Developmental Biology 327 (2009) 83–96

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Developmental Biology

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Plakoglobin has both structural and signalling roles in zebrafish development

Eva D. Martin a, Miriam A. Moriarty a, Lucy Byrnes b, Maura Grealy a,⁎ a Department of Pharmacology and Therapeutics and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Galway, Ireland b Department of Biochemistry and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Galway, Ireland article info abstract

Article history: Plakoglobin, or gamma-, is found in both and adherens junctions and participates in Received for publication 14 May 2008 Wnt signalling. Mutations in the human are implicated in the congenital disorder, Revised 21 November 2008 arrhythmogenic right ventricular (ARVC), but the signalling effects of plakoglobin loss in Accepted 25 November 2008 ARVC have not been established. Here we report that knockdown of plakoglobin in zebrafish results in Available online 10 December 2008 decreased heart size, reduced heartbeat, cardiac oedema, reflux of blood between heart chambers and a twisted tail. Wholemount in situ hybridisation shows reduced expression of the heart markers nkx2.5 at 24 Keywords: ARVC hours post fertilisation (hpf), and cmlc2 and vmhc at 48 hpf, while there is lack of restriction of the valve Adherens junctions markers notch1b and bmp4 at 48 hpf. Wnt target was examined by semi-quantitative RT- Desmosomes PCR and found to be increased in morphant embryos indicating that plakoglobin is antagonistic to Wnt Zebrafish signalling. Co-expression of the Wnt inhibitor, Dkk1, rescues the cardiac phenotype of the plakoglobin Wnt/beta-catenin signalling morphant. β-catenin expression is increased in morphant embryos as is its colocalisation with E- Plakoglobin in adherens junctions. Endothelial cells at the atrioventricular boundary of morphant have Morpholino an aberrant morphology, indicating problems with valvulogenesis. Morphants also have decreased numbers Heart development of desmosomes and adherens junctions in the intercalated discs. These results establish the zebrafish as a model for ARVC caused by loss of plakoglobin function and indicate that there are signalling as well as structural consequences of this loss. © 2008 Elsevier Inc. All rights reserved.

Introduction binds to members of the T-cell specific factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors (Behrens et al., 1996; Plakoglobin, or gamma-catenin, is a member of the catenin family Molenaar et al., 1996). The signalling role of plakoglobin was first of cell adhesion , containing the 42 amino acid armadillo indicated when its overexpression in Xenopus resulted in anterior repeat, the product of the segment polarity gene in Drosophila (Peifer axis duplication (Karnovsky and Klymkowsky, 1995). However, the and Wieschaus, 1990; Peifer et al., 1992, 1994). It is one of only a few mechanism by which plakoglobin exerts a signalling role is still proteins found in both desmosomes and adherens junctions. At the uncertain. Studies in keratinocytes have shown that plakoglobin from cell membrane, it associates with the cytoplasmic tail of cadherin adherens junctions can directly move to the nucleus and bind to molecules and links them to the filament network in adherens transcription factors (Hu et al., 2003). However, in a number of cell junctions or to the intermediate filament network in desmosomes. lines, nuclear plakoglobin prevented binding of TCF-4 or LEF-1 to The latter are found in tissues that undergo mechanical stress such as DNA resulting in a lack of transcriptional activity, whereas studies in epidermis and heart (for review Garrod and Chidgey, 2008). a β-catenin deficient cell line have shown plakoglobin to have direct In addition to their structural role in cell junctions, cytosolic TCF/LEF transcriptional activity (Zhurinsky et al., 2000; Miravet et al., plakoglobin and β-catenin participate in canonical Wnt signalling. In 2002; Maeda et al., 2004). In addition, plakoglobin may also have an the absence of a Wnt signal, they are phosphorylated and targeted indirect transcriptional activity as a result of replacing β-catenin in for degradation by the ubiquitin pathway. Binding of Wnt to a adherens junctions and allowing translocation of β-catenin to the Frizzled receptor results in stabilisation of the catenin degradation nucleus (Li et al., 2007). complex and translocation of the catenin to the nucleus where it A mutation leading to the insertion of at position 39 in the N-terminus of plakoglobin causes arrhythmogenic right ventricular cardiomyopathy (ARVC), which is characterised by irregular heartbeat and loss of myocardial tissue with replacement by adipose and fibrous Abbreviations: APC, adenomatous polyposis coli; ARVC, arrhythmogenic right tissue in the right (Asimaki et al., 2007), whereas a two base ventricular cardiomyopathy; BMP, bone morphogenic protein; TCF, T-cell specific pair (bp) deletion causing truncation of plakoglobin C-terminal results transcription factor; LEF, lymphocyte enhancer factor. ⁎ Corresponding author. Fax: +353 91 525700. in Naxos disease, which presents as palmoplantar and E-mail address: [email protected] (M. Grealy). woolly hair in addition to the ARVC (McKoy et al., 2000). Mutations in

0012-1606/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2008.11.036 84 E.D. Martin et al. / Developmental Biology 327 (2009) 83–96 other desmosomal proteins are also associated with ARVC (Rampazzo Western blotting and immunoprecipitation et al., 2002; Gerull et al., 2004; Pilichou et al., 2006). In an effort to model these lethal human diseases, plakoglobin Deyolked and dechorionated embryos were collected as described knockout mice were generated (Bierkamp et al., 1996; Ruiz et al., by Link et al. (2006) with protease inhibitor cocktail (Sigma) in all 1996). However, they showed embryonic lethality at day E10.5 or E12- solutions. Lysates were snap frozen in liquid nitrogen, thawed on ice and 15 due to ruptured ventricles. It is thought that the lack of proper 50 μl of lysis buffer (400 mM NaCl, 20 mM Tris pH 8.0, 20% Glycerol, desmosomes in cardiac tissue prevents it from withstanding the onset 2 mM DTT and 10% protease inhibitor cocktail) were added per 100 of cardiac contraction. In some genetic backgrounds, plakoglobin embryos. To aid yolk removal, Freon (100 μl) was added to lysates which knockout mice survived until late gestation but exhibited skin were vortexed for 30 s and centrifuged at 4°C for 15 min at 11,337×g . blistering (Bierkamp et al., 1996). These studies focused only on the Protein (25 μg) was resolved by 7.5% reducing SDS-PAGE followed by structural role of plakoglobin. Due to this embryonic lethality in mice, western blotting. Antibodies used included anti-plakoglobin (1:1000, we chose the zebrafish model system to examine plakoglobin function BD Transduction Laboratories), anti-α- (1:2000, Sigma), anti-β- because zebrafish embryos can survive for a few days without a catenin (1:1000, Sigma), and anti-FLAG antibody (1:2000, Sigma). A functioning cardiovascular system (reviewed by Stainier, 2001). cross-linking anti-mouse IgG antibody (1:2000, Sigma) was used with Previous studies in zebrafish have identified zebrafish plakoglobin the plakoglobin antibody. Secondary antibodies of goat anti-mouse IgG and localised the protein expression to cell–cell adhesion junctions in (1:8000, Sigma) and goat anti-rabbit IgG (1:140,000, Sigma) were used. follicle cells of the ovary (Cerdà et al., 1999). We have previously The cross-linking anti-mouse IgG antibody and Protein A beads (Sigma) localised plakoglobin protein expression in embryos from 1-cell to 72 were used to immunoprecipitate proteins. Following separation by SDS- hours post fertilisation (Martin and Grealy, 2004). We hypothesised PAGE, bands were excised and sequenced by ESI-tandem mass that plakoglobin has a signalling role in addition to its structural role spectrometry (Dr. S. Liddell, University of Nottingham). in embryo development. Here we report that the loss of plakoglobin results in embryos Preparation of mRNA for rescue experiments with severe cardiac defects. Increased colocalisation of proteins, E-cadherin and β-catenin, in the epithelial layer of Full length open reading frames (ORF) of plakoglobin-1a and morphant embryos confirms that plakoglobin co-operates with β- plakoglobin-1b were amplified using primers that included a FLAG- catenin in adhesion. Loss of plakoglobin results in an increase in tag label at the 5′ end of each construct (Supplementary Data, Table 1). Wnt target gene expression indicating that plakoglobin competes A second set of primers for each transcript was used to alter 5 bp in the with β-catenin in Wnt/β-catenin signalling. The atrioventricular morpholino target region without altering the resulting protein endothelial cells of morphant embryos have an abnormal morphol- sequence using a Quick-change site-directed mutagenesis kit (Strata- ogy indicating aberrant valvulogenesis. Adhesion junctions of the gene). Both constructs were subcloned into pCS2+vector. Capped intercalcated discs in the absence of plakoglobin are lower in mRNA was synthesized using mMessage mRNA kit (Ambion) accord- number and have a more diffuse ultrastucture. These results ing to manufacturer's instructions. indicate that plakoglobin has both signalling and structural roles in zebrafish development. Wholemount in situ hybridisation (WISH)

