(a-d) μCT scans of Ddr2fl/fl and Col2a1Cre;Ddr2fl/fl skulls (3-month-old) showing reduced anterior-posterior skull length, length of individual bones and increased anterior and, to a lesser extent, posterior skull width in conditional knockout mice. Note thinning of frontal bone in Col2a1Cre;Ddr2fl/fl skulls, but no effect on cranial sutures. Scale bars: 1 mm. (e) Quantification of frontal, parietal, and occipital bone thickness. (f–h) Quantification of μCT scans showing enlarged synchondroses associated with shortening in cranial base bone lengths in Col2a1Cre;Ddr2fl/fl skulls. Data are presented as mean ± SD. (n=10). *p<0.01, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant, two-tailed unpaired t test. (i) Alcian blue and alizarin red whole mount staining showing 2-week Col2a1Cre;Ddr2fl/fl skulls had wide cranial base synchondroses compared with Col2a1Cre and Ddr2fl/fl. Scale bar: 500 μm. (j) Immunofluorescence images showing reduced pDDR2 (red) immunostaining indicating loss of DDR2 signaling in Col2a1Cre;Ddr2fl/fl mice (Quantification in k). Scale bar: 50 μm. Boxed region is shown at higher magnification, lower panel. Cell nuclei were stained with DAPI (blue). (l-n) Analysis showing Col2a1Cre;Ddr2fl/fl mice exhibited time-dependent widening in resting zone, altered polarization and ectopic hypertrophy. (l) Time-course analysis using H&E staining shows no difference in histological structures of the ISS between Col2a1Cre;Ddr2fl/fl mice and their control littermates at P1, but during the first 2 weeks, the resting zone became abnormally wide (red lines) and exhibited ectopic hypertrophy on the ventral side of cranial base synchondrosis (red box). Scale bar: 50 μm. (m) Immunofluorescent images of GM130 staining (green) shows well-defined Golgi staining adjacent to the nucleus of cells in RZ of wild type synchondroses, but in mutant synchondroses, GM130 immunostaining is diffuse and ill-defined indicating disturbed cell organization. Boxed region is shown in higher magnification, lower panel. Cell nuclei were stained with DAPI (blue). Scale bar: 20 μm. (n) Linear measurements shows increased spacing between chondrocytes in resting zone of wildtype and mutant synchondroses. Spacing between chondrocytes was measured by drawing lines between chondrocytes in resting zone using ImageJ. (a–h) 3-month-old mice, (i–n) 2-week-old mice. Data are presented as mean ± SD. (n=3). ****p<0.0001, two-tailed unpaired t test.