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First published online April 10, 2009
doi: 10.1242/10.1242/dev.034066

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Non-canonical Wnt signaling regulates cell polarity in female reproductive tract development via van gogh-like 2

Myology Group, UMR S 787 Inserm, Université Pierre et Marie Curie Paris VI, 105 bd de l'Hôpital, 75634 Paris Cedex 13, France.

View larger version (47K): [in this window] [in a new window]   Fig. 1. Vangl2Lp mutants have gross morphological defects at E18.5: 50% of Vangl2Lp heterozygote adult females are infertile and have a septate vagina. Wild-type (A) and Vangl2Lp/Lp mutant (B,C) E18.5 mouse female reproductive tract (FRT). The mutant demonstrates a lack of oviduct coiling and a septate vagina (arrows in C). (D) Wild-type adult FRT. (E) About 50% of Vangl2Lp heterozygote FRTs are greatly enlarged and fluid-filled, indicating a vaginal blockage. ovi, oviduct; ov, ovary; ut, uterine horn; cx, cervix; vg, vagina; bl, bladder. Scale bars: 1 mm in A,B; 0.5 mm in C; 5 mm in D,E.

View larger version (56K): [in this window] [in a new window]   Fig. 2. Vangl2Lp mutants display alterations in cytoarchitecture at E18.5, including the loss of typical columnar epithelial cell morphology. (A,B) Wild-type (A) and Vangl2Lp mutant (B) mouse uterine semi-thin cross-sections reveal a change in uterine lumen shape in the mutant (it is more rounded) as well as disorganized mesenchyme and non-columnar epithelium. (C-F) Transmission EM of wild type (C,E) and Vangl2Lp mutant (D,F) reveals ultrastructural changes associated with the loss of columnar uterine epithelial cell morphology in mutants. Near the tight junctions, electron-dense fibrillar structures are seen in the mutant (F, arrows), but are rarely visible in wild type (E). (G-J) Immunofluorescent confocal staining of E18.5 wild-type (G,I) and mutant (H,J) uterine sections with E-cadherin antibody. Note the nearly detached epithelial cell (H, arrow). E-cadherin is enriched at the apical edge (I, arrows); note increased number of cell layers of E-cadherin-staining epithelium in the Vangl2Lp mutant (J, arrows). Scale bars: 10 µm in C,D,G,H; 5 µm in A,B,I,J; 1 µm in E,F.

View larger version (102K): [in this window] [in a new window]   Fig. 3. Vangl2 protein localizes to epithelial membranes and is enriched at the apical edge of lateral cell contacts in both immature and adult uterus. In E18.5 wild-type mouse uterine tissue, Vangl2 protein (green), as detected by confocal IF, localizes to epithelial cell membranes (A) and is enriched at cell junctions (C), whereas in the Vangl2Lp mutant at E18.5 (B) there is very little detectable expression. In 1-month-old wild-type tissue, Vangl2 is enriched at the apical edge of lateral cell contacts (D) and is similarly found at epithelial cell membranes (E) and in the epithelium of glands. We note that the wild type shown here (A) has a somewhat rounder lumen, reflecting the fact that it was harvested at a slightly earlier stage than E18.5 to allow for easier visualization of the IF staining. Scale bars: 50 µm in A,B; 5 µm in C,D; 10 µm in E.

View larger version (89K): [in this window] [in a new window]   Fig. 4. Vangl2Lp mutants have defective cytoskeletal actin polarization and scribble distribution is not restricted to the apical edge of epithelial cells at E18.5. (A-D) Wild-type (A,C) and Vangl2Lp/Lp mutant (B,D) mouse uterine sections were stained with Alexa 647-phalloidin. C,D are higher magnifications from A,B, with the lumen located towards the upper left in D. (E-H) Scrb1-stained (yellow) uterine sections of wild type (E,G), and Vangl2Lp/Lp mutant (F,H) with the lumen located to the right in G,H. Arrows delineate the apical edge (G,H), which displays increased Scrb1 signal in the Vangl2Lp mutant (H). Scale bars: 10 µm in A,B,E,F; 5 µm in G,H.