Materials and methods Antisense probes were synthesized from linearised plasmid using SP6, T7 or T3 polymerase and a DIG-labelling RNA kit (Roche). Zebrafish maintenance Embryos were fixed in 4% paraformaldehyde (PFA), dehydrated and hydrated through a methanol series and hybridized with probe as per Zebrafish, AB strain, Tg(fli1:EGFP) and Tg(TOP:GFP)w25 (Lawson Westerfield (1995) and Hauptmann (1999). The bound probe was and Weinstein, 2002; Dorksy et al., 2002) were obtained from the detected with BM-Purple AP-Substrate (Roche). Zebrafish International Resource Centre and maintained under standard conditions at 28 °C (Westerfield, 1995). O-dianisidine staining

Amplification of plakoglobin transcripts Dechorionated embryos were incubated in O-dianisidine staining solution (2.45 mM O-dianisidine, 0.01 M sodium acetate, pH 4.5, 0.65%

Transcripts of plakoglobin were amplified from 24 hpf zebrafish H2O2, and 40% ethanol) for 15 minutes in the dark at room cDNA using primers (Supplementary Data, Table 1) with the following temperature. Embryos were fixed in 4% PFA, briefly washed through conditions; 95 °C, 5 min; 95 °C, 60 s; 55 °C, 60 s; 72 °C, 3 min; 35 a methanol series and mounted for viewing (Ransom et al., 1996). cycles; 72 °C, 10 min. Products were subcloned in pGEM-TEasy vector (Promega) and sequenced in the forward and reverse directions by Immunofluorescence Agowa Sequencing Service, Germany. The predicted amino acid sequence was analysed using Predictprotein software (www.predict- For sectioning, embryos were embedded in 4% agarose, sectioned by protein.org), and the PSIPRED Protein Structure Prediction Server vibratome and blocked in blocking solution (0.3% Triton X-100, 4% BSA, (bioinf.cs.ucl.ac.uk/psipred/psiform.html). PBS). Antibodies used included anti-plakoglobin (1:100, BD Transduction Laboratories), anti- 2 and 3 (1:100, Zymed), anti-E-cadherin Plakoglobin knockdown (1:100, Sigma), monoclonal anti-β-catenin (1:500, Sigma) and poly- clonal anti-β-catenin (1:200, Sigma). Isotype-specificAlexaFluor-633or A morpholino against the AUG region common to plakoglobin- -546 secondary antibodies were also used (Molecular Probes). Embryos 1a and plakoglobin-1b was designed by and purchased from Gene were examined using Zeiss LSM 510 laser scanning microscope and Tools, Oregon (5′-GAGCCTCTCCCATGTGCATTTCCAT-3′). A five bp colocalisation was quantified using Zeiss LSM 5 software. mismatch control morpholino was also used (5′-GACCCTCTGCCA- TCTGGATTTGCAT-3′). Morpholinos were resuspended in nuclease- Semi-quantitative RT-PCR free water at a stock concentration of 1 mM. Plakoglobin morpholino, control morpholino or phenol red vehicle control (1 nl) was injected at Total RNA from morpholino injected embryos was extracted by the 1- to 2-cell stage. TriReagent (MRC) and was reversed transcribed. The cDNA was used E.D. Martin et al. / Developmental Biology 327 (2009) 83–96 85 as template in semi-quantitative PCR as described by O'Boyle et al. one way ANOVA and Student–Newman–Keuls. Chi-square tests were (2007). All primer sequences are provided in Supplementary Data, used to compare the distribution pattern of adhesion junctions. A P- Table 1. value of less than 0.05 was considered statistically significant.

Transmission electron microscopy (TEM) Results

Dechorionated embryos at the desired stage were fixed in 2.5% We used the previously published plakoglobin sequence to amplify gluteraldehyde / 2.5% PFA and post fixed in osmium tetraoxide. The the ORF of zebrafish plakoglobin (Cerdà et al., 1999). However, in embryos were dehydrated in an ethanol series and embedded in a low amplifying the ORF and a region of the 3′ untranslated region by PCR, viscosity resin (Agar Scientific Ltd.). Embryos were sectioned we obtained two products; one of 2.9 kb, the expected size for transversely using a Reichert Jung Ultracut instrument and ultrathin plakoglobin, and a second product of 1.9 kb. Both products were sections of interest were selected using light microscopy. Ultrathin subcloned and sequenced and these sequences were compared to sections were stained with uranyl acetate and lead citrate and Zebrafish ensembl database release 7. Following these sequence examined under a Hitachi H7000 electron microscope. searches, we changed the annotation of the gene previously known as plakoglobin to plakoglobin-1a. The shorter transcript was annotated Statistics as plakoglobin-1b (GenBank accession no. AM998809). Plakoglobin- 1b shares 99% identity with plakoglobin-1a over the 1248 bp of its Results were expressed as mean±SD. Normality was tested for ORF. The plakoglobin-1a transcript contains an additional 1089 bp in using Shapiro–Wilkes. Differences between groups were evaluated by the 3′ end which are absent from the plakoglobin-1b transcript. The predicted protein product of plakoglobin-1b has an expected molecular weight of 42 kDa compared to 83 kDa for plakoglobin-1a. The predicted protein contains only four domains compared to 13 repeats for plakoglobin-1a. A comparison of this transcript variant with the transcripts of armadillo-related proteins in other species by ClustalW showed plakoglobin-1b to be equally similar to the human plakoglobin transcript variant 1 and transcript variant 2 sequences (score 49) (Franke et al., 1989). In Drosophila,two variants of the armadillo protein have been reported (Peifer and Weischaus,1990; Loureiro and Peifer, 1998). Again, the plakoglobin-1b transcript is equally similar to the armadillo and the neural armadillo sequences by ClustalW (score 39).

