Evolutionarily conserved Tbx5–Wnt2/2b pathway orchestrates cardiopulmonary development Jeffrey D. Steimlea,b,c, Scott A. Rankind,e,f,g, Christopher E. Slagleh,i,j,k, Jenna Bekenya,b,c, Ariel B. Rydeena,b,c, Sunny Sun-Kin Chanl,m, Junghun Kweona,b,c, Xinan H. Yanga,b,c, Kohta Ikegamia,b,c, Rangarajan D. Nadadura,b,c, Megan Rowtona,b,c, Andrew D. Hoffmanna,b,c, Sonja Lazarevica,b,c, William Thomasn,o, Erin A. T. Boyle Andersonp, Marko E. Horbn,o, Luis Luna-Zuritaq,r, Robert K. Hom, Michael Kybal,m, Bjarke Jensens, Aaron M. Zornd,e,f,g, Frank L. Conlonh,i,j,k, and Ivan P. Moskowitza,b,c,1 aDepartment of Pediatrics, University of Chicago, Chicago, IL 60637; bDepartment of Pathology, University of Chicago, Chicago, IL 60637; cDepartment of Human Genetics, University of Chicago, Chicago, IL 60637; dCenter for Stem Cell and Organoid Medicine, Cincinnati Children’s Research Foundation, Cincinnati, OH 45229; eDepartment of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, OH 45229; fDivision of Developmental Biology, Perinatal Institute, Cincinnati Children’s Research Foundation, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, OH 45229; gDepartment of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, OH 45229; hDepartment of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; iDepartment of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; jIntegrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; kThe University of North Carolina McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; lDepartment of Pediatrics, University of Minnesota, Minneapolis, MN 55455; mLillehei Heart Institute, University of Minnesota, Minneapolis, MN 55455; nNational Xenopus Resource, Marine Biological Laboratory, Woods Hole, MA 02543; oEugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA 02543; pDepartment of Organismal Biology and Anatomy, University of Chicago, Chicago, IL 60637; qGladstone Institute of Cardiovascular Disease, San Francisco, CA 94158; rRoddenberry Center for Stem Cell Biology and Medicine, Gladstone Institutes, San Francisco, CA 94158; and sAmsterdam Cardiovascular Sciences, Department of Medical Biology, Amsterdam University Medical Center, University of Amsterdam, 1105AZ Amsterdam, The Netherlands Edited by Roeland Nusse, Stanford University School of Medicine, Stanford, CA, and approved September 27, 2018 (received for review July 9, 2018) Codevelopment of the lungs and heart underlies key evolutionary these structures comprise common forms of human congenital innovations in the transition to terrestrial life. Cardiac specializations heart disease. that support pulmonary circulation, including the atrial septum, are Recent work has highlighted the common developmental or- generated by second heart field (SHF) cardiopulmonary progenitors igin of multiple mesodermal derivatives in both the heart and the (CPPs). It has been presumed that transcription factors required in lung (5, 6). This lateral plate mesodermal population has been the SHF for cardiac septation, e.g., Tbx5,directlydriveacardiacmor- termed the “second heart field” (SHF) or “cardiopulmonary phogenesis gene-regulatory network. Here, we report instead that progenitors” (CPPs). This population originates dorsal to the TBX5 directly drives Wnt ligands to initiate a bidirectional signaling cardiac inflow tract and ventral to the anterior foregut and loop between cardiopulmonary mesoderm and the foregut endo- generates multiple structures in the heart, e.g., the atrial septum, derm for endodermal pulmonary specification and, subsequently, and in the lungs, e.g., smooth muscle and vascular endothelium atrial septation. We show that Tbx5 is required for pulmonary spec- (5, 6). This essential CPP region is labeled by expression of the ification in mice and amphibians but not for swim bladder develop- canonical Wnt signaling ligand Wnt2, the Hedgehog (Hh) ment in zebrafish. TBX5 is non–cell-autonomously required for Wnt2 pulmonary endoderm specification by directly driving and Significance Wnt2b expression in cardiopulmonary mesoderm. TBX5 ChIP- sequencing identified cis-regulatory elements at Wnt2 sufficient for In the 20 years since the discovery of the genetic link between endogenous Wnt2 expression domains in vivo and required for Wnt2 the transcription factor TBX5 and congenital heart defects, few expression in precardiac mesoderm in vitro. Tbx5 cooperated with direct targets of TBX5 in cardiac morphogenesis have been ShhsignalingtodriveWnt2b expression for lung morphogenesis. identified. In this work, we demonstrate that TBX5 directly Tbx5 haploinsufficiency in mice, a model of Holt–Oram syndrome, regulates canonical Wnt ligands required for initiation of lung caused a quantitative decrement of mesodermal-to-endodermal Wnt development. Lung endoderm forms a Hedgehog