TBX5 drives Aldh1a2 expression 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 that Tbx5 regulates an evolutionarily conserved retinoic acid (RA)-Hedgehog-Wnt signaling cascade coordinating cardiopulmonary development. We demonstrate that Tbx5 directly maintains expression of the RA-synthesizing enzyme Aldh1a2 in the foregut lateral plate mesoderm via an intronic enhancer that is evolutionarily conserved among terrestrial vertebrates. Tbx5 promotes 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 activates Shh transcription 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 of Wnt2/2b that 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. KEY FINDINGSO_LITbx5 regulates second heart field patterning and pulmonary development via retinoic acid (RA) and Hedgehog (Hh) signaling. C_LIO_LITbx5 directly maintains transcription of the RA-synthesizing enzyme Aldh1a2 in the posterior second heart field mesoderm via an evolutionarily conserved intronic enhancer. C_LIO_LIDownstream of Tbx5, RA directly promotes Shh transcription through the evolutionarily conserved MACS1 endoderm enhancer. C_LIO_LIDownstream of Tbx5, RA suppresses FGF signaling to pattern the second heart field while promoting a Hedgehog-Wnt2/2b signaling cascade that induces pulmonary fate. C_LI SUMMARY STATEMENTTbx5-dependent Retinoic Acid signaling regulates an evolutionarily conserved gene regulatory network that coordinates cardiac and pulmonary development.