeks of starvation without losing their fitness, but simply progressively reducing their size as a result of apoptosis and autophagy (Otto & Campbell 1977b; Bosch & David 1984; Chera et al. 2009a). By contrast when regularly fed, Hydra polyps make use of the excess of cycling cells by reproducing through budding, a form of asexual reproduction (Otto & Campbell 1977a). Moreover, as discovered 270 years ago, when bisected at any level along the body column, the animal regenerates all missing parts in a few days (Trembley 1744). This regeneration potential, which is not dependent on the feeding diet, actually goes to the point where animals can regenerate from dissociated tissues (Noda 1971; Gierer et al. 1972; Technau et al. 2000).
Initiation of head regeneration and the activation of the MAPK/CREB pathway
Four successive phases characterize head regeneration, the immediate wound healing response, the early setting-up of the head-organizer, the early-late patterning phase that precedes the emergence of the tentacle rudiments, and the late growth of the head once the tentacle rudiments have emerged (Galliot et al. 2006). Within the first 10 h following mi-gastric bisection, beside the immediate wound healing process, transplantation experiments have evidenced a steadily rising head-organizer activity in head-regenerated tips. Indeed grafting experiments have shown that head-regenerating tips grafted 10 h after amputation onto an intact host polyp induce ectopic head formation (Browne 1909; MacWilliams 1983). During this time period the cells located in the head-regenerating-tips upregulate numerous “early” genes, which are supposed to deploy this head-organizer activity (Galliot et al. 1995; Martinez et al. 1997; Gauchat et al. 1998; Technau & Bode 1999; Hobmayer et al. 2000; Shimizu et al. 2002; Chera et al. 2007; Rentzsch et al. 2007; Lengfeld et al. 2009). Among those, the cAMP response element binding protein (CREB) transcription factor is submitted in the minutes that follow amputation to an asymmetric ribosomal S6 kinase (RSK)-dependent phosphorylation event, which occurs exclusively in the head-regenerating tips (Kaloulis et al. 2004). In fact when the MAP Kinase (MEK) is inhibited, RSK that is no longer phosphorylated is inactive and CREB remains unphosphorylated, then head-, but not foot-regeneration is blocked. Systematic pharmacological approaches also showed the importance of the MAPK pathway in the formation of the head organizer activity (Arvizu et al. 2006; Manuel et al. 2006). All together these studies lent weight to a key function for the MAPK/RSK/CREB pathway in the initiation phase of head-regeneration.
Immediate apoptosis-induced compensatory proliferation in head-regenerating tips after mid-gastric bisection
Recently a detailed analysis of the immediate and early cellular remodeling that takes place in regenerating halves identified a massive wave of apoptosis in early head-regenerating tips (Chera et al. 2009b). Beside typical apoptotic nuclei (Fig. 1C), one can detect apoptotic bodies engulfed by the endodermal epithelial cells (Fig. 1D) that transiently lose their epithelial polarity (Fig. S3). Interestingly this wave of apoptosis affects 50% of the cells in the head-regenerating tips (region-1) at 1 h post-amputation (hpa) whereas in the sub-jacent regions (region-2 and region-3) less than 1% of the cells undergo apoptosis (Fig. 1E). By contrast the level of apoptosis does not exceed 7% in the foot-regenerating tips. Hence this injury-induced wave of cell death is asymmetrically activated and remains localized in the vicinity of the wound. Importantly these apoptotic cells were shown to transiently overexpress the Wnt3 protein that subsequently activates the beta-catenin pathway in the adjacent cycling cells, which in turn synchronously divide by 4 hpa (Chera et al. 2009b).
