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Tail regression in Ciona intestinalis (Prochordate) involves a Caspase-dependent apoptosis event associated with ERK activation

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

View larger version (136K): [in a new window]   Fig. 1. Swimming tadpole does not shown features of apoptosis. (A) Swimming tadpole of Ciona intestinalis was labeled with TUNEL. Digitized images were merged to superimpose the TUNEL labeling (green pseudocolor field) over the respective Nomarski field. No sign of apoptosis was observed at this stage (Ot, otolith; Oc, ocellus; N, notochord). Double arrowed line indicates the level of the ultra-thin section shown in B. (B) Electron micrograph of cross-section of a Ciona tadpole tail at 20 hours post fertilization (hpf) (T, tunic; EP, epidermis; SM, striated muscle cell; NT, neural tube; ES, endodermal strand; NC, notochord). (C) Cross-section of a striated muscle cell from the tail of a Ciona intestinalis tadpole. The myofibrils (My) seen in the cross section are arranged in a single peripheral layer. The nucleus (N) and mitochondria (m) are present in the internal core. (D) Striated muscle cell tangentially sectioned and showing one sarcomere. Scale bars: 380 µm in A; 7 µm in B; 3 µm in C; 0.4 µm in D.

View larger version (79K): [in a new window]   Fig. 2. TUNEL labeling of Ciona intestinalis tadpole at successive stages (A-C) of the tail regression. Apoptotic nuclei appear in green. Scale bars: 220 µm in A; 140 µm in B; 80 µm in C.

View larger version (160K): [in a new window]   Fig. 3. Apoptosis of striated muscle, notochord, epidermis and tunic cells 26 hours post-fertilization. (A) Superposition of TUNEL and wheat-germ agglutinin (WGA) labeling (apoptotic nuclei appear in green, tadpole in red). Double arrowed line indicates the level of the semi-thin section shown in B. (B) Transverse section performed in the tail observed using Nomarski differential-interference optics. One of the six muscle cells was clearly replaced by apoptotic body (ab) (T, tunic; TC, tunic cell; SM, striated muscle cells; NC, notochord). (C) Higher magnification of the apoptotic body shown in the rectangle in B (EP, epidermis). (D,E) Extracellular muscle apoptotic bodies, one with large vacuoles (lv) and a nuclear fragment (nf) containing condensed chromatin (cc). (E) At later stage of apoptosis, nuclear membrane disappears around the condensed chromatin. (F) Striated muscle cell at earlier stage of apoptosis. Note the presence of large vacuoles (lv) around the nucleus, the chromatin condensation (arrow) and the well preserved mitochondria (m). (G) NC showing chromatin condensation (arrow). (H) Highly vacuolated TC with condensed nucleus (arrow). Note the importance of the endoplasmic reticulum (er) in cytoplasm of epidermal cells (EP). (I) Typical feature of apoptotis in epidermal cell. Nuclear fragments (nf) containing condensed chromatin (cc) are wrapped by multiple membranes (mm). The membranes seem to be derived from endoplasmic reticulum as suggested by the presence of ribosome-like particle (rp, arrowhead). Some vacant spaces (v) appear to grow between the wrapped fragment resulting in their separation. Scale bars: 180 µm in A; 10 µm in B; 4 µm in C; 1.7 µm in D; 1.6 µm in E; 2.7 µm in F; 3 µm in G,H; 1.2 µm in I.

View larger version (133K): [in a new window]   Fig. 4. Extra-embryonic apoptosis in internal follicle cells at mid-tailbud stage (12 hpf). (A) Confocal optical section showing the superposition of TUNEL and WGA labeling in a mid-tailbud embryo. Apoptotic nuclei of internal follicle cell appear in green, cytoplasm in red. No apoptosis is seen in the head (H) or in the tail (T) of the future tadpole. (B) Optical section at a different focal plane of the embryo shown in (A). Note the accumulation of apoptotic internal follicle cells between the head and the tail (arrow). (C,D) Apoptotic internal follicle cells observed by TEM at mid-tailbud stage (12 hpf). The nuclear membrane disappears when apoptosis progresses to result in a rounded condensed chromatin (cc), while a new membrane system encloses narrow regions of the cytosol (arrow). Scale bars: 85 µm in A,B; 1.4 µm in C; 1.2 µm in D.

View larger version (81K): [in a new window]   Fig. 5. Partial sequence alignment of Ciona and human caspases. Ciona sequences were aligned and clustered using ClustalW (Thompson et al., 1994) with human sequences: HsCasp2, XP_036975 (amino acids 243-301); HsCasp9, XP_048851 (amino acids 220-285); HsCasp10, NP_001221 (amino acids 298-356); HsCasp8, XP_054990 (amino acids 285-343); HsCasp3, XP_054686 (amino acids 104-161); HsCasp7, NP_203125 (amino acids 127-184); HsCasp6, XP_003600 (amino acids 15-72). Three main clusters (framed) were obtained: CiCSP2a, b, c and d, related to human caspase 2; CiCSP9, related to human capsases 8, 9 and 10; and a larger cluster including three subgroups – CiCSP3 and human caspases 3 and 7, CiCSP7a-c and CiCSP6a-e. These latter are related to human caspase 6. For each cluster, residues shared by at least 80% and 60% sequences are indicated in red and blue, respectively. A consensus sequence is indicated below each frame. Conserved residues in each cluster or subgroup are shaded. Residues corresponding to the ICE pentapeptide are underlined.

