|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online May 30, 2008
Journal of Experimental Biology 211, 1874-1881 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.012690
Extensive apoptosis and abnormal morphogenesis in pro-caspase-3 transgenic zebrafish during development
National Research Institute of Fisheries Science, 2-12-4 Fukuura, Yokohama 236-8648, Japan
* Author for correspondence (e-mail: mic{at}affrc.go.jp)
Accepted 2 April 2008
 |
Summary |
|---|
 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Key words: caspase-3, apoptosis, transgenic zebrafish, eye and heart formation, stress response, antisense morpholino oligo
|
INTRODUCTION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
;
Sardella et al., 2004;
Sollid et al., 2003;
Uchida et al., 2002) and
higher vertebrates (Cohen et al.,
1992; Ellis et al.,
1991; Fernandes-Alnemri et al.,
1994; Nicholson and
Thornberry, 1997; Steller,
1995). The characterization of genes involved in apoptosis has
been intensively pursued and has led to the identification of two major
classes of genes, the bcl2 family and the caspase family. Caspases
are proteases that cleave target substrates with a specific peptide sequence
(Chou et al., 2000;
Enari et al., 1998). During
apoptosis, the activation of caspases, which can be induced by nuclear,
metabolic or externally activated stimuli, takes place in a cascade fashion
and leads to nuclear engulfment and cell death
(Chou et al., 2000;
Thornberry and Lazebnik,
1998). The caspase-activated endonuclease (CAD)1, which cleaves
and inactivates the inhibitor of CAD (ICAD), is responsible for DNA
fragmentation at the linker regions between the nucleosomes
(Enari et al., 1998;
Sakahira et al., 1998). There
is evidence that caspases contribute to the drastic morphological changes seen
during apoptosis by proteolysing and disabling a number of key substrates,
including Crk-associated substrate, focal adhesion kinase and rabaptin-5
(Cosulich et al., 1997;
Kook et al., 2000;
Liu et al., 1997;
Wen et al., 1997). The most
commonly activated caspase-3 mediates the limited proteolysis of these
proteins.
Recent studies have highlighted some of the prominent features of apoptosis
during development (Raff,
1996; Weil et al.,
1997). In mammals, cardiomyocyte apoptosis results from
cardiomyopathy and other cardiac disorders, and occurs in the myocardial
tissue of patients with heart failure
(Condorelli et al., 2001;
Narula et al., 1996). Mice
expressing caspase-3 in the muscle cells of the heart have increased rates of
heart failure and apoptosis (Condorelli et
al., 2001). Knockout mice lacking the caspase-3 gene showed skull
defects with ectopic masses of supernumerary cells, reflecting defective
programmed cell death during brain development and resulting in perinatal
lethality (Kuida et al.,
1996). Caspase-3-deficient mice showed an absence or delay of
apoptosis-associated morphological changes, such as cytoplasmic blebbing and
DNA fragmentation, in hepatocytes and thymocytes after Fas engagement
(Zheng et al., 1998). Thus,
caspase-3 is involved in the late stages of apoptosis in these cells
(Wang and Lenardo, 2000).
However, it is not clear whether apoptosis induced by overexpressed caspases
represents the physiological functions of these caspases in vivo. In
previous studies of zebrafish development, the fish embryo was demonstrated to
be a useful model for stress-induced apoptosis under conditions such as heat
shock and UV and 
-irradiation (Yabu
et al., 2001a; Yabu et al.,
2001b). Extensive apoptosis and caspase-3 activity were induced
under these stress conditions. In addition, the transient overexpression of
pro-caspase-3 in zebrafish embryos resulted in enhanced pro-apoptotic signals
(caspase activation and ceramide generation) under normal developmental
conditions in vivo. Therefore, the overexpression in transgenic
embryos would be expected to enhance the pro-apotoptic signalling and abnormal
morphogenesis regulated by a caspase-3-mediated mechanism under early
embryogenesis.
In order to characterize the importance of caspase-3 in embryogenesis, we generated transgenic zebrafish that overexpressed pro-caspase-3 in their embryos. These transgenic zebrafish were characterized under basal conditions at both the functional and molecular level. This study describes the effects of pro-caspase-3 overexpression and deficiency on the pro-apoptotic pathway regulating apoptosis in embryogenesis and stress tolerance during zebrafish development.
|
MATERIALS AND METHODS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
). Eggs and
embryos were collected and maintained at 28.5°C and were staged according
to the time that had elapsed since fertilization, and based on morphological
criteria (Westerfield, 1995).
