Desmosomal armadillo proteins in zebrafish cardiac development
|dc.description.abstract||Plakoglobin and plakophilin-2 are desmosomal armadillo proteins, required for correct cardiac development in zebrafish. Mutations in the human genes can cause the congenital heart defect arrhythmogenic right ventricular cardiomyopathy (ARVC). Both proteins have structural and signalling roles in development, with plakoglobin implicated as a negative regulator of Wnt signalling. Our lab has previously established zebrafish loss of function models for both proteins using morpholino antisense oligonucleotides. Loss of plakoglobin has previously been shown to up-regulate expression of Wnt target genes at early stages of development. This study shows the up-regulation to be transient and stage-dependent. Wnt target genes bmp4, ndr1 and dharma show a tendency towards increased expression in the early blastula stage, with ndr1 and dharma mRNA expression levels normalised by late blastula, and bmp4 by 48 hpf. Cardiac and developmental defects caused by loss of plakoglobin can be rescued by co-injection of Wnt inhibitor Dickkopf-1 (Dkk1). This study shows that though Dkk1 can restore cardiac patterning it is not sufficient to compensate for loss of plakoglobin in cell-cell junctions (desmosomes). Whole-mount in-situ hybridisation experiments and qPCR were used to investigate the expression of Wnt target genes in plakophilin-2 knockdown embryos. Wnt target gene expression was unaffected at the developmental stages examined. Left-right patterning defects previously observed in plakophilin-2 morphant embryos suggested the Nodal signalling pathway as a target for further investigation. Using the same methods, cardiogenic genes were examined in plakophilin-2 morphant embryos at the 18 somite stage. Expression of Nodal target genes lefty1, bmp4, southpaw, one-eyed pinhead and ndr1 was significantly increased, with concomitant down-regulation of lefty2. These results are the first reported incidence of an interaction between plakophilin-2 and the Nodal signalling pathway. Our group aims to establish a zebrafish model that recapitulates the cardiac and signalling defects of ARVC. To investigate if signalling aspects of the human condition are replicated in the morphant embryos, expression levels of ARVC-related genes connexin 43, desmoplakin-a, adipogenic transcription factor Wnt5b and adipogenic marker PPAR-¿ were examined by qPCR. All genes were decreased in plakoglobin morphant embryos, and unaffected in plakophilin-2 morphant embryos. Complete protein loss has failed to accurately mimic molecular features of the disease, hence TALEN targeting was used to induce mutations in exon 4 of the plakoglobin protein in vivo and the resulting mutant embryos were characterised and assessed as a model for ARVC. The achieved germline mutations are predicted to encode premature stop codons, truncating the plakoglobin protein. This putative shortened protein resulted in cardiac, signalling and developmental defects reminiscent of the plakoglobin knock-down phenotype, with additional epithelial defects observed in a small subset of mutant embryos. The cardiac defects arising from TALEN-induced mutations in plakoglobin indicate potential for the development of an improved zebrafish model for ARVC using this technique to introduce mutations in different regions of the gene.||en_US|
|dc.subject||Arrhythmogenic right ventricular cardiomyopathy||en_US|
|dc.title||Desmosomal armadillo proteins in zebrafish cardiac development||en_US|
|dc.local.note||Mutations in the human genes plakoglobin and plakophilin-2 can cause heart defects, including the congenital heart condition arrhythmogenic right ventricular cardiomyopathy (ARVC). This thesis examines the roles of plakoglobin and plakophilin-2 in early stages of zebrafish development, and how loss of the proteins these genes produce can affect cardiac development.||en_US|
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