Cardiac differentiation in Xenopus requires the cyclin-dependent kinase inhibitor, p27Xic1

AIMS Cyclin-dependent kinase inhibitors (CDKIs) play a critical role in negatively regulating the proliferation of cardiomyocytes, although their role in cardiac differentiation remains largely undetermined. We have shown that the most prominent CDKI in Xenopus, p27(Xic1)(Xic1), plays a role in neuronal and myotome differentiation beyond its ability to arrest the cell cycle. Thus, we investigated whether it plays a similar role in cardiomyocyte differentiation. METHODS AND RESULTS Xenopus laevis embryos were sectioned, and whole-mount antibody staining and immunofluorescence studies were carried out to determine the total number and percentage of differentiated cardiomyocytes in mitosis. Capped RNA and/or translation-blocking Xic1 morpholino antisense oligonucleotides (Xic1Mo) were microinjected into embryos, and their role on cardiac differentiation was assessed by in situ hybridization and/or PCR. We show that cell-cycling post-gastrulation is not essential for cardiac differentiation in Xenopus embryos, and conversely that some cells can express markers of cardiac differentiation even when still in cycle. A targeted knock-down of Xic1 protein by Xic1Mo microinjection decreases the expression of markers of cardiac differentiation, which can be partially rescued by co-injection of full-length Xic1 RNA, demonstrating that Xic1 is essential for heart formation. Furthermore, using deleted and mutant forms of Xic1, we show that neither its abilities to inhibit the cell cycle nor the great majority of CDK kinase activity are essential for Xic1's function in cardiomyocyte differentiation, an activity that resides in the N-terminus of the molecule. CONCLUSION Altogether, our results demonstrate that the CDKI Xic1 is required in Xenopus cardiac differentiation, and that this function is localized at its N-terminus, but it is distinct from its ability to arrest the cell cycle and inhibit overall CDK kinase activity. Hence, these results suggest that CDKIs play an important direct role in driving cardiomyocyte differentiation in addition to cell-cycle regulation.