Download Plakoglobin expression and localization in zebrafish embryo

Signalling Outwards and Inwards
Plakoglobin expression and localization in
zebrafish embryo development
E.D. Martin1 and M. Grealy
Pharmacology Department and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland
Abstract
Plakoglobin (γ -catenin) and β-catenin are major components of the adherens junctions and can be localized
to the nucleus by activation of the Wnt signalling pathway. In addition, plakoglobin is also found in
desmosomes, a vertebrate-specific cell–cell adhesion structure. Plakoglobin expression and localization
were examined at the protein level during zebrafish embryonic development by Western blotting and
confocal microscopy. Plakoglobin was expressed throughout embryo development at the protein level.
Western blotting revealed that embryonic plakoglobin protein content increased between 12- and 24-h
post-fertilization (hpf). Confocal microscopy showed that at stages up to 12 hpf, plakoglobin and β-catenin
were co-localized and expressed in both the nucleus and in cell–cell junctions. At 24- and 72-hpf, separate
patterns were seen for plakoglobin and β-catenin. These data indicate that plakoglobin localization in the
heart region shifts from adherens junctions to desmosomes during heart chamber development.
Introduction
Cell adhesion is important in many cellular processes including organ development. In vertebrates, cell–cell adhesion is
mediated by two main types of adhesion structures, adherens
junctions and desmosomes. Adherens junctions connect cells
to the actin filament network and play a role in signalling
in the cell. Plakoglobin (γ -catenin) and β-catenin are major
components of the adherens junctions and can be localized to
the nucleus by activation of the Wnt signalling pathway. In
addition, plakoglobin is also found in desmosomes, which
are vertebrate-specific cell–cell adhesion structures found
in tissues that undergo mechanical stress, such as skin and
heart [1].
Zebrafish has many advantages as a model species, including external fertilization of optically clear embryos that
develop rapidly and can survive for a few days without a
functional cardiovascular system allowing analysis of severe
cardiovascular defects [2,3]. Although much is known about
the morphological and genetic control of heart development,
little is known about the signalling molecules that are involved
in this control [4]. We hypothesized that plakoglobin is involved in the control of heart development and, in the
present study, we found its expression in normal zebrafish
development.
Materials and methods
Western immunoblotting
Zebrafish embryos at the appropriate developmental stage
were dechorionated and deyolked, rinsed in PBS and
sonicated for 1 s in lysis buffer [400 mM sodium chloride,
Key words: β-catenin, desmosomes, heart, plakoglobin, zebrafish.
Abbreviation used: hpf, hours post-fertilization.
1
To whom correspondence should be addressed (email [email protected]).
20 mM Tris (pH 8.0), 20% (v/v) glycerol, 2 mM dithiothreitol
and 1% protease inhibitor cocktail; Sigma]. Protein content
was estimated by Bradford assay and 25 µg of protein was
loaded on to 7.5% reducing SDS/polyacrylamide gels followed by transfer to nitrocellulose membranes. Membranes
were blocked in 5% (w/v) milk/Tris buffered saline for 1 h
at room temperature (20–25◦ C). The primary antibody was
diluted in appropriate blocking solution (anti-plakoglobin
1:1000; BD Biosciences) and incubated at 4◦ C overnight with
agitation. Goat anti-mouse peroxidase conjugate (1:8000;
Sigma) was diluted in blocking solution and incubated
with membrane for 2 h at room temperature followed by
chemiluminescence detection.
Confocal microscopy
Zebrafish embryos at the appropriate developmental stage
were fixed in 4% (w/v) paraformaldehyde and permeabilized
in a methanol series. Embryos were blocked for 2 h at
room temperature in a blocking solution [1 M glycine, 5%
(w/v) BSA, 1% fetal bovine serum in PBS/1% Tween] and
incubated in primary antibodies (plakoglobin 1:100 and βcatenin 1:200; Sigma) overnight at 4◦ C followed by secondary
antibodies, goat anti-rabbit FITC-labelled antibody (1:1000)
and goat anti-mouse monoclonal IgG2a Alexa Fluor 633
labelled antibody (1:500), for 2 h at room temperature. All
antibodies were diluted in blocking solution. Embryos were
mounted in 1% agarose and viewed using a Zeiss LSM 510
confocal microscope.
Results
Plakoglobin was detected by Western blotting in 6-, 12-, 24-,
48- and 72-hpf (hours post-fertilization) embryos with an
increase in expression between 12- and 24-hpf (Figure 1).
Plakoglobin was localized in 2-cell, 8-cell, sphere, 12-,
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Figure 1 Zebrafish plakoglobin expression in embryos by
Figure 2 Plakoglobin and β-catenin expression in the heart
Western blotting
Lane 1, molecular-mass standard; lane 2, 12 hpf; lane 3, 18 hpf; and
region of 72 hpf embryo by confocal microscopy
Plakoglobin (green) and β-catenin (red) have distinct expression
lane 4, 24 hpf.
patterns.
24-, 48- and 72-hpf embryos by confocal microscopy. It
was localized to the cell membrane and nucleus at all stages
examined. To determine whether plakoglobin was involved
in adherens junctions or desmosomes or both, β-catenin
localization was also examined. β-catenin was localized to
the cell membrane and nucleus at all developmental stages
examined. Embryos were examined after dual staining with
both anti-plakoglobin and anti-β-catenin antibodies. Up to
12 hpf, plakoglobin and β-catenin were co-localized and
expressed in both the nucleus and in cell–cell junctions.
However, by 24 hpf, there was a reduction in co-localization
and at 72 hpf, a clearly separate band was seen for plakoglobin and β-catenin in the heart region, with β-catenin band
being exterior to the plakoglobin band (Figure 2).
Discussion
In the present study, plakoglobin protein expression in
zebrafish embryos was analysed for the first time by Western
blotting and confocal microscopy. Zebrafish protein was
examined using antibodies raised against the human form
of the protein, which were shown to react specifically with
the zebrafish protein. Plakoglobin protein was detected at
all developmental stages with an increase in the level of the
protein between 12- and 24-hpf. This protein expression
corresponds well with the predictions of previous studies
of plakoglobin mRNA, which was ubiquitously expressed
at all stages examined [5]. The protein was localized to the
cell membrane in cell-adhesion junctions and to the nucleus,
indicating involvement in both cell adhesion and in Wnt
signalling. Up to 12 hpf, plakoglobin was extensively colocalized with β-catenin, which is found mainly in adherens
junctions. By 24 hpf, the pattern of expression for both
catenins in the heart region changed to two distinct expression
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patterns which were also found in 72 hpf embryos. The
change from co-localization to distinct expression patterns
may be as a result of plakoglobin moving from adherens
junctions to desmosomes. This may represent a requirement
for zebrafish heart to form desmosomes. Plakoglobin plays
an essential role in mouse heart development. Plakoglobin
null mutants die from embryonic day 10.5 onwards due to
severe heart abnormalities such as ventricle bursting. Desmosomes in these knockouts were significantly reduced and
structurally altered suggesting the importance of desmosomes
in vertebrate heart development [6]. In addition, mutation of
the human plakoglobin gene results in arrhythmogenic right
ventricular cardiomyopathy and palmoplantar keratoderma
[7]. The present work forms a foundation for the study
of the effects of loss of plakoglobin during zebrafish heart
development.
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Received 8 July 2004