WT1 transcriptionally upregulates Snail in VHL-deficient cells.

<p>(A) ChIP assays for WT1 were performed in the isogenic SN12C and ACHN cell lines. WT1 was immunoprecipitated and the bound DNA fragments were analyzed by PCR amplification for Snail. Histone H3 and rabbit IgG were used as positive and negative controls, respectively. Human RPL30 exon3 primers amplified a 160 bp fragment from immunoprecipitation of H3. Snail primers amplified a 120 bp fragment from immunoprecipitation using WT1 antibody. (B) <i>SNAI1</i> promoter luciferase activity was measured in the isogenic SN12C and ACHN cells. (C) SN12C-VHL and ACHN-VHL cells were cotransfected with either scrambled or siRNA-WT1 oligonucleotides, and <i>SNAI1</i> promoter luciferase activity was measured. Graphs depict mean±SD of one representative of three independent experiments. (D) Top, schematic representation of WT1 binding sequence within the Snail promoter with mutated residues highlighted with asterisks (***). Bottom, HEK293T cells were cotransfected with GFP or GFP-WT1 and either wild-type or mutated <i>SNAI1</i> promoter constructs and luciferase activity was measured. Graph depicts mean±SD of three independent experiments. *, <i>P</i><0.05.</p

WT1 regulates Snail expression.

<p>(A) Analysis of Snail protein (top) and mRNA (bottom) in the isogenic SN12C and ACHN cell lines. (B) HEK293T cells were transfected with scrambled oligonucleotides or VHL-specific siRNAs and Snail protein was measured. (C) HEK293T cells were transfected with GFP or GFP-WT1 and Snail protein (top) and mRNA (bottom) was measured. Graphs show mean±SD of one representative of three independent experiments. *, <i>P</i><0.05. (D) HEK293T and SN12C-VHL cells were transfected as indicated and protein expression was assessed by immunoblot.</p

WT1 upregulates E-cadherin expression in the presences of Snail.

<p>(A) Immunoblot analysis of E-cadherin and N-cadherin expression in the isogenic SN12C and ACHN cell lines. (B) VHL-knockdown RCC cells were transfected with scrambled or WT1-specifc siRNAs and E-cadherin expression was assessed by immunoblot. (C) Top, HEK293T cells were transfected as indicated and protein expression was assessed by immunoblot. Bottom, analysis of E-cadherin mRNA levels. (D, E) HEK293T (D) or MDCK (E) cells were cotransfected with the indicated plasmids and the <i>CDH1</i> promoter reporter construct, and luciferase activity was measured. Graphs represent mean±SD of one representative experiment. *, <i>P</i><0.05.</p

VHL-knockdown alters the expression of EMT markers in RCC cells.

<p>Representative phase-contrast and immunofluorescence images of the isogenic SN12C and ACHN cells. Scale bars = 100 µm in the phase-contrast images. Scale bars = 7.5 µm and 10 µm in the immunofluorescence images of the SN12C and ACHN cells, respectively.</p

WT1 preserves epithelial junctions and suppresses motility in renal cells.

<p>(A) MDCK cells were transfected as indicated and protein expression was assessed by immunoblot. (B) Representative immunofluorescene images of MDCK cells transfected with Snail alone or Snail and WT1. (C) Electron microscopy images of MDCK cells transfected with WT1, Snail, or WT1 and Snail. Red boxes indicate cell-cell contact regions showing intercellular junctions junctions enlarged in the inset (arrow). Note the absence of intercellular contacts and spindle morphology of Snail-transfected cells. (D) HEK293T cells were transfected with either GFP or GFP-WT1 and a scratch motility assay was performed. (E) Quantification of the width of the wound at the indicated time points. Graph depicts mean±SD of three independent experiments. *, <i>P</i><0.05.</p

Melanoblasts replace a proportion of the smooth muscle cells in the ctnnb1Δex3 DA.

<p>SMA-positive and X-gal-positive cells in transverse sections of E18.5 WT-Rosa and ctnnb1Δex3-Rosa DA were counted. Three categories of cells were distinguished: non-recombined SMC1 (SMA+ LacZ−), recombined SMC2 (SMA+ LacZ+), and recombined non-SMC (SMA− LacZ+), corresponding to melanoblasts (Mb). Note that the number of SMA+ LacZ+ SMC2 in WT-Rosa DA is similar to the number of SMA− LacZ+ Mb in ctnnb1Δex3-Rosa DA. Genotypes: WT-Rosa  =  <i>Tyr::Cre/</i>°; <i>+/+</i>; <i>Rosa26/</i>°, ctnnb1Δex3-Rosa  =  <i>Tyr::Cre/</i>°; <i>ctnnb1Δex3/+</i>; <i>Rosa26/</i>°. Note, the production of a mutated form of β-catenin in recombined cells did not seem to greatly affect the number of floxed cells, suggesting that there was no cell non-autonomous effect on the floxed SMC. In both panels, there were significant differences between the numbers of SMA+, LacZ+ cells and SMA−, LacZ+ cells (for each genotype, the number of cells were estimated from 5–8 sections per embryo using 4 embryos: ** p-value <0.01).</p

Histological analysis of WT and ctnnb1Δex3 lungs at P28.

