Tbx5 drives Aldh1a2 expression to regulate a RA-Hedgehog-Wnt gene regulatory network coordinating cardiopulmonary development

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 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 cardiopulmonary evolution and birth defects.

Modeling human multi-lineage heart field development with pluripotent stem cells

“…The cells generated from the ALDH + CXCR4 À pSHF mesoderm preferentially expressed the pSHF markers (Figure 3F), whereas those Resource derived from the ALDH À CXCR4 + aSHF mesoderm expressed higher levels of the aSHF markers (Figure 3G). Some of the FHF genes were expressed at comparable levels in the pSHF population, a finding consistent with studies in model organisms (Rankin et al, 2021;Ryan and Chin, 2003;Wiesinger et al, 2021).…”

The homeodomain transcription factor Ventx2 regulates respiratory progenitor cell number and differentiation timing duringXenopuslung development

“…BMP signaling can activate expression of human VENTX during the directed differentiation of human embryonic stem cells into mesoderm and Smad1 binding motifs have been identified in the human VENTX promoter region (Sumi et al 2008; von Bubnoff et al 2005). In the future it will be of interest to perform pSmad1 and B-catenin ChIP-seq during respiratory lineage induction from NF31-35 on isolated Xenopus foregut endoderm tissue to unambiguously define the genomic landscape of BMP and Wnt regulated enhancers at these key developmental stages, and couple this with RNA-seq and functional enhancer studies that are readily performed in Xenopus (Blitz and Cho 2021; Rankin et al 2021).…”

“…BMP signaling can activate expression of human VENTX during the directed differentiation of human embryonic stem cells into mesoderm and Smad1 binding motifs have been identified in the human VENTX promoter region(Sumi et al 2008;von Bubnoff et al 2005). In the future it will be of interest to perform pSmad1 and B-catenin ChIP-seq during respiratory lineage induction from NF31-35 on isolated Xenopus foregut endoderm tissue to unambiguously define the genomic landscape of BMP and Wnt regulated enhancers at these key developmental stages, and couple this with RNA-seq and functional enhancer studies that are readily performed in Xenopus(Blitz and Cho 2021;Rankin et al 2021).The phenotypes we observe when depleting Ventx2 in the developing foregut include elevated proliferation, elevated numbers of respiratory progenitors, premature onset and elevated levels of surfactant protein gene expression, and a failure of proper T-E separation. It is important to note that in many contexts premature differentiation of progenitor cells is either associated with or has been shown to be a causative driving force of a myriad of developmental defects or disease states in numerous organ systems including the heart, lung, kidney, and brain(Rowton et al 2021; Morrisey 2013; Chung et al 2018; Kopan et al 2014; Nakafuku and Del Águila 2020; Caracci et al 2021; Khanbabaei et al 2019).…”

The Long Noncoding RNA Playrr Regulates Pitx2 Dosage and Protects Against Cardiac Arrhythmias

“…8,14 Histological analyses reveal that Playrr Ex1sj mutant mice have perturbed distribution and lateralization of Connexin 43 (Cx43), the major cardiac gap junction protein, and increased atrial fibrosis. Finally, we show via transcriptomic analyses of adult Playrr Ex1sj mutant hearts that loss of Playrr leads to upregulation of the T-box family gene (Tbx5), a key regulator of heart development, [22][23][24] and Hcn1 (Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 1), first identified in the SAN as a key regulator of heart rate, 25,26 as well as calcium handling genes Ryr2 (Ryanodine Receptor 2) and Jph2 (Junctophilin 2) required for the generation of normal cardiac rhythm. Together our studies implicate a role for the lncRNA transcript Playrr located at the Pitx2-linked AF genomic locus in the maintenance of normal sinus rhythm and protection from atrial arrhythmias.…”