Fig. 2. Inactivation of VGLL4 in MSCs leads to impaired bone ossification.

(A) Skeletons of Vgll4^fl/fl and Vgll4^prx1 mice at P1 were double-stained by Alizarin red and Alcian blue. Scale bar, 5 mm. (B) Quantification of body length in (A) (n = 6). (C) Skull and clavicle preparation from Vgll4^fl/fl and Vgll4^prx1 mouse newborns were double-stained with Alizarin red and Alcian blue at P1. Scale bars, 5 mm. (D) Quantification of the defect area of skulls in (C) (n = 6). (E) Quantification of clavicle length in (C) (n = 6). (F) Alp staining and Alizarin red staining of BMSCs from Vgll4^fl/fl and Vgll4^prx1 mice after cultured in osteogenic medium. Scale bars, 3 mm. (G) Alp activity was measured by phosphatase substrate assay. (H) Relative mRNA levels were quantified by RT-PCR. (I) 3D μ-QCT images of trabecular bone (top) and cortical bone (bottom) of distal femurs. (J to N) μ-QCT analysis for trabecular bone volume per tissue volume (BV/TV, Tb) (J), trabecular number (Tb.N/mm) (K), trabecular thickness (Tb.Th/mm) (L), trabecular separation (Tb.Sp/mm) (M), and cortical bone thickness (Cor.Th/mm) (N). (O) Representative images of von Kossa staining of 12-week-old Vgll4^fl/fl and Vgll4^prx1 mice. Scale bar, 500 μm. (P) Representative images of calcein and Alizarin red S labeling of proximal tibia. Scale bar, 50 μm. (Q) Quantification of MAR. (R and S) ELISA analysis of serum PINP (ng ml⁻¹) and CTX-1 (ng ml⁻¹) from 10-week-old Vgll4^fl/fl and Vgll4^prx1 mice (n = 5). In (B), (D), (E), (G), (H), (J) to (N), and (Q) to (S), data were presented as means ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Student’s t test. Photo credit: Jinlong Suo, State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai.