BMP4 triggers regulatory circuits specifying the cardiac mesoderm lineage

Cardiac lineage specification in the mouse is controlled by TGFβ and WNT signaling. From fly to fish, BMP has been identified as an indispensable heart inducer. A detailed analysis of the role of Bmp4 and its effectors Smad1/5, however, was still missing. We show that Bmp4 induces cardiac mesoderm formation in murine embryonic stem cells in vitro. Bmp4 first activates Wnt3 and upregulates Nodal. pSmad1/5 and the WNT effector Tcf3 form a complex, and together with pSmad2/3 activate mesoderm enhancers and Eomes. They then cooperate with Eomes to consolidate the expression of many mesoderm factors, including T. Eomes and T form a positive feedback loop and open additional enhancers regulating early mesoderm genes, including the transcription factor Mesp1 establishing the cardiac mesoderm lineage. In parallel, the neural fate is suppressed. Our data confirm the pivotal role of Bmp4 in cardiac mesoderm formation in the mouse. We describe in detail the consecutive and cooperative actions of three signaling pathways, BMP, WNT and Nodal, and their effector transcription factors, during cardiac mesoderm specification. Transcriptomes (RNA-Seq) and chromatin accessibility (ATAC-Seq) from cardiac mesoderm differentiated WT, Smad4, Eomes, T, and double Eomes/T knockout mouse ESCs. ChIP-Seq of P-Smad1/5/9, P-Smad2, Eomes, and T from cardiac mesoderm differentiated WT mouse ESCs.