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.
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 .
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 . 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
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, 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.
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 β-catenin 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.
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-, 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).
Zebrafish plakoglobin expression in embryos by Western blotting
Plakoglobin and β-catenin expression in the heart region of 72 hpf embryo by confocal microscopy
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 . 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 co-localized 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 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 . In addition, mutation of the human plakoglobin gene results in arrhythmogenic right ventricular cardiomyopathy and palmoplantar keratoderma . The present work forms a foundation for the study of the effects of loss of plakoglobin during zebrafish heart development.
Signalling Outwards and Inwards: A Focus Topic at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by J. Challiss (Leicester, U.K.), A. Harwood (University College London, U.K.), M. Humphries (Manchester, U.K.), C. Isacke (Institute of Cancer Research, London, U.K.), R. Liddington (Burnham Institute, La Jolla, CA, U.S.A.), T. Palmer (Glasgow, U.K.), K. Siddle (Cambridge, U.K.), C. Sutherland (Dundee, U.K.), H. Wallace (Aberdeen, U.K.) and M. Welham (Bath, U.K.).