1 UMR 5539 Centre National de la Recherche Scientifique, Dynamique Moléculaire des Interactions Membranaires, Université Montpellier II place E. Bataillon 34095 Montpellier cedex 05, France
2 CNRS FRE 2376, Génétique Moléculaire et Biologie du Développement, Villejuif, France
3 Centre de Recherche en Biochimie Macromoléculaire, CNRS-UPR 1086, 1919 Route de Mende, 34293 Montpellier cedex 5, France
*Author for correspondence (e-mail: stbaghdi{at}univ-montp2.fr)
Accepted 9 April 2002
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SUMMARY |
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Movies available on-line
Key words: Ciona, Metamorphosis, Apoptosis, Caspase, ERK
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INTRODUCTION |
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The ascidian Ciona intestinalis, an invertebrate member of the Chordate phylum (Cameron et al., 2000), presents a spectacular metamorphosis. The swimming planctonic larval stage (tadpole) exhibits a remarkable chordate body plan, characterized by a dorsal neural tube and a typical notochord. Metamorphosis into a sessile filter feeder invertebrate involves an extensive reorganization of the body plan and most notably the regression of the tail. Tail regression during metamorphosis also occurs in amphibians and involves massive apoptosis. This raises the question of whether this mechanism is specific to amphibian or whether it was already present in early chordates.
Various mechanisms have been proposed to explain tail regression in ascidians. The first involved the contractile properties of the epithelial layer, which would induce the first phases of tail shortening. The second suggested that notochord cells develop contractile properties that would trigger tail shortening. The third proposed that tail regression would be caused by striated tail muscle contraction (reviewed by Cloney, 1978). However, the actual fate of these tail cells has remained unknown. As developmental events are rapid and highly reproducible in the context of a laboratory, Ciona is used as a very pertinent developmental model organism (Corbo et al., 2001
; Satoh, 2001
). We show that Ciona provides an exquisite model to study developmentally regulated apoptosis in the context of an organism that is equipped with the similar caspase complexity of a higher vertebrate.
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MATERIALS AND METHODS |
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TUNEL staining and indirect immunofluorescence
Tadpole larvae at the tail regression stage were fixed for 20 minutes with 3.7% formaldehyde in filtered seawater. Embryos were permeabilized with 0.2% Triton in phosphate-buffered saline (PBS) and washed once in PBS. The TUNEL staining was performed by two methods in order to visualize apoptotic cells in green or red fluorescence. The first TUNEL method (an in situ cell death fluorescein detection kit) was used according to the manufacturers instructions (Boehringer). In the second TUNEL method, apoptotic cells were detected by incubating larvae for 1 hour at 37°C with biotin-16-dUTP (Roche Molecular Biochemicals) and terminal deoxynucleotidyl transferase (Promega). After the TUNEL reaction, embryos were washed three times in PBS and incubated for 45 minutes at room temperature with Cy3-conjugated streptavidin (Jackson ImmunoResearch Laboratories) diluted 1:500 in PBS. Larvae were then washed three times in PBS, once in TBS, rinsed in distilled water and mounted in mowiol. Positive controls were made by incubating larvae with 0.5 mg/ml DNAse 1 (Sigma) at room temperature for 10 minutes prior to TUNEL staining. DNAse 1-treated sections incubated with fluorescein-labeled nucleotide mixture, without the addition of terminal deoxynucleotidyl transferase (TdT), were used as negative controls. The slides were analyzed with a Leica TCS 4D Laser Confocal Microscope. For double labeling studies, phosphorylated ERK was visualized with a polyclonal antibody raised in rabbit against the active form of MAP kinases ERK1 and ERK2 (di-phosphorylated HTGFLT(p)EY(p)VAT peptide) (Cell Signaling Technology) and a Texas Red-conjugated goat-anti-rabbit immunoglobulin (Jackson Laboratories) as secondary antibody.
Light and transmission electron microscopy (TEM)
Larvae were fixed in 2.5% glutaraldehyde in 0.2 M cacodylate buffer (pH=7.2), and post-fixed in 1% osmium tetroxide in 0.45 M cacodylate buffer (pH=7.2). The fixed material was dehydrated in a graded alcohol series and embedded in Epon 812. For light microscopy semi-thin sections were stained with Toluidine Blue and observed on a Reichert microscope equipped with Nomarski optics. Images were captured from a 12 bits CCD camera (Princeton Instruments). For TEM, ultra-thin sections were classically contrasted with uranyl acetate and lead citrate, and observed with a Jeol 1200X transmission electron microscope.
CaspACE in vivo incorporation
The CaspACE FITC-VAD-FMK In situ Marker (Promega) was added at the hatching stage at a final concentration of 100 µM in filtered seawater. Larvae were fixed and TUNEL stained as described above at the onset of tail regression [i.e. 24 hours post fertilization (hpf)].
