AIMS: Bicuspid aortic valve (BAV), where two valve leaflets are found instead of the usual three, affects 1-2% of the general population and is associated with significant morbidity and mortality. Despite its frequency, the majority of cases remain unexplained. This is, at least in part, because there are two types of valve leaflet primordia: endocardial cushions and intercalated valve swellings (ICVS). Moreover, multiple progenitors make distinct contributions to the formation of these primordia. Genomic studies in mouse and human have suggested a correlation between BAV and malfunctional primary cilia. However, the precise requirement for cilia during early embryonic valvulogenesis remains unknown. METHODS AND RESULTS: Here, we disrupted primary cilia by deleting the ciliary gene Ift88 in the main progenitor cells forming the aortic valve using specific Cre drivers: Wnt1-Cre for neural crest cells, Isl1-Cre for second heart field (SHF) cells, Tie2-Cre for endocardial-derived cells, and Tnnt2-Cre for direct-differentiating SHF in the ICVS. Loss of Ift88, and thus primary cilia, from neural crest cells and endocardium did not impact aortic valve formation. However, primary cilia were essential in SHF cells for aortic valve leaflet formation, with over half of Ift88f/f;Isl1-Cre mutants presenting with BAV. As the valve leaflets were forming, 50% of the Ift88f/f;Isl1-Cre mutants had two small leaflets in the position of the usual posterior leaflet, meaning that at this stage, the aortic valve was quadricuspid, which then remodelled to BAV by E15.5. Mechanistic studies demonstrated premature differentiation of SHF cells as the ICVS formed, leading to the formation of a broadened ICVS that formed two posterior leaflet precursors. This abnormality in the formation of the ICVS was associated with disruption of Notch-Jag1 signalling pathway, with Jag1f/f;Isl1-Cre mutants presenting with a similar phenotype. CONCLUSION: These data show that primary cilia, via the Notch-Jag1 signalling pathway, regulate differentiation of SHF cells in the aortic valve primordia. Additionally, we identify a mechanistic link between the developmental basis of quadricuspid and bicuspid arterial valve leaflets.

Bioscience Institute Newcastle University Centre for Life Central Parkway Newcastle upon Tyne NE1 3BZ UK

Institute of Anatomy 1st Faculty of Medicine Charles University U Nemocnice 3 Prague 2 12 800 Czech Republic

Comment In

Cardiovasc Res. 2025 Sep 29;121(11):1645-1646. doi: 10.1093/cvr/cvaf150. PubMed

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Pierpont  ME, Brueckner  M, Chung  WK, Garg  V, Lacro  RV, McGuire  AL, Mital  S, Priest  JR, Pu  WT, Roberts  A, Ware  SM, Gelb  BD, Russell  MW. Genetic basis for congenital heart disease: revisited: a scientific statement from the American Heart Association. Circulation  2018;138:e653–e711. PubMed PMC

Shinohara  K, Hamada  H. Cilia in left–right symmetry breaking. Cold Spring Harb Perspect Biol  2017;9:a028282. PubMed PMC

Versacci  P, Pugnaloni  F, Digilio  MC, Putotto  C, Unolt  M, Calcagni  G, Baban  A, Marino  B. Some isolated cardiac malformations can be related to laterality defects. J Cardiovasc Dev Dis  2018;5:24. PubMed PMC

Mill  P, Christensen  ST, Pedersen  LB. Primary cilia as dynamic and diverse signalling hubs in development and disease. Nat Rev Genet  2023;24:421–441. PubMed PMC

Hilgendorf  KI, Myers  BR, Reiter  JF. Emerging mechanistic understanding of cilia function in cellular signalling. Nat Rev Mol Cell Biol  2024;25:555–573. PubMed PMC

Li  Y, Klena  NT, Gabriel  GC, Liu  X, Kim  AJ, Lemke  K, Chen  Y, Chatterjee  B, Devine  W, Damerla  RR, Chang  C, Yagi  H, San Agustin  JT, Thahir  M, Anderton  S, Lawhead  C, Vescovi  A, Pratt  H, Morgan  J, Haynes  L, Smith  CL, Eppig  JT, Reinholdt  L, Francis  R, Leatherbury  L, Ganapathiraju  MK, Tobita  K, Pazour  GJ, Lo  CW. Global genetic analysis in mice unveils central role for cilia in congenital heart disease. Nature  2015;521:520–524. PubMed PMC