The zebrafish pro-caspase-3 cDNA, containing the complete 889 bp open reading
frame (ORF), was amplified by PCR using the isolated full-length cDNA as a
template with the sense primer 5'-TACTGTTTAAAAGGGCTCGTTAAGCGG-3'
and an antisense primer corresponding to six histidine residues in front of a
stop code, 5'-TTAGTGGTGGTGGTGGTGGTGAGGAGTGAAGTACATCTCTTTGGT-3',
according to a previous paper (Yabu et
al., 2001b). The reaction was carried out with ExTaq
polymerase (Takara, Kyoto, Japan) for 25 cycles of denaturation at 94°C
for 30 s, annealing at 60°C for 30s, and extension at 72°C for 2min.
The amplified product was subcloned downstream from the human cytomegalovirus
(CMV) promoter of the pTARGET mammalian expression vector (Promega, Madison,
WI, USA), and the vector was named pCaspase-3. This plasmid construct was
linked to the green fluorescent protein (GFP) gene (pEGFP-C1 vector; Clontech,
Palo Alto, CA, USA) and was used for the generation of transgenic zebrafish
(accession no. AB090853). Fertilized one-cell stage embryos were collected
from wild-type zebrafish and microinjected with plasmid DNA, according to a
previously described method (Yabu et al.,
2001b). The injected zebrafish embryos, which were identified by
green fluorescence from GFP expression, were cultured for 5 days at 28.5°C
in sterilized tap water. Juvenile fish were cultivated to adulthood on a diet
of paramecium, brine shrimp larvae and commercial feed. Because the transgenic
embryos displayed bright green (GFP) fluorescence, the embryos that stably
expressed the transgene were easily selected. Two independent transgenic
lines, casp-1 and casp-2, were used for further studies. The F2 transgenic
progeny from an F1 x wild-type cross were used for further analyses.
Other transgenic lines overexpressing GFP, introduced by the pEGFP-C1 vector,
were also used for this study as a negative control
(Imamura et al., 2004).
Microinjection of morpholino oligonucleotide
The morpholino antisense oligonucleotide
5'-TTGCGTCCACACAGTCTCCGTTCAT-3' for zebrafish pro-caspase-3
(caspase-MO) was synthesized (Gene Tools, Boston, MA, USA) and used for
knockdown experiments by microinjection at 1–10 ng into a one-cell stage
zebrafish embryo. A morpholino oligonucleotide with five mispaired bases
(5-mispaired control MO; 5'-TTGCCTCCAGACAGTGTCGGTTGAT-3') was used
for microinjection as a negative control.
Whole-mount terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining and immunostaining
Whole-mount TUNEL staining of zebrafish embryos was adapted from a
tissue-sectioning method (Yabu et al.,
2001a). Embryos were fixed overnight at 4°C in a 4% solution
of formaldehyde in phosphate-buffered saline (PBS). The samples were washed
twice in 100mmoll–1 Tris-HCl (pH 7.5) buffer that contained
150mmoll–1 NaCl and 0.1% Tween 20 (TBST) and then fixed in
methanol at –20°C for at least 1 day. The samples were rehydrated
following methanol fixation by washing three times for 15 min each in TBST at
room temperature. The samples were transferred to TUNEL buffer (25 mmol
l–1 Tris-HCl pH 6.6, 200 mmol l–1 sodium
cacodylate, 5 mmol l–1 cobalt chloride and 0.25% bovine serum
albumin) and washed for 30 min. Fluorescence labelling of fragmented DNA was
carried out for 1 h at 37°C in TUNEL buffer that contained 1 mmol
l–1 fluorescein-dUTP (Hoffmann-La Roche, Basel, Switzerland)
and 50unitsml–1 terminal deoxynucleotidyl transferase
(Hoffmann-La Roche). The reaction was stopped by washing the samples five
times in TBST buffer for 5 min each at room temperature. The embryos were
stained with alkaline phosphatase-labelled anti-fluorescein antibody with a
chromogenic BM Purple AP substrate (Hoffmann-La Roche) according to the
methods described by the manufacturer. The samples were observed after
mounting in a TBST/glycerol solution (1:1). For histological observation,
cross-sections of the TUNEL-stained embryos were prepared after paraffin
embedding.
