Regulation of cardiogenesis by putative WNT signalling pathways

Abstract

The Wnt/ -catenin and the Wnt/planar cell polarity (Wnt/PCP) signalling pathways have been shown to play important roles in cardiogenesis and their disruption has been shown to cause severe disturbances in heart development. Spatially and temporally complex interplays between the two pathways have been described. One component of the PCP pathway is Jnk, a member of the highly conserved mitogenactivated protein kinase (MAPK) family. This stress responsive mitogen is known to control a variety of cellular behaviours such as proliferation, apoptosis and cell migratory behaviour and as such, is likely to be of pivotal importance in cardiac development. The aim of this study was to investigate the role played by Jnk in vertebrate heart formation and the relationships between Jnk signalling and canonical Wnt signalling, using in silico and in vivo approaches in zebrafish and an in vitro approach on a mouse embryonic stem (ES) cell model of cardiogenesis. Firstly, using a range of bioinformatic methods, an analysis of jnk genes, splice variants and proteins, and an investigation of their phylogenetic relation with other species was undertaken. This suggested conservation of Jnk family members, but suggested that there were additional orthologues of jnk1 present in the zebrafish transcriptome. The spatial and temporal expression profiles of these genes were then examined by semi-quantitative PCR and in situ hybridisation. The functional role of Jnk proteins during zebrafish development was subsequently investigated using a specific chemical inhibitor, SP600125. Inhibition of Jnk signalling during gastrulation and somitogenesis caused a convergence extension-like phenotype and severe cardiac defects, including looping anomalies and alterations in atrial versus ventricular cell numbers. ES cells have the capacity to differentiate in vitro and give rise to cells of many different lineages, including cardiomyocytes. Canonical Wnt and Jnk components were manipulated during specific windows of differentiation as ES cells formed beating embryoid bodies. Examination of the spontaneous contractile behaviour of differentiating ES cells as they entered the cardiogenic lineage, and analysis of their developmental gene expression profiles, showed the beating behaviour of ES cellderived cardiac cells was enhanced in a temporally specific manner after inhibition of the non-canonical Wnt/Jnk pathway, while there was marked alteration of canonical Wnt signalling. To investigate whether there were reciprocal interactions between the two pathways, analysis of the system after activation of the canonical pathway was also undertaken. These studies indicated that the beating behaviour of ES cell-derived cardiac cells was enhanced in a temporally specific manner after inhibition of Jnk, while after activation of canonical Wnt/ -catenin signalling, the cardiogenic potential of differentiating ES cells was severely suppressed. The findings of this study extend our understanding of the role played by canonical and non-canonical Wnt signalling pathways in heart morphogenesis and highlight the interacting effects of related signalling pathways activity in cardiogenesis.