Treatments with caspase and MEK inhibitors
The pan-caspase inhibitors (z-VAD-FMK and CaspACE FITC-VAD-FMK) were added at a final concentration of 100 µM immediately after fertilization. Embryos were cultured at 18°C during 30 hours. Treated and control larvae were scored for signs of metamorphosis. The results were then statistically tested using variance analysis and were found to be significant at 0.05. Results were the mean of four independent experiments for a total of 2012 untreated and 1881 treated larvae.
In two independent experiments, MEK inhibitor U0126 (Promega) was added at two final concentrations (2 and 6 µM) at 13 hpf and embryos left to develop up to 30 hours. Results were scored from a total of 3493 (control), 2521 (2 µM U0126) and 2702 (6 µM U0126) larvae.
SDS-PAGE and western immunoblotting
Fertilized eggs were dechorionated with 1% sodium thioglycolate and 0.05% pronase E as described (Mita-Miyazawa et al., 1985) and thoroughly washed six times in 0.2 µm filtered sea water. For caspase 3 analysis, larvae were sonicated on ice in RIPA lysis buffer (150 mM NaCl, 50 mM Tris-Cl, pH 7.6, 5 mM EDTA, 0.5% NP-40, 1 mM PMSF, 1 mM orthovanadate, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with Complete Protease Inhibitor Cocktail Tablets (Roche Molecular Biochemicals). Lysates were then clarified by centrifugation. Samples were diluted in sample buffer (Laemmli, 1970
) and incubated at 85°C for 5 minutes. Total proteins were separated on 17.5% SDS gels and transferred onto PVDF membranes. The blots were blocked with 5% milk powder in PBS-Tween, incubated with a polyclonal rabbit anti-caspase 3 (raised against the pro and activated forms, Santa Cruz) diluted 1:500 in PBS-Tween for 1 hour, washed in TBS-Tween and then incubated with the secondary antibody (HRP-conjugated anti-rabbit IgG antibody diluted 1:10000) for 1 hour and washed in PBS-Tween. Labeled proteins were detected using the ECL chemiluminescence kit (Amersham Pharmacia Biotech).
Additional analysis by western blotting with different antibodies were performed similarly using 12.5% SDS polyacrylamide gels containing 0.13% bisacrylamide. The various primary antibodies used were: rabbit anti-rat ERK1/2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal antibody against the active diphosphorylated form of MAP kinases ERK1 and ERK2 (raised against the di-phosphorylated HTGFLT(p)EY(p)VAT peptide) (New England Biolabs), anti-rabbit skeletal myosin heavy chain and anti-chicken -tubulin (Sigma, Saint Louis, MO), anti-rabbit sarcomeric
-actinin (ICN Biomedicals, Aurora, OH), and anti-rat myogenin (BD Biosciences Pharmingen, San Diego, CA); the rabbit polyclonal directed against chicken
-actinin-binding PDZ-LIM protein (ALP) was as described (Pomiès et al., 1999
). Horseradish peroxidase linked to goat antibodies directed against rabbit immunoglobulins or mouse immunoglobulin Fc fragments (Sigma, Saint Louis, MO) were used as secondary antibodies.
Cell culture
The human Jurkat T cell line was cultured in RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were treated with 10% or 5% DMSO for 5 hours in order to induce apoptosis, then lysed with RIPA lysis buffer (150 mM NaCl, 50 mM Tris-Cl, pH 7.6, 5 mM EDTA, 0.5% NP-40, 1 mM PMSF, 1 mM orthovanadate, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with Complete Protease Inhibitor Cocktail Tablets (Roche Molecular Biochemicals).
The C2C12 myogenic cell line was grown as previously described (Pomiès et al., 1999).
Acridine Orange incorporation and time-lapse movie
Larvae at the early metamorphosis stage (24 hpf) were placed in a perfusion chamber continuously flowed with sea water containing 3-aminobenzoic acid ethyl ester (0.5 mM) and Acridine Orange (2 µg/ml). The pictures were collected every 5 minutes for 2 hours through the rhodamine channel of the Leica TCS 4D confocal microscope. The time lapse movie was made with Metamorph (Universal imaging).
Database searches
Searches in Ciona intestinalis genomic databases were done using TBLASTN and BLAST (Altschul et al., 1990) Department of Energy (DOE) web facilities. For all searches, the resulting sequences were deduced from at least three independent individual sequences.
MAPK
A single Ciona ERK2 protein sequence encoded by six exons was derived from the genomic sequences LQW190167.y1, LQW185341.x1, LQW78034.x1, LQW221663.y1, DEV44654.y1 and LQW243489.x1. p38 was deduced from the overlapping sequences LQW134919.y1, LQW208456.x1, LQW220041.y1, LQW259229.y2 and LQW42581.x1. Ciona ERK and p38 protein sequences were aligned with homologs from various species using ClustalW (Thompson et al., 1994).