Whole-mount antibody staining with anti-caspase-3
(Yabu et al., 2001b)
polyclonal antibody was performed as described previously
(Westerfield, 1995). A
fluorescent secondary antibody labelled with Cy3 (Amersham, Piscataway, NJ,
USA) was used for fluorescence immunostaining. Photographs were taken using an
Edge 3D microscope (Edge, Marina Del Rey, CA, USA).
For histological analysis, embryos were fixed in a 4% solution of formaldehyde in PBS, embedded in Historesin Plus (Leica, Wetzlar, Germany) and sectioned at 5 µm. Sections were stained with Methylene Blue–Azure II, and mounted with Permount (Fisher Scientific, Waltham, MA, USA).
Caspase assay
To detect the active form of caspase-3 in the zebrafish embryos, the
hydrolysis of acetyl (Ac)-DEVD-
-methylcoumaryl-7-amide (MCA, Peptide
Institute, Osaka, Japan) was assayed according to a method described
previously (Yabu et al.,
2001a). Each aliquot of five embryos was washed once with PBS and
lysed in 50 µl of lysis buffer (20 mmol l–1 Hepes-KOH pH
7.5, 250 mmol l–1 sucrose, 50 mmol l–1
potassium chloride, 2.5 mmol l–1 magnesium chloride, 1 mmol
l–1 dithiothreitol). The lysate was centrifuged at 10 000
g for 15 min at 4°C, and the supernatant was collected and
used for assays of Ac-DEVD-MCA hydrolysis. The protein concentration was
determined with the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules,
CA, USA). One unit of enzyme activity was defined as the release of 1 nmol AMC
per hour at 37°C.
Stress treatment of zebrafish embryos
Stress treatment of embryos was carried out according to a previously
described method (Yabu et al.,
2001a). Three groups, each containing 30 embryos, were incubated
at 28.5°C in 2 ml of sterilized tap water in a six-well tissue culture
dish (Corning, Corning, NY, USA). The dish was sealed with Parafilm (American
National Can Co., Menasha, WI, USA) and kept in a water bath at the same
temperature. The 12 h embryos were placed in a dish that contained 0.5 ml of
sterilized tap water without a cover and were irradiated at 254 nm in a UV
cross-linker (model FS-800, Funakoshi, Tokyo, Japan). The embryos were allowed
to recover at 28.5°C, and the survival rates were measured daily.
Statistical survival analysis was performed with Prism4 software (GraphPad
Software, San Diego, CA, USA).
|
|
RESULTS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
|
The pro-caspase-3 transgenic embryos showed stable overexpression of the transgene product by immunofluorescent chemical detection (Fig. 2). We introduced the gene construct consisting of pro-caspase-3 cDNA with a His-tag at the C-terminal end to detect the transgene product in the transgenic zebrafish. His-tagged pro-caspase-3 was detected in the whole embryos of pro-caspase-3 transgenic fish at 24 h post-fertilization (h.p.f.), but not in the GFP control transgenic fish (Fig. 2). This finding indicates that the full-length pro-caspase-3 was ubiquitously expressed under the control of the CMV promoter in the transgenic zebrafish embryos.
|
The casp-1 and casp-2 transgenic lines clearly showed similar phenotypic disruptions in the formation of the eyes, heart, notochord and yolk sac (Fig. 3, Table 1). The hearts of the transgenic embryos developed abnormally (Fig. 4). The heart cavity of the transgenic embryos was enlarged, and the ventricle formed an elongated structure and was reduced in size. The contractility of the ventricle was weaker in transgenic embryos than in wild-type embryos, and blood circulation was reduced or ceased at 48 h.p.f. Heart rate measurements (beats min–1) at 30 h.p.f. indicated that the heart rate of transgenic casp-1 embryos was approximately 82% that of wild-type embryos. The transgenic embryos were characterized by reduced eye size. Histological sections revealed the loss of retinal cells in the transgenic fish; this loss of retinal cells was not localized to any particular cell layer in the retina (Fig. 5). The photoreceptor cells displayed the most severe defects. However, pigmentation and lens formation in the eyes of transgenic fish were not affected. The pro-caspase-3 transgenic embryo showed abnormal (irregular) morphology of the notochord (Fig. 6). Therefore, overexpression of pro-caspase-3 in the transgenic embryos induced abnormal morphogenesis in the eyes, notochord, heart and yolk sac.
