1 Laboratory of Molecular Virology and Biotechnology, Institute of
Biotechnology, National Cheng-Kung University, Tainan 701, Taiwan
2 Laboratory of Marine Molecular Biology and Biotechnology, Institute of
Zoology, Academia Sinica, Nankang, Taipei 115, Taiwan
3 Graduate Institute of Life Sciences, National Defense Medical Center, Taipei
117, Taiwan
Author for correspondence (e-mail:
jlwu{at}gate.sinica.edu.tw)
Accepted 19 August 2004
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SUMMARY |
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Key words: Phosphatidylserine receptor, Apoptotic corpses, Zebrafish, Knockdown, Brain, Organogenesis
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Introduction |
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Recent advances in the study of the nematode Caenorhabditis
elegans (Chung et al.,
2000; Wang et al.,
2003
) and Drosophila melanogaster (France et al., 1999a)
illustrate the power of using genetically tractable systems to identify
necessary phagocytic genes. Major efforts to understand crucial pathways that
mediate programmed cell death have also led to the genetic and molecular
characterization of a number of genes involved in the recognition and
engulfment mechanisms of cells amongst invertebrates
(Chung et al., 2000
;
Lauber et al., 2003
;
Arur et al., 2003
;
Ravichandran, 2003). For C. elegans it is important to recognize that
phagocytosis is performed by cells that are non-specific phagocytes rather
than by specialized phagocytes such as macrophages, as tends to be the case in
D. melanogaster (Savill and
Fadok, 2000
).
Cell death that is morphologically and genetically distinct from apoptosis
is strongly implicated in some human disease
(Meier et al., 2000). Little
is known regarding the molecular mechanisms by which the resulting (cell)
corpses are eliminated, and the clearance of defective events for zebrafish.
We sought to define the genetic requirements for a potentially distinct death
paradigm associated with a loss-of-function of the cell-corpse receptor PSR in
the zebrafish model system, to elucidate gene function related to
organogenesis.
Morpholinos are chemically modified oligonucleotides with base-stacking
abilities similar to those of natural genetic material
(Summerton and Weller, 1997).
Morpholinos have been shown to bind to and block translation of mRNA during
cell genesis in zebrafish (Nasevicius and
Ekker, 2000
). Initially, we cloned the psr gene from the
24 hours post-fertilization (hpf) cDNA library, and designed PSR morpholinos
for the knockdown of PSR expression during the embryonic development of
zebrafish. We found that a lot of apoptotic cells accumulated in the furrow or
individual boundary of the whole somite, and interfered with the normal
interior and posterior somitic formation at the early segmentation stage. At
the post-segmentation and organogenesis developmental stages, the embryos were
phenotypically defective in the brain, heart, somite and notochord. In
addition, injection of psr mRNA with PSR morpholinos could compensate
for the defective phenotype. These observations are consistent with an
evolutionarily conserved pathway involving PSR that is responsible for the
removal of cell corpses during cell migration, and for cell-cell interaction
processes that are tightly linked to normal development during morphogenesis
and organogenesis in zebrafish.
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Materials and methods |
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Electron microscopy
Embryos at different developmental stages (30% epiboly, 50% epiboly,
shield, 90% epiboly to tailbud) were collected with plastic droppers, placed
in microtubes, washed twice with phosphate buffered saline (PBS), and then
fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for
2 hours. They were then washed with sodium cacodylate buffer, post-fixed in 1%
aqueous osmium tetroxide for 2 hours, and washed with the same buffer. The
embryos were dehydrated in a series of ethanol solutions and embedded in a
Spurr's resin mixture using standard protocols. Semi-thin sections were cut,
stained with Toluidine Blue, and examined by light microscopy (Nikon Eclipse
E600, Nikon Corporation, Japan) to identify morphological patterns. Ultrathin
sections, cut using a microtome, were stained with standard preparations of
lead citrate and uranyl acetate, and observed using an electron microscope
(Hitachi H-7000, Japan) (Hong et al.,
1998).
