Fig 2. Young ATC;Adgrg6^f/f mutant mice display alterations in IVDs consistent with disc degeneration pathology.

(A-D’) Representative IVD tissues stained Safranin-O/Fast green (SO/FG) (Induced from E0.5-P20, n = 3 for each group.) No overt structural defects were observed in ATC;Adgrg6^f/f mutant IVDs compared with controls at P2 at midline (A, B) or at 1.5 months of age (C-D’), except for the mild increase of acellular clefts in the CEP and GP at 1.5 months (yellow arrowheads, D’). Section in panel D is a little bit pass midline to show the obvious acellular clefts in CEP and GP. (E-O) IHC (E-N) and qPCR (O) analyses of common markers of degenerative disc in mice at 1.5 months (induced from E0.5-P20). ATC;Adgrg6^f/f mutant IVDs display reduced expression of markers of healthy disc: SOX9/Sox9 (blue arrows, E’) and PRG4/Prg4 (blue arrows, G), and mildly reduced COLII/Col2a1 (yellow arrows, K). They also display increased expression of hypertrophic marker COLX/Col10a1 (red arrows, N), extracellular matrix modifying enzymes (MMP-13/Mmp13 (red arrows, J), Adamts4, and Adamts5), fibrosis markers (Col1a1 and Col3a1). (E-N, n = 3 for each group. O, n = 3 biological replicates and representative result is shown. Bars represent mean ± SD. *p≤0.05, two-tailed Student's t Test.) (P, Q) Mechanical testing using 5% strain cyclic loading (stiffness mean w/95% CI, *p < 0.05) (P), and 50% monotonic overloading (stiffness mean w/95% CI, **p < 0.01) (Q), demonstrating increased stiffness in ATC;Adgr6g^f/f mutant lumbar IVDs (induced from E0.5-P20, n = 4 for each group, 4 IVDs were analyzed /mouse). Scale bars: 100μm in (A-D’); 50μm in (E-N). AF- annulus fibrosis, CEP- cartilaginous endplate, GP- growth plate, and NP- nucleus pulposus.