Four types of functional evidences currently support the importance of apoptosis-induced compensatory proliferation in head regeneration in Hydra: First, treatment of head-regenerating Hydra with the pan-caspase inhibitor Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (ZVAD) inhibits apoptosis and head regeneration while, as expected, foot regeneration is not affected. Second, it is possible to force foot-regenerating tips to regenerate a head by briefly heating them locally. In this context the level of apoptosis is increased, Wnt3 is produced and the beta-catenin pathway is activated in the cycling cells. Third upon RNAi knockdown of the Wnt3 or beta-catenin genes, cell proliferation is abolished and the animals no longer regenerate their head. Fourth head-regeneration in ZVAD-treated animals can be fully rescued (and even speed up) by adding some Wnt3 protein in the medium at the time of amputation. All together these data indicate that apoptosis-induced compensatory proliferation is necessary and sufficient to launch head regeneration in Hydra (Chera et al. 2009b).
One key question regarding apoptosis-induced head regeneration concerns the characterization of the injury-dependent signaling that activates it. Given the immediate asymmetric activation of the MAPK/CREB pathway in head-regenerating tips (Galliot et al. 1995; Kaloulis et al. 2004; Arvizu et al. 2006; Manuel et al. 2006), this pathway appeared as an obvious candidate. In this paper we have investigated with pharmacological and genetic tools the role of the MAPK/CREB pathway in apoptosis-induced compensatory proliferation during head regeneration after mid-gatric bisection. The results indicate that the activation of this pathway plays an essential role for launching injury-induced apoptosis.
Materials and methods
Culture of animals, chemical treatments of live Hydra and regeneration experiments
Hydra vulgaris (Hv, Basel strain) and Hydra magnipapillata (Hm, 105 strain) were cultured as in (Gauchat et al. 2004). Regeneration experiments were performed at 18°C on mature budding Hv polyps, gently detached from their parents, bisected at mid-gastric position after 2–5 days of starvation and kept in Hydra Medium (HM, 1 mL/polyp). For pharmacological treatments, triplicates were pretreated for 90 min in U0126 (Cell Signaling, 20 μmol/L) or ZVAD (OMe)-FMK (Alexis, 20 μmol/L), bisected and left to regenerate in the same medium up to 90 min. These regenerating halves were either exposed to BrdU and fixed as indicated, or macerated for Hoechst and immunostaining to evaluate the level of apoptosis.
Hydra maceration and immunofluorescence
At indicated time-points, 30 regenerating halves were sliced off to provide each region as described in Figure 1E and gastric regions of 10 intact Hydra were used as control. Tissues maceration and immunodetection were performed as in (Chera et al. 2007) with the anti-RSK antibody (BD Bioscience N°610226, 1:1000) mixed either with the anti-hyCREB (Galliot et al. 1995; Kaloulis et al. 2004) (1:4000) or the anti-CBP (CREB binding protein) (Santa Cruz, sc-1211, 1:1000) antibodies.
Detection of apoptosis on whole mounts
The in situ cell death detection kit fluorescein (Roche) was used for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Hydra were fixed at 1 hpa in 4% paraformaldehyde (PFA) for 1 day, washed 3× 10 min in phosphate-buffered saline (PBS) and permeabilized for 10 min in 0.1% sodium citrate, 0.5% triton X100 at room temperature (RT). The samples were then heated for 10 min at 70°C, washed in PBS for 5 min, incubated in the TUNEL mixture at 37°C for 90 min, washed 2× 10 min in PBS, stained in Hoechst 33258.
BrdU-labeling, immunofluorescence on whole mounts and cell density
Hydra were incubated in 5 mmol/L BrdU/HM (Sigma) for 2 h as indicated, then relaxed in 2% urethane and fixed in 4% PFA overnight at 4°C. Samples were dehydrated in 100% methanol for at least 1 day, stepwise rehydrated in 75%, 50%, 25% methanol/PBS (10 min each step), then washed 2× 10 min in PBS, treated 20 min at RT in 2N HCl, washed in PBS 3× 5 min and blocked 2 h in bovine serum albumin (BSA) 2%. After overnight incubation in the mixed anti-BrdU (1:20, Roche Kit III) and anti-hyCREB (1:4000 clone 81 524) antibodies at 4°C, samples were washed in PBS 3× 15 min and exposed to the mixed AlexaFluor 488 and AlexaFluor 555 (Molecular Probes 1:400) secondary antibodies. After 3× 15 min PBS washes, samples were stained in Hoechst for 5 min, briefly washed, mounted in Mowiol and analyzed with the Leica SP2 confocal. Nuclei were counted on squared confocal snapshots with l = 190 μm. Cell density was established as the ratio between the number of CREB+ nuclei counted in a given region of the regenerating half over the number of CREB+ nuclei counted in the body column of intact Hydra. The BrdU-labeling index (BLI) was measured as the percentage of BrdU+ nuclei over the CREB+ nuclei.
