Figure 1

Generation and characterization of Nestin-Cre; LRP6^f/f mice. (a) Schematic diagram to generate Nestin-Cre transgenic mice on Lrp6-flox background. LoxP sites in Lrp6 gene are indicated by dark triangles. Primers used for the allele-specific PCR in panel b are indicated by black arrows. (b and c) Expression of LRP6 in bone tissues from male Cre^−/−; LRP6^F/f (Lrp6^+/+), Cre^+/−; Lrp6^wt/f (Lrp6^+/−) and Cre^+/−; Lrp6^f/f (LRP6^−/−) mice tested by genomic PCR (b) and qRT-PCR (c). The bands in upper panel represent the insertion of loxP site in exons 2. The bands in lower panel show Cre-mediated recombination of the Lrp6^flox allele only occurred in bone tissue of Lrp6^+/− and Lrp6^−/− mice. The primers used for the PCR amplification in upper and lower panels were indicated in a. (d–f) Immunohistochemical analysis of LRP6 expression in femur sections of 3-month-old male Lrp6^+/+ Lrp6^+/− and Lrp6^−/− mice. Number of total LRP6-positive cells per mm² tissue area (N.LRP6⁺ cells/T.Ar) (d). Representative double-immunofluorescence staining of LRP6 (green) and nestin (red) in femur sections from 3-month-old male Lrp6^+/+ , Lrp6^+/− and Lrp6^−/− mice (e). Nuclei were counterstained with DAPI (blue). Yellow arrows, perivascular nestin⁺ cells that express LRP6; green arrows, nestin⁻ cells that express LRP6; red arrows, nestin⁺ cells that do not express LRP6. Scale bars=100 µm. Quantitative analysis of the percentage of nestin⁺LRP6⁺ double=positive cells out of total nestin⁺ cells (f). A total of three femur sections from each mouse and five mice per treatment group were analyzed. *P<0.05, **P<0.001, vs. Lrp6^+/+ group. (g) Representative images showing the sizes of 3-month-old male mice with different genotypes. DAPI, 4′,6-diamidino-2-phenylindole; qRT-PCR, quantitative reverse transcriptase-PCR.