Chromatin Profiling of Micro-dissected Mouse Embryonic Heart and CPP regions

Co-development of the lungs and heart underlies key evolutionary innovations in the transition to terrestrial life. Cardiac specializations that support pulmonary circulation, including the atrial septum, are generated by second heart field (SHF) cardiopulmonary progenitors (CPPs). It has been presumed that transcription factors required in the SHF for cardiac septation, e.g. Tbx5, directly drive a cardiac morphogenesis gene regulatory network. Here, we report instead that TBX5 directly drove Wnt ligands to initiate a bi-directional signaling loop between cardiopulmonary mesoderm and the foregut endoderm for endodermal pulmonary specification, and subsequently, atrial septation. TBX5 ChIP-seq identified cis-regulatory elements at Wnt2 sufficient for endogenous Wnt2 expression domains in vivo and required for Wnt2 expression in pre-cardiac mesoderm in vitro. Thus, Tbx5 initiated a mesoderm-endoderm-mesoderm signaling loop in lunged vertebrates that provides a molecular basis for the co-evolution of pulmonary and cardiac structures required for terrestrial life. For ATAC-seq, 4 biological replicates were generated for each the micro-dissected hearts and CPP regions from 5 E9.5 CD-1 mouse embryos per replicate. For ChIP-seq, 2 biological replicates were generated for each the micro-dissected hearts and CPP regions from 50 E9.5 CD-1 mouse embryos per replicate. For immunoprecipitation, the chromatin extract was incubated with 5ug of the anti-TBX5 antibody (Santa Cruz Biotechnology sc-17866; Lot #G1516), 1μg of anti-H3K4me3 (Wako Chemicals #305-34819; Lot #14004), or 1μg of anti-H3K4me1 (Abcam ab8895; Lot #GR257926-1) at 4°C for >12 hours in a total volume of 200 μL.