<i>Lp</i> mice display a spectrum of outflow tract abnormalities.

<p><b>A,B</b>) <i>In situ</i> hybridisation on E10.5 <i>Lp/+</i> and <i>Lp/Lp</i> embryos reveals normal expression of <i>Tbx20</i> in the mutant embryo, but illustrates the abnormal heart loop (the outline of the outflow tract and ventricular chambers is indicated by the dotted lines). <b>C,D</b>) H&E sections of E14.5 <i>Lp/+</i> and <i>Lp/Lp</i> embryos show the double outlet right ventricle in the mutant embryo (the arrows indicate the communication between and the aorta and the ventricle). <b>E–H</b>) β-gal staining (blue) of wholemount stained <i>Lp/+</i> and <i>Lp/Lp</i> E10.5 embryos shows that NCC migration (labelled by <i>Wnt1-Cre</i> based lineage tracing) appears normal in the mutants. Transverse sections (G,H) show that although the OFT is reduced in length, there is normal migration of NCC into the outflow vessel (arrow). The bars in G,H indicate the characteristic shortened outflow tract seen in the mutant. <b>I–L</b>) β-gal staining of wholemount stained <i>Lp/+</i> and <i>Lp/Lp</i> E9.5 embryos shows that the SHF, labelled by <i>Isl1-Cre</i> based lineage tracing, appears normal in the mutants, however the cells appear disorganised (arrows). <b>M,N</b>) Isl1 antibody labels SHF cells in the distal outflow tract (brown staining – arrows). These cells appear disorganised in the <i>Lp/Lp</i> embryo at E9.5 (N′ arrow, compare to M′). Ao – aorta, LV - left ventricle, OFT - outflow tract, RV - right ventricle.</p

SHF-specific loss of <i>Vangl2</i> results in outflow tract defects.

<p><b>A,B,E,F</b>) Targeted deletion of Vangl2 by <i>Wnt1-Cre</i>, in NCC, does not result in neural tube (A,E) or outflow tract defects (B,F). <b>C,D,G,H</b>) In contrast, although there are no neural tube defects when <i>Vangl2</i> is deleted in the <i>Isl1-Cre</i> expressing SHF (G), the resultant embryos do have double outlet right ventricle (H – compare with D). <b>I–P</b>) No defects were seen when <i>Vangl2</i> was deleted in either <i>Nkx2.5-Cre</i> expressing cardiac progenitors or <i>Mlc2v-Cre</i> expressing differentiated cardiomyocytes. In each case the arrows show the communication between the ventricle and the aorta. All embryos are E14.5. Also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004871#pgen.1004871.s004" target="_blank">S4 Fig</a>. Ao – aorta, LV - left ventricle, RV - right ventricle, <i>Vangl2^f</i> – <i>Vangl2^flox</i>. Scale bar  = 2 mm (white), 500 µm (black).</p

Disruption of epithelial organisation in the distal outflow tract of <i>Vangl2^flox/flox; Isl1-Cre</i> embryos.

<p><b>A–H</b>) At E9.0 in control embryos, β-catenin (green; B,D) is localised to the basolateral domain of the cells in the transition zone of the distal outflow wall and laminin (red; C,D) is becoming localised to the basement membrane underlying this. In <i>Vangl2^flox/flox; Isl1-Cre</i> littermates, β-catenin (F,H) and laminin (G,H) are less abundant and the tissue appears disorganised (n = 3). <b>I–P</b>) By E9.5, immunofluorescent staining for β-catenin is localised to the basolateral region of cells in the control embryo and shows the pseudo-stratified epithelium of the transition zone (J,L). In contrast, although β-catenin expression is still abundant in the transition zone of <i>Vangl2^flox/flox; Isl1-Cre</i> embryos, the cells appear disorganised and it is difficult to determine its subcellular distribution (N - arrows). Laminin is found basally to the cells of the transition zone in control embryos (K - arrows), but is lost in some places and surrounds other cells within the transition zone of <i>Vangl2^flox/flox; Isl1-Cre</i> embryos (O – arrows, n = 3). Note that whereas the distal outflow wall is 2-3 cell layers thick in the control embryo (L), in some places it is 4-5 cell layers thick in the mutant (P). <b>Q–T</b>) γ-tubulin staining of MTOCs at E9.5 shows that these are localised to the apical side of the cells in the distal outflow wall in control embryos (Q and rose plot S). In contrast, the position of the MTOC is much more variable in <i>Vangl2^flox/flox; Isl1-Cre</i> embryos (R and rose plot T), frequently localising to the basolateral side of the cell layer (n = 5) Chi-square, p<0.001. Ap =  Apical, Ba =  Basal, Dis  =  distal, Prox  =  proximal, <i>Vangl2^f</i>  =  <i>Vangl2^flox</i>. Quantification of γ-tubulin performed on 10 embryos (5 control, 5 mutant), with a total of 178 and 193 cells from control and mutant embryos respectively. Scale bar  = 20 µm.</p