Phenotype generated by knocking down plakoglobin in zebrafish

To examine the role of plakoglobin in zebrafish, we injected 2.5, 5 or 7.5 ng of plakoglobin morpholino and analysed the phenotypes at 24-, 48-, and 72 hpf (Figs. 1A–F). Embryos were classified depending on their phenotype into normal, mild, intermediate or severely affected. At 24 hpf, mildly affected plakoglobin morpholino injected embryos had delayed midbrain-hindbrain border formation. Intermediately affected embryos also had reduced heart size with either oedema or a kinked tail. Severely affected embryos had all these defects. At 48 hpf and 72 hpf, mildly affected embryos had kinked tail, oedema and a

Fig. 1. Effects of plakoglobin knockdown on zebrafish development. (A, C, E) Control 5 bp mismatch morpholino or (B, D, F) plakoglobin morpholino, 5 ng, were injected into embryos at 1- to 2-cell stage. Phenotypes were observed at (A and B) 24 hpf, (C and D) 48 hpf, and (E and F) 72 hpf. At 24 hpf, (B) morphant embryos displayed oedema (arrowhead), reduced heart size and a delay in midbrain–hindbrain boundary formation. At 48 hpf, (D) morphant embryos displayed cardiac oedema, reduced heart size, kinked tail, blood pooling (asterisk) compared to (C) controls. At 72 hpf, (F) morphant embryos displayed defects including cardiac oedema, blood pooling and a kinked tail. The numbers of embryos that displayed the above phenotypes were (A) 120/ 129 (B) 111/137 (C) 120/129 (D) 20/137 (E) 120/129 (F) 24/137. (G) Either 2.5 ng, 5 ng, or 7.5 ng, of plakoglobin morpholino was injected into embryos at 1- to 2-cell stage and the phenotypes scored at 24-, 48-, and 72-hpf. The percentage of embryos classed as normal (blue), mild (maroon), intermediate (yellow), severely affected (light blue) or dead (purple) for each dose at each timepoint are displayed. At 24 hpf, a similar percentage of embryos was affected at all doses. However, by 48 hpf, embryos injected with 2.5 ng appeared normal. 50 embryos were injected per experiment, n=3 separate experi- ments. (H–K) Lateral views of 72 hpf plakoglobin morphant embryo hearts with anterior to left and dorsal to top. The cardiac phenotype ranged from a (H) normal phenotype similar to control injected embryos 49.15%, (I) mildly affected 16.62%, (J) intermediate 14.61% and (K) severely affected 19.65% morphant embryos alive at 72 hpf n=129. (L) Western blot analysis of 72 hpf plakoglobin- or control-morpholino injected embryos, phenol red injected vehicle control and wildtype embryos using anti- plakoglobin antibody. Plakoglobin was detected in control morpholino injected embryos, phenol red and wildtype embryos (arrow) but not in morphant embryos confirming knockdown of the protein by the morpholino. An anti-α-tubulin antibody was used as a loading control. 86 E.D. Martin et al. / Developmental Biology 327 (2009) 83–96 reduced heartbeat. Intermediately affected embryos also had severe 0.91 bpm respectively, Pb0.05). The knockdown of plakoglobin in oedema, reduced heart size, blood pooling, reduction in blood morpholino injected embryos was confirmed by western blotting circulation and reflux of blood between the chambers of the heart. (Fig. 1L). A band of approximately 83 kDa, the expected size for Severely affected embryos typically had a complete absence of blood in zebrafish plakoglobin-1a, was detected in control morpholino injected, a heart that failed to loop. At 24 hpf, at least 90% of embryos were phenol red injected and wildtype embryos. No band was detected in affected at all doses. However, the majority of embryos receiving the the plakoglobin morphant embryos, indicating that the morpholino lowest dose appeared normal by 48 hpf, whereas over 65% of those inhibits plakoglobin-1a protein translation. A second band of approxi- receiving the two higher doses remained affected at 48 and 72 hpf (Fig. mately 70 kDa was detected with the plakoglobin antibody. This band 1G). A 5 bp mismatch morpholino was injected as a control and at the was isolated by immunoprecipitation and sequenced by mass spectro- 2.5 and 5 ng doses these embryos were phenotypically indistinct from metry and identified as vitellogenin-1, an abundant yolk precursor sibling wildtype embryos, whereas 3% of embryos injected with 7.5 ng protein. These results indicate that the morpholino prevents transla- of control morpholino showed non-specific defects at 72 hpf. There- tion of plakoglobin-1a mRNA resulting in morphant embryos. fore, we chose 5 ng as the dose for further experiments. At this dose, morphant embryos exhibited a range of cardiac phenotypes, from mild The morphant phenotype can be rescued by co-injection of oedema, to reduced heart size and a failure to loop correctly (Figs. 1H– plakoglobin-1a RNA K; Supplementary data, videos 1–4). In addition, the mean heart rate of morphants was reduced compared to control morpholino injected To confirm that the observed phenotype was specific to the loss of embryos at 48 hpf (106.2±0.58 beats per minute (bpm) vs 117.5± plakoglobin-1a, FLAG-tagged zebrafish plakoglobin-1a mRNA with a

Fig. 2. Plakoglobin-1a RNA rescues the morphant phenotype in a dose-dependent manner. (A) In each experiment, 5 ng of control morpholino (yellow) or plakoglobin morpholino was injected on its own (purple) or with 62.5 pg (white), 125 pg (green) or 250 pg (black) of plakoglobin-1a RNA. At 72 hpf, injection of plakoglobin morpholino alone resulted in 71% affected embryos while co-injection of 62.5 pg of morpholino and plakoglobin-1a resulted in 67% affected embryos. Co-injection of 125 pg of plakoglobin-1a resulted in only 11% of embryos exhibiting a morphant phenotype. Co-injection of 250 pg of plakoglobin-1a and plakoglobin morpholino resulted in all embryos showing either an abnormal phenotype or death. (B) Control morpholino, (C) plakoglobin morpholino, or (D) plakoglobin morpholino and 125 pg of plakoglobin-1a, were injected into embryos at 1- to 2-cell stage. At 72 hpf, the (C) morphant phenotype, including cardiac oedema (arrows), was rescued in (D) plakoglobin morpholino and plakoglobin-1a co-injected embryos. (E) Translation of injected plakoglobin-1a mRNA was confirmed by western blotting of 24 hpf embryo lysate with an anti-FLAG antibody. A band of approximately 83 kDa was detected corresponding to the expected size of FLAG tagged plakoglobin-1a (arrow). Blotting with an anti-plakoglobin antibody confirmed knockdown of plakoglobin in morphant embryos and partial rescue of expression in co-injected embryos (arrowhead). An anti-α-tubulin antibody was used as a loading control. E.D. Martin et al. / Developmental Biology 327 (2009) 83–96 87 mutated morpholino-binding region was co-injected with the mor- We altered the morpholino binding region to make the plakoglobin- pholino. A dose response study determined the optimal dose to be 1b mRNA resistant to the plakoglobin morpholino. This mRNA 125 pg of plakoglobin-1a (Fig. 2A). This dose normalised the plakoglobin (62.5 pg, 125 pg or 250 pg) was co-injected with 5 ng of plakoglobin morphant phenotype (Figs. 2B–E) whereas embryos co-injected with morpholino. Co-injection of plakoglobin-1b at any of the doses failed 62.5 pg showed the morphant phenotype, while those receiving 250 pg to rescue the morphant phenotype (Pb0.05 vs. control; Figs. 3A–D). were severely disrupted or dead. Western blot analysis using an anti- Plakoglobin-1b injected embryos were subject to western blotting FLAG antibody detected a band of approximately 83 kDa in co-injected with an anti-FLAG antibody to detect the protein and confirm the embryos that was not detected in control morpholino injected or translation of the injected mRNA. A band of approximately 42 kDa, the morphant embryos indicating the translation of the injected plakoglo- predicted size of plakoglobin-1b, was detected (Fig. 3E). Embryos bin-1a (Fig. 2E). These results confirmed that the morphant phenotype co-injected with both plakoglobin morpholino and plakoglobin-1b is specific to the loss of plakoglobin-1a as co-injection of plakoglobin-1a RNA were phenotypically morphant indicating that plakoglobin-1b mRNA with the morpholino resulted in normal embryos. failed to rescue the effects of plakoglobin knockdown.

