Disrupted Slit-Robo signalling results in membranous ventricular septum defects and bicuspid aortic valves

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.

Slit-Robo signaling

“…Way beyond its most well-studied function in axon guidance in the developing nervous system, it has become obvious that Slit-Robo signaling additionally plays a role in cell proliferation (Borrell et al, 2012), organogenesis, and reproductive tract and mammary (Mommersteeg et al, 2013(Mommersteeg et al, , 2015 development. Without pretense for completeness, this following section provides an overview of how Slit-Robo signaling functions in a variety of developmental processes.…”

“…Phenotypic analyses of knockout mice show that Robo1/2 and Slit2/3 contribute differentially to heart development: the ventricular septum is absent in Robo1;2 and Slit3 mutants, whereas septum defects were less severe in Slit2 mutants (Mommersteeg et al, 2015). By contrast, Slit2 is more important for proper aortic valve formation (Mommersteeg et al, 2015).…”

“…Phenotypic analyses of knockout mice show that Robo1/2 and Slit2/3 contribute differentially to heart development: the ventricular septum is absent in Robo1;2 and Slit3 mutants, whereas septum defects were less severe in Slit2 mutants (Mommersteeg et al, 2015). By contrast, Slit2 is more important for proper aortic valve formation (Mommersteeg et al, 2015). Robo1 knockout mice also lack parts of the pericardium, a phenotype probably caused by the ectopic formation of a pericardial cavity (celom) at E12.5 due to increased apoptosis of cardiac neural crest cells (Mommersteeg et al, 2013).…”

Probing chromatin landscape reveals roles of endocardial TBX20 in septation

“…Taken together, these results link TBX20 binding to a distant enhancer that regulates Vcan in endocardial lineages, uncovering a mechanism by which Vcan expression is TBX20 dependent and providing a framework for how downregulation of Vcan in TBX20 endocardial mutants may contribute to congenital heart defects. had defects in valve development, including immature atrioventricular valves and bicuspid aortic valves (24). Myh10-null mice and mice that carry a point mutation in Myh10 have defects strikingly similar to those seen in Tie2-Cre Tbx20 fl/null mutants, including DORV, decreased OFT myocardialization, and defects in the fusion of atrioventricular cushions (25,26).…”

“…In Genes grouped by the axon guidance term may be of significance to the observed defects in endocardial cell migration as well as myocardialization defects. Axon guidance genes significantly (P < 0.05) downregulated in mutants included roundabout 1 (Robo1) and slit homolog 2 (Slit2), components of the Slit/roundabout signaling pathway that regulate cardiac septation and valve development (24). Myh10, encoding a nonmuscle myosin required for cell motility (25), was also downregulated in Tie2-Cre Tbx20 fl/null mutants.…”