Cloning the RSK and CBP Hydra genes
The Hv_RSK-609 cDNA was isolated with the RSK-for1 (gattgaaatcatgccacttgc), RSK-rev1 (ctacatctaacagaatattctctg) primers designed from the Hm_RSK EST-clone tac30b10.y1 (Genbank, Hydra EST sequencing project). This cDNA that encodes the N-terminus domain (200 AAs), is highly conserved, 66% identical (75% similar) to the rat RSK2 N-domain expressed to raise the anti-RSK antibody used here (see Fig. S1). The TAZ-KIX-332 Hm cDNA that encodes the full KIX domain was obtained with the CBP-TAZ-for2 (YtWYtWcatgcWcataaRtgY) and CBP-KIX-rev3 (tgggggttaatcatatttgaactg) primers derived from the Podocoryne CBP sequence (kindly provided by V. Schmid). The Hydra KIX domain is 56% identical (72% similar) to the mouse KIX domain that was expressed as immunogen to raise the anti-CBP antibody used here (see Fig. S2). The CBP-620 Hv cDNA was obtained with the CBP-for1 (ctaccgcattcaacacaagttaca) and CBP-rev1 (cataaagtttggactgttgaagag) primers designed from the Hm_CBP EST-clone tab 48d04.x1 (Genbank, Hydra EST sequencing project). Accession numbers: FR796474, FR796475.
RNA interference experiments
The Hv_CREBα-628, Hv_RSK-609, Hv_CBP-620 cDNAs were inserted into the pPD129.36 (L4440) double T7 vector. Hydra were exposed to dsRNAs as in (Chera et al. 2006; Buzgariu et al. 2008). Control animals were similarly exposed to the 183 bp dsRNAs produced from the L4440 vector. Briefly, 2× 2 mL bacteria suspension were pelleted after isopropylthio-β-d-galactopyranoside (IPTG) induction, resuspended in 200 μL 0.5% low melting point (LMP) agarose in HM, 5 mmol/L Tris pH 7.5 (HMT), solidified on ice, ground and given to 50 to 70 Hydra previously transferred in 0.5 mL HMT and exposed to glutathione (50 μmol/L). Bacteria feeding was conducted every other day. For regeneration experiments, Hydra were bisected 1 day after the last dsRNA feeding (Fig. 2A) and gene silencing was confirmed by reverse transcription–polymerase chain reaction (RT–PCR) analysis on 10 regenerating halves per condition.
Semi-quantitative RT–PCR experiments
At indicated time points, the mRNA of 10 head-regenerating halves was prepared after removing the basal disc with the QuickPrep micro-mRNA Purification Kit (GE Healthcare) and resuspended in 10 μL H2O. 50 ng mRNA was used for Sensiscript Reverse Transcription (Qiagen) and specific cDNAs were PCR amplified over 20 and 24 cycles with the pairs of primers RSK-for1/RSK-rev1 (609 bp), CREB-upfish2 (agcatccaagaaagcgtaaca)/CREB-rev564 (tgcgagttgatggggcgatgct) (628 bp), CBP-for1/CBP-rev1 (620 bp), hyBra1-f1 (gacattgatggagttgcgcatc)/hyBra1-r1 (gatttaaggcatctctttcgc) (680 pb) and Act-4HF (aaggattcctacgtcggtgacgaa)/ Act-4HR (ggataccahctgattccataccaa) (683 bp).