Plakoglobin-1b cannot compensate for the loss of plakoglobin-1a in Role of plakoglobin in cardiac development morphant embryos As the cardiac phenotype in morphant embryos was the most To investigate the relative contribution of plakoglobin-1b in prevalent, we used several cardiac marker to identify the stage at zebrafish development a FLAG-tagged plakoglobin-1b construct was which cardiac development was perturbed in plakoglobin morphants. co-injected with the plakoglobin morpholino. The plakoglobin anti- First, we examined cardiac precursor migration and heart tube body could not be used as plakoglobin-1b lacked the antibody epitope. formation using nkx2.5, one of the earliest cardiac precursor markers

Fig. 3. Plakoglobin-1b RNA fails to rescue morphant phenotype at three doses. (A) Phenotypic analysis of embryos injected with control morpholino (yellow), plakoglobin morpholino (purple), co-injection of morpholino and either 62.5 pg of plakoglobin-1b (white), 125 pg of plakoglobin-1b (green), or 250 pg of plakoglobin-1b (black). Co-injection of plakoglobin-1b failed to rescue morphant phenotype at any of the doses injected (Pb0.05, vs. control). In 125 pg and 250 pg doses, co-injection of plakoglobin-1b increased the number of embryos with an abnormal phenotype at 48- and 72-hpf (Pb0.05, vs. morphant). (B) Control injected embryos were unaffected and (C) plakoglobin morpholino injected embryos displayed the morphant phenotype. (D) Co-injection of morpholino and plakoglobin-1b RNA failed to rescue the morphant phenotype. (E) Western blot analysis of 24 hpf plakoglobin morpholino, control morpholino or plakoglobin morpholino and plakoglobin-1b co-injected embryos. An anti-FLAG antibody was used to detect FLAG-tagged plakoglobin-1b protein (arrow). An anti α-tubulin antibody was used as a loading control. The presence of FLAG protein confirms plakoglobin-1b was translated. 88 E.D. Martin et al. / Developmental Biology 327 (2009) 83–96

(Lyons et al., 1995). Its expression was reduced in plakoglobin morphant embryos at 24 hpf but in the correct location, to the left of the midline, indicating that the loss of plakoglobin results in fewer cardiac precursors but does not affect precursor migration or heart tube formation (Fig. 4B). To investigate heart chamber formation we examined expression of chamber specific genes, cardiac light chain2 (cmlc2) and ventricular myosin heavy chain (vmhc; Yelon et al., 1999). These were both reduced in 48 hpf morphant embryos compared to control morpholino injected embryos (Figs. 4C–F). Expression of these chamber markers also indicated that only the severely affected morphant hearts failed to loop correctly. The abnormal reflux of blood between the chambers of the heart of morphant embryos could be as a result of disrupted valve formation. We examined this using bone morphogenic protein-4 (bmp-4) and notch 1b. Both genes are expressed throughout the anterior-posterior axis of the heart prior to 36 hpf and, in normal embryos, expression becomes restricted to future cardiac valve regions by 37 hpf for bmp4 and 48 hpf for notch1b (Walsh and Stainier, 2001; Westin and Lardelli, 1997). At 30 hpf, expression of notch 1b was similar to controls in 73% of plakoglobin morpholino injected embryos (38/52, data not shown). At 48 hpf, notch 1b expression failed to restrict in 79% of morphant embryos and continued to be expressed throughout the entire heart (Fig. 4H). Bmp-4 expression at 30 hpf was increased throughout the embryo in 86% of morphant embryos (49/57, data not shown) and at 48 hpf had failed to restrict to the valve forming regions in 92% of morphant embryos (Fig. 4J). Therefore, the absence of plakoglobin results in an increase in bmp-4 and notch1b expression and a loss of the restriction of these genes that is required for cardiac valve formation.

Cardiac valve morphology is altered in morphant embryos

Following the expanded expression of genes important in cardiac valve development, we examined cardiac valve morphology by TEM in morphant embryos. Using electron microscopy, the structure of 72 hpf control and morphant hearts was examined. In control hearts, the cuboidal endothelial cells in the future valve region between the atrium and ventricle were detected (Figs. 5A and C). However, in morphant hearts, no cuboidal cells were observed. Instead the endothelial cells in the area between the atrium and ventricle had an irregular morphology (Figs. 5B and D). The number of cell junctions between the endothelial cells in morphants (24 junctions in 33 borders) was reduced compared to control embryos (65 junctions in 34 borders; Pb0.001; see supplementary Table 2). These results indicate that endothelial cells have an aberrant morphology with reduced adhesion, indicating abnormal valvulogenesis in the absence of plakoglobin.

Haematopoiesis and angiogenesis in plakoglobin morphants

We wished to examine if the reduced circulation and blood pooling observed in plakoglobin morphant embryos were as a result of abnormal cardiac function or abnormal haematopoiesis. Firstly, we confirmed the morphological defects by O-dianisidine staining of haemoglobin in 48 hpf embryos ( Figs. 6A, B). The levels of immature

Fig. 4. Expression of cardiac marker genes in plakoglobin morphant and control morpholino injected embryos. (A and B) lateral views of 24 hpf and (C–J) ventral views of 48 hpf wholemount embryos. (A) Nkx2.5 at 24 hpf was expressed to the left of the midline in control injected embryos. (B) Nkx2.5 expression is reduced in morphant embryos but in the correct location. (C) Cmlc2 is expressed in both atrium and ventricle in 48 hpf control embryos and there is decreased expression in (D) morphant embryos. (E) Vmhc delineates ventricle specification at 48 hpf and (F) is decreased in morphant embryos. (G and I) Expression of both notch1b and bmp4 expression was restricted to the atrio-ventricular boundary (black arrows) in control morpholino injected embryo hearts. (H and J) In morphant embryos, both cardiac valve markers were expressed throughout the heart and were not restricted to the atrio-ventricular boundary (white arrows). The numbers of embryos with the displayed phenotype were (A) 62/65 (B) 55/ 63 (C) 72/75 (D) 53/72 (E) 68/70 (F) 40/72,(G) 77/80, (H) 67/85, (I) 74/78 and (J) 68/74. E.D. Martin et al. / Developmental Biology 327 (2009) 83–96 89