Karp  N, Grosse-Wortmann  L, Bowdin  S. Severe aortic stenosis, bicuspid aortic valve and atrial septal defect in a child with Joubert Syndrome and Related Disorders (JSRD)—a case report and review of congenital heart defects reported in the human ciliopathies. Eur J Med Genet  2012;55:605–610. PubMed

Watkins  WS, Hernandez  EJ, Wesolowski  S, Bisgrove  BW, Sunderland  RT, Lin  E, Lemmon  G, Demarest  BL, Miller  TA, Bernstein  D, Brueckner  M, Chung  WK, Gelb  BD, Goldmuntz  E, Newburger  JW, Seidman  CE, Shen  Y, Yost  HJ, Yandell  M, Tristani-Firouzi  M. PubMed PMC

Braun  DA, Hildebrandt  F. Ciliopathies. Cold Spring Harb Perspect Biol  2017;9:a028191. PubMed PMC

Fulmer  D, Toomer  K, Guo  L, Moore  K, Glover  J, Moore  R, Stairley  R, Lobo  G, Zuo  X, Dang  Y, Su  Y, Fogelgren  B, Gerard  P, Chung  D, Heydarpour  M, Mukherjee  R, Body  SC, Norris  RA, Lipschutz  JH. Defects in the exocyst-cilia machinery cause bicuspid aortic valve disease and aortic stenosis. Circulation  2019;140:1331–1341. PubMed PMC

Toomer  KA, Yu  M, Fulmer  D, Guo  L, Moore  KS, Moore  R, Drayton  K'D, Glover  J, Peterson  N, Ramos-Ortiz  S, Drohan  A, Catching  BJ, Stairley  R, Wessels  A, Lipschutz  JH, Delling  FN, Jeunemaitre  X, Dina  C, Collins  RL, Brand  H, Talkowski  ME, Del Monte  F, Mukherjee  R, Awgulewitsch  A, Body  S, Hardiman  G, Hazard  ES, da Silveira  WA, Wang  B, Leyne  M, Durst  R, Markwald  RR, Le Scouarnec  S, Hagege  A, Le Tourneau  T, Kohl  P, Rog-Zielinska  EA, Ellinor  PT, Levine  RA, Milan  DJ, Schott  J-J, Bouatia-Naji  N, Slaugenhaupt  SA, Norris  RA. Primary cilia defects causing mitral valve prolapse. Sci Transl Med  2019;11:eaax0290. PubMed PMC

Burns  TA, Deepe  RN, Bullard  J, Phelps  AL, Toomer  KA, Hiriart  E, Norris  RA, Haycraft  CJ, Wessels  A. A novel mouse model for cilia-associated cardiovascular anomalies with a high penetrance of total anomalous pulmonary venous return. Anat Rec  2019;302:136–145. PubMed PMC

Toomer  KA, Fulmer  D, Guo  L, Drohan  A, Peterson  N, Swanson  P, Brooks  B, Mukherjee  R, Body  S, Lipschutz  JH, Wessels  A, Norris  RA. A role for primary cilia in aortic valve development and disease. Dev Dyn  2017;246:625–634. PubMed PMC

Pazour  GJ, Baker  SA, Deane  JA, Cole  DG, Dickert  BL, Rosenbaum  JL, Witman  GB, Besharse  JC. The intraflagellar transport protein, IFT88, is essential for vertebrate photoreceptor assembly and maintenance. J Cell Biol  2002;157:103. PubMed PMC

Clement  CA, Kristensen  SG, Møllgård  K, Pazour  GJ, Yoder  BK, Larsen  LA, Christensen  ST. The primary cilium coordinates early cardiogenesis and hedgehog signaling in cardiomyocyte differentiation. J Cell Sci  2009;122:3070–3082. PubMed PMC