<p>(A) WT ( = <i>Tyr::Cre</i>/°; +/+) and (B) ctnnb1Δex3 mice. Note the disorganized alveolae of the mutant lung. Nonetheless, the lung cells do not express the ctnnb1Δex3 transgene, suggesting that the effect is cell non-autonomous. Scale bars, (A, C, D)  = 50 µm, (B)  = 20 µm.</p

Closure of the ligamentum arteriosum in ctnnb1Δex3 adult heart.

<p>The <i>Tyr::Cre; ctnnb1Δex3</i>/+ ( =  ctnnb1Δex3) ligamentum arteriosum (LigA) is not fully closed, rendering it a patent ductus arteriosus. At P28, the LigA does not usually show macroscopic hyperpigmentation in wildtype (WT) mice (arrows, A), whereas <i>Tyr::Cre; ctnnb1Δex3</i>/+ ( =  ctnnb1Δex3) LigA does (B). Transverse sections show that the WT LigA (C) is fully closed and does not contain any Mc, whereas ctnnb1Δex3 LigA (D–F) is only partially closed, containing both blood in the lumen and numerous pigmented melanocytes in the wall (E, F). The areas occupied by the intimal cushion (ic) and lumen (l) are shown in G and H, respectively. Cross-sections of the LigA show that the outer tunica is dense, while the inner ellipsoid part, known as the intimal cushion, has a distinct aspect. In ctnnb1Δex3 mice, a lumen is observable inside the ic. Ao: aorta, LigA: ligamentum arteriosum, Pa: pulmonary artery, Mc: melanocyte. Scale bars, (A, B)  = 0.25 mm, (C, D)  = 100 µm, (E)  = 50 µm and (F)  = 20 µm. For each genotype, the number of cells were estimated from 8-10 sections per LigA using 4 mice. *: p-value <0.05.</p

Melanoblasts are numerous in ctnnb1Δex3 DA.

<p>Ventral view of WT-Dct (A) and ctnnb1Δex3-Dct (B) E18.5 hearts stained with X-gal. Note that ctnnb1Δex3-Dct samples contain numerous β-galactosidase-stained cells (arrow) in the ductus arteriosus (DA). High magnification of the WT-Dct (C) and ctnnb1Δex3-Dct (D) DA regions, including the aorta (Ao) and the pulmonary trunk (PT). Scale bar (A, B)  = 1 mm.</p

A Subpopulation of Smooth Muscle Cells, Derived from Melanocyte-Competent Precursors, Prevents Patent Ductus Arteriosus

<div><h3>Background</h3><p>Patent ductus arteriosus is a life-threatening condition frequent in premature newborns but also present in some term infants. Current mouse models of this malformation generally lead to perinatal death, not reproducing the full phenotypic spectrum in humans, in whom genetic inheritance appears complex. The <em>ductus arteriosus</em> (DA), a temporary fetal vessel that bypasses the lungs by shunting the aortic arch to the pulmonary artery, is constituted by smooth muscle cells of distinct origins (SMC1 and SMC2) and many fewer melanocytes. To understand novel mechanisms preventing DA closure at birth, we evaluated the importance of cell fate specification in SMC that form the DA during embryonic development. Upon specific Tyr::Cre-driven activation of Wnt/β-catenin signaling at the time of cell fate specification, melanocytes replaced the SMC2 population of the DA, suggesting that SMC2 and melanocytes have a common precursor. The number of SMC1 in the DA remained similar to that in controls, but insufficient to allow full DA closure at birth. Thus, there was no cellular compensation by SMC1 for the loss of SMC2. Mice in which only melanocytes were genetically ablated after specification from their potential common precursor with SMC2, demonstrated that differentiated melanocytes themselves do not affect DA closure. Loss of the SMC2 population, independent of the presence of melanocytes, is therefore a cause of patent ductus arteriosus and premature death in the first months of life. Our results indicate that patent ductus arteriosus can result from the insufficient differentiation, proliferation, or contractility of a specific smooth muscle subpopulation that shares a common neural crest precursor with cardiovascular melanocytes.</p> </div