Grant: Operating Grant

The conversion of epithelia to mesenchyme (EMT) is essential for heart formation. The cadherin switch, ie, switch from expression of epithelial cell-cell adhesion protein, E-cadherin, to mesenchymal N-cadherin, is an integral part of EMT. In ES cells, the cadherin switch occurs as cells exit from pluripotency and commit to a cell fate. Calreticulin (CRT) is a fetal cardiac gene: its expression is induced as cardiogenesis starts, peaks at the midpoint of it and is then repressed. CRT regulates activation of calcineurin (CaN), which controls translocation of a transcription factor, NFAT to the nucleus. HYPOTHESIS: CRT and CaN control cardiogenesis by affecting the cadherin switch and ES cell community behaviour. This occurs parallel to NFAT nuclear translocation and it is intra-nuclear NFAT that regulates cadherin-specific transcription factors. The kinetics of E-to-N cadherin switch suggest a relationship between adhesion, expression of CRT, activation of CaN, nuclear translocation of NFAT and cardiomyogenesis. The cell surface properties affect cardiogenesis: hence we will first elucidate cell community behaviour of ES cells with manipulated CRT and CaN genes using microfluidics assays. Inhibiting NFAT nuclear translocation induces a "super KO" phenotype in all ES cells, while expressing activated calcineurin induces a "super WT" phenotype. Using this system we will elucidate the mechanism of regulation of exit from pluripotency and subsequent progression of cardiomyogenesis from ES by CRT-CaN signalling. As we postulate that intranuclear NFAT regulates cadherins, we will next work out mechanisms of the inferred but so far undiscovered regulation of cadherin transcription factors by NFAT using EMSA and chromatin immunoprecipitation. CRT is a modular protein, with a chaperoning and a Ca handling module, so by gain-of-function approach we will elucidate which of CRT's functional modules affects the cadherin switch and cardiac differentiation.