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Updated: Jun 26, 2026
High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function
Published on: October 19, 2013
Plakoglobin has both structural and signalling roles in zebrafish development
Eva D Martin1, Miriam A Moriarty, Lucy Byrnes
1Department of Pharmacology and Therapeutics and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Galway, Ireland.
Insights
Plakoglobin loss in zebrafish causes heart defects and developmental issues, revealing its crucial role in heart development and Wnt signaling. This study establishes zebrafish as a model for arrhythmogenic right ventricular cardiomyopathy (ARVC).
Area of Science:
- Cardiovascular Biology
- Developmental Biology
- Molecular Signaling
Background:
- Plakoglobin (gamma-catenin) is a key protein in desmosomes and adherens junctions, also involved in Wnt signaling.
- Mutations in human plakoglobin are linked to arrhythmogenic right ventricular cardiomyopathy (ARVC), but its signaling role in ARVC is unclear.
Purpose of the Study:
- To investigate the functional and signaling consequences of plakoglobin loss in a vertebrate model.
- To establish the zebrafish as a model for studying ARVC caused by plakoglobin deficiency.
Main Methods:
- Zebrafish were utilized, with plakoglobin knockdown achieved via morpholino injection.
- Phenotypic analysis included morphological assessment, whole-mount in situ hybridization for cardiac markers, and semi-quantitative RT-PCR for Wnt target gene expression.
- Rescue experiments involved co-expression of the Wnt inhibitor Dkk1.
Main Results:
- Plakoglobin knockdown resulted in cardiac abnormalities including reduced heart size, impaired heartbeat, edema, and valve defects.
- Gene expression analysis showed altered cardiac markers (nkx2.5, cmlc2, vmhc) and valve markers (notch1b, bmp4).
- Plakoglobin deficiency led to increased Wnt target gene expression, indicating an antagonistic role in Wnt signaling. beta-catenin levels and localization were also affected.
Conclusions:
- Plakoglobin is essential for normal heart development and function in zebrafish, with loss leading to structural and signaling defects.
- The study confirms plakoglobin's antagonistic role in Wnt signaling and establishes zebrafish as a relevant model for ARVC research.
- Both structural integrity (desmosomes, adherens junctions) and signaling pathways are impacted by plakoglobin loss.
Abstract:
Plakoglobin, or gamma-catenin, is found in both desmosomes and adherens junctions and participates in Wnt signalling. Mutations in the human gene are implicated in the congenital heart disorder, arrhythmogenic right ventricular cardiomyopathy (ARVC), but the signalling effects of plakoglobin loss in ARVC have not been established. Here we report that knockdown of plakoglobin in zebrafish results in 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 hours post fertilisation (hpf), and cmlc2 and vmhc at 48 hpf, while there is lack of restriction of the valve markers notch1b and bmp4 at 48 hpf. Wnt target gene expression was examined by semi-quantitative RT-PCR and found to be increased in morphant embryos indicating that plakoglobin is antagonistic to Wnt signalling. Co-expression of the Wnt inhibitor, Dkk1, rescues the cardiac phenotype of the plakoglobin morphant. beta-catenin protein expression is increased in morphant embryos as is its colocalisation with E-cadherin in adherens junctions. Endothelial cells at the atrioventricular boundary of morphant hearts have an aberrant morphology, indicating problems with valvulogenesis. Morphants also have decreased numbers 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.
