Generation and analysis of Wnt11-LoxP and Wnt11-Del mutant models. (A) Schematic demonstrating the mutations made to the miR-140-5p binding site of the 3′-UTR of the Wnt11 gene. The Wnt11-Del model was created by deleting the 14 bp sequence including the miR-140-5p binding site. The Wnt11-LoxP mutation was created by inserting a loxP sequence in the 3′-UTR ablating the miR-140-5p binding site. Both models were designed to diminish miR-140-5p binding. (B) Relative expression of Wnt11 in rib chondrocytes of homozygous Wnt11-LoxP and Wnt11-Del models. (Left) Wnt11 expression is significantly increased in both models. Mean ± SEM with individual data is shown. n = 6–8 from three biological replicates, Student's t-test versus WT. (Right) Wnt11 up-regulation in Wnt11-LoxP rib chondrocytes was confirmed by immunoblot analysis. The relative signal intensity after normalization to histone 3 (H3) is indicated. (C,D) Representative images of hematoxylin and eosin-stained proximal tibias of Wnt11-LoxP (C), Wnt11-Del (D) and respective littermate wild-type controls (+/+). Both models had a modest acceleration of secondary ossification development (blue arrows) (C,D). Scale bar represents 500 µm.