|
|
|
|
|
|
|
-irradiation, induce extensive apoptosis through the
caspase-3-mediated pro-apoptotic signalling pathway at the early stages of
zebrafish development (Yabu et al.,
2001a; Yabu et al.,
2001b). Thus, UV irradiation can be used for bioassaying apoptosis
during development to assess stress sensitivity and tolerance in
vivo. In order to understand the relationship between cellular
pro-caspase-3 levels and stress tolerance in vivo, we examined the
survival rates of transgenic zebrafish that overexpressed pro-caspase-3 under
different stress conditions. When pro-caspase-3 transgenic fish embryos at 12
h.p.f. were exposed to UV irradiation at 0, 2 and 5mJcm–2,
the embryos displayed significantly lower survival rates than the GFP control
embryos in a dose-dependent manner (Fig.
10). The survival rate of the pro-caspase-3 transgenic fish on the
4th day after UV irradiation at 5 mJ cm–2 was 17.6%, while
that of the GFP control fish was 77.8%. These findings indicate that
pro-caspase-3 expression levels in the embryos determine stress
sensitivity.
|
To confirm that caspase-3-dependent apoptosis is essential for stress tolerance during zebrafish development, the specific effects of caspase-3 repression were observed after the microinjection of antisense morpholino oligonucleotide (MO) into zebrafish embryos. We designed a MO spanning the region +1 to + 25 (caspase-3-MO) of the zebrafish pro-caspase-3 mRNA, as predicted from its cDNA sequence, and injected caspase-3-MO into one-cell stage embryos. When a higher dose (10 ng) of caspase-3-MO was introduced into the caspase-3 transgenic and wild-type embryos, epiboly was arrested at 8–12 h.p.f., indicating that complete caspase deficiency caused the arrest of early embryogenesis (data not shown). When 1 ng of caspase-3-MO was introduced into wild-type embryos, the embryos showed normal or slightly dorsalized phenotypes. The embryos in which a 5-mispaired control MO sequence had been introduced showed no apparent abnormal phenotypes. We examined the efficiency of the targeted knockdown by measuring Ac-DEVD-MCA hydrolysing activity and through the immunochemical detection of the active form of caspase-3 using anti-active caspase-3 antibody in the MO-injected embryos (Fig. 11). The MO-injected wild-type embryos had significantly lower activity than the control embryos. Thus, the caspase-3-MO was effective for the repression of caspase-3 expression in both wild-type embryos in vivo. We used embryos injected with 1 ng of caspase-3-MO for further experiments involving exposure to UV irradiation.
|
To examine the relationship between cellular pro-caspase-3 levels and stress tolerance in vivo, we exposed the caspase-3-deficient embryos (through MO injection) to UV irradiation at 5mJcm–2. When embryos were exposed to UV irradiation at 12 h.p.f., the caspase-3-MO-injected embryos displayed a significantly higher survival rate than the control embryos (Fig. 12). Therefore, the present findings indicate that pro-caspase-3 expression levels in the embryos determine stress sensitivity and tolerance.
|
|
DISCUSSION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
;
Thornberry and Lazebnik,
1998). Caspase-3 activity levels are markedly increased in
zebrafish embryos that have been exposed to heat shock, UV or
-irradiation (Yabu et al.,
2001a), and it has been suggested that caspase-3 is responsible
for early development through ceramide signalling
(Yabu et al., 2001b).
Apoptosis has been characterized in zebrafish embryos under both normal
developmental and severe stress conditions
(Yabu et al., 2001b).
Therefore, it was of interest to consider whether developmental functions
could be modified by pro-caspase-3 overexpression and deficiency. Our findings
showed that pro-caspase-3 overexpression induced extensive apoptosis and
abnormal formation of the eye, notochord and cardiac system. These findings
suggest critical roles for pro-caspase-3 in regulating multiple processes
during early embryogenesis. Interestingly, tissue-specific expression patterns
of apoptotic cells and abnormal morphology were observed in the pro-caspase-3
transgenic zebrafish.