PSR cloning
Two pairs of degenerate primers derived from human and Drosophila
sequences (GenBank) (Fadok, 2000) were used to synthesize a 0.5 kb probe (by
RT-PCR) to screen a 24-hour-old wild-type zebrafish (Danio rerio)
embryo cDNA library (Stratagene). The degenerate primers used were as
follows:
The cDNA library was screened using low stringency conditions. The positive clones obtained were sequenced using a single-base reaction with the ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's protocol. The following PSR homologs were acquired from GenBank: the human homolog KIAA 0585 (Nagase, 1998) (Accession number BAA25511); the mouse homolog AAH 06067 (Accession number AAG27719); the D. melanogaster homolog CG5383 (Accession number AF401485); the zebrafish homolog (Accession number AF401485. The C. elegans homolog was acquired from the cosmid F29B9.4 (Accession number AAF99922).
Morpholinos
Morpholinos were obtained from Gene Tools, LLC (Corvallis, OR, USA). All
morpholinos were arbitrarily designed to bind to sequences flanking and
including the initiating methionine. We selected sequences based on design
parameters according to the manufacturer's recommendations (21-25mer
antisense), and tested each design sequence for representation elsewhere in
the genome (Nasevicius and Ekker,
2000). Sequences were as follows (the sequence complimentary to
the predicted start codon is shown in bold in all cases): PSR-MO,
5'-TCCgTTTCTTgCTTTTATggTTCAT-3'; and control-MO,
5'-TCCCTTACTTgCATTTATCgTACAT-3'.
The five sites in the control sequence that are subject to point mutation in
the PSR-MO sequence are underlined.
Injection of psr morpholinos
Morpholino oligonucleotides were solubilized in water at a concentration of
1mM, and diluted with water to 0.5, 0.25 and 0.125 mM prior to injection
(1.5-3 nl) into the yolk (Ekker et al.,
1995).
In situ hybridization
Digoxigenin-labeled antisense RNA probes were synthesized from linearized
DNA templates, including psr, pax2.1 and nkx2.5, using T7
RNA polymerase (Boehringer Mannheim, Germany). Whole-mount in situ
hybridization was performed as previously described
(Xu et al., 1994). The in situ
hybridization assay used embryos injected with PSR-MO or control-MO at 12 hpf,
36 hpf or 3 days post-fertilization (dpf).
Apoptotic cell staining
Embryos at the one- or two-cell stage were injected with PSR-MO or
control-MO. They were harvested at 12 and 24 hpf, and fixed with 4%
paraformaldehyde in PBS (pH 7.4) at room temperature for 30 minutes. The
embryos were stained with Acridine Orange (1 µg ml-1) for 3-5
minutes, washed twice in PBS, and evaluated under fluorescence microscopy
(using incident light at 488 nm excitation, with a 515 nm longpass filter for
detection) (Hong et al.,
1998). For the TdT-dUTP labeling step, the embryos were fixed in
paraformaldehyde at the end of the incubation period (12 and 24 hpf),
dechorionated, and incubated in blocking solution (0.1%
H2O2 in methanol) for 30 minutes at room temperature.
Embryos were rinsed with PBS, incubated on ice in a solution of 0.1% Triton
X-100 in 0.1% sodium citrate for 30 minutes, to increase permeability, and
rinsed twice with PBS. Afterwards, 50 µl of TUNEL reaction mixture (in-situ
cell death-detection Kit, Boehringer Mannheim, Germany) was added and the
embryos were incubated in a humidified chamber for 1 hour at 37°C. Embryos
were analyzed for positive apoptotic cells under a fluorescence microscope
equipped with a spot II cool CCD (Diagnostic Instruments, Sterling Heights,
MI, USA).
Western blotting
Embryos were injected with PSR-MO or control-MO at the one- or two-cell
stage. They were harvested at 24 hpf and lyzed in 150-200 µl sodium dodecyl
sulfate (SDS) sample buffer [0.63 ml 1 M Tris-HCl (pH 6.8), 1.0 ml glycerol,
0.5 ml ß-mercaptoethanol, 1.75 ml 20% SDS, 6.12 ml H2O in a
total of 10 ml]. Protein from 40 µg of 24 hpf embryos was loaded on to each
lane. Standard western-blot analysis was conducted using a human anti-PSR
monoclonal antibody (Cascade Bioscience, Winchester, MA, USA) and a mouse
anti-actin monoclonal antibody (Chemicon, Temecula, CA, USA). PSR was
visualized using horseradish peroxidase-conjugated anti-mouse immunoglobulin
(IgG) and the ECL detection kit (Amersham Pharmacia Biotech, Denmark)
(Hong et al., 1999).