Fig. 5. The structures of control and morphant cardiac valves were examined. (A) In control hearts, endothelial cells in the atrioventricular boundary have cuboidal morphology. (B) In morphant hearts, endothelial cells have a squamous morphology. (C) Higher magnification of the boxed area in (A). (D) Higher magnification of the boxed area in (B). Atria are indicated by (A) and ventricles by (V). The number of embryos with the displayed phenotype were (A) 2/2 and (B) 2/2. Scale bar is 10 μm for A and B and 2 μm for C and D. blood cells in the heart were compared in morphant and control encodes a basic helix–loop–helix transcription factor and knockdown embryos by labelling E-cadherin, which is expressed by developing of scl in zebrafish results in a lack of haemangioblasts (Patterson et al., erythrocytes (Armeanu et al., 1995). Confocal microscopy of 72 hpf 2005). The gata 1 transcription factor is downstream of scl and is a embryo heart sections showed a reduction in the amount of E- marker for primitive erythoid lineage (Detrich et al., 1995). These cadherin-labelled immature blood cells in the morphant heart markers for haematopoiesis had normal expression patterns at 20 compared to control embryos (Figs. 6C, D). To investigate whether somites in morphant embryos (data not shown). This suggests that the this reduced blood cell expression was due to altered blood reduction in blood cells was not due to disrupted blood formation. We development, we examined expression of the haematopoiesis next examined if the reduced blood circulation was due to defects in markers stem cell leukaemia (scl) and gata1. Stem cell leukaemia the vasculature of the morphant embryo. Transgenic fli1:EGFP fish

Fig. 6. Reduced circulation in morphant embryos. (A, B) Lateral view of O-dianisidine stained 48 hpf (A) control and (B) morpholino injected embryos. Blood was present in the anterior blood vessels, the heart, the common cardinal vein, the dorsal aorta, and posterior cardinal vein. In the morphant embryo, blood was pooled as it exited the common cardinal vein and there was a reduction in blood cells in the anterior blood vessels. (C, D) Laser-scanning immunofluorescence microscopy images of transverse sections of 72 hpf embryo hearts. Fluorescence images with anti-E-cadherin antibody (red), a marker for and haematocytes, in (C) control morpholino injected embryo. Haematocyte numbers were reduced in (D) morphant heart. Hearts are outlined by white dotted lines. (E–H) The endothelial specific transgenic fli1:EGFP embryos injected with (G) control or (H) plakoglobin morpholino were analysed under light (E and F) and fluorescence microscopy (G and H) for vascular defects at 72 hpf. There was no alteration in vasculature in morphant embryos despite evident blood pooling (arrow). 90 E.D. Martin et al. / Developmental Biology 327 (2009) 83–96 were injected with the plakoglobin morpholino or control morpholino at earlier stages in the morphant embryos we examined the to examine angiogenesis. The vasculature was examined at 24 hpf, expression of known Wnt target genes, bozozok and bmp4, and 48 hpf and 72 hpf. Vascular development was normal in plakoglobin other signalling pathway components. At the shield stage, bmp-4 morphants (Figs. 6E–H). This indicates that any circulation defects or expression was increased in 86% of morphant embryos (Fig. 7B). blood pooling were not as a result of defects in vasculature. From our However, bmp-7 expression was similar in morphant and control study of blood and vasculature formation, we can deduce that injected embryos (n=57) (data not shown). Bmp2b was upregulated in haematopoiesis and angiogenesis are not affected in morphant 62% of shield stage morphant embryos (Fig. 7D). Wnt/β-catenin embryos but the later abnormal cardiac valve function results in signalling is required for organiser formation in vertebrate embryos. defects to blood circulation. These results indicate that loss of We examined this using early dorsal markers bozozok (boz) and squint plakoglobin results in reduced cardiac chambers with impaired (sqt) in control-injected and morphant embryos. Bozozok, a home- cardiac valve formation. Although morphants have normal vascula- odomain protein and direct target of β-catenin, is expressed in the ture, these defects result in reduced blood circulation and oedema. dorsal yolk syncitial layer and dorsal blastomeres, and co-operates with squint, a nodal related protein, in organiser formation (Shimizu Plakoglobin is antagonistic to Wnt/β-catenin signalling et al., 2000 ;Solnica-Krezel and Driever, 2001). Bozozok expression was increased in 62% and sqt in 78% of sphere stage plakoglobin The increased expression of bmp-4 at 30 hpf indicated that morphant embryos (Figs. 7F and H). The relative transcript levels of signalling was altered in morphant embryos. To investigate signalling bmp-4, bmp-2b, boz and sqt were examined by semi-quantitative RT-

Fig. 7. Loss of plakoglobin increases expression of signalling genes in early stage embryos. Plakoglobin morphant embryos have increased expression of (B) bmp4, (D) bmp2b, (F) bozozok and (H) sqt compared to their respective control injected embryos (A, C, E and G). The numbers of embryos with the above phenotype were (A) 49/53 (B) 47/55, (C) 85/ 91 (D) 54/87 (E) 50/55 (F) 38/62(G) 61/65 (H) 49/63. (I) Semi-quantitative RT-PCR amplification from shield and sphere stage plakoglobin and control morpholino injected embryos. Lane order: 100 bp ladder, PCR cycle 13, 18, 23, 28, 33, positive plasmid control, negative template control and 1 Kb ladder. β-actin was used as a template control. Expression of bmp4, bmp2b and squint were detected five cycles and bozozok was detected ten cycles earlier in morphants than control embryos. Analysis of GFP expression in morpholino injected TOPdGFP fish. (J) In control morpholino injected fish, GFP expression is detected at the atrioventricular boundary (black arrowhead). (K) In morphant embryos, GFP is expressed throughout the heart (white arrowhead). The number of embryos with the displayed phenotype were (J) 31/39 and (K) 34/43. Co-expression study of Dkk1 and control or plakoglobin morpholino. (L) Control morpholino injected embryos at 72 hpf with normal hearts (165/165). (M) Plakoglobin morphants at 72 hpf with abnormal hearts including oedema, reflux of blood between chambers (88/149). (N) Co-injection of 20 pg of DKK1 and control morpholino resulted in oedema and reflux of blood between chambers (30/128). (O) Co-injection of 20 pg of Dkk1 and plakoglobin morpholino with normal cardiac phenotypes (10/162). E.D. Martin et al. / Developmental Biology 327 (2009) 83–96 91

