AIMS: The cardiac conduction system (CCS) is progressively specified during development by interactions among a discrete number of transcription factors (TFs) that ensure its proper patterning and the emergence of its functional properties. Meis genes encode homeodomain TFs with multiple roles in mammalian development. In humans, Meis genes associate with congenital cardiac malformations and alterations of cardiac electrical activity; however, the basis for these alterations has not been established. Here, we studied the role of Meis TFs in cardiomyocyte development and function during mouse development and adult life. METHODS AND RESULTS: We studied Meis1 and Meis2 conditional deletion mouse models that allowed cardiomyocyte-specific elimination of Meis function during development and inducible elimination of Meis function in cardiomyocytes of the adult CCS. We studied cardiac anatomy, contractility, and conduction. We report that Meis factors are global regulators of cardiac conduction, with a predominant role in the CCS. While constitutive Meis deletion in cardiomyocytes led to congenital malformations of the arterial pole and atria, as well as defects in ventricular conduction, Meis elimination in cardiomyocytes of the adult CCS produced sinus node dysfunction and delayed atrio-ventricular conduction. Molecular analyses unravelled Meis-controlled molecular pathways associated with these defects. Finally, we studied in transgenic mice the activity of a Meis1 human enhancer related to an single-nucleotide polymorphism (SNP) associated by Genome-wide association studies (GWAS) to PR (P and R waves of the electrocardiogram) elongation and found that the transgene drives expression in components of the atrio-ventricular conduction system. CONCLUSION: Our study identifies Meis TFs as essential regulators of the establishment of cardiac conduction function during development and its maintenance during adult life. In addition, we generated animal models and identified molecular alterations that will ease the study of Meis-associated conduction defects and congenital malformations in humans.

1st Faculty of Medicine Institute of Anatomy Charles University U Nemocnice 3 Praha 2 128 00 Czech Republic

Cardiovascular Regeneration Program Centro Nacional de Investigaciones Cardiovasculares 3 Melchor Fernández Almagro Madrid 28029 Spain

Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares 3 5 Av Monforte de Lemos Madrid 28029 Spain

Institut für Experimentelle und Klinische Pharmakologie und Toxikologie Friedrich Alexander Universität Erlangen Nürnberg 17 Fahrstraße Erlangen 91054 Germany

Institute of Neurogenomics Helmholtz Zentrum 1 Ingolstädter Landstraße Neuherberg 85764 Germany

Instituto de Investigación Sanitaria del Hospital Clínico San Carlos Calle del Prof Martín Lagos Madrid 28040 Spain

Novel Arrhythmogenic Mechanisms Program Centro Nacional de Investigaciones Cardiovasculares 3 Melchor Fernández Almagro Madrid 28029 Spain

See more in PubMed

Longobardi  E, Penkov  D, Mateos  D, De Florian  G, Torres  M, Blasi  F. Biochemistry of the tale transcription factors PREP, MEIS, and PBX in vertebrates. Dev Dyn  2014;243:59–75. PubMed PMC

Moscow  JA, Huang  H, Carter  C, Hines  K, Zujewski  J, Cusack  G, Chow  C, Venzon  D, Sorrentino  B, Chiang  Y, Goldspiel  B, Leitman  S, Read  EJ, Abati  A, Gottesman  MM, Pastan  I, Sellers  S, Dunbar  C, Cowan  KH. Engraftment of MDR1 and NeoR gene-transduced hematopoietic cells after breast cancer chemotherapy. Blood  1999;94:52–61. PubMed

Oulad-Abdelghani  M, Chazaud  C, Bouillet  P, Sapin  V, Chambon  P, Dollé  P. Meis2, a novel mouse Pbx-related homeobox gene induced by retinoic acid during differentiation of P19 embryonal carcinoma cells. Dev Dyn  1997;210:173–183. PubMed

Nakamura  T, Jenkins  NA, Copeland  NG. Identification of a new family of Pbx-related homeobox genes. Oncogene  1996;13:2235–2242. PubMed

Delgado  I, Giovinazzo  G, Temiño  S, Gauthier  Y, Balsalobre  A, Drouin  J, Torres  M. Control of mouse limb initiation and antero-posterior patterning by Meis transcription factors. Nat Commun  2021;12:3086. PubMed PMC