Silencing of the RSK, CREB and CBP genes in Hydra
To test whether RSK, CREB and CBP are involved in injury-induced apoptosis in head-regenerating tips, we knocked-down their expression by exposing the animals up to three times prior to amputation to dsRNAs produced from the empty L4440 vector or the RSK-, CREB-, and CBP-L4440 constructs (Fig. 2A). A single exposure to dsRNAs was sufficient to knockdown RSK, CREB and CBP expression in Hydra vulgaris as detected by RT–PCR (Fig. 2B) while several exposures were required in Hydra magnipapillata (Fig. 2C). Indeed whole mount in situ hybridization confirmed that the early upregulation of RSK, CREB and CBP observed after mid-gastric bisection, was no longer detected after several exposures to dsRNAs (Fig. 2C). Therefore all subsequent RNAi experiments were conducted in Hydra vulgaris. As the anti-CREB, anti-RSK and anti-CBP antibodies specifically cross-react with the cognate proteins in most cell types from intact animals (Fig. 3A and not shown), we also verified the level of protein silencing after one or three exposures to dsRNAs in Hydra vulgaris (Fig. 3B, C). This analysis showed that the RSK, CREB and CBP proteins were actually present after a single exposure to dsRNAs (Fig. 3B). In the absence of detectable transcripts (Fig. 2B), we concluded that these proteins were produced prior to dsRNA feeding. By contrast after three dsRNAs feedings, RSK, CREB and CBP were undetectable (Fig. 3C) while the levels of RSK, CREB and CBP were unaltered in control cells (from L4440(RNAi)3× animals –Fig. 3C, first row). This result was confirmed by western blot analysis (Fig. S4).
These cellular and biochemical analyses also showed that these three proteins likely interact in an epistatic fashion as in RSK(RNAi)3x cells RSK but also CREB and CBP were fully depleted, whereas in CREB(RNAi)3x cells both CREB and CBP were depleted (Fig. 3C). Therefore a sufficient level of RSK protein appears necessary to maintain detectable levels of CREB and CBP, whereas CREB seemingly sustains the CBP protein level. At the functional level these three proteins are essential to maintain homeostasis, as their full depletion was lethal in few days (not shown).
The pro-apoptotic function of the MAPK/CREB pathway in head-regenerating Hydra
To test whether RSK, CREB and CBP need to be expressed at the time of amputation to observe injury-induced apoptosis in head-regenerating tips, we knocked-down their expression by feeding once or repeatedly the animals with unspecific (L4440) or specific (RSK, CREB, CBP) dsRNAs prior to amputation. As a result neither apoptosis nor engulfment was detected in cells from head-regenerating tips (Fig. 4). TUNEL assays performed on whole mounts showed that apoptotic nuclei were indeed very few in head-regenerating tips of RSK(RNAi), CREB(RNAi) and CBP(RNAi) Hydra, although still present in foot-regenerating tips (Fig. 4A). The quantification of these apoptotic cells was performed on macerated tissues and indeed proved that <8% of the cells from region-1 were apoptotic at 1 hpa (Fig. 4B). The pharmacological approach confirmed this pro-apoptotic function of the MAPK/CREB pathway as Hydra exposed to the MEK-inhibitor U0126 that prevents RSK and CREB phosphorylation, (Kaloulis et al. 2004) also exhibited a drastic reduction in the number of apoptotic cells after bisection (Fig. 4C). Both assays thus indicate that the MAPK/CREB pathway is required for immediate apoptosis in head-regenerating tips. However a single exposure to dsRNAs was sufficient to prevent injury-induced apoptosis, indicating that the RSK, CREB and CBP proteins produced in homeostatic conditions prior to RNAi silencing do not suffice to induce apoptosis. The regulation of this pathway might actually be even more complex as RT–PCR analyses detected the transient presence of an injury-induced truncated CREB isoform (Kaloulis 2000, L. Ghila unpubl. data, 2009). This suggests that a stress-induced alternative splicing event is involved in this regulation as previously reported in vertebrates (Hua et al. 2006).