PCR and all showed increased levels of expression in morphant boundary, indicating Wnt/β-catenin activity in these cells (Fig. 7J). embryos. Bmp-4 and bmp-2b appeared at cycle 28 in morphants vs However in morphant embryos, Wnt/β-catenin activity was detected cycle 33 in controls; boz appeared at cycle 23 in morphants vs cycle 33 throughout the heart indicating an increased level of Wnt signalling in in controls and sqt appeared at cycle 23 in morphants vs cycle 28 in the heart (Fig. 7K). To further examine the relationship of plakoglobin controls (Fig. 7I). The increased expression of Wnt target genes, such and Wnt/β-catenin signalling, we co-injected dickkopf-1 (Dkk1), an as bozozok and BMP4, in morphant embryos confirms that plakoglobin inhibitor of Wnt signalling, and examined the resulting phenotype is antagonistic to Wnt/β-catenin signalling. To ascertain if this (Hashimoto et al., 2000). At 24 hpf, 85% (109/128) of embryos co- increase in Wnt/β-catenin signalling in morphant embryos results in injected with control morpholino and Dkk1 displayed the Dkk1 the cardiac phenotype, we examined GFP expression in the TCF overexpression phenotype of an enlarged head, increased eye size and reporter TOPdGFP transgenic line (Dorsky et al., 2002). GFP expression shortened body axis, with 17% of embryos also showing somite in control embryos was detected only at the atrioventricular defects. While 68% (112/163) of plakoglobin morpholino and Dkk1 co-

Fig. 8. Increased colocalisation of β-catenin and E-cadherin in adherens junctions of plakoglobin morphant embryos. (A–R) Laser-scanning immunofluorescence microscopy images of transverse sections of the eye region of 72 hpf embryos. Dual label fluorescence images with anti-E-cadherin antibody as red and anti-β-catenin antibody as green are shown as (A–F and J–O) single and as (G–I and P–R) merged images. Images are 400× magnification. (G) In control morpholino injected embryos, there was little colocalisation of E-cadherin and β-catenin. (H) By contrast, in plakoglobin morphant embryos there was extensive colocalisation (yellow). (I) This colocalisation was reduced in plakoglobin morpholino and plakoglobin-1a co-injected embryos. (R) However, this colocalisation was still evident in plakoglobin morpholino and plakoglobin-1b co-injected embryos. (S) Quantification of co-localisation of E-cadherin and β-catenin in control, morphant, plakoglobin morpholino and plakoglobin-1a, plakoglobin morpholino and plakoglobin-1b co-injected embryos. There was increased colocalisation of the proteins in morphant embryos compared to control morpholino injected embryos. (S) This co-localisation in co-injected morpholino and plakoglobin-1a RNA injected embryos was similar to control injected embryos. (T) In contrast, levels of co-localisation in plakoglobin morpholino and plakoglobin-1b co-injected embryos were similar to morphant embryos. Pb0.05. Asterisks indicate statistical significance. 92 E.D. Martin et al. / Developmental Biology 327 (2009) 83–96 injected embryos displayed increased head and eyes, none displayed cycle 33 in both control and morphant embryos. β-actin was somite defects. At 72 hpf, control morpholino injected embryos had detected at cycle 23 in both the plakoglobin morphant and control normal cardiac phenotype while plakoglobin morpholino injected injected samples. These results show that β-catenin-1 and β-catenin- embryos displayed the morphant phenotype (Figs. 7L and M). By 2 levels are similar in morphant and control injected embryos 72 hpf, 24% of control morpholino and Dkk1 co-injected embryos (Supplementary Fig. 1G) indicating that increased expression of β- exhibited cardiac defects including failure to loop and reflux of blood catenin in morphant embryos was not due to increased transcrip- between chambers (Fig. 7N). However, in plakoglobin morpholino and tion but may be due to increased translation or increased stability of Dkk1 co-injected embryos, only 6% of embryos exhibited cardiac the β-catenin protein. defects (10/162; Pb0.001 vs plakoglobin morpholino; Fig. 7O). These results indicate that in the absence of plakoglobin, Wnt/β-catenin β-catenin compensates for loss of plakoglobin in adherens junctions but signalling is increased. Co-injection of DKK1 rescues this defect. These not in desmosomes results highlight the importance of the antagonistic signalling role of plakoglobin in embryo development. We examined the effect of the loss of plakoglobin and the increased β-catenin protein expression on the localisation of other Loss of plakoglobin does not increase β-catenin mRNA expression but adherens junction proteins by confocal immunofluorescence micro- increases protein expression scopy. β-catenin and E-cadherin had distinct localisation patterns in control-injected 72 hpf embryos (Fig. 8G). However, in plakoglobin As Wnt signalling is increased in morphant embryos, we wished morphant embryos these two adhesion proteins were extensively to examine if the loss of plakoglobin resulted in changes in β- colocalised (Fig. 8H). Quantification of colocalisation, using Pearson's catenin mRNA or protein expression. β-catenin protein expression correlation coefficient, confirmed the increase in co-localisation of E- was examined by western blotting using an antibody that cadherin and β-catenin in morphant embryos compared to control recognises both β-catenin-1 and β-catenin-2. Total β-catenin protein embryos (Pb0.05, Fig. 8S). Embryos co-injected with morpholino and was increased in plakoglobin morphant embryos (Supplementary plakoglobin-1a had a similar level of co-localisation to control Fig. 1A). Protein levels were normalised to α-tubulin. The relative embryos, indicating that co-injecting plakoglobin-1a with morpholino levels of total β-catenin to α-tubulin in morphant embryos was could rescue the morphant phenotype (Fig. 8I). However, co-injection 147.66±15.67 relative densitometric units compared to 78.10±3.84 of plakoglobin-1b failed to rescue the phenotype and co-localisation relative densitometric units for control injected embryos (Pb0.05, was similar in co-injected plakoglobin-1b and morpholino to mor- Supplementary Fig. 1B, n=3). β-catenin mRNA was then examined phant embryos (Fig. 8R). Therefore, in the absence of plakoglobin-1a, to determine if this increase was due to increased transcription or β-catenin increased its binding with E-cadherin in adherens junctions. translation of the β-. β-catenin-1 and β-catenin-2 mRNA We next examined the effect of loss of plakoglobin on desmosomal localisation was normal in morphant embryos as examined by WISH proteins. β-catenin is not normally expressed in desmosomes and (Supplementary Figs. 1C–F). The relative transcript levels of β- there was little co-localisation of β-catenin and the desmosomal catenin-1 and β-catenin-2 were examined by semi-quantitative RT- , desmocollin 2 and 3 in control injected embryos. In PCR. β-catenin-1 was first detected at cycle 28 in both control and plakoglobin morphant embryos, there was no change in the expres- plakoglobin morphant embryos. β-catenin-2 was first detected at sion pattern of β-catenin or 2 and 3 (Supplementary

Fig. 9. Loss of plakoglobin results in fewer adhesion junctions and with an altered structure. (A and B) Electron microscopy of the intercalated discs of zebrafish embryo hearts at 72 hpf. (A) In control morpholino injected embryos, desmosomes (black arrowheads) were in series. (B) In morphant embryos, diffuse adhesion junctions (white arrowhead) were detected. (C) A table of indicating the cell–cell borders numbers, the number of adhesion junctions, and the number of each type of junction in control and morphant embryos. Unclassified indicates adhesion junctions that could not be definitively classed as adherens junction, or . N=2 embryos per sample. Scale bar is 100 nm. E.D. Martin et al. / Developmental Biology 327 (2009) 83–96 93