In this study, we introduced pro-caspase-3 with a His-tag at the C-terminal
end to allow detection of the protein in the transgenic zebrafish. Previously,
we characterized the biochemical properties of recombinant His-tagged
caspase-3 expressed in Escherichia coli
(Yabu et al., 2001b). The
bacterially produced recombinant caspase-3 showed specific activity against
substrate Ac-DEVD-MCA, similar to mammalian caspase-3
(Yabu et al., 2001b). In
addition, the transiently expressed pro-caspase-3 with a His-tag sequence at
the C-terminus in cultured fish cells was activated and induced apoptosis and
ceramide generation by limited proteolytic processing under stress conditions
(Yabu et al., 2001b). Thus,
the gene construct is well designed for examining caspase function and
stress-induced apoptosis in vivo. In this study, the full-length
pro-caspase-3 cDNA was stably overexpressed during zebrafish development under
the control of the CMV promoter, which exhibits ubiquitous expression in
zebrafish embryos (Fig. 2).
From these previous studies and the present findings, the His-tagged zebrafish
pro-caspase-3 used in this study was considered to have the ability to trigger
endogenous pro-apoptotic signalling activation in vivo. Therefore,
the differential effects of apoptosis and abnormal morphogenesis in the eyes
and heart could be the result of a tissue-specific enhanced processing of
pro-caspase-3 in these organs, rather than increased expression.
The activation of pro-caspase-3 is strictly repressed in vitro and
in vivo under normal physiological conditions
(Nicholson and Thornberry,
1997). These findings suggest a differential regulation of the
increased sensitivity to stress stimuli
(Yabu et al., 2001a), the
increased expression of Bcl2 (Chou et al.,
2000), and/or the decreased expression of Bax
(Nicholson and Thornberry,
1997) in the tissues of developing transgenic embryos. Therefore,
the zebrafish model overexpressing pro-caspase-3 is important and useful for
delineating structural changes mediated by differential regulation of
apoptosis in zebrafish embryos.
A phenotype found to be associated with pro-caspase-3 over-expression was
the defective formation of the eye and notochord. Several mutations that
affected the development of the retina, brain and heart were noted in previous
screens for genetic defects in zebrafish embryogenesis
(Chen et al., 1996;
Malicki et al., 1996;
Stainier et al., 1996). The
retinal mutations were classified into six phenotypic categories: neuronal
patterning defect, cyclopia, defect of the outer retina, growth retardation,
non-specific retinal degradation, and retinal degradation associated with
defective pigmentation. In this study, the pro-caspase-3 transgenic fish had
reduced eye size and degeneration of the retina and photoreceptor cell layers,
but had normal pigmentation and lens formation, suggesting that the specific
generation of retinal cells was induced by excessive caspsase-3 activation.
These defects in the transgenic fish were similar to those of previously
reported mutant phenotypes, such as turbulent and ziemniok
(Malicki et al., 1996).
Therefore, caspase-3-mediated pro-apoptotic signalling may regulate retinal
differentiation and development, and retinal degeneration may result from
enhanced apoptotic signalling in response to different environmental stimuli,
such as stress and treatment with apoptosis-inducing reagents.
Another phenotype observed in the pro-caspase-3 transgenic embryos was
abnormal cardiac formation and function. We observed heart failure due to a
weakened heart and reduced circulation, which has also been seen in fish with
known mutations, such as pipe heart
(Stainier et al., 1996) and
pipe line (Chen et al.,
1996). Heart-targeted overexpression of caspase-3 in mice
increased infarct size and depressed cardiac contractility after
ischaemia–reperfusion injury
(Condorelli et al., 2001).
Therefore, the caspase-3-mediated pro-apoptotic pathway may play an important
role in the development of the heart, eye and notochord. Although many
zebrafish mutants with neural degeneration and developmental defects have been
generated (Chen et al., 1996;
Malicki et al., 1996;
Stainier et al., 1996), the
genes associated with these mutations have rarely been identified. There is
the possibility that these morphological defects may be a secondary
consequence of general retardation caused by extensive apoptosis during early
development.