Global loss of Vangl2 recapitulates the <i>Lp/Lp</i> phenotype.

<p><b>A–D</b>) At E14.5, <i>Lp/Lp</i> embryos exhibit gross abnormalities in body patterning including the severe neural tube defect craniorachischisis (arrows in C). Sectioning of these embryos revealed double outlet right ventricle (the arrows show the communication between the ventricle and the aorta). <b>E–H</b>) The phenotype of the <i>Vangl2^flox/flox; Sox2-Cre</i> embryos (G,H) was indistinguishable from <i>Lp/Lp</i> (C,D). The <i>Vangl2^f/+; Sox2-Cre</i> however did not have a looped tail, whereas this can be seen in <i>Lp/+</i> embryos (compare E with A white arrow). <b>I</b>) Breakdown of the cardiac defects seen in the <i>Vangl2^flox/flox; Sox2-Cre</i> embryos at E14.5. Also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004871#pgen.1004871.s003" target="_blank">S3 Fig</a>. Ao – aorta, LV - left ventricle, RV - right ventricle, <i>Vangl2^f</i> – <i>Vangl2^flox</i>. Scale bar  = 2 mm (white), 500 µm (black).</p

Targeting strategy and confirmation of knockdown.

<p><b>A</b>) Cartoon indicating the targeting strategy. Disruption of the <i>Vangl2</i> gene was achieved by modification of the wild type allele to insert <i>LoxP</i> sites flanking exon 4. Expression of <i>Cre</i> recombinase results in the excision of exon 4 and subsequent loss of the transmembrane domains. <b>B</b>) RT-PCR on RNA isolated from whole E10.5 <i>Vangl2^flox/flox; Sox2-Cre</i> embryos showed that there was no <i>Vangl2</i> transcript produced in the mutants, although this was abundant in controls. <i>Actin</i> was used as a loading control. <b>C</b>) Western blotting using protein isolated from whole E15.5 <i>Vangl2^flox/flox; Sox2-Cre</i> embryos showed that there was a major reduction in Vangl2 protein in the mutant embryos, although the presence of a faint band suggested that the <i>Cre</i> was not 100% efficient at later stages. Gapdh was used as a loading control. <b>D–K</b>) Immunohistochemistry for <i>Sox2-Cre</i> (using eYFP as a reporter for <i>Cre</i> expression) showed that recombination was variable across the embryo in the mutants (E,I). However, immuno-staining for Vangl2 showed that the protein was lost from the outflow tract (J, compare to F; in F strong staining is apparent within the OFT and neural tube - arrows). Also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004871#pgen.1004871.s002" target="_blank">S2 Fig</a>. OFT - outflow tract, <i>Vangl2^f</i> – <i>Vangl2^flox</i>. Scale bar  = 200 µm.</p

Loss of Vangl2 results in loss of SHF progenitor phenotype and premature differentiation in the distal outflow tract of <i>Vangl2^flox/flox; Isl1-Cre</i> embryos.