Inhibition of MAPK/CREB pathway alters the formation of the proliferative zone
To assess the role of the MAPK/CREB pathway in apoptosis-induced compensatory proliferation, the abundance of cycling cells was evaluated in Hydra knocked-down for CREB or pharmacologically inhibited for MEK signaling. In Hydra exposed once to CREB(RNAi), head-regenerating tips contained few apoptotic cells but exhibited a surprisingly high density of CREB+/BrdU+ cells (Figs 5A,B and 6A–C). When BrdU labeling was performed for 2 h before bisection, <40% of the cells were BrdU labeled at 4 hpa, showing similar BrdU-labeling index to that measured in untreated animals (Figs 5C, 6G,H). By contrast, at 8 hpa CREB+/BrdU+ cells were twice more numerous in region-1 (76%), significantly higher than that measured in region-2 in untreated animals (34%, Figs 5C, 6G,H). This high density of cycling cells in region-1 at 8 hpa was likely obtained at the expenses of lower regions that appeared strongly depleted in BrdU+ cells (9% in region-3), indicating a massive migration of cycling cells from the lower regions to the tip. When BrdU labeling was performed after bisection, the number of BrdU+ cells in head-regenerating tips at 4 hpa was extremely high (83% and 91%, Fig. 6G, H). If we assume that the period of BrdU exposure does not influence the speed of migration towards the wound after amputation, then the striking difference observed between untreated and CREB(RNAi)1× cells suggest that CREB(RNAi)1× cells massively re-enter the cell cycle between 0 and 4 hpa. Hence despite the absence of apoptosis, cell migration and cell cycle re-entry were not inhibited in CREB(RNAi)1x leading to the formation a proliferative zone in region-1 instead of region-2.
However, the proliferative regions in CREB(RNAi)1× and untreated animals are likely not equivalent: despite an extremely high density of BrdU+ cells in CREB(RNAi)1×Hydra the observed increase in cell density at 8 hpa in head-regenerating tips never exceeded 2× when this increase reached 3× in the wild-type context (Fig. 6G, H). This indicates that the synchronous mitotic activity observed between 2 and 4 hpa in untreated Hydra (Fig. 7A) was not observed in CREB(RNAi)1×Hydra. The analysis of the size of the nuclei at 4 hpa indeed confirmed this lack of synchronous cell division (Fig. 7B, C). When RNAi was continued for three feedings, CREB+/BrdU+ cells were either very rare and clustered, or completely absent (Figs 5D and 6D). Therefore when CREB silencing was partial, the amputation-driven apoptosis was abolished but the CREB+ cells were still able to enter S-phase and gather close to the wound although unable to synchronously divide (Fig. 7B, C). When CREB was completely silenced, apoptosis and cell cycling were no longer observed. Moreover, a defect in the wound healing process was noted in both contexts, still present at 8 hpa (Fig. 5C, D).
The importance of apoptosis to induce compensatory proliferation is also evidenced in ZVAD-treated Hydra that never form any proliferative zone (Figs 5E and 6E). Indeed when apoptosis is inhibited by a short ZVAD treatment, the cell density remains basically unregulated in head-regenerating halves, except a mild increase to 1.5× noted at the 8 hpa in region-1 (Fig. 6I, upper). Accordingly the density of CREB+/BrdU+ cells stays low, whatever the region, whatever the time-point, except a slight increase at 4 hpa in region-1 (52%) when animals are exposed to BrdU immediately after bisection (Fig. 6I, lower). This suggests that, despite the absence of apoptosis, a limited number of cells are still able to re-enter the cell cycle after amputation, although not dividing at 4 hpa (Fig. 7B). In both ZVAD and CREB(RNAi)1× phenotypes the cycling cells appear shifted to the tip of head-regenerating halves.
Finally the role of the MAPK pathway in apoptosis-induced compensatory proliferation is confirmed by exposing wild-type Hydra to the U0126 MAPK inhibitor (Figs 5F and 6F, J). This treatment causes a dramatic reduction in the number of BrdU+ cells, possibly linked to the strong decrease in the level of CREB expression (Figs 5F and 6F). As expected the regeneration-induced increase in cell density is not observed in U0126-treated Hydra where, in all three regions and at all time points, the cell density remains similar to that measured in intact untreated Hydra (Fig. 6J, upper). Indeed in these animals BrdU-labeling indexes and mitotic activity at 4 hpa are dramatically low (BLI <20%, Fig. 6I lower, Fig. 7B, C), indicating a strongly altered cell cycling response.