Figs. 2A–F). There was no statistical difference in colocalisation of the desmosomes. The region of arm repeats 6–10 has been identified as the proteins in control morpholino and plakoglobin morpholino injected minimum required for axis duplication in Xenopus mutation experi- embryos (Supplementary Fig. 2G). Controls for the desmocollin ments and so indicates that the domain is important in plakoglobin antibody are included in Supplementary Fig. 2. These results indicate signalling (Rubenstein et al.,1997). The absence of this domain suggests that β-catenin compensates for the loss of plakoglobin in adherens that plakoglobin-1b does not play a signalling role in zebrafish. This junctions but not in desmosomes in plakoglobin morphant embryos. protein also lacks desmosomal cadherin binding domains that modulate the interaction of desmosomal cadherins (Wahl et al., 1996). Loss of plakoglobin results in reduced and altered adhesion junctions The presence of a second immunoreactive band in western immuno- blotting with the plakoglobin antibody raised the possibility of the We next examined the ultrastructure of adhesion junctions in the presence of additional transcript variants of plakoglobin-1a as it was not intercalated discs of control and morphant 72 hpf embryos by electron of the predicted size for plakoglobin-1b. However, the band was microscopy. In control embryos, adherens junctions and desmosomes, identified as vitellogenin-1, and vitellogenin was found to contaminate with their distinctive dense tripartite composition, could be easily zebrafish lysates in proteomic analysis of zebrafish proteins (Link et al., distinguished and were often observed in a series at the cell borders 2006; Tay et al., 2006). The presence of this abundant yolk protein may (Fig. 9A). However in morphant embryos (Fig. 9B), there was a striking hinder the identification of plakoglobin isoforms. loss in the number and proportion of adherens junctions and Plakoglobin morphant embryos have defective cardiac valves with desmosomes, despite similar numbers of cell–cell borders (pb0.05) increased expression of bmp4 and notch1b. Hurlstone et al. (2003) (Fig. 9C). Adhesion junctions, when present, were more diffuse in proposed a role for Wnt/β-catenin signalling in cardiac valve structure than either the adherens junctions or desmosomes observed development. Loss of adenomatous polyposis coli (APC), a component in control embryos and therefore were difficult to distinguish. These of the catenin destruction complex, in zebrafish caused an upregula- results indicate that the absence of plakoglobin results in a reduced tion of Wnt/β-catenin signalling, resulting in an excess of endocar- number of adhesion junctions. dium. In our study, the loss of plakoglobin also resulted in a loss of restriction of bmp4 and notch1b, increased Wnt signalling and altered Discussion cardiac valve formation. However we did not observe the excessive endocardial cushion outgrowth reported by Hurlstone and colleagues. In this study, we have shown that plakoglobin-1a has both Instead we found that whereas the endothelial cells of the atrio- structural and signalling roles in zebrafish embryo development. ventricular canal of control embryos were cuboidal in shape, Loss of plakoglobin-1a resulted in cardiac oedema, abnormal cardiac plakoglobin morphants lacked these cuboidal cells. This is similar to development and abnormal cardiac valve formation. β-catenin the findings of Beis et al. (2005) following ectopic expression of the compensated for the loss of plakoglobin in adherens junctions but Notch intracellular domain in all endothelial cells. These authors also not in desmosomes. Loss of plakoglobin resulted in an increase in Wnt found that loss of APC increased expression of cuboidal endothelial signalling in the heart and throughout the embryo. cells in the ventricle of APC mutant. They argue that increased notch Theabsenceofplakoglobininzebrafish resulted in cardiac defects signalling maintains squamous endothelial cells and prevents transi- which are broadly similar to those found when desmocollin-2 was tion to the cuboidal shape. An alternative possibility is that increased knocked down in zebrafish (Heuser et al., 2006), or when plakoglobin notch signalling in the plakoglobin morphants caused increased was knocked down in mice (Bierkamp et al.,1996; Ruiz et al.,1996). All of endothelial to mesenchymal transition, which is characterised by loss these manipulations recapitulate ARVC in humans. Due to the embryonic of junctions and altered shape of endothelial cells, both of which were lethality of plakoglobin null mice, Kirchhof et al. (2006) studied the seen in the plakoglobin morphants. This effect was observed by effects of reduced plakoglobin in heterozygous plakoglobin deficient Timmerman et al. (2004) in mice with increased Notch signalling. mice; these mice also had enlarged right ventricles with . However, recent studies suggest that in zebrafish endocardial Mutation of plakoglobin in humans also results in arrhythmias and cushions do not form, but leaflets are formed directly by invagination replacement of ventricle muscle tissue by adipose tissue (McKoy et al., (Scherz et al., 2008). In the light of this report, it appears that in 2000). In addition, a few plakoglobin knockout mice in some genetic plakoglobin morphants endothelial cells fail to transition from a backgrounds exhibited skin blisters which were not detected in the squamous to a cuboidal shape. zebrafish morphant (Bierkamp et al., 1996, 1999). However, the primary Despite having transcriptional activity through the T-cell specific cardiac phenotype was similar in mice and zebrafish. family of transcription factors, plakoglobin does not appear to play a We have identified a transcript variant of plakoglobin in zebrafish, role in haematopoiesis. Expression of haematopoietic markers scl and plakoglobin-1b. Splice variants of the human plakoglobin have been gata 1 appeared normal in morphant embryos. Later defects in identified in carcinoma cell lines (Franke et al.,1989; Ozawa et al.,1995). circulation may be as a result of secondary effects due to a non- However, this second zebrafish transcript was identifi ed in wild type functioning cardiovascular system. This agrees with the findings of embryos. The armadillo protein in Drosophila has an isoform which Koch et al. (2008) who showed that the conditional knockout of β- functions in differentiating neurons, which may indicate a specific catenin in plakoglobin knockout mice did not affect haematopoiesis. tissue expression of plakoglobin-1b at a later stage in embryo Our findings indicate that the loss of plakoglobin results in an development (Loureiro and Peifer, 1998). As the transcript lacks the increase in β-catenin protein expression. Although this is not due to plakoglobin antibody epitope, its protein expression could not be increased transcription, it may be due to increased translation or an localised in the embryo. It does not function redundantly with increase in protein stability due to less degradation via the ubiquitin plakoglobin-1a as co-injection of plakoglobin-1b mRNA with morpho- pathway. This is in contrast to the findings in plakoglobin knockout lino failed to rescue the morphant phenotype or change the colocalisa- mice where there was no increase in β-catenin levels in keratinocytes tion of β-catenin and E-cadherin at adherens junctions. This may be due (Bierkamp et al., 1999). These authors also found that β-catenin to the structure of the plakoglobin-1b protein. The amino terminal and localised to the desmosomes in mice lacking plakoglobin, which we first armadillo (arm) repeat of the human plakoglobin protein did not find in plakoglobin morphant zebrafish embryos. However, we comprises the α-catenin binding domain. The first three armadillo found increased colocalisation of β-catenin with E-cadherin, suggest- repeats contain a binding domain, while arm repeats 4 and ing β-catenin increases its role in adherens junctions to compensate 5 mediate E-cadherin binding (Chitaev et al., 1996; Troyanovsky et al., for the loss of