Abstract
AimsThe mesenchymal cushions lining the early embryonic heart undergo complex remodelling to form the membranous ventricular septum as well as the atrioventricular and semilunar valves in later life. Disruption of this process underlies the most common congenital heart defects. Here, we identified a novel role for Slit-Robo signalling in the development of the murine membranous ventricular septum and cardiac valves.Methods and resultsExpression of Robo1 and Robo2 receptors and their ligands, Slit2 and Slit3, was present in or adjacent to all cardiac cushions/valves. Loss of Robo1 or both Robo1 and Robo2 resulted in membranous ventricular septum defects at birth, a defect also found in Slit3, but not in Slit2 mutants. Additionally, Robo1;Robo2 double mutants showed thickened immature semilunar and atrioventricular valves as well as highly penetrant bicuspid aortic valves. Slit2 mutants recapitulated the semilunar phenotype, whereas Slit3 mutants displayed thickened atrioventricular valves. Bicuspid aortic cushions were already observed at E12.5 in the Robo1;Robo2 double mutants. Expression of Notch- and downstream Hey and Hes genes was down-regulated in Robo1 mutants, suggesting that reduced Notch signalling in mice lacking Robo might underlie the defects. Luciferase assays confirmed regulation of Notch signalling by Robo.ConclusionCardiac defects in mutants for Robo or Slit range from membranous ventricular septum defects to bicuspid aortic valves. These ligands and receptors have unique functions during development of specific cardiac cushion derivatives, and the Slit-Robo signalling pathway likely enforces its role by regulating Notch signalling, making these mutants a valuable new model to study cardiac valve formation.
Highlights
Cardiac septation and valve formation are complex processes requiring precise gene regulation, and even the smallest disruption can lead to congenital defects such as ventricular septum defects or malformed and malfunctioning valves
We have identified a broad spectrum of cardiac defects in mutants for Robo and Slit, ranging from membranous ventricular septum defects to bicuspid aortic valves
The presence of these genes in and surrounding the cardiac cushions prompted us to study their expression in these regions in more detail
Summary
Cardiac septation and valve formation are complex processes requiring precise gene regulation, and even the smallest disruption can lead to congenital defects such as ventricular septum defects or malformed and malfunctioning valves. In addition to the epithelial-to-mesenchymal transformation, parts of the cardiac cushions, including the future membranous ventricular septum, receive contributions from neural crest cells invading the arterial pole of the heart, adding to the complexity of the region.[1] Correct formation and alignment of the cushions requires accurate signalling from within as well as from neighbouring tissues such as the myocardium, endocardium, second heart field, and neural crest. Major signalling pathways, such as Notch, Bmp, and Wnt, are crucial for cushion development and, in turn, regulate and are regulated by a plethora of transcription factors such as the Tbox and Sox families.1,2 – 5
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