View larger version (39K): [in a new window]   Fig. 6. Caspase 3 like protein is expressed and activated during Ciona intestinalis metamorphosis. Comparative western blot analysis of Ciona tissue homogenates, with antibody against human caspase 3. Coomassie Blue-stained gel (CB) showing the total proteins from Ciona (top). Western blotting (WB) was performed at various stages of embryonic development and metamorphosis (bottom).

View larger version (36K): [in a new window]   Fig. 7. Direct detection of caspase activity in situ using the viable reagent CaspACETM FITC-VAD-FMK. (A-C) Double-detection of activated caspase and apoptosis in the tail of Ciona at metamorphosis stage (28 hpf). TUNEL labeling was performed after in vivo incorporation of caspACE/FITC-VAD-FMK. Digitized images were merged to superimpose the caspACE-labeled cells (green pseudo-color field) over the respective TUNEL-labeled field (TUNEL-positive nuclei appear in red pseudo-color). Note the presence of caspACE-positive/TUNEL-positive cells (yellow nuclei/green cytoplasm) that co-exist with caspACE-positive/TUNEL-negative cells (green nuclei/green cytoplasm) and CaspACE-negative/TUNEL-positive cells (red nuclei). (D) In vivo incorporation of caspACE in striated muscle cells of Ciona tail at 28 hpf. (E) Pan-caspase inhibitor treatment blocks the metamorphosis of Ciona intestinalis. Data represent the mean of four independent experiments expressed as a percentage. Vertical bars correspond to the interval of confidence for percentage at 5%. The results were assessed by variance analysis and were found to be significant (P<0.05). Scale bars: 80 µm.

View larger version (74K): [in a new window]   Fig. 8. (A) Detection of a MAP kinase isoform and tubulin in protein extracts from Ciona intestinalis. Coomassie Blue-stained gel showing the total proteins from Ciona intestinalis before (18 hpf) and after (24 hpf) induction of metamorphosis (upper panel; lanes 1 and 2, respectively). Corresponding western immunoblots probed with the anti-ERK1/ERK2, anti-activated ERK1/ERK2 and anti-tubulin antibodies (lower panels). Western immunoblot analysis shows the presence of equivalent amount of a MAP kinase isoform in both Ciona intestinalis extracts, whereas activation of this MAP kinase is detected only after metamorphosis induction. -Tubulin breakdown is also observed at this stage. C2 C12 cells were used as control (lane 3). (B) Alignment of Ciona and human -tubulin. Ciona -tubulin (Ci -tub) was aligned with human -tubulin (Hs -tub, NP_006073). Conserved residues are indicated by a dash. Non-conserved residues are shaded. (C) Alignment of Ciona ERK and p38 with MAPK from other species. Ciona sequences were aligned with ERK2 from frog (XlERK2, CAA42482.1), human (HsERK2, NP_002736.1), mouse (MmERK2, NP_036079.1) and fish (DrERK2, BAB11813.1), with ERK1 from human (HsERK1, AAH13992.1) and mouse (MmERK1, S28184), and with p38 from human (Hsp38, Q16539) and frog (Xlp38, P47812). Red indicates residues shared by all proteins; blue indicates residues shared by all ERKs; gray indicates consensus p38 residues; yellow indicates specific ERK1 residues; green indicates residues shared by ERK1 and CiERK2; the blue shading indicates epitope recognized by the ERK monoclonal anti-phosphotyrosine antibody.

View larger version (70K): [in a new window]   Fig. 9. Inactivation of Ciona ERK blocks metamorphosis. (A,B) Nuclei of cells of the tail are negative for ERK activation (red fluorescence) and apoptosis (green fluorescence) at 18 hpf (A is the phase contrast image of the same fluorescent field shown in B). (C) At 28 hpf, numerous nuclei of cells of the tail extremity are labeled either for ERK activation (red), apoptosis (green) or very rarely for both (yellow, arrow). Scale bars: 40 µm in A,B; 25 µm in C. (D) Extracts from untreated larvae at 18 hpf (lane 1) and at 24 hpf (lane 2), and larvae at 24 hpf treated with 2 µM U0126 MEK inhibitor (lane 3) were run on SDS-PAGE, stained with Coomassie brilliant blue (upper panel) and western blotted with the anti-phosphorylated ERK monoclonal antibody (lower panel). (E) U0126 MEK inhibitor blocks the metamorphosis of Ciona intestinalis. Data represent the mean of two independent experiments expressed as a percentage of total number of larvae. Vertical bars correspond to the interval of confidence for percentage at 5%. The results were assessed by variance analysis and were found to be significant (P<0.01). Note that during these experiments, a significant number of larvae did not hatch, but no effect of U0126 was detected at this stage.

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