plakoglobin. Similarly, in the absence of β-catenin in 1996). Plakoglobin-1b has these domains but the lack of arm repeats 6– mouse embryos, plakoglobin becomes more co-localised with E- 13 may affect the targeting of the complex to adherens junctions and cadherin (Huelsken et al., 2000). In β-catenin deleted cell lines, 94 E.D. Martin et al. / Developmental Biology 327 (2009) 83–96 plakoglobin can compensate for lack of β-catenin at adherens mechanism for ARVC in which mutated desmosomal genes result in junctions (Fukunaga et al., 2005). Cardiac specific deletion of β- the release of plakoglobin from desmosomes and translocation to the catenin in adult mouse cardiomyocytes resulted in increased nucleus, suppressing Wnt signalling. Loss of Wnt signalling resulted in plakoglobin expression at intercalated discs and did not affect any myoblasts changing to adipocytes in vitro (Ross et al., 2000). It is other adherens junctions proteins (Zhou et al., 2007). These results, unclear how mutations in plakoglobin itself, which results in ARVC, together with the increased co-localisation of β-catenin and E- could be incorporated into this model. We propose that the loss cadherin in plakoglobin morphants confirm that β-catenin and of both the structural and the signalling roles of plakoglobin results plakoglobin share a function in adhesion junctions. Therefore, β- in ARVC, and that the signalling defects are due to increased Wnt catenin can fulfil some of roles of plakoglobin in the cell by signalling. compensating for the loss of plakoglobin in adherens junctions. However, the use of zebrafish as a model system for the study of Morphant embryos showed a striking loss of desmosomes and ARVC has a number of limitations. The use of morpholinos restricts adherens junctions compared to control embryos, with a concomitant examination of phenotype to early developmental stages and increased expression of diffuse junctions, which were not seen in the subsequent steps in disease progression, such as replacement of controls. These likely represent altered adherens junctions and cardiac tissue with fatty tissue cannot be examined. Another desmosomes. These results are in general agreement with findings limitation is the possible presence of a second copy of a gene due to when desmosomal proteins were ablated. In desmocollin-2 zebrafish genome duplication in zebrafish after separation of fish from the morphants, desmosomes were also reduced in number and lacked the mammalian lineage. Recent studies have identified differences in the midline structure between the cell–cell membranes (Heuser et al., structure of adhesion junctions in cardiomyocytes of lower verte- 2006). Ultrastructural studies in the plakoglobin knockout mouse also brates compared to mammals. Fish cardiomyocytes contain discrete showed a reduced number of desmosomes with an altered structure structures of adherens junction components and desmosomal and the absence of the cytoplasmic plaque (Bierkamp et al., 1999). components compared to the composite junctions detected in higher Another study of the intercalated discs of plakoglobin knockout mice vertebrates (Pieperhoff and Franke, 2008). However, formation of showed the presence of extended adhesive structures that contained these composite junctions occurs late in mammalian development, both desmosomal and adhesion junction components rather than mainly post-natally, and at the stages examined in the current study distinctive desmosomes (Ruiz et al., 1996). These results indicate the separate adherens junctions and desmosomes are present in mam- loss of plakoglobin disrupts adhesion junction structure in morphant malian embryonic hearts similar to those in zebrafish (Pieperhoff and embryos. Franke, 2007). Thus, plakoglobin knockdown in zebrafish provides an The absence of plakoglobin results in an increase in Wnt target excellent model to investigate the underlying molecular mechanisms gene expression. Bozozok acts downstream of β-catenin and is of ARVC. required for organiser formation and development of axial mesoderm From this study, we can conclude that plakoglobin co-operates (Fekany et al., 1999; Leung et al., 2003). Here we show that, in the with β-catenin in adherens junctions but acts antagonistically with it absence of plakoglobin, expression of these genes is increased. The in Wnt signalling. We have provided evidence for the possible absence of the characteristic phenotype associated with overexpres- mechanism of ARVC involving plakoglobin. We identified both sion of the genes may be explained by the negative feedback loop such signalling and structural roles for plakoglobin in zebrafish embryo as for bozozok (Solnica-Krezel and Driever, 2001). Crosstalk between development. signalling pathways is a complex control mechanism for organ formation. The current study shows that an increase in Wnt signalling Acknowledgments resulted in an increase in BMP and Notch signalling pathways. In mice, a mutation in APC resulted in increased Wnt signalling and also The authors wish to thank Dr. D. Yelon for providing cmlc2 and increased Notch signalling (van Es et al., 2005). Wnt signalling has vmhc probes, Dr. M. Fishman for nkx2.5, Dr. J. Yost for bmp4,Dr.M. been shown to increase BMP-2 signalling resulting in mesenchymal Lardelli for notch1b, Dr. T. Hirano for boz and Dkk1, Dr. I. Dawid for sqt, differentiation (Nakashima et al., 2005). Our study shows that Dr. L. Zon for bmp2b and gata1, Dr. M. Hammerschmidt for bmp7,Dr.A. inhibition of the increase in Wnt signalling in morphant embryos by Green for scl probe, Dr. R. Dorsky for TopdGFP and Dr. E. Weinberg for co-injecting Dkk1 rescued the cardiac phenotype. Studies by Hurlstone β-catenin-1 and β-catenin-2. We wish to thank the Stainier lab for et al. (2003) have shown that inhibition of endogenous Wnt/β-catenin technical assistance. We would like to thank Mr. P. Lalor and Prof. P. signalling, by Dkk1 overexpression, results in an absence of endo- Dockery of the Anatomy Department, NUI, Galway for their expert cardial cushion formation. Recent studies have uncovered the help with TEM. We wish to thank Catherine M. Glynn for fish importance of Wnt signalling at particular time points in cardiac husbandry. This work was funded by the Millennium Fund, NUI, development. While previously considered an inhibitor of cardiac Galway, Health Research Board of Ireland and Irish Research Council formation, Wnt signalling has been found to have an important pro- for Science Engineering and Technology. cardiac role (Lickert et al., 2002). Expression of Wnt signalling prior to gastrulation promoted cardiac differentiation, but was inhibitory to Appendix A. Supplementary data cardiac formation if expressed after gastrulation, indicating the critical temporal-specific expression of Wnt in cardiac formation (Naito et al., Supplementary data associated with this article can be found, in 2006; Ueno et al., 2007). It is likely that the continual upregulation of the online version, at doi:10.1016/j.ydbio.2008.11.036. Wnt signalling observed in plakoglobin morphant embryos disrupted this important temporal-specific expression of Wnt and thereby References altered cardiac formation. Heuser et al. (2006) have previously used zebrafish with disrupted Armeanu, S., Buhring, H.J., Reuss-Borst, M., Muller, C.A., Klein, G., 1995. E-cadherin is desmosomes as a model for ARVC. The failure of plakoglobin to functionally involved in the maturation of the erythroid lineage. J. Cell Biol. 131, – localise to desmosomes when mutated and when other desmosomal 243 249. Asimaki, A., Syrris, P., Wichter, T., Matthias, P., Saffitz, J.E., McKenna, W.J., 2007. A novel components are mutated has been proposed to identify a common dominant mutation in plakoglobin causes arrhythmogenic right ventricular mechanism for ARVC (Tsatsopoulou et al., 2006). Garcia-Gras et al. cardiomyopathy. Am. J. Hum. Genet. 81, 964–973. (2006) have shown that a reduction in Wnt signalling can result in Behrens, J., von Kries, J.P., Kuhl, M., Bruhn, L., Wedlich, D., Grosschedl, R., Birchmeier, W., fi 1996. 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