Nevertheless, no epicardial derived myocardial compartment has been described during chicken cardiogenesis, advocating the chicken as a legitimate model system to investigate processes associated with PE -Epi lineage divergence. By comparing the changes in gene-expression profiles of the different stages of cultured proepicardial cells with the different stages of embryonic epicardial cells, we were able to identify many genes in these two INCB28060 lineages that had divergent profiles and therefore may be associated with the epicardial lock. Of particular interest are genes that, in addition to displaying divergent expression profiles, are also associated with cardiac specification. i.e., that show a transient increase in expression early during PE differentiation towards cardiomyocytes in explant cultures. Our analyses showed that Wnt signaling components were one group of molecules that were prominently present in this subset of genes, in addition to the many other Wnt-related components that our array analysis had identified, like Wnt2a and Wnt5b, Frizzled receptors Fzd1, 2 and 7, Frzb, dickkopf homolog 1, Wnt1 inducible signaling pathway protein-1 and b-catenin. Specifically, the extracellular Wnt antagonist Wif1, was chosen as a follow-up candidate to delineate its role during cardiomyogenesis in models for the first and second heart fields using the p19cl6 cell line and PE explant cultures, respectively. Little is known about the role of Wif1 in cardiogenesis. Schneider et al. found that injecting mRNA coding for Wif1 in Xenopus ventral marginal zone explants only weakly induced Nkx2.5 expression. In our p19cl6 intervention studies, limiting Wif1 exposure to the first 4 days during during culture, lead to induced Gata4 expression at day 8 of culture, while the prolonged exposure up to day 8 blocked the increase in Gata4, suggesting an increase in the cardiomyocyte progenitor pool through early exposure. Western blot analysis for sarcomeric myosin on day 12 samples confirmed this biphasic effect at the protein level. Moreover, in vivo Wif1 CUDC-907 incubation indicated that the Tbx18 positive cardiac progenitor pool upstream of the heart expands and differentiates into cardiomyocytes precociously. Studies have shown canonical Wnt signaling to be biphasic in nature in embryonic stem cell like models, i.e., early Wnt activation stimulates while late Wnt activation inhibits cardiomyocyte differentiation. The Gata4 and Mesp1 expression profiles in response to Wif1 in p19cl6 cells would indeed imply an early phase of activated Wnt signaling, followed by a phase of Wnt signaling inhibition. In the chicken PE-explant cultures we observed that both Wnt activation and Wif1 incubation resulted in a significant increase in the fraction of cardiomyocytes after 5 days in culture.