Fibroblast growth factor receptor-1 phosphorylation requirement for cardiomyocyte differentiation in murine embryonic...

Fibroblast growth factor receptor-1 (Fgfr1) gene knockout impairs cardiac and haematopoietic development in murine embryonic stem cells (mESC). In FGFR1, tyrosine residues Y653 and Y654 are responsible for its tyrosine kinase (TK) activity whereas phosphorylated Y463 and Y766 represent docking sites for intracellular substrates. Aim of this study was the characterization of FGFR1 signalling requirements necessary for cardiomyocyte differentiation in mESC. To this purpose, fgfr1(-/-) mESC were infected with lentiviral vectors harbouring human wild-type hFGFR1 or the Y653/654F, Y463F and Y766F hFGFR1 mutants. The resulting embryonic stem (ES) cell lines were differentiated as embryoid bodies (EBs) and beating foci formation was evaluated. In order to appraise the presence of cells belonging to cardiovascular and haematopoietic lineages, specific markers were analysed by quantitative PCR, whole mount in situ hybridization and immunofluorescence. Transduction with TK(+) hFGFR1 or the TK(+) Y766F-hFGFR1 mutant rescued cardiomyocyte beating foci formation in fgfr1(-/-) EBs whereas the TK(-) Y653/654F-hFGFR1 mutant and the TK(+) Y463F-hFGFR1 mutant were both ineffective. Analysis of the expression of early and late cardiac markers in differentiating EBs confirmed these observations. At variance with cardiomyocyte differentiation, all the transduced TK(+) FGFR1 forms were able to rescue haematopoietic differentiation in EBs originated by infected fgfr1(-/-) mESC, only the TK(-) Y653/654F-hFGFR1 mutant being ineffective. In keeping with these observations, treatment with different signalling pathway inhibitors indicates that protein kinase C and ERK activation are essential for cardiomyocyte but not for haematopoietic differentiation in EBs generated by fgfr1(+/-) approximately mESC. In conclusion, our results suggest that, although FGFR1 kinase activity is necessary for both cardiac and haematopoietic lineage maturation in mESC, phosphorylation of Y463 in the intracellular domain of the receptor is a specific requirement for cardiomyocyte differentiation.