Expression of RSK, CREB and CBP is required for head regeneration
To monitor the kinetics of head regeneration in RNAi Hydra, the progression through the four morphologically distinct phases of head regeneration was recorded (Fig. 8A). In RSK(RNAi), CREB(RNAi) and CBP(RNAi) Hydra head-regeneration was dramatically delayed as phase-1 lasted from 2.8× to 4× longer after a single exposure to dsRNAs, over 5× longer after two. After two dsRNAs exposures, the early apical differentiation process (phase-3) was also significantly lengthened. After three feedings, Hydra were no longer able to regenerate and died (not shown). In animals exposed to L4440 dsRNAs, the RSK, CREB and CBP genes were found expressed at each phase of the head-regeneration process, although with some variations (Fig. 8B). Because non-lethal RNAi conditions only delay head-regeneration, we looked at whether gene silencing was merely transient. Indeed in RSK(RNAi), CREB(RNAi) and CBP(RNAi) Hydra, the corresponding genes were re-expressed after several days: after a single dsRNA exposure, RSK was first re-expressed around 72 hpa, CREB 12 h later and finally CBP reached a significant level at 96 hpa (Fig. 8C–E). Hence their respective re-expressions preceded by about 12 h the first morphological modifications appearing in head-regenerating tips. As previously noticed (Fig. 2B), we found RSK downregulated in CBP(RNAi) Hydra, re-expressed only when CBP transcripts were back (Fig. 8E).
We searched for a possible delayed apoptotic process at the time genes were re-expressed and screened the cells from head-regenerating tips every 3 h over the 2 days preceding the predicted emergence of tentacle buds. However, we failed to detect any sign of apoptosis and/or metaplasia (not shown), suggesting that these cellular events do not occur in the absence of amputation. If confirmed, we anticipate that head regeneration once RSK, CREB, CBP expression resumes occurs through a different mechanism, possibly by-passing the injury-induced apoptosis and the apoptosis-induced compensatory proliferation phases.
Injury-induced apoptosis requires a functional MAPK/CREB pathway in Hydra
Previous work had shown the importance of apoptosis-induced compensatory proliferation to specifically launch a head regeneration process in Hydra after mid-gastric bisection (Chera et al. 2009b). Beside ZVAD that inhibits injury-induced apoptosis, we have now shown that inhibition of the MAPK/CREB pathway either pharmacological (U0126 treatment) or through RNAi silencing of the RSK, CREB and CBP genes also prevent injury-induced apoptosis. This function of the MAPK pathway in Hydra apoptosis is not surprising as the MAPK/c-Jun N-terminal kinase (JBK) pathway was shown to regulate apoptosis in a variety of contexts (Kuranaga & Miura 2002; Lin 2003) either during development as in C. elegans (Gumienny et al. 1999) and Drosophila larvae (Kuranaga et al. 2002; Moreno et al. 2002), or during tissue repair as in Drosophila larvae (Ryoo et al. 2004; Bosch et al. 2005; Bergantinos et al. 2010a) or in mammalian cells (Wang et al. 2007). Although initially reported as exhibiting an anti-apoptotic effect (Xia et al. 1995), the MAPK/ERK pathway can also promote apoptosis as in the ascidian Ciona larva losing its tail during metamorphosis (Chambon et al. 2007) or in mammals differentiating their thymocytes (Sohn et al. 2008). Despite the fact that the role of JNK during apoptosis in Hydra is currently unknown, these data indicate that the injury-dependent regulation of the MAPK activity was already established in cnidarians, where it can contribute to apoptosis.
