TBX5 drivesAldh1a2expression to regulate a RA-Hedgehog-Wnt gene regulatory network coordinating cardiopulmonary development

The gene regulatory networks that coordinate the development of the cardiac and pulmonary systems are essential for terrestrial life but poorly understood. The T-box transcription factor Tbx5 is critical for both pulmonary specification and heart development, but how these activities are mechanistically integrated remains unclear. We show thatTbx5regulates an evolutionarily conserved retinoic acid (RA)-Hedgehog-Wnt signaling cascade coordinating cardiopulmonary development. We demonstrate that Tbx5 directly maintains expression of the RA-synthesizing enzymeAldh1a2in the foregut lateral plate mesoderm via an intronic enhancer that is evolutionarily conserved among terrestrial vertebrates.Tbx5promotes posterior second heart field identity in a positive feedback loop with RA, antagonizing a Fgf8-Cyp regulatory module and restricting FGF activity to the anterior. Tbx5/Aldh1a2-dependent RA signaling also directly activatesShhtranscription in the adjacent foregut endoderm through the conserved MACS1 enhancer. Epithelial Hedgehog then signals back to the mesoderm, where together with Tbx5 it activates expression ofWnt2/2bthat ultimately induce pulmonary fate in the foregut endoderm. These results provide mechanistic insight into the interrelationship between heart and lung development informing cardiopulmonary evolution and birth defects.