Lehman  JM, Michaud  EJ, Schoeb  TR, Aydin-Son  Y, Miller  M, Yoder  BK. The Oak Ridge Polycystic Kidney mouse: modeling ciliopathies of mice and men. Dev Dyn  2008;237:1960–1971. PubMed PMC

Willaredt  MA, Gorgas  K, Gardner  HAR, Tucker  KL. Multiple essential roles for primary cilia in heart development. Cilia  2012;1:23. PubMed PMC

Eley  L, Alqahtani  AMS, Macgrogan  D, Richardson  RV, Murphy  L, Salguero-Jimenez  A, Sintes Rodriguez San Pedro  M, Tiurma  S, McCutcheon  L, Gilmore  A, de La Pompa  JL, Chaudhry  B, Henderson  DJ. A novel source of arterial valve cells linked to bicuspid aortic valve without raphe in mice. eLife  2018:7:e34110. PubMed PMC

Mifflin  JJ, Dupuis  LE, Alcala  NE, Russell  LG, Kern  CB. Intercalated cushion cells within the cardiac outflow tract are derived from the myocardial troponin T Type 2 (Tnnt2) Cre lineage. Dev Dyn  2018;247:1005–1017. PubMed PMC

Haycraft  CJ, Zhang  Q, Song  B, Jackson  WS, Detloff  PJ, Serra  R, Yoder  BK. Intraflagellar transport is essential for endochondral bone formation. Development  2007;134:307–316. PubMed

Kiernan  AE, Xu  J, Gridley  T. The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear. PLoS Genet  2006;2:e4. PubMed PMC

Danielian  PS, Muccino  D, Rowitch  DH, Michael  SK, McMahon  AP. Modification of gene activity in mouse embryos PubMed

Kisanuki  YY, Hammer  RE, Miyazaki  J, Williams  SC, Richardson  JA, Yanagisawa  M. Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Dev Biol  2001;230:230–242. PubMed

Jiao  K, Kulessa  H, Tompkins  K, Zhou  Y, Batts  L, Baldwin  HS, Hogan  BLM. An essential role of Bmp4 in the atrioventricular septation of the mouse heart. Genes Dev  2003;17:2362–2367. PubMed PMC

Verzi  MP, McCulley  DJ, De Val  S, Dodou  E, Black  BL. The right ventricle, outflow tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field. Dev Biol  2005;287:134–145. PubMed

Yang  L, Cai  CL, Lin  L, Qyang  Y, Chung  C, Monteiro  RM, Mummery  CL, Fishman  GI, Cogen  A, Evans  S. Isl1Cre reveals a common Bmp pathway in heart and limb development. Development  2006;133:1575. PubMed PMC

Muzumdar  MD, Tasic  B, Miyamichi  K, Li  N, Luo  L. A global double-fluorescent cre reporter mouse. Genesis (United States)  2007;45:593–605. PubMed

Srinivas  S, Watanabe  T, Lin  CS, William  CM, Tanabe  Y, Jessell  TM, Costantini  F. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol  2001;1:1–8. PubMed PMC

Phillips  HM, Mahendran  P, Singh  E, Anderson  RH, Chaudhry  B, Henderson  DJ. Neural crest cells are required for correct positioning of the developing outflow cushions and pattern the arterial valve leaflets. Cardiovasc Res  2013;99:452–460. PubMed PMC

Garza-Lopez  E, Vue  Z, Katti  P, Neikirk  K, Biete  M, Lam  J, Beasley  HK, Marshall  AG, Rodman  TA, Christensen  TA, Salisbury  JL, Vang  L, Mungai  M, AshShareef  S, Murray  SA, Shao  J, Streeter  J, Glancy  B, Pereira  RO, Abel  ED, Hinton  A  Jr. Protocols for generating surfaces and measuring 3d organelle morphology using amira. Cells  2022;11:65. PubMed PMC

Van Der Heiden  K, Groenendijk  BCW, Hierck  BP, Hogers  B, Koerten  HK, Mommaas  AM, Gittenberger-de Groot  AC, Poelmann  RE. Monocilia on chicken embryonic endocardium in low shear stress areas. Dev Dyn  2006;235:19–28. PubMed

