Neural crest cells are required for correct positioning of the developing outflow cushions and pattern the arterial valve leaflets

AIMS: Anomalies of the arterial valves, principally bicuspid aortic valve (BAV), are the most common congenital anomalies. The cellular mechanisms that underlie arterial valve development are poorly understood. While it is known that the valve leaflets derive from the outflow cushions, which are populated by cells derived from the endothelium and neural crest cells (NCCs), the mechanism by which these cushions are sculpted to form the leaflets of the arterial valves remains unresolved. We set out to investigate how NCCs participate in arterial valve formation, reasoning that disrupting NCC within the developing outflow cushions would result in arterial valve anomalies, in the process elucidating the normal mechanism of arterial valve leaflet formation. METHODS AND RESULTS: By disrupting Rho kinase signalling specifically in NCC using transgenic mice and primary cultures, we show that NCC condensation within the cardiac jelly is required for correct positioning of the outflow cushions. Moreover, we show that this process is essential for normal patterning of the arterial valve leaflets with disruption leading to a spectrum of valve leaflet patterning anomalies, abnormal positioning of the orifices of the coronary arteries, and abnormalities of the arterial wall. CONCLUSION: NCCs are required at earlier stages of arterial valve development than previously recognized, playing essential roles in positioning the cushions, and patterning the valve leaflets. Abnormalities in the process of NCC condensation at early stages of outflow cushion formation may provide a common mechanism underlying BAV, and also explain the link with arterial wall anomalies and outflow malalignment defects.