An Endocardial Pathway Involving Tbx5, Gata4, and Nos3 Required for Atrial Septum Formation

An endocardial pathway involving Tbx5, Gata4, and Nos3 required for atrial septum formation Mathieu Nadeaua,b,1, Romain O. Georgesa,b,1, Brigitte Laforesta,b, Abir Yamaka,c, Chantal Lefebvrea,b, Janie Beauregarda,c, Pierre Paradisa,2, Benoit G. Bruneaud, Gregor Andelfingera,e, and Mona Nemera,c,3 aResearch Unit in Cardiac Growth and Differentiation and bMolecular Biology Program, Université de Montréal, Montréal, QC, Canada H3C 3J7; cDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada K1N 6N5; dGladstone Institute of Cardiovascular Disease, Department of Pediatrics, University of California, San Francisco, CA 94158; and eDepartment of Cardiovascular Genetics, Hôpital Ste-Justine, Montréal, QC, Canada H3T 1C5 Edited* by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved October 5, 2010 (received for review January 5, 2010) In humans, septal defects are among the most prevalent congen- thelial gene expression and their knockdown in fish disrupted ital heart diseases, but their cellular and molecular origins are not vascular development (15). Others like the basic helix-loop-helix fully understood. We report that transcription factor Tbx5 is pres- transcription factor SCL (16) or Tbx20 (17) and Twist1 (18) are ent in a subpopulation of endocardial cells and that its deletion involved in early migration or proliferation of endocardial pro- therein results in fully penetrant, dose-dependent atrial septal genitors. Transcription factors associated with later stages of defects in mice. Increased apoptosis of endocardial cells lacking endocardial cell differentiation include Sox9 (19), Gata5 (20), Tbx5, as well as neighboring TBX5-positive myocardial cells of and NFATc (8, 21, 22). Last, GATA4 is expressed in early en- the atrial septum through activation of endocardial NOS (Nos3), docardial progenitors and persists therein throughout de- is the underlying mechanism of disease. Compound Tbx5 and Nos3 velopment (20, 23). The exact role of Gata4 in the endocardium is not fully understood but is thought to involve proliferation and haploinsufficiency in mice worsens the cardiac phenotype. The remodeling of the endocardial cushion (24). In this study, we data identify a pathway for endocardial cell survival and unravel Tbx5 fi Nos3 show that the transcription factor Tbx5, a member of the Tbox a cell-autonomous role for therein. The nding that , family of important developmental regulators, is expressed in a gene regulated by many congenital heart disease risk factors endocardial cells destined to form the atrial septum, where it BIOLOGY including stress and diabetes, interacts genetically with Tbx5 pro- plays a cell-autonomous role in endocardial cell survival, thus DEVELOPMENTAL vides a molecular framework to understand gene–environment identifying a regulatory pathway in endocardial growth and dif- interaction in the setting of human birth defects. ferentiation. In human, TBX5 mutations are linked to Holt– Oram syndrome, an autosomal-dominant disorder characterized heart development | Holt–Oram syndrome by upper-limb defects and a large spectrum of cardiac malformations ranging from simple arrhythmia to complex structural n humans, the incidence of congenital heart disease (CHD) is malformations (25, 26). Conduction defects are thought to be Inow estimated to be nearly 5% of live births (1), and CHD is a consequence of Cx40 dysregulation. ASDs are the most com- fi mon cardiac malformations found in patients with Holt–Oram the major noninfectious cause of death in infants within the rst +/− year of life. Moreover, undiagnosed less severe defects increase syndrome (27) and in Tbx5 mice (28), but the cellular origin the risks of morbidity and premature mortality and constitute of the defects remains unknown. We now show that endocardial- fi risk factors for stroke, ischemic heart disease, and sudden death speci c mutation of Tbx5 causes dose-dependent ASDs and that (2). Secundum atrial septal defect (ASD) is the third most endocardial Tbx5 interacts genetically with Gata4 and endocar- common congenital heart malformation (3) and occurs as an dial NOS (Nos3) to regulate cell survival and atrial septum for- isolated defect or as a feature of more complex syndromes (4). A mation. The data provide a mechanism to explain synergy complex multifactorial inheritance model involving alterations in between a transcription factor and an enzyme in CHD. multiple genes and interactions with environment factors has Results been suggested to account for the lower penetrance and variable expressivity of familial ASDs (5). Heart development requires Loss of Tbx5 from the Endocardium Leads to CHD. To better identify differentiation, proliferation and cell–cell