Vangl2 as a key regulator of cell behaviour within the developing cardiac outflow tract :elaborating specific roles in second heart field and neural crest cells

Abstract

ii Abstract Vangl2 is a key member of the multi-protein planar cell polarity (PCP) pathway. Previous studies using the loop-tail (Lp) mouse, which carries a mutation in the Vangl2 gene, have shown that PCP is required for normal development of the cardiac outflow tract. The main cell types involved in development of the outflow tract are neural crest cells (NCC) and cells derived from the second heart field (SHF). The PCP pathway plays important roles in polarisation of cells within tissues and in directional cell movements. I hypothesised that PCP signalling is required for efficient movement of progenitor cells into the developing heart and that an abnormality in these processes is sufficient to cause common outflow tract defects. Whilst loss of Vangl2 in NCC has no affect on outflow tract development, deletion of Vangl2 from SHF cells (using Vangl2flox crossed with Isl1-Cre mice) recapitulates the shortened outflow tract and malalignment defects seen in Lp mice. The cellular distribution of Vangl2 changes as SHF cells pass from a progenitor state, still expressing Isl1 protein, to differentiated myocardium. When Vangl2 is lost from the cells derived from the SHF, the cells within the distal walls of the outflow tract show altered localisation of polarised molecules such as β-catenin, fibronectin and laminin, as well as PCP proteins including Dvl2 and Celsr1, suggesting disrupted cellular polarity. The expression of PKCζ and E-cadherin is also altered in the distal outflow tract walls of Vangl2flox/flox;Isl1-Cre embryos, supporting the idea that Vangl2 may regulate the polarity of this tissue. Together, these studies suggest that Vangl2 plays a role in imparting polarity on SHF cells as they contribute to the outflow and that this is important for its lengthening. Confirmation of the importance of the PCP pathway in regulating the polarity of the cells in the distal outflow tract, and its importance for outflow tract development, was obtained by examining upstream components (Wnt5a and Ror2) and downstream targets (Rac1 and ROCK) of the pathway, showing outflow defects and a similar expression pattern of polarised molecules.