Mann  RS, Affolter  M. Hox proteins meet more partners. Curr Opin Genet Dev  1998;8:423–429. PubMed

Azcoitia  V, Aracil  M, Martinez-A  C, Torres  M. The homeodomain protein Meis1 is essential for definitive hematopoiesis and vascular patterning in the mouse embryo. Dev Biol  2005;280:307–320. PubMed

Carramolino  L, Fuentes  J, García-Andrés  C, Azcoitia  V, Riethmacher  D, Torres  M. Platelets play an essential role in separating the blood and lymphatic vasculatures during embryonic angiogenesis. Circ Res  2010;106:1197–1201. PubMed

Hisa  T, Spence  SE, Rachel  RA, Fujita  M, Nakamura  T, Ward  JM, Devor-Henneman  DE, Saiki  Y, Kutsuna  H, Tessarollo  L, Jenkins  NA, Copeland  NG. Hematopoietic, angiogenic and eye defects in Meis1 mutant animals. EMBO J  2004;23:450–459. PubMed PMC

Stankunas  K, Shang  C, Twu  KY, Kao  SC, Jenkins  NA, Copeland  NG, Sanyal  M, Selleri  L, Cleary  ML, Chang  CP. Pbx/Meis deficiencies demonstrate multigenetic origins of congenital heart disease. Circ Res  2008;103:702–709. PubMed PMC

Gonzalez-Lazaro  M, Rosello-Diez  A, Delgado  I, Carramolino  L, Angeles Sanguino  M, Giovinazzo  G, Torres  M. Two new targeted alleles for the comprehensive analysis of Meis1 functions in the mouse. Genesis  2014;52:967–975. PubMed

Machon  O, Masek  J, Machonova  O, Krauss  S, Kozmik  Z. Meis2 is essential for cranial and cardiac neural crest development. BMC Dev Biol  2015;15:40. PubMed PMC

Unnisa  Z, Clark  JP, Roychoudhury  J, Thomas  E, Tessarollo  L, Copeland  NG, Jenkins  NA, Grimes  HL, Kumar  AR. Meis1 preserves hematopoietic stem cells in mice by limiting oxidative stress. Blood  2012;120:4973–4981. PubMed PMC

Simsek  T, Kocabas  F, Zheng  J, Deberardinis  RJ, Mahmoud  AI, Olson  EN, Schneider  JW, Zhang  CC, Sadek  HA. The distinct metabolic profile of hematopoietic stem cells reflects their location in a hypoxic niche. Cell Stem Cell  2010;7:380–390. PubMed PMC

Kocabas  F, Zheng  J, Thet  S, Copeland  NG, Jenkins  NA, DeBerardinis  RJ, Zhang  C, Sadek  HA. Meis1 regulates the metabolic phenotype and oxidant defense of hematopoietic stem cells. Blood  2012;120:4963–4972. PubMed PMC

Mahmoud  AI, Kocabas  F, Muralidhar  SA, Kimura  W, Koura  AS, Thet  S, Porrello  ER, Sadek  HA. Meis1 regulates postnatal cardiomyocyte cell cycle arrest. Nature  2013;497:249–253. PubMed PMC

Louw  JJ, Corveleyn  A, Jia  Y, Hens  G, Gewillig  M, Devriendt  K. MEIS2 involvement in cardiac development, cleft palate, and intellectual disability. Am J Med Genet A  2015;167:1142–1146. PubMed

Verheije  R, Kupchik  GS, Isidor  B, Kroes  HY, Lynch  SA, Hawkes  L, Hempel  M, Gelb  BD, Ghoumid  J, D'Amours  G, Chandler  K, Dubourg  C, Loddo  S, Tumer  Z, Shaw-Smith  C, Nizon  M, Shevell  M, Van Hoof  E, Anyane-Yeboa  K, Cerbone  G, Clayton-Smith  J, Cogne  B, Corre  P, Corveleyn  A, De Borre  M, Hjortshoj  TD, Fradin  M, Gewillig  M, Goldmuntz  E, Hens  G, Lemyre  E, Journel  H, Kini  U, Kortum  F, Le Caignec  C, Novelli  A, Odent  S, Petit  F, Revah-Politi  A, Stong  N, Strom  TM, van Binsbergen  E, DDD study; Devriendt  K, Breckpot  J. Heterozygous loss-of-function variants of MEIS2 cause a triad of palatal defects, congenital heart defects, and intellectual disability. Eur J Hum Genet  2019;27:278–290. PubMed PMC