Apoptosis-induced compensatory proliferation requires a functional MAPK/CREB pathway in Hydra
Thanks to the three tools that inhibit injury-induced apoptosis (ZVAD, U0126 or CREB[RNAi]), we could compare what is left of the compensatory proliferation process in head-regenerating halves when injury-induced apoptosis is inhibited. Two criteria were considered: (i) the injury-induced increase in the number of cycling interstitial cells close to the wound as deduced from the comparison of the number of BrdU+ cells labeled for 2 h either immediately before or immediately after bisection; (ii) the synchronous division of the cycling interstitial cells as evidenced by the dividing cells observed at 4 hpa, the resulting increase in cell density recorded at 8 hpa and the dramatically low BrdU labeling index when BrdU is given between 2 and 4 hpa.
In all three inhibitory contexts the synchronous mitosis of the interstitial precursors is abolished as evidenced by the lower increase in cell densities recorded at 8 hpa: 2× in CREB(RNAi)1× tips, 1.5× after ZVAD, 1.2× after U0126 versus 3× in untreated polyps. However, the injury-induced increase in the number of cycling interstitial progenitors is fully efficient in CREB(RNAi)1×Hydra, limited in ZVAD-treated Hydra and absent in U0126-treated Hydra. In fact, a partial CREB silencing (CREB(RNAi)1×) where the CREB transcripts are undetectable but the CREB+/BrdU+ cells still numerous, is sufficient to prevent the activation of apoptosis and to modify the proliferative response: first the proliferative zone is mislocalized at the regenerating tip (where apoptosis normally takes place) and these cells are unable to divide in time and to drive head regeneration until CREB expression resumed. When the CREB and CBP proteins are no longer detectable (CREB(RNAi)3×, Fig. 3C), cell proliferation is totally abolished in most animals that survive amputation for only a few hours.
These data are in agreement with the following scenario (Fig. 9): in the absence of apoptosis, the transient source of Wnt3 signal produced by the apoptotic cells is missing and the subsequent activation of beta-catenin signaling in the surrounding cycling cells cannot take place. As a consequence the cycling cells present at the tip of regenerating halves do not undergo synchronous mitotic division as observed in region-2 of control animals. These results indicate that CREB that is overexpressed in cycling cells in homeostatic conditions (Chera et al. 2007), is actually playing an additional function in the head regenerative context. Beside its positive effect on cell proliferation, CREB that is submitted to RSK-dependent phosphorylation in head-regenerating tips immediately after mid-gastric bisection, exerts a transient pro-apoptotic function.
One possible mechanism for this injury-induced pro-apoptotic function might be linked to the production of injury-specific isoforms. In vertebrates, multiple CREB isoforms were identified with sometimes opposite activities (Mayr & Montminy 2001). As an example a stress-induced truncated isoform of the CREB-related gene activating transcription factor 3 (ATF3) exhibits a pro-apoptotic function by reducing the CBP-dependent activation of anti-apoptotic genes (Chen et al. 1994; Hua et al. 2006). As in vertebrates, several CREB isoforms were identified in Hydra including one truncated isoform that is transiently produced after mid-gastric bisection (Galliot et al. 1995; Kaloulis 2000). Further experiments will tell us whether this amputation-induced CREB isoform also plays a pro-apoptotic function in Hydra.