In transgenic mice, overexpression of the human caspase-3 gene did not
induce morphogenetic defects during normal development but leads to greater
apoptosis under stress conditions (Kerr et
al., 2004). The transgenic mice showed significantly larger
lesions when they were subjected to focal cerebral ischaemia–reperfusion
injury. These results indicate that mice overexpressing human caspase-3 are
essentially normal; however, they have increased susceptibility to
degenerative insults. On the other hand, the present findings provide evidence
that pro-caspase-3 overexpression, even in a partly activated state, is lethal
under normal developmental conditions. Although in the case of the transgenic
embryos with extremely high caspase activity almost all of the embryos
survived under normal conditions, abnormal morphogenesis was found especially
in the eyes, heart and spinal chord. In addition, approximately 60% of the F2
fluorescent progeny did not exhibit developmental abnormalities. Thus,
caspase-3 activation and apoptosis may occur in only a limited number of
specific tissue and cell types in the embryos.
In the human breast cancer cell line MCF-7, pro-caspase-3 overexpression
showed a 3.7-fold higher specific enzyme activity but was not toxic and did
not affect background apoptosis (Friedrich
et al., 2001). Interestingly, the pro-caspase-3-transfected cells
were more sensitive to cytotoxic drugs compared with control cells. Thus,
overexpression of caspase-3 in the transgenic zebrafish embryos may affect
only a limited number of apoptotic cells under normal growing conditions and
may enhance stress sensitivity especially in situations where activation of
pro-apoptotic signalling is disturbed.
Apoptosis may be regulated for the purpose of eliminating unnecessary cells
after cell differentiation during embryogenesis, as was shown in the nematode
ced-3 mutant, which has many extra cells due to the prevention of
almost all programmed cell death (Avery and
Horvitz, 1987). Therefore, the proper temporal and spatial
regulation of apoptosis may be maintained by the tissue-specific expression of
intercellular morphogenetic signalling factors, such as BMP4 and other
TGF-β superfamily members, as pointed out in the case of digit formation
in mouse and chicken (Monsoro-Burq, 1996;
Yokouchi et al., 1996).
Furthermore, the overexpression of pro-caspase-3 in transgenic embryos
under stress conditions of low-dose UV irradiation enhanced mortality
(Fig. 10), while caspase-3
deficiency decreased mortality (Fig.
12). Environmental stresses have been shown to be critical
mediators of apoptosis during zebrafish embryogenesis
(Yabu et al., 2001a). The
pro-caspase-3 transgenic fish were very sensitive to low doses (2 and 5 mJ
cm–2) of UV irradiation
(Fig. 10). This suggests that
the activation of caspase-3 in apoptosis can be affected by UV irradiation and
that UV irradiation and pro-caspase-3 may act as mediators of the same
pathway. Therefore, pro-caspase-3 may regulate the progression of
pro-apoptotic signalling and play a critical role in the survival of fish
embryos under normal and stress conditions in vivo.
In conclusion, the stable overexpression of pro-caspase-3 induced extensive apoptosis and abnormal morphogenesis in the eyes, notochord, heart and yolk sac during zebrafish development. The enhanced processing of pro-caspase-3 appears to trigger significant apoptotic responses in these specific target tissues. High pro-caspase-3 levels may enhance stress sensitivity and death of the organism in vivo. As a model for the characterization and analysis of the pro-apoptotic pathway, the transgenic zebrafish offers a high-quality, high-throughput bioassay tool for determining the biological effects of chemical compounds as well as for dissecting biological pathways.
|
Acknowledgments |
|---|
|
References |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Avery, L. and Horvitz, H. R. (1987). A cell that dies during wild-type C. elegans development can function as a neuron in a ced-3 mutant. Cell 51,1071 -1078.[CrossRef][Medline]
Chen, J. N., Haffter, P., Odenthal, J., Vogelsang, E., Brand, M., van Eeden, F. J., Furutani-Seiki, M., Granato, M., Hammerschmidt, M., Heisenberg, C. P. et al. (1996). Mutations affecting the cardiovascular system and other internal organs in zebrafish. Development 123,293 -302.[Abstract]
Chou, K. C., Tomasselli, A. G. and Heinrikson, R. L. (2000). Prediction of the tertiary structure of a caspase-9/inhibitor complex. FEBS Lett. 470,249 -256.[CrossRef][Medline]
Cohen, J. J., Duke, R. C., Fadok, V. A. and Sellins, K. S. (1992). Apoptosis and programmed cell death in immunity. Annu. Rev. Immunol. 10,267 -293.[CrossRef][Medline]
Condorelli, G., Roncarati, R., Ross, J., Jr, Pisani, A., Stassi,
G., Todaro, M., Trocha, S., Drusco, A., Gu, Y., Russo, M. A. et al.
