IVD expression of Pax and Sox genes and proposed model. (A) Sagittal section of E13.5 WT vertebral column showing Pax1 expression in the IVD anlagen detected by SISH assay. (B) Mallory's tetrachrome staining of E15.5 WT vertebral column; IAF is stained blue and OAF is stained dark red. (C,D) Immunohistochemistry and Alcian blue staining of vertebral column showing (C) Pax1 and (D) Pax9 protein expression restricted to the OAF at E15.5. (E-I) Expression of Pax1, Pax9, Smad3, Tgfb3 and Bmp4 in the IVD anlagen at E12.5. (J-M) Expression of Pax1, Pax9, Sox5 and Sox9 in the IVD anlagen at E13.5. (N-R) Expression of Pax1, Pax9, Sox9 at E15.5 and Tgfb3 and Bmp4 at E14.5 in the IVD. (S) Proposed negative feedback loop mechanism between Pax, Sox, Bmp4 and TGF-B pathway at E12.5. Black arrows indicate the final site of expression at E14.5. Brackets indicate IVD region. VB, vertebral body; n, notochord; IVD, intervertebral disc; NP, nucleus pulposus; IAF, inner annulus fibrosus; OAF, outer annulus fibrosus; WT, wild type; SISH, sectioned in situ hybridization; D, dorsal; V, ventral.