Sensitivity to cell death signals seems cell lineage-dependent in Hydra
In Hydra, where the apoptosis machinery is well conserved (Lasi et al. 2010a,b), apoptosis was characterized in a variety of physiological and developmental contexts: (i) in starving Hydra where the proliferative rate is maintained for weeks, the excess of produced cells is eliminated by apoptosis (Bosch & David 1984); (ii) during oogenesis the nurse cells undergo partial apoptosis and engulfment by the oocyte (Honegger et al. 1989; Miller et al. 2000; Technau et al. 2003), whereas during spermatogenesis a subset of sperm precursors become apoptotic (Kuznetsov et al. 2001); (iii) after wound-healing or bisection differentiating nematocytes located along the body column are submitted to cell death (Fujisawa & David 1984); (iv) during head regeneration after mid-gastric bisection, interstitial cell derivatives and progenitors located in head-regenerating tips are rapidly eliminated (Chera et al. 2009b); (v) in the thermosensitive sf-1 mutant, heat shock rapidly induces the apoptosis of the S-phase cells (Terada et al. 1988; Cikala et al. 1999). Apoptosis can also be induced in Hydra polyps either by pharmacological treatments that affect the mitotic spindle as colchicine (Campbell 1976) or inhibit the Pi-3 kinase as Wortmannin (David et al. 2005), or by physical treatments as grafting polyps from different species together (Kuznetsov et al. 2002), or briefly heating the cells of foot-regenerating tips (Chera et al. 2009b). Except mid-gastric head regeneration and heat-induced apoptosis in foot-regenerating tips, it is currently not known whether apoptosis might lead to compensatory proliferation in some of the contexts listed above. However in each of these contexts, the cells that are mostly sensitive to the death signals belong to the interstitial cell lineage and not to the epithelial cell lineages. Indeed those appear extremely resistant to the apoptosis-inducing signals although they quickly respond to the presence of apoptotic bodies by engulfing them. This is in sharp contrast with the Drosophila studies where the epithelial cells of the imaginal discs were shown to easily enter apoptosis upon irradiation, heat-shock or genetic manipulation of the cell death signaling pathway (Bergantinos et al. 2010b).
Injury-induced apoptosis, an evolutionarily-conserved process to launch a regenerative response?
We previously reported that apoptosis-induced compensatory proliferation is necessary and sufficient to drive head regeneration after mid-gastric bisection (Chera et al. 2009b; Galliot & Chera 2010). The data shown here indicate that the asymmetric activation of the MAPK/CREB pathway likely participates in the signaling cascade that leads to apoptosis. Cell death was actually reported as a driving force in several regenerative contexts (Bergmann & Steller 2010): in humans repairing their tissue after irradiation (Kondo 1988), in rodents regenerating their skin or their liver (Li et al. 2010), in Drosophila larvae regenerating their imaginal discs after induction of cell death (Bergantinos et al. 2010b), in Xenopus regenerating its tail (Tseng et al. 2007). Also in newt regenerating its limb (Vlaskalin et al. 2004) and in planarians regenerating their body (Hwang et al. 2004; Pellettieri et al. 2010), cell death was reported but its role in the regenerative process is unknown. More generally these studies point to the non-apoptotic functions of caspases as reported during developmental processes (Kuranaga & Miura 2007).
In Drosophila imaginal discs, “undead” cells (cells that are induced to enter apoptosis but cannot proceed to complete the cell death process) were shown to release Wnt or Dpp signals to induce compensatory proliferation of their neighboring cells and regenerate the missing tissue (Huh et al. 2004; Perez-Garijo et al. 2004; Ryoo et al. 2004; Fan & Bergmann 2008; Smith- Bolton et al. 2009). However, this release of signaling molecules might be linked to the “undead” status of the cells (Perez-Garijo et al. 2009) and the activation of the JNK pathway might suffice to trigger the compensatory growth (Bergantinos et al. 2010a; Warner et al. 2010). In intact Hydra JNK is predominantly expressed in differentiating nematocytes (Philipp et al. 2005) but JNK also appears to be involved in the stress response (Bridge et al. 2010). Therefore JNK might play a role in apoptosis-induced compensatory proliferation in Hydra. If identified, then the balance between CREB and jun would also need to be tested in Hydra as the balance between ATF and jun regulates the sensitivity of mammalian cells to apoptosis (Bhoumik et al. 2002). Comparative investigations in phylogenetically distant contexts where initiation of regeneration relies on apoptosis-induced compensatory proliferation should help identify some shared mechanisms of interest for regenerative medicine.
The authors thank Philippe Jean and Kevin Dobretz for their work on the characterization of the CBP gene. This study was supported by the Canton of Geneva, the Swiss National Foundation (FNS 3100A0-116784), the National Center for Competence in Research (NCCR) “Frontiers in Genetics” Stem Cells & Regeneration Pilot project, the Claraz Donation and the Academic Society of Geneva.