Microinjection of psr mRNA
Zebrafish psr was cloned into the pCDNA3 vector, which contains a
T7 RNA polymerase promoter site. Linearized plasmid DNA was used a template
for in vitro transcription with the Message Machine Kit (Ambion, Austin, TX,
USA), according to the manufacturer's instructions. For rescue of defective
morphants, 0.1 nl of 200 ng/µl mRNA encoding soluble psr mRNA and
PSR-MO (0.5 µM) were injected into the one-cell stage of each embryo using
a gas-driven microinjector (Medical System Corporation)
(Ekker, 1995).
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Results |
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|
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The predicted molecular weight (Fig.
2A) for F29B9.4 was 44.3 kDa, based on sequence analysis
(Fig. 2A). This is similar to
the corresponding molecular weight for the mouse species
(Fadok et al., 2000), slightly
smaller than the corresponding figure for the gene products in humans (45.5
kDa) and D. melanogaster (45 kDa), but larger than the analogous
figure for C. elegans (38.4 kDa). In addition, the consensus sequence
for the PSR-binding motif (FxFxLKxxxKxR) found in protein kinase C isoforms
indicates that a 12-amino acid peptide motif is responsible for the specific
interaction with PSR (Igarashi et al.,
1995
). A potential tyrosine phosphorylation site is indicated by
box A (Fig. 2A), corresponding
to residues 100-108 (KCGEDNDGY), which is well within the predicted
intracellular domain (Schultz et al.,
2000
). We found that the sequence indicated by box B (residues
143-206) resembled the jmjC domain that is part of the cupin metalloenzyme
superfamily that can regulate the chromatin-reorganization process
(Clissold and Ponting, 2001
).
Presently, the function(s) of the PSR region is unknown.
The protein sequence of topology programs varied slightly in their specific
assignments (see box C; FVPGGWWHVVLNLDTTIAITQNF, residues 257-287 of PSR-F),
based on an assessment of topology and possible hydrophobicity (SMART-TMHMM2)
(Schultz et al., 2000). The
predicted extracellular domain (indicated by box D) of the membrane-associated
domain (residues 340-359) contains a serine-rich sequence (342-355) that may
be glycosylated sites.
psr expression pattern
psr was expressed in embryos from the one-cell developmental stage
(30 minutes; data not shown) to the 3 dpf larval stage
(Fig. 2B, panels a-f). After
the somite segmentation period, psr was apparent throughout the
embryo (Fig. 2B, panels c-e)
and the hatching grand (Fig.
2B, panel e). At the larval (3 dpf) stage, psr expression
was detected in the heart and kidney (Fig.
2B, panel f).
Effects of psr knockdown
PSR morpholino oligonucleotides (MO; 40 ng) were injected into embryos to
accomplish knockdown of psr expression
(Fig. 3). psr
knockdown at the epiboly stage strongly affected embryonic morphological
formation at 12 hpf, and produced a severe delay in epiboly formation
(Fig. 3F), when compared with
control embryos (Fig. 3E). In
addition, cell corpses accumulated between the somite boundaries, close to the
notochord, at the six-to-seven segmentation stage
(Fig. 3D). Embryos treated with
control-MO did not display cell corpses in the somite or near the notochord
(Fig. 3B). PSR-MO embryos
appeared to be thin from an anterior lateral view, with the loss of furrow
from within the somite (Fig.
3C), when compared with control embryos
(Fig. 3A).
|
We next examined whether the cell corpses underwent apoptotic death (Fig. 4). Apoptotic corpses were covered in the furrow (indicated by arrows in Fig. 4), and lay close to the surface of individual somites at 14 hpf (Fig. 4C). After this time, a gradual increase in numbers and accumulation throughout embryos treated with psr oligonucleotides was evident (Fig. 4A,B, 17 hpf). Cell corpses leaked out from the 17 hpf embryos (Fig. 4B) that displayed chromatin condensation (Fig. 4D,E) in the corpse cells. Positive apoptoic cells were also evident in whole embryos at 17 hpf upon examination by the TUNEL assay (Fig. 4F-I).
|
|
We estimated the effect of different individual doses of PSR-MO (5 ng, n=209; 10 ng, n=238; 20 ng, n=160; 40 ng, n=224; control-MO, 40 ng, n=194) on embryonic development. As summarized in Table 1, developmental effects correlated to PSR-MO dosage, and were significant when compared with control-MO-injected embryos.
|
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Rescue of defective morphants with psr mRNA
In a previous study (Bauer et al.,
2001), 10-20 pg of psr mRNA compensated for the
developmental blockage imposed by PSR-MO when co-injected with 20 ng of PSR-MO
at the embryonic one- or two-cell stage. We observed that a similar
application of 20 ng of PSR-MO and 20 pg of psr mRNA reproduced the
earlier findings. In the PSR-MO group, embryos had accumulated many corpse
cells in the whole embryo by 12 hpf (Fig.