<p><b>A–F</b>) At E9.0, cardiac troponin I expression is low distally and increases proximally through the outflow tract of control embryos (A). In contrast, high-level expression is found more distally in <i>Vangl2^flox/flox; Isl1-Cre</i> embryos (D, n = 2). Desmin, which is expressed at high level in cardiomyocytes and at lower level by smooth muscle cells (B) is also increased within the distal outflow tract of <i>Vangl2^flox/flox; Isl1-Cre</i> embryos (E, n = 3). Whereas Isl1 is localised to the nucleus of control embryos throughout an extended region of the distal outflow tract, defining the transition zone (C - arrows), it is significantly reduced in the nuclei of cells in the distal outflow of <i>Vangl2^flox/flox; Isl1-Cre</i> embryos (F – arrows point to the proximal extent of the staining, n = 3). G–J) Similar to E9.0, at E10.5, both αSMA (G,I, n = 3) and MF20 (H,J; staining myosin heavy chain, n = 3) are expressed more distally in the outflow tract of <i>Vangl2^flox/flox; Isl1-Cre</i> embryos than in stage-matched littermates. K,L) Cartoon showing distribution of Vangl2 (bright green) at the boundary of the transition zone in the distal outflow tract of control embryos, where it is localised to the membrane through the transition zone, but is cytoplasmic (green stars) more proximally. Basolateral markers are represented in red and the MTOC, localising to the apical side of the cell, in dark green (K). In the absence of Vangl2, basolateral marker domains are expanded and the MTOC, although still apically positioned, is rotated in many cells. The wall is also thickened (L). M) Model showing how loss of epithelial phenotype of the cells within the distal outflow tract wall at E9.5 could result in a shortened outflow tract and double outlet right ventricle by E14.5. A =  Apical, B =  Basal, D =  distal, P =  proximal, <i>Vangl2^f</i>  =  <i>Vangl2^flox</i>. Scale bar  = 100 µm.</p

Loss of Vangl2 results in disrupted polarity in the distal outflow tract in <i>Vangl2^flox/flox; Isl1-Cre</i> embryos.

<p><b>A</b>) Cartoon representation of a sagittal view of the heart at E9.5, showing both the outflow and inflow regions and the dorsal pericardial wall in between. <b>B</b>) Vangl2 is found at the membrane of the distal outflow tract and dorsal pericardial wall, but is cytoplasmic in the myocardium of the heart tube (arrows). <b>C,D</b>) E-cadherin and N-cadherin are both found in the distal outflow tract although only N-cadherin is found in the inflow region (C- lower arrow). Neither are expressed at a high level in the dorsal pericardial wall although E-cadherin is expressed strongly in the columnar epithelium of the pharyngeal endoderm (D - arrowheads). <b>E–J</b>) Within the transition zone of the distal outflow tract, E-cadherin is enriched apically (E,G – see arrows in E); this enrichment is lost in the mutant embryos (H,J, n = 3). PKCζ is apically restricted in control embryos (F,G – see arrows in G). This apical restriction is lost in the <i>Vangl2^flox/flox; Isl1-Cre</i> embryos (I,J, n = 3). <b>K–P</b>) Similar to E-cadherin, Scrib and N-cadherin are apically enriched in control embryos (K-M - arrows). This enrichment is generally lost in the cells from the mutant embryos (N-P - arrowheads), although some can still be observed (P – arrow, n = 3) <i>Vangl2^f</i>  =  <i>Vangl2^flox</i>. Scale bar  = 100 µm (B-D), 50 µm (E<b>–</b>P).</p

Vangl2 is expressed in the distal outflow region.

<p><b>A</b>) Cartoon showing the region encompassing the dorsal pericardial wall and the distal outflow tract, including the region we describe as the transition zone. <b>B–F</b>) Vangl2 protein (red), labelled by immunofluorescence, is expressed in the distal outflow region (B), localising to the basal part of the membrane of the cells (as shown by co-localisation with β-catenin, a baso-lateral marker; green) in the dorsal pericardial wall and transition zone (B,C,E), but is found diffusely in the cytoplasm more proximally (B,D,F). <b>G–H</b>) Cardiac troponin I staining (red; labelling cardiomyocytes) is initially weak within the distal outflow but is upregulated more proximally (I). Vangl2 (green) and cardiac troponin I are co-expressed in the transition zone (J - TZ and arrows) of the outflow tract with the membrane-localisation of Vangl2 gradually lost (H) as cardiac troponin I staining becomes stronger. <b>K–N</b>) Vangl2 and Isl1 are also co-expressed in the cells of the transition zone (N - TZ and arrows), with the loss of Vangl2 from the membrane proximally coinciding with the loss of nuclear Isl1 localisation (N - lower white arrowhead). All images shown are of Vangl2^f/+ embryos. A =  Apical, B =  Basal, D =  distal, endo  =  endocardium, myo  =  myocardium, P =  proximal, TZ =  transition zone. Scale bar  = 25 µm.</p