Levay  AK, Peacock  JD, Lu  Y, Koch  M, Hinton  RB, Kadler  KE, Lincoln  J. Scleraxis is required for cell lineage differentiation and extracellular matrix remodeling during murine heart valve formation in vivo. Circ Res  2008;103:948–956. PubMed PMC

Tian  H, Feng  J, Li  J, Ho  T-V, Yuan  Y, Liu  Y, Brindopke  F, Figueiredo  JC, Magee  W, Sanchez-Lara  PA, Chai  Y. Intraflagellar transport 88 (IFT88) is crucial for craniofacial development in mice and is a candidate gene for human cleft lip and palate. Hum Mol Genet  2017;26:860–872. PubMed PMC

Black  BL. Transcriptional pathways in second heart field development. Semin Cell Dev Biol  2007;18:67–76. PubMed PMC

Engleka  KA, Manderfield  LJ, Brust  RD, Li  L, Cohen  A, Dymecki  SM, Epstein  JA. Islet1 derivatives in the heart are of both neural crest and second heart field origin. Circ Res  2012;110:922–926. PubMed PMC

Wheway  G, Nazlamova  L, Hancock  JT. Signaling through the primary cilium. Front Cell Dev Biol  2018;6:8. PubMed PMC

Niehrs  C, Da Silva  F, Seidl  C. Cilia as Wnt signaling organelles. Trends Cell Biol  2025;35:24–32. PubMed

High  FA, Jain  R, Stoller  JZ, Antonucci  NB, Lu  MM, Loomes  KM, Kaestner  KH, Pear  WS, Epstein  JA. Murine Jagged1/Notch signaling in the second heart field orchestrates Fgf8 expression and tissue-tissue interactions during outflow tract development. J Clin Invest  2009;119:1986–1996. PubMed PMC

Luna-Zurita  L, Flores-Garza  BG, Grivas  D, De La Pompa  JL. A cooperative response to endocardial NOTCH signaling stimulation regulates transcriptional activity during cardiac valve development and disease running title: NOTCH in cardiac valve transcriptional activity. Circ Res  2023;133:1022–1039. PubMed PMC

MacGrogan  D, Luxán  G, de la Pompa  JL. Genetic and functional genomics approaches targeting the Notch pathway in cardiac development and congenital heart disease. Brief Funct Genomics  2014;13:15–27. PubMed

Henderson  DJ, Eley  L, Chaudhry  B. New concepts in the development and malformation of the arterial valves. J Cardiovasc Dev Dis  2020;7:38. PubMed PMC

Soto-Navarrete  MT, López-Unzu  MÁ, Durán  AC, Fernández  B. Embryonic development of bicuspid aortic valves. Prog Cardiovasc Dis  2020;63:407–418. PubMed

Hurwitz  LE, Roberts  WC. Quadricuspid semilunar valve. Am J Cardiol  1973;31:623–626. PubMed

Fernández  B, Fernández  MC, Durán  AC, López  D, Martire  A, Sans-Coma  V. Anatomy and formation of congenital bicuspid and quadricuspid pulmonary valves in Syrian hamsters. Anat Rec  1998;250:70–79. PubMed

Nomura-Kitabayashi  A, Phoon  CKL, Kishigami  S, Rosenthal  J, Yamauchi  Y, Abe  K, Yamamura  K-I, Samtani  R, Lo  CW, Mishina  Y. Outflow tract cushions perform a critical valve-like function in the early embryonic heart requiring BMPRIA-mediated signaling in cardiac neural crest. Am J Physiol Heart Circ Physiol  2009;297:1617–1628. PubMed PMC

Nakamura  E, Makita  Y, Okamoto  T, Nagaya  K, Hayashi  T, Sugimoto  M, Manabe  H, Taketazu  G, Kajino  H, Fujieda  K. 5.78 Mb terminal deletion of chromosome 15q in a girl, evaluation of NR2F2 as candidate gene for congenital heart defects. Eur J Med Genet  2011;54:354–356. PubMed

Nakamura  Y, Taniguchi  I, Saiki  M, Morimoto  K, Yamaga  T. Quadricuspid aortic valve associated with aortic stenosis and regurgitation. Jpn J Thorac Cardiovasc Surg  2001;49:714–716. PubMed

