Increased osteclastogenesis in Fzd8-deficient mice. (A) Quantification of osteoclast number per bone perimeter (Oc.N/B.Pm) and osteoclast surface per bone surface (OcS/BS) in 24-wk-old wild-type and Fzd8-deficient mice. Error bars represent mean ± SD (n = 6). Asterisks indicate statistically significant differences between the two groups. (B) Western blot analysis for phosphorylated Lrp6 and β-catenin after stimulation of wild-type and Fzd8-deficient osteoblasts by Wnt3a at day 10 of differentiation. The black arrows indicate the position and molecular weight of the nearest marker. (C) qRT-PCR expression analysis of the indicated genes in Fzd8-deficient osteoblasts relative to wild-type cultures. Error bars represent mean ± SD (n = 4). (D) qRT-PCR expression analysis of the same genes in Fzd8-deficient marrow-flushed bones relative to wild-type littermates. Error bars represent mean ± SD (n = 4). (E) Serum levels of Opg and Rankl in 24-wk-old wild-type and Fzd8-deficient littermates. Error bars represent mean ± SD (n = 6). (F) TRAP activity staining of bone marrow cells differentiated in the presence of M-Csf and Rankl with or without Wnt3a as indicated. Bars, 50 µm. Quantification of TRAP-positive multinucleated cells in wild-type and Fzd8-deficient cultures is given on the right. Error bars represent mean ± SD (n = 6). Asterisks indicate statistically significant differences compared with control-treated cells.