Giliberti  A, Currò  A, Papa  FT, Frullanti  E, Ariani  F, Coriolani  G, Grosso  S, Renieri  A, Mari  F. MEIS2 gene is responsible for intellectual disability, cardiac defects and a distinct facial phenotype. Eur J Med Genet  2020;63:103627. PubMed

Pfeufer  A, van Noord  C, Marciante  KD, Arking  DE, Larson  MG, Smith  AV, Tarasov  KV, Muller  M, Sotoodehnia  N, Sinner  MF, Verwoert  GC, Li  M, Kao  WH, Kottgen  A, Coresh  J, Bis  JC, Psaty  BM, Rice  K, Rotter  JI, Rivadeneira  F, Hofman  A, Kors  JA, Stricker  BH, Uitterlinden  AG, van Duijn  CM, Beckmann  BM, Sauter  W, Gieger  C, Lubitz  SA, Newton-Cheh  C, Wang  TJ, Magnani  JW, Schnabel  RB, Chung  MK, Barnard  J, Smith  JD, Van Wagoner  DR, Vasan  RS, Aspelund  T, Eiriksdottir  G, Harris  TB, Launer  LJ, Najjar  SS, Lakatta  E, Schlessinger  D, Uda  M, Abecasis  GR, Muller-Myhsok  B, Ehret  GB, Boerwinkle  E, Chakravarti  A, Soliman  EZ, Lunetta  KL, Perz  S, Wichmann  HE, Meitinger  T, Levy  D, Gudnason  V, Ellinor  PT, Sanna  S, Kaab  S, Witteman  JC, Alonso  A, Benjamin  EJ, Heckbert  SR. Genome-wide association study of PR interval. Nat Genet  2010;42:153–159. PubMed PMC

Smith  JG, Magnani  JW, Palmer  C, Meng  YA, Soliman  EZ, Musani  SK, Kerr  KF, Schnabel  RB, Lubitz  SA, Sotoodehnia  N, Redline  S, Pfeufer  A, Müller  M, Evans  DS, Nalls  MA, Liu  Y, Newman  AB, Zonderman  AB, Evans  MK, Deo  R, Ellinor  PT, Paltoo  DN, Newton-Cheh  C, Benjamin  EJ, Mehra  R, Alonso  A, Heckbert  SR, Fox  ER; Candidate-gene Association Resource (CARe) Consortium . Genome-wide association studies of the PR interval in African Americans. PLoS Genet  2011;7:e1001304. PubMed PMC

van Eif  VWW, Devalla  HD, Boink  GJJ, Christoffels  VM. Transcriptional regulation of the cardiac conduction system. Nat Rev Cardiol  2018;15:617–630. PubMed

Frank  DU, Carter  KL, Thomas  KR, Burr  RM, Bakker  ML, Coetzee  WA, Tristani-Firouzi  M, Bamshad  MJ, Christoffels  VM, Moon  AM. Lethal arrhythmias in Tbx3-deficient mice reveal extreme dosage sensitivity of cardiac conduction system function and homeostasis. Proc Natl Acad Sci U S A  2012;109:E154–E163. PubMed PMC

Takeda  M, Briggs  LE, Wakimoto  H, Marks  MH, Warren  SA, Lu  JT, Weinberg  EO, Robertson  KD, Chien  KR, Kasahara  H. Slow progressive conduction and contraction defects in loss of Nkx2-5 mice after cardiomyocyte terminal differentiation. Lab Invest  2009;89:983–993. PubMed PMC

Madisen  L, Zwingman  TA, Sunkin  SM, Oh  SW, Zariwala  HA, Gu  H, Ng  LL, Palmiter  RD, Hawrylycz  MJ, Jones  AR, Lein  ES, Zeng  H. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci  2010;13:133–140. PubMed PMC

Delgado  I, López-Delgado  AC, Roselló-Díez  A, Giovinazzo  G, Cadenas  V, Fernández-de-Manuel  L, Sánchez-Cabo  F, Anderson  MJ, Lewandoski  M, Torres  M. Proximo-distal positional information encoded by an Fgf-regulated gradient of homeodomain transcription factors in the vertebrate limb. Sci Adv  2020;6:eaaz0742. PubMed PMC

