First published online March 1, 2007
doi: 10.1242/10.1242/dev.002220
Development 134, 1203-1219 (2007)
Published by The Company of Biologists 2007
ERK- and JNK-signalling regulate gene networks that stimulate metamorphosis and apoptosis in tail tissues of ascidian tadpoles
Jean-Philippe Chambon1,*,
,
Akie Nakayama1,2,*,
Katsumi Takamura3,
Alex McDougall4 and
Noriyuki Satoh1,
1 Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku,
Kyoto 606-8502, Japan.
2 Department of Biology, Graduate School of Science, Osaka University, 1-1
Machikaneya-cho, Toyonaka, Osaka 560-0043, Japan.
3 Department of Marine Biotechnology, Faculty of Life Science and Biotechnology,
Fukuyama University, Fukuyama 729-0292, Japan.
4 UMR 7009, Centre National de la Recherche Scientifique/Université
Pierre et Marie Curie, Biologie du Développement, Observatoire
Océanologique de Villefranche-sur-Mer, quai de la Darse-06234
Villefranche-sur-Mer Cedex, France.
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Fig. 1. MAPK activation in swimming larvae of Ciona intestinalis.
(A) Comparative western blot analysis of Ciona-tissue
homogenates with antibodies against dual-phosphorylated ERK, JNK and p38, and
non-phosphorylated ERK, JNK and p38. Top: Coomassie Blue-stained gel showing
the total protein. Bottom: western blotting was performed at various stages of
the swimming larval phase and during metamorphosis. ERK- and JNK-proteins were
expressed and activated during the acquisition of metamorphosis competence.
(B) The CNS and papillae in swimming larvae of C.
intestinalis. OC, ocellus; OT, otolith. (C) Ci-JNK activation was
localized in the CNS. Ci-JNK phosphorylation (green) was detected in the
sensory vesicles, the neck region, the visceral ganglion and along the nerve
cord. (D) Ci-ERK activation was localized in the papillae of C.
intestinalis larvae. Ci-ERK phosphorylation (green) was detected in the
three posterior palps of the papillae of the tadpole. Scale bars: 120
µm.
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Fig. 2. Inactivation of MAPK blocks metamorphosis and associated
apoptosis-dependent tail regression. (A) Double detection of
apoptosis and nuclei in the tail of Ciona intestinalis during
metamorphosis (at 28 hpf). Digitized images were merged to superimpose nuclei
(blue) over the respective TUNEL-labelled field (TUNEL-positive nuclei appear
in green). Notice that the nuclei of numerous cells of the tail extremity are
TUNEL positive in the control panel (DMSO) and in p38 inhibitor
(SB203580)-treated larvae. By contrast, TUNEL-positive nuclei were detected
very rarely in the presence of the ERK inhibitor (U0126) or the JNK inhibitor
(SP600125) in treated larvae. The white square corresponds to the region of
higher magnification displayed in the lower panel. (B) Double detection
of ERK phosphorylation (green) or JNK phosphorylation (red) and nuclei (blue)
in larvae at 22 hpf. ERK phosphorylation was detected in papillae and JNK
phosphorylation was detected in the CNS of larvae treated with DMSO at 22 hpf.
Larvae treated with U0126 MEK inhibitor were negative for ERK activation in
papillae, and the larvae treated with SP600125 JNK inhibitor were negative for
JNK activation in the CNS (red). (C) Extracts from untreated larvae at
22 hpf and larvae treated with U0126 or SP600125 at this time were run on
SDS-PAGE and western blotted with the anti-phosphorylated ERK and
anti-phosphorylated JNK monoclonal antibodies. (D) U0126 MEK inhibitor
and SP600125 JNK inhibitor blocked metamorphosis of C. intestinalis.
From hatching, larvae were treated with 6 µm of U0126 or 10 µm of
SP600125. In one condition (U0126*) the treatment was repeated
every 6 hours due to the loss of activity of the MEK inhibitor U0126. Data
represent the mean of three independent experiments (400 animals per
experiment) expressed as a percentage of the total number of larvae. Scale
bars: 225 µm in A, upper panel; 50 µm in A, lower panel; 45 µm in
B.
