N-cadherin-deleted mouse embryos show low Wnt pathway activity and reduced downstream target gene expression during EMT.

<p>(A) Western blot analysis of membrane fraction of HEK293 cells transfected with control or N-cadherin siRNA. Transferrin receptor serves as a loading control. N-cadherin siRNA attenuates N-cadherin expression. β-catenin is also slightly reduced. (B) TOPflash reporter assay in HEK293 cells, after transfection of N-cadherin siRNA. Cells were either treated with control medium (C) or Wnt3a medium (W), or transfected with β-catenin (β). N-cadherin siRNA attenuates the effect of β-catenin transfection. *<i>P</i><1×10⁻⁶. (C–F) TOP-LacZ reporter mouse embryos at 8.5 dpc with genotypes of wild type (+/+) or deleted (−/−) N-cadherin. The reporter expression is detected by an in situ hybridisation probe against LacZ. At the level of the primitive streak in the posterior region (arrowheads in C,D), the reporter expression is much weaker in the −/− mutant, while the midbrain (short arrows) and anterior somites (long arrows) show a comparable level of expression. (E) and (F) are transverse sections of (C) and (D), respectively, at the level of the primitive streak (arrowheads), where epiblast cells delaminate at the midline and migrate laterally to form mesoderm. (G–J) Mouse embryos at 8.25 dpc with wild type (G,I) or N-cadherin −/− (H,J) genotypes, stained with <i>Tbx6</i>, a Wnt signal target gene, by RNA in situ hybridisation. (G) and (H) are close-ups of the open neural plate region in the posterior. N-cadherin mutant embryos show a weaker Tbx6 expression. Transverse sections at the level indicated by arrowheads are shown in I and J. The expression level of <i>Tbx6</i> in delaminated mesodermal cells in the N-cadherin −/− embryo is significantly low compared to that of wild type. Scale bars, 100 µm.