Yuan  SM. Quadricuspid aortic valve: a comprehensive review. Braz J Cardiovasc Surg  2016;31:454–460. PubMed PMC

Ingham  PW. Hedgehog signaling. Curr Top Dev Biol  2022;149:1–58. PubMed

Smoak  IW, Byrd  NA, Abu-Issa  R, Goddeeris  MM, Anderson  R, Morris  J, Yamamura  K, Klingensmith  J, Meyers  EN. Sonic hedgehog is required for cardiac outflow tract and neural crest cell development. Dev Biol  2005;283:357–372. PubMed

Alfieri  CM, Cheek  J, Chakraborty  S, Yutzey  KE. Wnt signaling in heart valve development and osteogenic gene induction. Dev Biol  2010;338:127–135. PubMed PMC

Pala  R, Alomari  N, Nauli  SM. Primary cilium-dependent signaling mechanisms. Int J Mol Sci  2017;18:2272. PubMed PMC

Yanardag  S, Pugacheva  EN. Primary cilium is involved in stem cell differentiation and renewal through the regulation of multiple signaling pathways. Cells  2021;10:1428. PubMed PMC

Ezratty  EJ, Pasolli  HA, Fuchs  E. A presenilin-2–ARF4 trafficking axis modulates Notch signaling during epidermal differentiation. J Cell Biol  2016;214:89–101. PubMed PMC

Kong  JH, Yang  L, Dessaud  E, Chuang  K, Moore  DM, Rohatgi  R, Briscoe  J, Novitch  BG. Notch activity modulates the responsiveness of neural progenitors to sonic hedgehog signaling. Dev Cell  2015;33:373–387. PubMed PMC

MacGrogan  D, D’Amato  G, Travisano  S, Martinez-Poveda  B, Luxán  G, del Monte-Nieto  G, Papoutsi  T, Sbroggio  M, Bou  V, Gomez-del Arco  P, Gómez  MJ, Zhou  B, Redondo  JM, Jiménez-Borreguero  LJ, de la Pompa  JL. Sequential ligand-dependent notch signaling activation regulates valve primordium formation and morphogenesis. Circ Res  2016;118:1480–1497. PubMed

Rochais  F, Kelly  RG, Rammah  M, Théveniau-Ruissy  M, Sturny  R. PPARγ and NOTCH regulate regional identity in the murine cardiac outflow tract. Circ Res  2022;131:842–858. PubMed

Seya  D, Ihara  D, Shirai  M, Kawamura  T, Watanabe  Y, Nakagawa  O. A role of Hey2 transcription factor for right ventricle development through regulation of Tbx2-Mycn pathway during cardiac morphogenesis. Dev Growth Differ  2021;63:82–92. PubMed

Van Dam  TJP, Kennedy  J, van der Lee  R, de Vrieze  E, Wunderlich  KA, Rix  S, Dougherty  GW, Lambacher  NJ, Li  C, Jensen  VL, Leroux  MR, Hjeij  R, Horn  N, Texier  Y, Wissinger  Y, van Reeuwijk  J, Wheway  G, Knapp  B, Scheel  JF, Franco  B, Mans  DA, van Wijk  E, Képès  F, Slaats  GG, Toedt  G, Kremer  H, Omran  H, Szymanska  K, Koutroumpas  K, Ueffing  M, Nguyen  T-MT, Letteboer  SJF, Oud  MM, van Beersum  SEC, Schmidts  M, Beales  PL, Lu  Q, Giles  RH, Szklarczyk  R, Russell  RB, Gibson  TJ, Johnson  CA, Blacque  OE, Wolfrum  U, Boldt  K, Roepman  R, Hernandez-Hernandez  V, Huynen  MA. CiliaCarta: an integrated and validated compendium of ciliary genes. PLoS One  2019;14:e0216705. PubMed PMC

Calvo  SE, Clauser  KR, Mootha  VK. MitoCarta2.0: an updated inventory of mammalian mitochondrial proteins. Nucleic Acids Res  2016;44:D1251–D1257. PubMed PMC