Abstract-Loop-tail (Lp) mice develop double outlet right ventricle and aortic arch defects, and the defects are caused by mutations in the Vangl2 gene.

We provide, for the first time, objective evidence of the mechanisms of closure of an AP foramen that exists distally between the lumens of the developing intrapericardial arterial trunks. Our findings provide insights into the formation of AP windows and the variants of common arterial trunk.

AimsAnomalies 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 resultsBy 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.ConclusionNCCs 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.

Planar cell polarity (PCP) is the mechanism by which cells orient themselves in the plane of an epithelium or during directed cell migration, and is regulated by a highly conserved signalling pathway. Mutations in the PCP gene Vangl2, as well as in other key components of the pathway, cause a spectrum of cardiac outflow tract defects. However, it is unclear why cells within the mesodermal heart tissue require PCP signalling. Using a new conditionally floxed allele we show that Vangl2 is required solely within the second heart field (SHF) to direct normal outflow tract lengthening, a process that is required for septation and normal alignment of the aorta and pulmonary trunk with the ventricular chambers. Analysis of a range of markers of polarised epithelial tissues showed that in the normal heart, undifferentiated SHF cells move from the dorsal pericardial wall into the distal outflow tract where they acquire an epithelial phenotype, before moving proximally where they differentiate into cardiomyocytes. Thus there is a transition zone in the distal outflow tract where SHF cells become more polarised, turn off progenitor markers and start to differentiate to cardiomyocytes. Membrane-bound Vangl2 marks the proximal extent of this transition zone and in the absence of Vangl2, the SHF-derived cells are abnormally polarised and disorganised. The consequent thickening, rather than lengthening, of the outflow wall leads to a shortened outflow tract. Premature down regulation of the SHF-progenitor marker Isl1 in the mutants, and accompanied premature differentiation to cardiomyocytes, suggests that the organisation of the cells within the transition zone is important for maintaining the undifferentiated phenotype. Thus, Vangl2-regulated polarisation and subsequent acquisition of an epithelial phenotype is essential to lengthen the tubular outflow vessel, a process that is essential for on-going cardiac morphogenesis.