We also found a biphasic pattern of Mzf1 expression during in vitro. Other factors like Myf-6 or D-mef2 that influence lineage specification also act in a biphasic manner during embryonic development. Our hypothesis that Mzf1 plays a role in cardiogenesis via an interaction with the Nkx2.5 CE was further supported by the differential expression of Mzf1 in purified Nkx2.5 CE positive CPCs at days five and seven of differentiation as well as in mouse embryonic hearts at E 9.5 but to a much lower extent in mature adult cardiomyocytes. These results indicate that the main influence of Mzf1 on Nkx2.5 CE labelled CPCs takes place during early cardiomyocyte differentiation but not after terminal differentiation of these cells. Since Mzf1 appears to regulate gene expression in CPCs, we examined the effect of Mzf1 BIBW2992 overexpression using a murine tetOMzf1-Nkx2.5 CE eGFP ES cell line. Flow cytometry results clearly indicated an increased frequency of CPCs induced by an Mzf1 overexpression from day five of in vitro differentiation. In contrast, continuous overexpression of Mzf1 from day 0-8 resulted in significant reduction of CPC formation. We furthermore found evident morphological changes during differentiation under permanent dox-addition. Settled EBs showed globular clusters which were closely packed while no beating areas could be observed. It can be assumed that the permanent Mzf1 overexpression led to a different migration behavior of cells in these EBs since it is well known that Mzf1 plays a role in migration and invasion. However, Mzf1 overexpression from day 5 exhibited an EB-morphology typical for undirected murine ES-cell differentiations and a regular appearance of beating areas. Based on this observation, we concluded that Mzf1 overexpression can induce cardiac lineage expansion in a temporal-specific fashion. Taken together, our results implicate a role for Mzf1 in the control of cardiac commitment by an interaction with the Nkx2.5 cardiac enhancer. As Mzf1 was significantly enhanced in a CPC population in vitro as well as in embryonic heart tissue and late overexpression of Mzf1 promoted cardiac lineage commitment we propose that Mzf1 may be a novel regulator of embryonic heart development. Figure 7 summarizes the physiological biphasic kinetics of Mzf1 expression. The first peak of Mzf1 up-regulation occurs early during specification of pluripotent cells: Around day two of in vitro differentiation, corresponding with the epiblast stage during murine development on E 6.0 or 6.5. At this time Mzf1 seems to have an inhibitory effect on cardiac lineage commitment as shown by our results. Mzf1 may inhibit the generation of cardiac mesoderm by suppressing Mesp1 and Flk1 expression. Runx1 and Nestin are virtually unaffected by a permanent overexpression of Mzf1. The second physiological peak of Mzf1 expression occurs during differentiation of pluripotent cells around day eight of in vitro differentiation. An overexpression of Mzf1 at the beginning of this peak, in parallel with the endogenous upregulation of the Nkx2.5 expression which is initiated at day four of in vitro differentiation and is highly increased at day five to seven.