Agah  R, Frenkel  PA, French  BA, Michael  LH, Overbeek  PA, Schneider  MD. Gene recombination in postmitotic cells. Targeted expression of Cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo. J Clin Invest  1997;100:169–179. PubMed PMC

Hoesl  E, Stieber  J, Herrmann  S, Feil  S, Tybl  E, Hofmann  F, Feil  R, Ludwig  A. Tamoxifen-inducible gene deletion in the cardiac conduction system. J Mol Cell Cardiol  2008;45:62–69. PubMed

Spieler  D, Kaffe  M, Knauf  F, Bessa  J, Tena  JJ, Giesert  F, Schormair  B, Tilch  E, Lee  H, Horsch  M, Czamara  D, Karbalai  N, von Toerne  C, Waldenberger  M, Gieger  C, Lichtner  P, Claussnitzer  M, Naumann  R, Muller-Myhsok  B, Torres  M, Garrett  L, Rozman  J, Klingenspor  M, Gailus-Durner  V, Fuchs  H, de Angelis  MH, Beckers  J, Holter  SM, Meitinger  T, Hauck  SM, Laumen  H, Wurst  W, Casares  F, Gomez-Skarmeta  JL, Winkelmann  J.  Restless legs syndrome-associated intronic common variant in Meis1 alters enhancer function in the developing telencephalon. Genome Res  2014;24:592–603. PubMed PMC

Mercader  N, Tanaka  EM, Torres  M. Proximodistal identity during vertebrate limb regeneration is regulated by Meis homeodomain proteins. Development  2005;132:4131–4142. PubMed

Teichholz  LE, Kreulen  T, Herman  MV, Gorlin  R. Problems in echocardiographic volume determinations: echocardiographic-angiographic correlations in the presence of absence of asynergy. Am J Cardiol  1976;37:7–11. PubMed

Sankova  B, Benes  J  Jr, Krejci  E, Dupays  L, Theveniau-Ruissy  M, Miquerol  L, Sedmera  D. The effect of connexin40 deficiency on ventricular conduction system function during development. Cardiovasc Res  2012;95:469–479. PubMed

Rivera-Torres  J, Calvo  CJ, Llach  A, Guzman-Martinez  G, Caballero  R, Gonzalez-Gomez  C, Jimenez-Borreguero  LJ, Guadix  JA, Osorio  FG, Lopez-Otin  C, Herraiz-Martinez  A, Cabello  N, Vallmitjana  A, Benitez  R, Gordon  LB, Jalife  J, Perez-Pomares  JM, Tamargo  J, Delpon  E, Hove-Madsen  L, Filgueiras-Rama  D, Andres  V. Cardiac electrical defects in progeroid mice and Hutchinson-Gilford progeria syndrome patients with nuclear lamina alterations. Proc Natl Acad Sci U S A  2016;113:E7250–E7259. PubMed PMC

Filgueiras-Rama  D, Vasilijevic  J, Jalife  J, Noujaim  SF, Alfonso  JM, Nicolas-Avila  JA, Gutierrez  C, Zamarreno  N, Hidalgo  A, Bernabe  A, Cop  CP, Ponce-Balbuena  D, Guerrero-Serna  G, Calle  D, Desco  M, Ruiz-Cabello  J, Nieto  A, Falcon  A. Human influenza A virus causes myocardial and cardiac-specific conduction system infections associated with early inflammation and premature death. Cardiovasc Res  2021;117:876–889. PubMed PMC

Mitchell  GF, Jeron  A, Koren  G. Measurement of heart rate and Q-T interval in the conscious mouse. Am J Physiol  1998;274:H747–H751. PubMed

Nguyen  NUN, Canseco  DC, Xiao  F, Nakada  Y, Li  S, Lam  NT, Muralidhar  SA, Savla  JJ, Hill  JA, Le  V, Zidan  KA, El-Feky  HW, Wang  Z, Ahmed  MS, Hubbi  ME, Menendez-Montes  I, Moon  J, Ali  SR, Le  V, Villalobos  E, Mohamed  MS, Elhelaly  WM, Thet  S, Anene-Nzelu  CG, Tan  WLW, Foo  RS, Meng  X, Kanchwala  M, Xing  C, Roy  J, Cyert  MS, Rothermel  BA, Sadek  HA. A calcineurin-Hoxb13 axis regulates growth mode of mammalian cardiomyocytes. Nature  2020;582:271–276. PubMed PMC