(2001). Heart-targeted overexpression of caspase-3 in mice
increases infarct size and depresses cardiac function. Proc. Natl.
Acad. Sci. USA 98,9977
-9982.
Cosulich, S. C., Horiuchi, H., Zerial, M., Clarke, P. R. and Woodman, P. G. (1997). Cleavage of rabaptin-5 blocks endosome fusion during apoptosis. EMBO J. 16,6182 -6191.[CrossRef][Medline]
Ellis, R. E., Yuan, J. and Horvitz, H. R. (1991). Mechanisms and functions of cell death. Annu. Rev. Cell Biol. 7,663 -698.[CrossRef][Medline]
Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A. and Nagata, S. (1998). A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391,43 -50.[CrossRef][Medline]
Fernandes-Alnemri, T., Litwack, G. and Alnemri, E. S.
(1994). CPP32, a novel human apoptotic protein with homology to
Caenorhabditis elegans cell death protein Ced-3 and mammalian
interleukin-1ß-converting enzyme. J. Biol. Chem.
269,30761
-30764.
Friedrich, K., Wieder, T., von Haefen, C., Radetzki, S., Janicke, R., Schulze-Osthoff, K., Dorken, B. and Daniel, P. T. (2001). Overexpression of caspase-3 restores sensitivity for drug-induced apoptosis in breast cancer cell lines with acquired drug resistance. Oncogene 20,2749 -2760.[CrossRef][Medline]
Imamura, S., Ojima, N. and Yamashita, M. (2004). In vivo regulation of the CDC48 gene promoter in zebrafish embryos. Mar. Biotechnol. 6, S1-S7.[CrossRef]
Kerr, L. E., McGregor, A. L., Amet, A. L. E., Asada, T., Spratt, C., Allsopp, T. E., Harmar, A. J., Shen, S., Carlson, G., Logan, N. et al. (2004). Mice overexpressing human caspase 3 appear phenotypically normal but exhibit increased apoptosis and larger lesion volumes in response to transient focal cerebral ischaemia. Cell Death Differ. 11,1102 -1111.[CrossRef][Medline]
Kook, S., Shim, S. R., Choi, S. J., Ahnn, J., Kim, J. I., Eom,
S. H., Jung, Y. K., Paik, S. G. and Song, W. K. (2000).
Caspase-mediated cleavage of p130cas in etoposide-induced apoptotic Rat-1
cells. Mol. Biol. Cell
11,929
-939.
Kuida, K., Zheng, T. S., Na, S., Kuan, C., Yang, D., Karasuyama, H., Rakic, P. and Flavell, R. A. (1996). Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature 384,368 -372.[CrossRef][Medline]
Lesser, M. P., Farrell, J. H. and Walker, C. W. (2001). Oxidative stress, DNA damage and p53 expression in the larvae of atlantic cod (Gadus morhua) exposed to ultraviolet (290–400 nm) radiation. J. Exp. Biol. 204,157 -164.[Abstract]
Liu, X., Zou, H., Slaughter, C. and Wang, X. (1997). DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89,175 -184.[CrossRef][Medline]
Malicki, J., Neuhauss, S. C., Schier, A. F., Solnica-Krezel, L., Stemple, D. L., Stainier, D. Y., Abdelilah, S., Zwartkruis, F., Rangini, Z. and Driever, W. (1996). Mutations affecting development of the zebrafish retina. Development 123,263 -273.[Abstract]
Monsoro-Burq, A. H., Duprez, D., Watanabe, Y., Bontoux, M., Vincent, C., Brickell, P. and Le Douarin, N. (1996). The role of bone morphogenetic proteins in vertebral development. Development 122,3607 -3616.[Abstract]
Narula, J., Haider, N., Virmani, R., DiSalvo, T. G., Kolodgie,
F. D., Hajjar, R. J., Schmidt, U., Semigran, M. J., Dec, G. W. and Khaw, B.
A. (1996). Apoptosis in myocytes in end-stage heart failure.
N. Engl. J. Med. 335,1182
-1189.
