Fig. 3.
Generation of non-graded Wnt5a expression. (A) Generation of an inducible Wnt5a mouse strain (Rosa5a/+). Mouse Wnt5a cDNA following a PGK-Neo cassette flanked by loxp sequences was knocked into the Rosa26 locus. IRES-GFP sequence was cloned downstream of Wnt5a cDNA. SA, splice acceptor sequence. (B) Schematic of the experimental strategy. The blue arrows represent Fgf signaling from distal limb AER. (C) Wnt5a whole-mount in situ hybridization in E11.5 mouse forelimb buds. A, anterior; P, posterior. Scale bars: 200 μm. (D) Levels of total or endogenous Wnt5a mRNA from E11.5 forelimb buds were quantified by qPCR. Wnt5a mRNA levels were normalized to Gapdh expression. Error bars represent s.d., n=3 repetitions. (E) The E11.5 forelimb buds were dissected into three parts, and each part was subjected to western blot analysis and qPCR. (F,G) Quantification of Wnt5a protein and mRNA levels of the three parts, which were normalized to Gapdh protein and mRNA levels, respectively. Two-tailed t-test, *P<0.05, **P<0.01, ***P<0.001. N.S, no significance. Error bars represent s.d., n=2 repetitions for western blotting, n=3 repetitions for qPCR.