Vangl2-Regulated Polarisation of Second Heart Field-Derived Cells Is Required for Outflow Tract Lengthening during Cardiac Development

Abstract

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.

Spatial regulation of cell cohesion by Wnt5a during second heart field progenitor deployment

“…In the mouse, we and others have carried out tissue-specific gene ablation to demonstrate that PCP genes Dvl1/2 and Vangl2 are required specifically in the SHF lineage for OFT elongation and morphogenesis (Ramsbottom et al, 2014; Sinha et al, 2012). Furthermore, loss of either Wnt5a or Wnt11 also leads to severe conotruncal defects in mice (Schleiffarth et al, 2007; Zhou et al, 2007).…”

The core planar cell polarity gene,Vangl2, directs adult corneal epithelial cell alignment and migration

“…CAGG-CreERTM Tg/− mice ( Tg ( CAG-cre/Esr1 )) which constitutively express a transgene encoding tamoxifen-inducible Cre recombinase were bred to Vangl2 flox/flox mice [24] and the CAGG-CreERTM Tg/− Vangl2 flox/+ progeny backcrossed to Vangl2 flox/flox to yield CAGG-CreERTM Tg/− Vangl2 flox/flox conditional knockouts and CAGG-CreERTM Tg/− Vangl2 flox/+ controls. When monolayers of corneal epithelial cells were cultured from these mice, it was found that addition of 10 nM 4-OH tamoxifen to the medium caused nuclear relocalization of the CreERTM within 24 h, knocking out Vangl2 and efficiently removed Vangl2 protein from the cells within 48 h (electronic supplementary material, figure S6).…”

“…Hemizygous Le-Cre Tg/− mice ( Tg(Pax6-cre,GFP)1Pgr ), driving expression of Cre recombinase in the lens and corneal epithelia [50] and CAGG-CreERTM Tg/− mice ( Tg ( CAG-cre/Esr1 )) driving constitutive expression of tamoxifen-inducible Cre  [51,52] were bred with Vangl2 flox/flox mice [24] and H253 (‘XLacZ’) reporter mice carrying X-linked nLacZ under the control of a housekeeping promoter [30]. Vangl2 Lp/+ mice were as described in [36].…”

Glypican4 promotes cardiac specification and differentiation by attenuating canonical Wnt and Bmp signaling

“…Canonical and non-canonical Wnt signaling pathways can be simultaneously controlled by Syndecan-4, which inhibits canonical Wnt signaling through interaction with LRP6 and R-spondin 3 and activates the non-canonical Wnt pathway (Ohkawara et al, 2011;Astudillo et al, 2014). The importance of non-canonical Wnt signaling for cardiac development has been shown in numerous studies (Phillips et al, 2005;Schleiffarth et al, 2007;Zhou et al, 2007;Ramsbottom et al, 2014). Whether Glypican4 also affects non-canonical Wnt signaling in the anterior LPM is not addressed here.…”