Galang  G, Mandla  R, Ruan  H, Jung  C, Sinha  T, Stone  NR, Wu  RS, Mannion  BJ, Allu  PKR, Chang  K, Rammohan  A, Shi  MB, Pennacchio  LA, Black  BL, Vedantham  V. ATAC-seq reveals an Isl1 enhancer that regulates sinoatrial node development and function. Circ Res  2020;127:1502–1518. PubMed PMC

Martin  M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J  2011;17:10–12.

Langmead  B, Salzberg  SL. Fast gapped-read alignment with Bowtie 2. Nat Methods  2012;9:357–359. PubMed PMC

Zhang  Y, Liu  T, Meyer  CA, Eeckhoute  J, Johnson  DS, Bernstein  BE, Nusbaum  C, Myers  RM, Brown  M, Li  W, Liu  XS. Model-based analysis of ChIP-Seq (MACS). Genome Biol  2008;9:R137. PubMed PMC

Lawrence  M, Huber  W, Pagès  H, Aboyoun  P, Carlson  M, Gentleman  R, Morgan  MT, Carey  VJ. Software for computing and annotating genomic ranges. PLoS Comput Biol  2013;9:e1003118. PubMed PMC

Li  H, Handsaker  B, Wysoker  A, Fennell  T, Ruan  J, Homer  N, Marth  G, Abecasis  G, Durbin  R; 1000 Genome Project Data Processing Subgroup . The sequence alignment/map format and SAMtools. Bioinformatics  2009;25:2078–2079. PubMed PMC

Asatryan  B, Medeiros-Domingo  A. Molecular and genetic insights into progressive cardiac conduction disease. Europace  2019;21:1145–1158. PubMed

Goodyer  WR, Beyersdorf  BM, Paik  DT, Tian  L, Li  G, Buikema  JW, Chirikian  O, Choi  S, Venkatraman  S, Adams  EL, Tessier-Lavigne  M, Wu  JC, Wu  SM. Transcriptomic profiling of the developing cardiac conduction system at single-cell resolution. Circ Res  2019;125:379–397. PubMed PMC

van Eif  VWW, Protze  SI, Bosada  FM, Yuan  X, Sinha  T, van Duijvenboden  K, Ernault  AC, Mohan  RA, Wakker  V, de Gier-de Vries  C, Hooijkaas  IB, Wilson  MD, Verkerk  AO, Bakkers  J, Boukens  BJ, Black  BL, Scott  IC, Christoffels  VM. Genome-wide analysis identifies an essential human TBX3 pacemaker enhancer. Circ Res  2020;127:1522–1535. PubMed PMC

Li  T, Li  W, Lu  J, Liu  H, Li  Y, Zhao  Y. SH2D4A regulates cell proliferation via the ERalpha/PLC-gamma/PKC pathway. BMB Rep  2009;42:516–522. PubMed

Ploeger  C, Huth  T, Sugiyanto  RN, Pusch  S, Goeppert  B, Singer  S, Tabti  R, Hausser  I, Schirmacher  P, Desaubry  L, Prohibitin  RS. STAT3 and SH2D4A physically and functionally interact in tumor cell mitochondria. Cell Death Dis  2020;11:1023. PubMed PMC

Yuki  R, Ikeda  Y, Yasutake  R, Saito  Y, Nakayama  Y. SH2D4A promotes centrosome maturation to support spindle microtubule formation and mitotic progression. Sci Rep  2023;13:2067. PubMed PMC

van Eif  VWW, Stefanovic  S, Mohan  RA, Christoffels  VM. Gradual differentiation and confinement of the cardiac conduction system as indicated by marker gene expression. Biochim Biophys Acta Mol Cell Res  2020;1867:118509. PubMed

Rentschler  S, Vaidya  DM, Tamaddon  H, Degenhardt  K, Sassoon  D, Morley  GE, Jalife  J, Fishman  GI. Visualization and functional characterization of the developing murine cardiac conduction system. Development  2001;128:1785–1792. PubMed PMC