-Tubulin
-tubulin exonic sequences were detected by TBLASTN. The complete gene was deduced from a contig reconstructed from the overlapping sequences LQW162562.y1, DEV14186.x1, LQW79054.y1, DEV7363.x1 and LWQ19150.y1.
Caspases
Ciona sequences homologous to the p20 domain of mammalian caspases were searched using TBLASTN. Fifteen independent Ciona caspase-like sequences were detected: CiCSP2a (LQW46573.x1), CiCSP2b (LQW202502.x1), CiCSP2c (LQW264446.x2), CiCSP2d (LQW190259.y1), CiCSP9 (LQW171787.x1), CiCSP3 (LQW237174.y1), CiCSP7c (LQW35987.y1), CiCSP7a (LQW160906.x1), CiCSP7b (DEV3140.y1), CiCSP6a (LQW237347.x2), CiCSP6b (LQW112173.x1), CiCSP6c (LWG743.y1), CiCSP6d (LQW134527.y01), CiCSP6e (DEV25998.y1) and CiCSP2f (LQW269851.x1).
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RESULTS |
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Tail regression is associated with tubulin down regulation and ERK phosphorylation
We next examined whether the expression of other proteins was affected during tail regression, among which we chose a set of cytoskeletal and signaling proteins. A differential screening of Ciona protein extracts was performed using antibodies directed against sarcomeric -actinin, myosin heavy chain,
-actinin-binding PDZ-LIM protein (ALP), myogenin (not shown),
-tubulin and ERK1/2 (Fig. 8). Only the two latter proteins were positively detected at an expected molecular weight. A highly conserved
-tubulin sequence was also found in the genome database (Fig. 8B). Interestingly,
-tubulin expression level was significantly reduced during metamorphosis (Fig. 8A, lane 2 lower panel), presumably as the result of the massive cell disorganization during the apoptosis-dependent tail regression stage.
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DISCUSSION |
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Whereas the ultrastructure of the swimming tadpole is well documented (reviewed by Satoh, 1994), little is known about the fate of Ciona intestinalis tail cells at metamorphosis stage. Using light and transmission electron microscopy, we showed unambiguously that during metamorphosis, these cells died by apoptosis. These included tunic, epidermal, notochord and striated muscle cells. This is in agreement with the degenerating figures observed during the metamorphosis of the solitary ascidian Herdmania momus (Degnan et al., 1996
). Intriguingly, no sign of apoptosis was ever found in the neural tube. This observation is in agreement with a previous report showing a general absence of cell death in the nervous system of Ciona during embryonic development (Bollner and Meinertzhagen, 1993
). The most striking result is the polarized origin of apoptosis that triggers cell regression. Through sequential TUNEL pictures, apoptosis started at the very tail extremity and was propagated through cell to cell up to the tail base. This was most obvious when in vivo Acridine Orange incorporation coupled to time-lapse videomicroscopy was viewed. This observation raises interesting questions: (1) what is the nature of the inductive signal that triggers apoptosis at the tip of the tail, and (2) what secondary signals transmit cell death from cell to cell. The microscopic data also document that no phagocytic event is linked to apoptosis as no cell cleaning process was observed. Instead, apoptotic cells accumulated in contact with the tunic before being shed into the surrounding medium.
One striking observation is that in general, metamorphosis is correlated with the definitive fixation of the larvae to the substratum. Upon adhesion, three types of larvae are successively observed: larvae with active tail, immobile larvae and metamorphosed larvae. It is tempting to correlate the induction of metamorphosis to the fixation step. However, in agreement with earlier reports (Just et al., 1981), a significant number of unfixed larvae were observed to undergo metamorphosis, bypassing the fixation step. If the general picture is that metamorphosis follows fixation, the correlation is not physiologically relevant. In other words, there were enough observed exceptions to rule out that the fixation step is the inductive trigger of metamorphosis. Our data favor the existence of a still ignored internal inductive mechanism that is independent of the fixation step. Recently, Patricolo et al. (Patricolo et al., 2001
) demonstrated the presence of thyroxine in Ciona intestinalis larvae at the metamorphosis stage, suggesting that thyroxine might be one of the inductive signals.
Using complementary methods, we provided evidence that tadpole tail histolysis is a caspase-triggered programmed cell death response. By contrast, the molecular events controlling the early extra-embryonic cell death program appear to be caspase independent.
Using an antibody against human caspase 3, a specific immunological signal was detected in extracts from larvae that had undergone metamorphosis. The antibody was sensitive enough to detect a band of lower Mr compatible of what is expected for an activated form of caspase. That caspase signal was not detected at any other earlier stage, meaning that, upon metamorphosis, there is a specific expression of that particular protein.
