Fig. 6. Suppressed expression of sclerostin by loss of BMP signaling ex vivo. (A) Newborn mouse calvariae were cultured for 5 days treated with 4OH-TM (100 ng/ml). (a) Confirmation of Cre activity in the calvariae from CreER:R26R mice assessed by β-gal staining. (b) Upregulation of canonical Wnt signaling in the calvariae from CreER:Bmpr1afx/fx:TOPGAL mice assessed by β-gal staining. Broken lines, areas of parietal bones (P). (c) Expression of Wnt inhibitors Sost, Dkk1, Dkk2 and Lrp5 assessed by QRT-PCR. (d) Expressions of bone resorption markers Mmp9, Ctsk, TRAP and Bmpr1a. Values are expressed relative to those of wild type (Cre-, TM-). (B) Sclerostin treatment of wild-type (WT+Scl) and cKO (cKO+Scl) calvariae ex vivo. The ratio of β-gal to DAPI-positive cells was evaluated from 50 fields in frontal sections (n=3, in each condition). Broken line, parietal bones. Scale bars: 50 µm. (C) Expression of bone resorption markers Rankl and Opg, and relative ratio of Rankl to Opg using wild-type and cKO calvariae in sclerostin treated versus untreated groups. Values are expressed relative to those of wild type without sclerostin treatment. (D) TRAP staining of cKO calvariae treated with sclerostin and non-treated ex vivo. Values in A-C represent mean±s.d. from three independent experiments. Student's t-test; ^*P<0.05.