Gurjarpadhye  A, Hewett  KW, Justus  C, Wen  X, Stadt  H, Kirby  ML, Sedmera  D, Gourdie  RG. Cardiac neural crest ablation inhibits compaction and electrical function of conduction system bundles. Am J Physiol Heart Circ Physiol  2007;292:H1291–H1300. PubMed

Winkelmann  J, Schormair  B, Lichtner  P, Ripke  S, Xiong  L, Jalilzadeh  S, Fulda  S, Putz  B, Eckstein  G, Hauk  S, Trenkwalder  C, Zimprich  A, Stiasny-Kolster  K, Oertel  W, Bachmann  CG, Paulus  W, Peglau  I, Eisensehr  I, Montplaisir  J, Turecki  G, Rouleau  G, Gieger  C, Illig  T, Wichmann  HE, Holsboer  F, Muller-Myhsok  B, Meitinger  T. Genome-wide association study of restless legs syndrome identifies common variants in three genomic regions. Nat Genet  2007;39:1000–1006. PubMed

VanOudenhove  J, Yankee  TN, Wilderman  A, Cotney  J. Epigenomic and transcriptomic dynamics during human heart organogenesis. Circ Res  2020;127:e184–e209. PubMed PMC

O'Meara  CC, Wamstad  JA, Gladstone  RA, Fomovsky  GM, Butty  VL, Shrikumar  A, Gannon  JB, Boyer  LA, Lee  RT. Transcriptional reversion of cardiac myocyte fate during mammalian cardiac regeneration. Circ Res  2015;116:804–815. PubMed PMC

Burnicka-Turek  O, Broman  MT, Steimle  JD, Boukens  BJ, Petrenko  NB, Ikegami  K, Nadadur  RD, Qiao  Y, Arnolds  DE, Yang  XH, Patel  VV, Nobrega  MA, Efimov  IR, Moskowitz  IP. Transcriptional patterning of the ventricular cardiac conduction system. Circ Res  2020;127:e94–e106. PubMed PMC

Li  H, Li  D, Wang  Y, Huang  Z, Xu  J, Yang  T, Wang  L, Tang  Q, Cai  CL, Huang  H, Zhang  Y, Chen  Y. Nkx2-5 defines a subpopulation of pacemaker cells and is essential for the physiological function of the sinoatrial node in mice. Development  2019;146:dev178145. PubMed PMC

Dupays  L, Shang  C, Wilson  R, Kotecha  S, Wood  S, Towers  N, Mohun  T. Sequential binding of MEIS1 and NKX2-5 on the Popdc2 gene: a mechanism for spatiotemporal regulation of enhancers during cardiogenesis. Cell Rep  2015;13:183–195. PubMed PMC

Yang  Y, Mlodzik  M. Wnt-frizzled/planar cell polarity signaling: cellular orientation by facing the wind (Wnt). Annu Rev Cell Dev Biol  2015;31:623–646. PubMed PMC

Schleiffarth  JR, Person  AD, Martinsen  BJ, Sukovich  DJ, Neumann  A, Baker  CV, Lohr  JL, Cornfield  DN, Ekker  SC, Petryk  A. Wnt5a is required for cardiac outflow tract septation in mice. Pediatr Res  2007;61:386–391. PubMed

Zhou  W, Lin  L, Majumdar  A, Li  X, Zhang  X, Liu  W, Etheridge  L, Shi  Y, Martin  J, Van de Ven  W, Kaartinen  V, Wynshaw-Boris  A, McMahon  AP, Rosenfeld  MG, Evans  SM. Modulation of morphogenesis by noncanonical Wnt signaling requires ATF/CREB family-mediated transcriptional activation of TGFbeta2. Nat Genet  2007;39:1225–1234. PubMed PMC

Yu  H, Smallwood  PM, Wang  Y, Vidaltamayo  R, Reed  R, Nathans  J. Frizzled 1 and frizzled 2 genes function in palate, ventricular septum and neural tube closure: general implications for tissue fusion processes. Development  2010;137:3707–3717. PubMed PMC

Yu  H, Ye  X, Guo  N, Nathans  J. Frizzled 2 and frizzled 7 function redundantly in convergent extension and closure of the ventricular septum and palate: evidence for a network of interacting genes. Development  2012;139:4383–4394. PubMed PMC