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

<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

<i>CEP290</i> is expressed in the developing kidney.

<p>Hybridisation with <i>CEP290</i> antisense RNA probe (A–F) to transverse sections. (A) CS12 and (B) CS14; (C) CS15; (D) CS16. <i>CEP290</i> transcripts are abundant in the mesonephros (Meso) and metanephros (Meta) of the developing kidney as well as the spinal cord (Sc). (E, F) A series of sections through mesonephric and metanephric development, respectively, from CS14 to 9 PCW. Hybridisations were also carried out with <i>CEP290</i> sense probe as a negative control and no signals were detected (data not shown). CD, collecting duct; Gl, glomerulus; H, hindgut; Meso, mesonephros; DMeso, degenerating mesonephros; Meta, metanephros; Sc, spinal cord; St, stomach; UL, upper limb; LL, lower limb. P, renal pelvis. Scale bar: A–D = 1 mm; E: CS12–CS16 = 500 µm; CS23 = 125 µm F: CS 14 = 250 µm; CS17 = 125 µm; CS20 = 250 µm; CS22 and 9 PCW = 125 µm.</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

Inversin/Nephrocystin-2 Is Required for Fibroblast Polarity and Directional Cell Migration

<div><p>Inversin is a ciliary protein that critically regulates developmental processes and tissue homeostasis in vertebrates, partly through the degradation of Dishevelled (Dvl) proteins to coordinate Wnt signaling in planar cell polarity (PCP). Here, we investigated the role of Inversin in coordinating cell migration, which highly depends on polarity processes at the single-cell level, including the spatial and temporal organization of the cytoskeleton as well as expression and cellular localization of proteins in leading edge formation of migrating cells. Using cultures of mouse embryonic fibroblasts (MEFs) derived from <i>inv^−/−</i> and <i>inv^+/+</i> animals, we confirmed that both <i>inv^−/−</i> and <i>inv^+/+</i> MEFs form primary cilia, and that Inversin localizes to the primary cilium in <i>inv^+/+</i> MEFs. In wound healing assays, <i>inv^−/−</i> MEFs were severely compromised in their migratory ability and exhibited cytoskeletal rearrangements, including distorted lamellipodia formation and cilia orientation. Transcriptome analysis revealed dysregulation of Wnt signaling and of pathways regulating actin organization and focal adhesions in <i>inv^−/−</i> MEFs as compared to <i>inv^+/+</i> MEFs. Further, Dvl-1 and Dvl-3 localized to MEF primary cilia, and β-catenin/Wnt signaling was elevated in <i>inv^−/−</i> MEFs, which moreover showed reduced ciliary localization of Dvl-3. Finally, <i>inv^−/−</i> MEFs displayed dramatically altered activity and localization of RhoA, Rac1, and Cdc42 GTPases, and aberrant expression and targeting of the Na⁺/H⁺ exchanger NHE1 and ezrin/radixin/moesin (ERM) proteins to the edge of cells facing the wound. Phosphorylation of β-catenin at the ciliary base and formation of well-defined lamellipodia with localization and activation of ERM to the leading edge of migrating cells were restored in <i>inv^−/−</i> MEFs expressing Inv-GFP. Collectively, our findings point to the significance of Inversin in controlling cell migration processes, at least in part through transcriptional regulation of genes involved in Wnt signaling and pathways that control cytoskeletal organization and ion transport.</p> </div

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

<i>AHI1</i> expression during nephrogenesis.

<p>(A, C, E) <i>AHI1</i> sense RNA probe and (B, D, F) <i>AHI1</i> antisense RNA probe hybridised to sections from (A, B) CS16; (C, D) CS22 and (E, F) 9 PCW. <i>AHI1</i> transcripts are abundant in the mesonephros (Meso) and metanephros (Meta) of the developing kidney as well as the spinal cord (Sc), liver and embryonic gonad. (G, H). A series of sections at different stages of mesonephric and metanephric development. <i>AHI1</i> expression is seen in (G) mesonephric excretory unit - a mesonephric tubule, glomerulus and duct at CS14, CS16 and CS22. By CS23 expression is visible in degenerating glomeruli (DG). By 9 PCW expression is detectable in the mesonephric tubule, ducts and paramesonephric duct. <i>AHI1</i> expression is seen in (H) early permanent metanephric kidney with intense staining in the metanephric cap and ureteric bud (CS14 and CS16). By CS22 and later developing glomeruli, tubules and collecting ducts are seen to strongly express <i>AHI1</i> (CS22, CS23 and 9 PCW). In 9 PCW human fetal sections, <i>AHI1</i> transcripts are abundant in the developing nephrons and collecting ducts of renal cortex and there is weak expression in medulla/renal pelvis. CD, collecting duct; DG, degenerating glomerulus; Gl, glomerulus; H, hindgut; LL, lower limb; Mc, metanephric cap; Md, mesonephric duct; Med, renal medulla; Meso, mesonephros; Meta, metanephros; Mt, mesonephric tubule; P, renal pelvis; Pmeso, paramesonephric duct; Sc, spinal cord; St, stomach; UB, ureteric bud; UL, upper limb. Scale bars: A–F = 2 mm; G: CS16 = 500 µm; CS14, CS23 & 9WPC = 250 µm; CS22 = 125 µm; H: CS22 & 9WPC = 500 µm; CS14, CS16 & CS23 = 250 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

Development of the arterial roots and ventricular outflow tracts.

The separation of the outflow tract of the developing heart into the systemic and pulmonary arterial channels remains controversial and poorly understood. The definitive outflow tracts have three components. The developing outflow tract, in contrast, has usually been described in two parts. When the tract has exclusively myocardial walls, such bipartite description is justified, with an obvious dogleg bend separating proximal and distal components. With the addition of non-myocardial walls distally, it becomes possible to recognise three parts. The middle part, which initially still has myocardial walls, contains within its lumen a pair of intercalated valvar swellings. The swellings interdigitate with the distal ends of major outflow cushions, formed by the remodelling of cardiac jelly, to form the primordiums of the arterial roots. The proximal parts of the major cushions, occupying the proximal part of the outflow tract, which also has myocardial walls, themselves fuse and muscularise. The myocardial shelf thus formed remodels to become the free-standing subpulmonary infundibulum. Details of all these processes are currently lacking. In this account, we describe the anatomical changes seen during the overall remodelling. Our interpretations are based on the interrogation of serially sectioned histological and high-resolution episcopic microscopy datasets prepared from developing human and mouse embryos, with some of the datasets processed and reconstructed to reveal the specific nature of the tissues contributing to the separation of the outflow channels. Our findings confirm that the tripartite postnatal arrangement can be correlated with the changes occurring during development

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