signaling signaling and subsequent endodermal-to-mesodermal Shh signaling source required for morphogenesis of both the lungs and the required for cardiac morphogenesis. Thus, Tbx5 initiates a mesoderm– cardiac inflow septum. Our work expands the role of TBX5 to endoderm–mesoderm signaling loop in lunged vertebrates that include a non–cell-autonomous component for atrial septation. provides a molecular basis for the coevolution of pulmonary and We find the mesoderm–endoderm–mesoderm signaling loop cardiac structures required for terrestrial life. initiated by TBX5 is evolutionarily conserved from amphibians to mammals. This work suggests that the evolutionary origin lung development | heart development | TBX5 | Wnt signaling | of lungs may have involved the recruitment of cardiac TBX5. Hedgehog signaling Author contributions: J.D.S., S.A.R., C.E.S., E.A.T.B.A., M.E.H., R.K.H., B.J., A.M.Z., F.L.C., tilization of atmospheric oxygen revolutionized the ability of and I.P.M. designed research; J.D.S., S.A.R., C.E.S., J.B., A.B.R., J.K., K.I., R.D.N., M.R., Uvertebrates to adapt to terrestrial life (1). At the center of A.D.H., S.L., W.T., E.A.T.B.A., L.L.-Z., and B.J. performed research; S.S.-K.C. and M.K. con- tributed new reagents/analytic tools; J.D.S., S.A.R., C.E.S., and X.H.Y. analyzed data; and this revolution are the lungs, a foregut-derived gas-exchange J.D.S., S.A.R., A.M.Z., F.L.C., and I.P.M. wrote the paper. BIOLOGY structure (1, 2). The derived cardiovascular system, utilizing The authors declare no conflict of interest. pulmonary oxygen, must manage blood from both the body and DEVELOPMENTAL This article is a PNAS Direct Submission. the lungs simultaneously (2). While most lungfish, amphibians, Published under the PNAS license. and reptiles exhibit a three-chambered heart with an atrial Data deposition: RNA-sequencing, ChIP-sequencing, and ATAC-sequencing data have septum separating pulmonary and systemic circulation enter- been deposited in the Gene Expression Omnibus databank (accession nos. GSE75077 ing the heart (3), the two-sided, four-chambered crocodilian, and GSE119885). avian, and mammalian hearts have independently evolved to 1To whom correspondence should be addressed. Email: [email protected]. completely separate pulmonary from systemic circulation (4). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. The proper development and placement of the cardiac septa 1073/pnas.1811624115/-/DCSupplemental. are critical for the efficient handling of blood, and defects in Published online October 23, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1811624115 PNAS | vol. 115 | no. 45 | E10615–E10624 Downloaded by guest on September 28, 2021 signaling-responsive transcription factor Gli1, and the T-box A Transcriptional Profiling of Microdissected CPP-containing Region family transcription factor Tbx5 (5, 7–9). Mate Tbx5+/- Mice Mutations in TBX5 have been implicated as the primary genetic Tbx5 cause of Holt–Oram syndrome (HOS), a human syndrome that Dissect E9.5 Embryos includes cardiac septal defects (10–14). Previous work has demon- Heart strated that Tbx5 is required in the posterior SHF (pSHF) for atrial Pool Tissue from 4 Embryos septation (7, 9, 15). In addition, Sonic hedgehog (Shh) signaling has each Tbx5+/+ and Tbx5-/- been implicated in cardiac septation (7–9, 16). Shh, expressed in the pulmonary endoderm (PE), activates GLI-mediated transcription in CPP RNA-Sequencing BCE9.5 CPP Tbx5+/+ and Tbx5-/- RNA-seq the CPPs (7, 8). Shh and Tbx5 genetically interact for cardiac sep- Spearman’s ρ tation, and constitutive activation of Hh signaling in CPPs rescues 50 atrial septal defects caused by reduced Tbx5 dose (7, 9). Further- 0.95 1.00 Tbx5+/+ Tbx5-/- Nkx2-1 more, TBX5 and GLI transcription factors directly cooperate at Shh 0.95 0.95 0.95 0.97 0.96 0.99 1 Wnt2 enhancers for genes required for cardiac septation (7, 9). This has 0.97 0.96 0.96 0.98 0.98 1 0.99 NANCI generated a model in which TBX5 and GLI transcription fac- 5 Tbx4 0.99 0.99 0.98 0.99 1 0.98 0.96 Wnt2b tors directly activate gene expression in the CPPs of the pSHF for Fzd10 −log10 FDR Gli1 cardiac morphogenesis. 0.99 0.99 0.98 1 0.99 0.98 0.97 Wnt7b 1 CPPs are an important source of signals that induce the pul- 0.99 0.99 1 0.98 0.98 0.96 0.95 monary lineage in the ventral foregut endoderm and contribute – 0.99 1 0.99 0.99 0.99 0.96 0.95 directly to the atrial septum and cardiopulmonary vasculature (17 −5 −2 −1 0 1 2 5 19). An evolutionarily conserved paracrine signaling cascade in- 1 0.99 0.99 0.99 0.99 0.97 0.95 log2 fold change volving retinoic acid, Hh, Wnt signaling, and bone morphogenic D qRT-PCR Validation of Early Pulmonary Development Genes 1.5 protein (BMP) regulates the induction of pulmonary progenitors * p<0.05 NS from amphibians to mammals (5, 17–21).