View larger version (61K): [in this window] [in a new window]   Fig. 5. Heterozygous and homozygous Vangl2Lp mutant grafted postnatal uterine tissues demonstrate various histological abnormalities, especially in the epithelium. Wild-type (A,B), Vangl2Lp/+ (C-H) and Vangl2Lp/Lp mutant (I-N) mouse tissues were grafted in the kidney capsule of ovarectomized nude mice for 2 weeks, cryosectioned and H&E stained. Bright-field images are shown. Images in the right-hand column are magnifications of those in the left column. Arrows indicate acellular material (D). Note the presence of glands in wild type (A,C) and heterozygous mutant (E,G). The double-headed arrow in F indicates the expanded layer of pseudostratified epithelium. Arrows in H indicate cell projections. Note the increased number and size of lipid droplets or vacuoles (J, arrows), the presence of cells that appear hyperpolarized (L), and epithelial cells that have detached from the underlying mesenchyme (N). Scale bars: 10 µm in A,C,E,G,I,K,M; 5 µm in B,D,F,H,J,L,N.

View larger version (94K): [in this window] [in a new window]   Fig. 6. Mutant Vangl2Lp grafted postnatal uterine tissue displays hyperpolarized actin and abnormal E-cadherin distribution. (A-F) The wild type (A-C) has a thin and even domain of actin polarization towards the lumen, as compared with the Vangl2Lp mutant (D-F) in which the actin staining (red) is uneven (compare B with E, arrows). E-cadherin (green) is enriched at the apical domain of lateral cell junctions in wild-type epithelium (C, arrows), but this localization is lost in the mutant (F, arrows). (G-L) Scrb1 localization is perturbed in grafted postnatal Vangl2Lp mutant uterine tissue. The wild type (G,J) demonstrates discrete points of Scrb1 localization (green) to apical regions (lumen to the left in G,J) of epithelial cell-cell contact (J, arrows). By contrast, in Vangl2Lp heterozygotes (H,K,I,L), the expanded pseudostratified layer of epithelial cells localize Scrb1 in a non-polarized fashion, including some Scrb1+ epithelial cells in the middle of the lumen (H, arrowhead) and uniform membrane localization (H,I,K,L). Scrb1 localization is abnormal in the highly pseudostratified Vangl2Lp/Lp mutant epithelium (I,L). (M-O) Increase in smooth muscle layer with corresponding decrease in number of mesenchymal cells in Vangl2Lp mutants. Wild type (M), Vangl2Lp/+ (N) and Vangl2Lp/Lp (O) grafted mouse uterine postnatal tissue sections were stained for laminin (green) and smooth muscle actin (red). Laminin delineates the basal lamina at the border between epithelium and mesenchyme. Double-headed arrows indicate the width of the mesenchyme (M,N). The homozygous mutant (O) displays smooth muscle directly adjacent to the basal lamina (no mesenchyme). Scale bars: 10 µm in G-I,M-O; 5 µm in A-F,J-L.

View larger version (20K): [in this window] [in a new window]   Fig. 7. Wnt7a expression is reduced in both heterozygous and homozygous Vangl2Lp mutants. Wnt7a (A), Wnt5a (B) and Wnt4 (C) expression was measured relative to P0 (acidic ribosomal phosphoprotein) control gene by quantitative RT-PCR of cDNA obtained from total RNA of mouse uterine horns from different animals. Error bars indicate s.d. of the mean; P-values were calculated using Student's t-test (wild type, n=3; Vangl2Lp/+, n=3; Vangl2Lp/Lp, n=5): *P=0.873, **P=0.837.

View larger version (19K): [in this window] [in a new window]   Fig. 8. Model showing how the Vangl2Lp mutation functions in neonatal and postnatal female reproductive tissues. (A) Wild-type neonatal mouse uterine epithelium localizes Vangl2 protein in the apical domain and Scrb1 in the basolateral domain. When Vangl2Lp is mutated, Scrb1 is no longer restricted to the basolateral domain. Wild-type epithelium remains columnar (beneath), whereas Vangl2Lp mutant epithelium is no longer columnar. (B) In postnatal uterine epithelium, Vangl2 is still membraneous, but is enriched at the apical domain, whereas Scrb1 is concentrated at discrete puncta near tight junctions. Wild-type postnatal epithelium remains columnar (beneath), whereas in the Vangl2Lp mutant it becomes highly pseudostratified, indicative of a change in the plane of cell division.

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