Fig 2: YAP signaling promotes Procr+ cells expansion at rupture sites through a combination of promoting cell division and enhancing Procr expression.Illustration of superovulation and analysis strategy as indicated using Yap1fl/fl (Ctrl) and Procr-CreER;Yap1fl/fl (Yap1-cKO) mice (A). At ovulation stage, the percentage of Procr+ ovarian surface epithelium (OSE) cells in Ctrl and Yap1-cKO were FACS analyzed (B) and quantified (C). n = at least three mice in each group and displayed as mean ± standard error of the mean (SEM). Unpaired two-tailed t-test is used for comparison. ***p < 0.001. Illustration of superovulation and analysis strategy as indicated using Procr-rtTA;TetO-H2B-GFP+/- (Ctrl) and Procr-rtTA;TetO-H2B-GFP+/-;TetO-Vgll4+/- (Vgll4-OE) mice (D). At ovulation stage, ovary section imaging showed that at the rupture sites, the number of H2B-GFP+ (Procr+) cells in Ctrl (arrowheads in E) is higher than those in Vgll4-OE (arrowheads in F). Scale bar, 100 µm. Quantification was shown in (G). n = 3 pairs of mice and 15 images in each group. Unpaired two-tailed t-test is used for comparison. ***p < 0.001. The percentage of Procr+ OSE cells were analyzed by FACS at ovulation stage (H). The percentage of Procr+ cells in Ctrl are higher than that in Vgll4-OE (H, I). n = at least 3 mice and displayed as mean ± SEM. Unpaired two-tailed t-test is used for comparison. ***p < 0.001. Illustration of Tead4 motif in Procr promoter region (J). TEAD4 chromatin immunoprecipitation-qPCR (ChIP-qPCR) analysis using cultured primary OSE cells showed the enrichment of Procr promoter, and Ctgf promoter was used as positive control (K). n = 2 biological repeats. Unpaired two-tailed t-test is used for comparison. **p < 0.01, *p < 0.05, n.s., not significant. Analysis of luciferase reporter activity driven by WT (L) and Tead4 motif (-1486 to -1481 bp) deleted Procr promoter (M) in HEK293T cells transfected with increased amount of Yap1 overexpression plasmids. Data are pooled from three independent experiments and displayed as mean ± SEM. Unpaired two-tailed t-test is used for comparison. ***p < 0.001, **p < 0.01, n.s., not significant. (N) A proposed model of which YAP signaling promotes Procr+ cells expansion at rupture site through a combination of promoting cell division and enhancing Procr expression. Figure 4—source data 1.Numeric data for Figure 4C, G, I, and K–M.

Fig 3: RUNX2 rescues Vgll4 deletion–mediated inhibition of osteogenesis differentiation.(A) Coimmunoprecipitation experiments of RUNX2, TEAD1, VGLL4, and VGLL4 HF4A in HEK-293T cells. The arrow indicated IgG heavy chain. (B) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2, VGLL4, VGLL4 HF4A, and TEAD1 (n = 3). (C) RT-PCR analysis of Alp expression in calvarial cells. Cells isolated from WT mice were infected with GFP, TEAD1, VGLL4, or VGLL4 HF4A lentivirus. (D) Coimmunoprecipitation experiments of RUNX2, TEAD1, and an increasing amount of VGLL4 or VGLL4 HF4A in HEK-293T cells. The arrow indicated IgG heavy chain. (E) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2, TEAD1, and an increasing amount of VGLL4 or VGLL4 HF4A. (F) Competitive GST pull-down assay to detect the effect of VGLL4 Super-TDU on the interaction between RUNX2 and TEAD4. (G) Cells isolated from Vgll4fl/fl and Vgll4prx1 mice were infected with GFP and RUNX2 lentivirus. Osteoblast differentiation was evaluated by Alp staining and Alizarin red staining after culture in osteoblast differentiation medium for 7 days (top) and 14 days (bottom). Data are representative of three independent experiments. Scale bars, 3 mm. (H) Alp activity quantification was measured by phosphatase substrate assay (n = 3). (I) Relative mRNA levels of Vgll4, Runx2, Alp, Col1a1, and Osterix were quantified by RT-PCR. (J) Schematic model of VGLL4/TEADs/RUNX2 in regulating osteogenic differentiation. In (B), (C), (E), (H), and (I), data were presented as means ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Student’s t test.

Fig 4: Inactivation of VGLL4 in MSCs leads to impaired bone ossification.(A) Skeletons of Vgll4fl/fl and Vgll4prx1 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 Vgll4fl/fl and Vgll4prx1 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 Vgll4fl/fl and Vgll4prx1 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 Vgll4fl/fl and Vgll4prx1 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 Vgll4fl/fl and Vgll4prx1 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.

Fig 5: VGLL4 promotes RUNX2 activity by competing for its binding to TEADs.(A) Coimmunoprecipitation experiments of RUNX2, TEADs, and VGLL4 in HEK-293T cells. The arrow indicated IgG heavy chain. (B) Coimmunoprecipitation experiments of RUNX2 and TEADs in BMSCs cells of Vgll4fl/fl mice treated with GFP and Cre lentivirus. (C) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2, TEADs, and VGLL4. (D) Cells isolated from Vgll4fl/fl and Vgll4prx1 mice were infected with GFP and TEAD shRNA lentivirus. Osteoblast differentiation was evaluated by Alp staining and Alizarin red staining after culture in osteoblast differentiation medium for 7 days (top) and 14 days (bottom). Data are representative of three independent experiments. Scale bars, 3 mm. (E) Alp activity quantification was measured by phosphatase substrate assay (n = 3). (F) Relative mRNA levels of Vgll4, Runx2, Alp, Col1a1, and Osterix were quantified by RT-PCR. In (B), (D), and (E), data were presented as means ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Student’s t test.