In vivo incorporation of fluorescent caspACE confirmed that a caspase-dependent event occurred during tail regression. This inhibitor is known to associate with activated forms of caspases, and, indeed, a high proportion (50%) of caspACE positive cells were also TUNEL positive. However, caspACE-positive and TUNEL-negative cells or caspACE-negative and TUNEL-positive cells were also detected during tail regression but at lower frequencies. CaspACE-positive, TUNEL-negative cells may be in the early stages of apoptosis; CaspACE-negative, TUNEL-positive cells may be in the final stage of apoptosis once caspase activation is terminated (Miho et al., 1999).
The addition in the medium, after egg fertilization, of the same fluorescent inhibitor or of a non fluorescent analog led to delayed metamorphosis as quantified 30 hours later by a significant increase in the number of swimming larvae and a reduction in the number of larvae that underwent metamorphosis when compared with untreated controls. These data added a definitive argument in the involvement of caspase(s) in polarized apoptosis and tail regression.
Whereas metamorphosis was blocked by addition of caspase inhibitor to the medium, it did not affect hatching (data not shown), a result in agreement with the recorded absence of caspase 3-like protein expression in the extracts from mid-tailbud stage embryos. Previous studies have supported a role for proteolytic enzymes leading to hatching of the tadpoles (Berrill, 1929; Caggegi et al., 1974
; Denuce, 1975
). We propose that these proteolytic enzymes are linked to, and presumably induced by, this apparent caspase-independent apoptotic event.
In silico genome anlysis led conclusively to the presence of 15 potential caspases, each with a high degree of homology to the vertebrates ones, a result with several implications. First, it adds a strong genetic support to the immunological data obtained with heterologous antibodies. Indeed, it validates the use of antibodies raised against human caspases, and gives additional confidence that the pro- and activated forms of Ciona caspase 3 have been correctly identified. Second, Ciona has the genomic capacity of controlling cell death with a comparable caspase equipment as higher vertebrates.
The use in Ciona of antibodies raised against proteins of higher vertebrates is of importance, because a very high number of them are immediately available. Especially interesting is the expression pattern obtained after analysis of extracts from pre- and post-metamorphosis larvae. Among a various set of antibodies that were tested, we identified two additional proteins that were significantly affected at metamorphosis.
The first protein that has been identified in our survey is Ciona tubulin. Tubulin protein expression was found to be strongly downregulated at metamorphosis. Tubulin turnover in cells is generally a slow event. Microtubule turnover in non-neuronal cells is, by comparison, very rapid. The downregulation of tubulin observed at metamorphosis presumably reflects the high level of cell disorganization that affects apoptotic cells.
Second, using an antibody raised against mammalian ERK1/2, a single band was identified in Ciona extracts and, more interestingly, the activated phosphorylated form of Ciona ERK was uniquely detected in the metamorphic extract, i.e. at a massive apoptosis stage. Additional in silico analysis provided a genomic confirmation of the existence of a single ERK gene made of a mixture of sequences found to be highly related to both human ERK1 and 2. The Ciona ERK gene is highly conserved, especially within the sequence from which the diphosphorylated peptide-directed antibody was generated. Therefore, Ciona ERK activation at metamorphosis, detected by this antibody, is fully compatible with in silico analysis. These results suggest that the phosphorylated form of ERK transduces the death-activating signal at the metamorphosis stage. Additional experimental data confirmed this conclusion, namely, the nuclear localization of activated ERK in cells of the tail and the effectiveness of MEK inhibitor U0126 to block ERK phosphorylation and metamorphosis. Double labeling with TUNEL staining indicated that TUNEL-positive nuclei very rarely colocalized with phosphorylated-ERK positive nuclei. This led to the suggestion that in normal larvae phosphorylation of ERK is a required step that precedes the onset of apoptosis.
The MAP kinase (ERK) pathway is usually involved in the suppression of apoptosis in mammalian somatic cells; however, ERK activation has been clearly linked to apoptosis in non-fertilized starfish eggs (Sasaki and Chiba, 2001). In this latter model, fertilization inactivates the MAP kinase (ERK) pathway and suppresses apoptosis. In addition, ERK has been reported both as an inducer of cytochrome c release with subsequent activation of the caspase pathway (Wang et al., 2000
), as well as a controller of anoikis, the anchorage-dependent form of apoptosis (Zugasti et al., 2001
). In ascidians, EGF signaling in the anterior of the larva has been linked to settlement and potassium ions have been implicated in the development of an anterior signal center (Davidson and Swalla, 2001
; Degnan et al., 1997
; Eri et al., 1999
). Therefore ERK activation in the tail could be a consequence of that signaling anterior pathway(s). If true, it will be interesting to understand how the signal is conveyed up to the tip of the tail where both ERK activation and apoptosis start.
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ACKNOWLEDGMENTS |
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