Corrections MEDICAL SCIENCES DEVELOPMENTAL BIOLOGY Correction for “Irradiation induces bone injury by damaging Correction for “Cardiac-specific transcription factor genes Smad4 bone marrow microenvironment for stem cells,” by Xu Cao, and Gata4 cooperatively regulate cardiac valve development,” by Xiangwei Wu, Deborah Frassica, Bing Yu, Lijuan Pang, Lingling Ivan P. Moskowitz, Jun Wang, Michael A. Peterson, William Xian, Mei Wan, Weiqi Lei, Michael Armour, Erik Tryggestad, T. Pu, Alexander C. Mackinnon, Leif Oxburgh, Gerald John Wong, Chun Yi Wen, William Weijia Lu, and Frank C. Chu, Molly Sarkar, Charles Berul, Leslie Smoot, Elizabeth J. Frassica, which appeared in issue 4, January 25, 2011, of Proc J. Robertson, Robert Schwartz, Jonathan G. Seidman, and Natl Acad Sci USA (108:1609–1614; first published January 10, Christine E. Seidman, which appeared in issue 10, March 8, 2011; 10.1073/pnas.1015350108). 2011, of Proc Natl Acad Sci USA (108:4006–4011; first published The authors note that their conflict of interest statement was February 17, 2011; 10.1073/pnas.1019025108). omitted during publication. The authors declare that John Wong The authors note that the title appeared incorrectly. The title has a research service contract with Gulmay Medical, Inc. in the should instead appear as “Transcription factor genes Smad4 and transfer of the SARRP technology from Johns Hopkins to the Gata4 cooperatively regulate cardiac valve development.” The company. The SARRP was used by Dr. Xu Cao to irradiate his online version has been corrected. study animals, and was only peripherally related to the subject matter of the manuscript. Additionally, the specific unit was www.pnas.org/cgi/doi/10.1073/pnas.1103973108 constructed at Johns Hopkins with grant support from the National Cancer Institute (US), and not by Gulmay. CORRECTIONS www.pnas.org/cgi/doi/10.1073/pnas.1104040108 www.pnas.org PNAS | April 5, 2011 | vol. 108 | no. 14 | 5921 Downloaded by guest on September 27, 2021 Cardiac-specific transcription factor genes Smad4 and Gata4 cooperatively regulate cardiac valve development Ivan P. Moskowitza,1,2, Jun Wangb, Michael A. Petersona, William T. Puc, Alexander C. Mackinnona, Leif Oxburghd, Gerald C. Chue, Molly Sarkarf, Charles Berulc, Leslie Smootc, Elizabeth J. Robertsong, Robert Schwartzb, Jonathan G. Seidmanh,1, and Christine E. Seidmanh,i,1,2 aDepartments of Pediatrics and Pathology, University of Chicago, Chicago, IL 60637; bInstitute of Biosciences and Technology, Texas A&M University System Health Science Center, Houston, TX 77030; cDepartment of Cardiology, Children’s Hospital Boston, Boston, MA 02115; dMaine Medical Center Research Institute, Scarborough, ME 04074; eDepartment of Pathology, Harvard Medical School, Boston, MA 02115; fCardiology Department, Cordis Corporation, Miami Lakes, FL 33014; gWellcome Trust Centre for Human Genetics, Oxford OX3 7BN, United Kingdom; hDepartment of Genetics, Harvard Medical School, Boston, MA 02115; and iHoward Hughes Medical Institute, Brigham and Women’s Hospital, Boston, MA, 02115 Contributed by Christine E. Seidman, January 4, 2011 (sent for review July 1, 2010) We report that the dominant human missense mutations G303E tae (19). The abnormalities produce substantial hemodynamic and G296S in GATA4, a cardiac-specific transcription factor gene, consequences that contribute to poor prognosis in affected pa- cause atrioventricular septal defects and valve abnormalities by tients and a virtually uniform requirement for surgical correction. disrupting a signaling cascade involved in endocardial cushion de- Human genetic studies have demonstrated that mutations in velopment. These GATA4 missense mutations, but not a mutation the gene encoding cardiac transcription factor GATA4 cause causing secundum atrial septal defects (S52F), demonstrated im- familial atrial secundum defects and, less commonly, sporadic paired protein interactions with SMAD4, a transcription factor re- AVSDs (20–23). quired for canonical bone morphogenetic protein/transforming We evaluated two families with dominant inheritance of AVSDs and identified two GATA4 missense mutations, G303E growth factor-β (BMP/TGF-β) signaling. Gata4 and Smad4 geneti- BIOLOGY β cally interact in vivo: atrioventricular septal defects result from en- and G296S. We investigated the canonical TGF- /BMP pathway DEVELOPMENTAL dothelial-specific Gata4 and Smad4 compound haploinsufficiency. effector SMAD4 and showed that G303E and G296S altered the transcriptional response to TGF-β/BMP activation. Deletion of Endothelial-specific knockout of Smad4 caused an absence of valve- Smad4 forming activity: Smad4-deficient endocardium was associated from the endocardium of mice caused severe maldevel- with acellular endocardial cushions, absent