Nicholson, D. W. and Thornberry, N. A. (1997). Caspases: killer proteases. Trends Biochem. Sci. 22,299 -306.[CrossRef][Medline]
Raff, M. C. (1996). Size control: the regulation of cell numbers in animal development. Cell 86,173 -175.[CrossRef][Medline]
Sakahira, H., Enari, M. and Nagata, S. (1998). Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391,96 -99.[CrossRef][Medline]
Sardella, B. A., Matey, V., Cooper, J., Gonzalez, R. J. and
Brauner, C. J. (2004). Physiological, biochemical and
morphological indicators of osmoregulatory stress in `California' Mozambique
tilapia (Oreochromis mossambicus x O. urolepis
hornorum) exposed to hypersaline water. J. Exp.
Biol. 207,1399
-1413.
Sollid, J., De Angelis, P., Gundersen, K. and Nilsson, G. E.
(2003). Hypoxia induces adaptive and reversible gross
morphological changes in crucian carp gills. J. Exp.
Biol. 206,3667
-3673.
Stainier, D. Y., Fouquet, B., Chen, J. N., Warren, K. S., Weinstein, B. M., Meiler, S. E., Mohideen, M. A., Neuhauss, S. C., Solnica-Krezel, L., Schier, A. F. et al. (1996). Mutations affecting the formation and function of the cardiovascular system in the zebrafish embryo. Development 123,285 -292.[Abstract]
Steller, H. (1995). Mechanisms and genes of
cellular suicide. Science
267,1445
-1449.
Thornberry, N. A. and Lazebnik, Y. (1998).
Caspases: enemies within. Science
281,1312
-1316.
Uchida, D., Yamashita, M., Kitano, T. and Iguchi, T.
(2002). Oocyte apoptosis during the transition from ovary-like
tissue to testes during sex differentiation of juvenile zebrafish.
J. Exp. Biol. 205,711
-718.
Wang, J. and Lenardo, M. J. (2000). Roles of caspases in apoptosis, development, and cytokine maturation revealed by homozygous gene deficiencies. J. Cell Sci. 113,753 -757.[Abstract]
Weil, M., Jacobson, M. D. and Raff, M. C. (1997). Is programmed cell death required for neural tube closure? Curr. Biol. 7,281 -284.[CrossRef][Medline]
Wen, L.-P., Fahrni, J. A., Troie, S., Guan, J.-L., Orth, K. and
Rosen, G. D. (1997). Cleavage of focal adhesion kinase by
caspases during apoptosis. J. Biol. Chem.
272,26056
-26061.
Westerfield, M. (1995). The Zebrafish Book. Oregon: University of Oregon Press.
Yabu, T., Todoriki, S. and Yamashita, M. (2001a). Stress-induced apoptosis by heat shock, UV and g-ray irradiation in zebrafish embryos detected by increased caspase activity and whole mount TUNEL staining. Fish Sci. 67,333 -340.[CrossRef]
Yabu, T., Kishi, S., Okazaki, T. and Yamashita, M. (2001b). Characterization of zebrafish caspase-3 and induction of apoptosis through ceramide generation in fish fathead minnow tailbud cells and zebrafish embryo. Biochem. J. 360, 39-47.[CrossRef][Medline]
Yokouchi, Y., Sakiyama, J., Kameda, T., Iba, H., Suzuki, A., Ueno, N. and Kuroiwa, A. (1996). BMP-2/-4 mediate programmed cell death in chicken limb buds. Development 122,3725 -3734.[Abstract]
Zheng, T. S., Schlosser, S. F., Dao, T., Hingorani, R., Crispe,
I. N., Boyer, J. L. and Flavell, R. A. (1998). Caspase-3
controls both cytoplasmic and nuclear events associated with Fas-mediated
apoptosis in vivo. Proc. Natl. Acad. Sci. USA
95,13618
-13623.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