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Fig. 3. Gene expression of MAPK pathway components in Ciona
intestinalis larvae. (A) Microarray analysis of MAPK gene
expression during C. intestinalis swimming larval phase. Diagram of
mammalian MAPK pathways superimposed with oligonucleotide-based chip data. The
microarray data are represented by a square composed of two rows (two
independent experiments) and eight smaller squares corresponding to each
experimental point (every 2 hours from 18 hpf to 30 hpf, the last experimental
point is at 50 hpf). Downregulated genes are displayed in green and
upregulated genes are displayed in red. The expression level of each gene at
each experimental point was normalized with its expression level at 18 hpf
(first small black square). (B) Expression level of the SRF,
c-jun and MKP genes from hatching to metamorphosis.
(C) Semi-quantitative RT-PCR for SRF, c-jun and MKP. mRNA was extracted
from embryos, reverse transcribed and semi-quantitative specific PCR was
performed (see Materials and methods). S26 ribosomal protein RNA represents an
internal control for the level of expression
(Vincent et al., 1993 ).
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Fig. 4. Genes controlled by the MAPK pathways. (A) mRNA expression of
ERK target genes at 25 hpf. mRNA was extracted from untreated embryos or
embryos treated with 6 µM U0126, reverse transcribed and semi-quantitative
specific PCR was performed (see Materials and methods). (B) mRNA
expression of JNK target genes at 25 hpf. mRNA was extracted from untreated
embryos or embryos treated with 10 µM SP600125, reverse transcribed and
semi-quantitative specific PCR was performed (see Materials and methods).
(C) Whole-mount in situ hybridization of genes controlled by ERK and
JNK displaying, respectively, specific expression in papillae and in the
nervous system (arrows) of Ciona intestinalis larvae.
Ci-Vwa1 and Ci-GNRH were detected in sensory vesicle
(arrows); Ci-endogl was observed in visceral ganglion and
Ci-oatp in the neck region (arrows).
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Fig. 5. Sushi antisense morpholino blocks apoptosis-dependent tail regression
during metamorphosis. (A) Whole-mount in situ hybridization of
Ci-sushi and Ci-Sccpb displaying, respectively, specific expression in tail
and at the tip of the tail of Ciona intestinalis larvae. The white
square corresponds to the region of higher magnification displayed in the
right panel. (B) JNK activation controls Ci-sushi expression.
Ci-sushi mRNA expression from hatching to metamorphosis. mRNA was
extracted from embryos at various time points, reverse transcribed and
semi-quantitative specific PCR was performed (see Materials and methods).
Ci-sushi mRNA expression at 25 hpf was extracted from untreated
embryos or from those treated with 10 µM SP600125, reverse transcribed and
semi-quantitative specific PCR was performed (see Materials and methods).
(C) Detection of apoptosis in the tail of C. intestinalis
tadpoles at the onset of metamorphosis (28 hpf). Apoptotic cells were TUNEL
labelled (TUNEL-positive nuclei appear green). Notice that numerous nuclei of
cells of the tail extremity were TUNEL-positive in the control panel. By
contrast, TUNEL-positive nuclei were detected very rarely in
Ci-sushi-morpholino antisense-injected larva. At this time, tunic
cells are TUNEL-positive in both cases (arrows), as described in our previous
work (Chambon et al., 2002).
(D) Model of the role played by the CNS in the regulation of apoptosis
during metamorphosis. Ci-JNK activation in the CNS leads to Ci-sushi
and Ci-Sccpb gene expression in epithelia. These genes are essential
for initiating apoptosis at the onset of metamorphosis. ECM modification is
also a result of Ci-JNK activation in the CNS, which could promote the
induction of apoptosis through Ci-ERK activation in adjacent tissues. Scale
bars: 200 µm.
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© The Company of Biologists Ltd 2007
