Figure 4. Differential expression of Vangl2 in different murine tissues and mouse cochlea.

(A) Expression profile of Vangl2 from murine tissues (brain, kidney and lung) detected with 2G4 antibody in western blot. (B) Vangl2 protein expression in mouse cochlear tissue deriving from homozygous Lp and heterozygous Vangl2^Lp/+ heterozygous mice compared to wild-type littermates, using 2G4 mAb antibody. Corresponding densitometry measurements representing relative Vangl2 protein expression normalized to GADPH protein levels. (C) Absence of staining with 2G4 mAb in Vangl2 mutant. Surface views of cochleae from WT (A-A’) and Looptail homozygote (B-B’) from E17.5 mice processed for immunocytochemistry with 2G4 mAb. At this stage, the cochlea comprises a single row of inner hair cells (#) and three rows of outer Hair Cells (OHC1 *, OHC2 **, OHC3 ***) surrounded by supporting cells. Phalloidin staining (actin, red) reveals hair cells borders. The image is taken at the level of the zonula adherens for the second and third row of OHC2 and OHC3, where Vangl2 accumulates. In the WT sample, we observe accumulation of Vangl2 at the junction between hair cells and supporting cells (A-A’, arrows). In contrast, in Lp/Lp cochlea, there is a complete absence of Vangl2 staining at the membrane of the cells (B,B’). Scale bar = 3 µm. (D) Co-localization of staining with 2G4 mAb and Vangl1 Ab.

Surface view of a cochlea from a newborn mouse processed for immunocytochemistry with 2G4 mAb and Vangl1 Ab. The two antibodies reveal a co-localization of Vangl1 (A”) and Vangl2 (A’”) proteins at the junction between hair cells and supporting cells (arrows). Phalloidin is in blue, Vangl1 in red, and Vangl2 in green. Scale bar = 3 µm. Note: in the green channel, remains of the tectorial membrane covering normally the cochlear epithelium lead spots of non-specific green labeling. (E) Schematic diagram representing the two possible models of Vangl1/Vangl2 interaction.