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Specification of the primary heart field in mouse embryos requires signaling from the anterior visceral endoderm (AVE). The nature of these signals is not known. We hypothesized that the TGFβ-activated kinase (TAK1/Map3k7) may act as a cardiogenic factor, based on its expression in heart-inducing endoderm and its requirement for cardiac differentiation of p19 cells. To test this, mouse embryonic stem (ES) cells overexpressing Map3k7 were isolated and differentiated as embryoid bodies (EBs). Map3k7-overexpressing EBs showed increased expression of AVE markers but interestingly, showed little effect on mesoderm formation and had no impact on overall cardiomyocyte formation. To test whether the pronounced expansion of endoderm masks an expansion of cardiac lineages, chimeric EBs were made consisting of Map3k7-overexpressing ES and wild type ES cells harboring a cardiac reporter transgene, MHCα::GFP, allowing cardiac differentiation to be assessed specifically in wild type ES cells. Wild type ES cells co-cultured with Map3k7-overexpressing cells had a 4-fold increase in expression of the cardiac reporter, supporting the hypothesis that Map3k7 increases the formation of cardiogenic endoderm. To further examine the role of Map3k7 in early lineage specification, other endodermal markers were examined. Interestingly, markers that are expressed in both the VE and later in gut development were expanded, whereas transcripts that specifically mark the early definitive (streak-derived) endoderm (DE) were not. To determine if Map3k7 is necessary for endoderm differentiation, EBs were grown in the presence of the Map3k7 specific inhibitor 5Z-7-oxozeaenol. Endoderm differentiation was dramatically decreased in these cells. Western blot analysis showed that known downstream targets of Map3k7 (Jnk, Nemo-like kinase (NLK) and p38 MAPK) were all inhibited. By contrast, transcripts for another TGFβ target, Sonic Hedgehog (Shh) were markedly upregulated, as were transcripts for Gli2 (but not Gli1 and Gli3). Together these data support the hypothesis that Map3k7 governs the formation, or proliferation of cardiogenic endoderm.

In vivo, cardiomyocytes comprise a heterogeneous population of contractile cells defined by unique electrophysiologies, molecular markers and morphologies. The mechanisms directing myocardial cells to specific sub-lineages remain poorly understood. Here we report that overexpression of TGFβ-Activated Kinase (TAK1/Map3k7) in mouse embryonic stem (ES) cells faithfully directs myocardial differentiation of embryoid body (EB)-derived cardiac cells toward the sinoatrial node (SAN) lineage. Most cardiac cells in Map3k7-overexpressing EBs adopt markers, cellular morphologies, and electrophysiological behaviors characteristic of the SAN. These data, in addition to the fact that Map3k7 is upregulated in the sinus venous-the source of cells for the SAN-suggest that Map3k7 may be an endogenous regulator of the SAN fate.