communication be- the cellular distribution of TBX5 within the heart, we performed tween two cell layers composed of myocardial and endocardial high-resolution immunohistochemical analysis of TBX5 protein in cells. Significant progress was accomplished in the past decade in the developing heart. Labeling with two different TBX5-specific identifying the molecules and mechanisms involved in myocar- antibodies revealed strong nuclear labeling in atrial and left ven- dial cell differentiation (reviewed in refs. 6, 7). Several key reg- tricular myocytes, consistent with previous reports on the distri- ulators of myocardial patterning and chamber specification have bution of Tbx5 transcripts (27, 28). In addition, labeling was found been identified. They include transcription factors GATA4, in cells of the endocardial cushions and in endocardial cells lining NKX2.5, and TBX5, mutations of which have been associated the atrial septal walls starting at approximately embryonic day with human ASDs (4). In contrast, molecular pathways involved in differentiation of the endocardium are only beginning to be elucidated. Valvuloseptal tissues arise from endocardial cells Author contributions: M. Nemer designed research; M. Nadeau, R.O.G., A.Y., and J.B. that undergo an epithelial mesenchymal transformation; this performed research; M. Nadeau, R.O.G., B.L., C.L., P.P., B.G.B., and G.A. contributed process is regulated by myocardial-derived growth factors such as new reagents/analytic tools; M. Nadeau, R.O.G., A.Y., P.P., G.A., and M. Nemer analyzed data; and M. Nadeau, R.O.G., and M. Nemer wrote the paper. bone morphogenetic proteins (BMP-2/-4) and VEGF (8, 9). fl Because genes linked to septal defects (in human or animal The authors declare no con ict of interest. models of disease) are either expressed predominantly in myo- *This Direct Submission article had a prearranged editor. cardial cells or are coexpressed in myocytes and endocardial Freely available online through the PNAS open access option. cells, the cellular basis of septal defects remains undefined. 1M. Nadeau and R.O.G. contributed equally to this work. The molecular pathways underlying endocardial development 2Present address: Lady Davis Institute for Medical Research, McGill University, Montreal, remain incompletely understood (10–13). A recent report iden- QC, Canada H3T 1E2. tifies the Ets-related protein-71 as obligatory for endothelial/ 3To whom correspondence should be addressed. E-mail: [email protected]. fi endocardial speci cation (14). Consistent with this, FoxC and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Ets transcription factors are sufficient to induce ectopic endo- 1073/pnas.0914888107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0914888107 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 12.5. In contrast to GATA4, whose expression marked all cells of frequency of ASD was 3% (one of 40) in control and Tie2Cre- the endocardial cushion, TBX5 localized in only a subset of EC Tbx5wt/wt mice. Direct measurements of chamber mass confirmed right heart enlargement with increased weight of the right cells (Fig. S1). To assess the role of Tbx5 in endocardial cells, mice − − with endocardium-specific deletion of Tbx5 were generated by atrium and ventricle in eTbx5 / mice, whereas no change was breeding Tbx5 flox mice (28, 29) with Tie2-cre transgenic mice found in the left atrium and ventricle (Fig. 2B). Mice lacking (30). This breeding removes Tbx5 alleles from all endothelial cells, Tbx5 in the endocardium were compared with mice lacking one Tbx5 allele in all cell types produced by crossing with CMV-Cre but because Tbx5 is not expressed in vascular endothelium, the loss − transgenic mice. Tbx5+/ mice (n = 20) showed an intermediary of Tbx5 function is restricted to the endocardium. Genotyping of +/− −/− resulting offspring showed that all the expected genotypes were phenotype between eTbx5 and eTbx5 . Consistent with present in a Mendelian ratio, indicating that removal of Tbx5 from previously published work (28), they displayed right atrial hy- endocardial cells is not embryonic-lethal. This contrasts with the pertrophy, ASD, and larger hearts (Fig. 1B, Right). Histologic embryonic lethality of Tbx5 null mice (28). Immunohistochemistry analysis (Fig. 2A) revealed ASDs and thinning of the valvula of tissue sections confirmed loss of TBX5 from EC but not myo- foramen ovalis (VFO). These results suggest that myocardial cardial cells (Fig. 1A). Tbx5 also contributes to proper formation of the atrial septum. As heterozygote Tbx5 mice have conduction defects (28), Anatomical examination of adult mice with homozygous de- −/− − − electrophysiology of eTbx5 mice was assessed using both letion of Tbx5

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