7C). Accumulation was particularly evident in the furrow between
somites. Conversely, in rescued embryos only a few corpse cells were evident
in the interior somite (Fig.
7D). Wild-type or control-MO embryos did not show accumulated
corpse cells (Fig. 7A,B).
|
We estimated the survival ratio and morphants phenotype ratio in the PSR-MO plus psr mRNA group (n=64), and in the PSR-MO group (n=93) at 2 dpf. Addition of psr mRNA reduced mortality from 12.5 to 1.5% (Fig. 7M). Addition of psr mRNA reduced the morphants phenotype ratio from 38 to 28% for the strongly defective embryos, and from 9 to 1.5% for the weakly defective embryos (Fig. 7M). The rescued group showed a normal morphogenesis and kidney-development pattern (Fig. 7O,R), when compared to the control-MO group (Fig. 7N,Q) at the 3-dpf stage. At the same stage, the weakly defective embryos in the PSR-MO group displayed patterns of morphogenesis and psr expression that were indicative of delayed kidney development (Fig. 7P,S).
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Discussion |
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Zebrafish as a model system for studying the engulfing gene from early to late development
The zebrafish (Danio rerio) has several advantages as a model for
studying development (ZFIN website,
http://zfish.uoregon.edu/)
(Dooley and Zon, 2000). In our
temporal study, we traced the path of abnormal development during the
knockdown of psr. We detected three phenotypically different embryos
(weakly defective, strongly defective and death type) during examinations at
12 and 36 hpf and 3 dpf. Although the weakly defective embryo was not apparent
at 12 hpf, the defect was apparent at 3 dpf as a mild abnormality that
included an enlarged heart cavity and defective notochord formation. The
strongly defective embryo type at 12 hpf was characterized by the accumulation
of a large number of apoptotic corpses in the posterior section of the embryo
that interfered with the posterior development. This was also the case at 36
hpf, and the embryo failed to hatch out at 3 dpf. The death type embryo was
characterized by an accumulation of a large number of cell corpses in the
whole embryo at 12 hpf. The embryos were developmentally delayed at 36 hpf and
died before the 3-dpf stage. Interestingly, the rescue studies that
demonstrate that the death type, and the weakly and strongly defective
phenotypes could be corrected or compensated for by the injection of
psr mRNA (Fig. 7)
suggest that psr mRNA could potentially be used to correct diseases
arising from psr gene defects.
How apoptotic cell corpses interfere with cell migration and embryonic development
The present study offers support for our idea that the accumulation of cell
corpses interferes with normal embryonic development by altering cell movement
(Fig. 3E,F) and cell-cell
interaction (Fig. 8).
|
PSR is a professional clearer of cell corpses
PSR mediates the engulfment of apoptotic cells in mice
(Fadok et al., 2000). A defect
of PSR in the early stages of organogenesis may be involved in respiratory
distress syndromes and congenital brain malformation
(Li et al., 2003
). In
addition, studies in C. elegans
(Wang et al., 2003
) and D.
melanogaster illustrate the power of using genetically tractable systems
to identify essential phagocytic genes
(Chung et al., 2000
;
Fares and Greenwald, 2001
;
Conradt, 2002
). It is important
to recognize that phagocytosis is performed by cells that are classified as
`non-professional' phagocytes, rather than by specialized phagocytes, such as
macrophages in D. melanogaster. Here, we shed more light upon the
role of this emerging phagocytosis receptor (PSR) in the vertebrate system
(Savill and Fadok, 2000
;
Henson et al., 2001
;
Schlegel and Williamson, 2001
;
Li et al., 2003
). Our results
indicate that phagocytosis during the development of zebrafish is performed by
PSR-mediated phagocytes, which are distributed throughout the entire embryo,
especially between the 2- and 3-somite stage and 24 hpf, and that these are
`professional' phagocytes rather than non-specialized phagocytes.
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ACKNOWLEDGMENTS |
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Footnotes |
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