epithelial-to-mesenchy- opment of endocardial cushions and diminished expression of the Id2 gene. Together these studies define a transcriptio- mal transformation, reduced endocardial proliferation, and loss of nal network directing endocardial cushion formation involving Id2 expression in valve-forming regions. We show that Gata4 and Gata4, Smad4, and Id2. Smad4 cooperatively activated the Id2 promoter, that human GATA4 mutations abrogated this activity, and that Id2 deficiency Results in mice could cause atrioventricular septal defects. We suggest that Human Mutations in GATA4 Cause Endocardial Cushion Defects. From one determinant of the phenotypic spectrum caused by human a cohort of 103 probands with congenital heart defects, we se- GATA4 mutations is differential effects on GATA4/SMAD4 interac- quenced all protein-encoding exons of candidate genes, in- tions required for endocardial cushion development. cluding GATA4. Two GATA4 mutations were identified in probands from unrelated families (Fig. 1) with dominantly in- he atrioventricular canal, the valve-forming region between herited AVSDs. Tthe atria and ventricles, adopts a unique molecular and mor- Family A had six members with a spectrum of AVSDs (Fig. phological program during cardiac development that requires 1A). Sequence analyses of the proband revealed a heterozygous coordinated activity of myocardial and endocardial lineages. The G-to-A substitution at nucleotide 4 in exon 4 of GATA4, which cardiac valve anlage is the endocardial cushion, a swelling com- was also present in all affected Family A members (Fig. 1A), but posed of myocardial-derived extracellular matrix that becomes absent from over 500 ethnically matched controls. This variant populated primarily by endocardial-derived mesenchymal cells encodes replacement of a conserved glycine with glutamate at (1). The development of a mature valve structure from the en- amino acid position 303 (denoted G303E), just distal to the docardial cushion requires multiple distinct steps (2), including carboxyl-terminal zinc finger of the transcription factor. We activation and proliferation of endocardial cells, endothelial-to- concluded that GATA4 G303E caused AVSDs in Family A. mesenchymal transformation (EMT) of activated endocardial Family B had five individuals with AVSDs and four individuals cells, and maturation of the cellularized cushions into functional with electrophysiologic abnormalities (Fig. 1B). Sequence analy- valve leaflets. ses of the proband revealed a heterozygous G-to-A transition at Development of the atrioventricular endocardial cushions into nucleotide 886 of GATA4, which was also present in all affected the atrioventricular valves (tricuspid and mitral) and adjacent members of Family B (Fig. 1B), but absent from over 500 eth- portions of the atrial and ventricular septae requires Tgf-β and nically matched controls. This variant is predicted to substitute Bmp signaling. At least eight Tgf-β or Bmp ligands are expressed a conserved glycine with serine at amino acid position 296 during valve formation (3, 4), and gene ablation of individual (denoted G296S), located directly adjacent to the nuclear local- ligands in model organisms produces phenotypes that range from pronounced valvuloseptal malformations to subtle valve matura- tion defects (5–18). Compound gene ablations of Bmp5/Bmp7 or Author contributions: I.P.M. and J.G.S. designed research; I.P.M., J.W., M.A.P., A.C.M., and Bmp6/Bmp7 produced more severe endocardial cushion defects M.S. performed research; I.P.M., W.T.P., L.O., G.C.C., C.B., L.S., E.J.R., and R.S. contributed than either single mutant did (16–18). These observations imply new reagents/analytic tools; I.P.M., J.G.S., and C.E.S. analyzed data; and I.P.M. and C.E.S. considerable redundancy of Tgf-β/Bmp signaling in the early wrote the paper. functions of this pathway in endocardial cushion development. The authors declare no conflict of interest. Human atrioventricular septal defects (AVSDs) are defined by 1I.P.M., J.G.S., and C.E.S. contributed equally to this study. variable abnormalities of the mitral and/or tricuspid valves in 2To whom correspondence may be addressed. E-mail: [email protected] conjunction with defects in the adjacent atrial or ventricular sep- or [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1019025108 PNAS Early Edition | 1of6 Fig. 1. GATA4 mutations cause endocardial cushion defects. (A) Family A pedigree with cosegregation of GATA4 G303E mutation (+) with congenital heart disease (Left). Note that five mutation carriers had endocardial cushion defects (Right). (B) Family B pedigree with cosegregation of GATA4 G296S muta- tion (+) with congenital heart disease (Left). Note that six mutation carriers had endocardial cushion