Abstract
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. Here using Xenopus and mouse embryos, we establish molecular links between Tbx5 and retinoic acid (RA) signaling in the mesoderm and between RA signaling and sonic hedgehog expression in the endoderm to unveil a conserved RA-Hedgehog-Wnt signaling cascade coordinating cardiopulmonary (CP) development. We demonstrate that Tbx5 directly maintains expression of aldh1a2, the RA-synthesizing enzyme, in the foregut lateral plate mesoderm via an evolutionarily conserved intronic enhancer. Tbx5 promotes posterior second heart field identity in a positive feedback loop with RA, antagonizing a Fgf8-Cyp regulatory module to restrict FGF activity to the anterior. We find that Tbx5/Aldh1a2-dependent RA signaling directly activates shh transcription in the adjacent foregut endoderm through a conserved MACS1 enhancer. Hedgehog signaling coordinates with Tbx5 in the mesoderm to activate expression of wnt2/2b, which induces pulmonary fate in the foregut endoderm. These results provide mechanistic insight into the interrelationship between heart and lung development informing CP evolution and birth defects.
Highlights
Proper integration of the cardiac and pulmonary systems begins during early embryogenesis and is essential for terrestrial life
We identify the following aspects of this SHF mesoderm – pulmonary endoderm signaling network: 1) Direct Tbx[5] activation of an aldh1a2 enhancer, which maintains aldh1a2 transcription in posterior SHF (pSHF) mesoderm; retinoic acid (RA) is in turn required to maintain tbx[5] expression in the pSHF, establishing a positive feedback loop between Tbx[5] and RA; 2) Tbx5-RA and FGF437 Cyp form mutually antagonistic modules, with the Tbx5-RA loop promoting pSHF/cardiopulmonary progenitors (CPPs) identity and suppressing anterior SHF (aSHF) fate, and Cyp-mediated RA degradation refining the spatial domain of RA activity; and 3) Direct RXR/RAR activation of the MACS1 enhancer at the shh locus, which provides a mechanism underlying the cell non-autonomous activation of endodermal Hh ligand expression by Tbx5/Aldh1a2-dependent RA signaling in the pSHF
We show that Tbx[5] and RA form a positive feedforward loop in the pSHF; in this domain Tbx[5] directly maintains Aldh1a2-dependent RA production while RA maintains tbx[5] expression
Summary
Proper integration of the cardiac and pulmonary systems begins during early embryogenesis and is essential for terrestrial life. A key feature of cardiopulmonary development is evolutionarily conserved bi-directional paracrine signaling between the foregut endoderm, which gives rise to pulmonary epithelium, and the cardiogenic mesoderm (Xie et al 2012; Rankin et al 2016; Steimle et al 2018). The interplay between these signals and lineage specific transcription factors (TFs) to control lineage specific gene regulatory networks (GRNs) for heart and lung morphogenesis are poorly understood. The anterior SHF (aSHF) is characterized by expression of Fgf[8], Fgf[10], Tbx[1] and generates the right ventricle, portions of the outflow tract, and pharyngeal mesoderm (Rochais et al 2009; Kelly et al 2014). CPPs are both the recipient and source of reciprocal signaling with the adjacent pulmonary endoderm essential for heart and lung development
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