Fig. 7. Deficient development of the notochord in Sox5/Sox6 compound mutants. (A) Histological analysis of notochord sheath formation in E12.5 embryos with all combinations of Sox5 and Sox6 wild-type and mutant alleles. The notochord sheath (arrow) becomes thinner as the number of mutant alleles increases. The mutation of Sox6 is more detrimental than that of Sox5. (B) Histological analysis of nucleus pulposus formation in control and Sox5^+/–/Sox6^–/– littermates. Mid-sagittal sections were photographed in the thoracic region. At E13.5, the notochord is starting to bulge in intervertebral spaces in both embryos (arrows). By E14.5, notochord cells have been completely removed from vertebral bodies (VB) in the control embryo, but incompletely removed in the mutant (arrow). The formation of cartilage matrix is delayed in E13.5 and E14.5 mutant embryos. By E16.5, the notochord has completed its transformation into nuclei pulposi in both embryos. The notochord sheath (arrow) is still visible in the mutant vertebral bodies. The mutant nuclei pulposi are smaller, eccentric and often fragmented. IA, inner annulus; IM, intervertebral mesenchyme; NP, nucleus pulposus. (C) High-magnification pictures of nuclei pulposi in E16.5 embryos. Control cells are highly swollen, as indicated by a large volume of cytoplasm (white) per cell nucleus (red). Sox5^–/–/Sox6^+/– cells are incompletely swollen and Sox5^+/–/Sox6^–/– cells hardly swollen. (D) Skeletal preparations of the vertebral column of newborn mice (P0) with various combinations of Sox5 and Sox6 null alleles. Non-mineralized cartilages and nuclei pulposi are stained with Alcian Blue, and mineralized cartilages and bones with Alizarin Red. Pictures show the L1 lumbar vertebra and flanking intervertebral discs. The nuclei pulposi (arrows) of Sox5^+/–/Sox6^+/– and Sox5^–/–/Sox6^+/+ and Sox5^–/–/Sox6^+/– mice are less expanded in width than those of the control (Sox5^+/–/Sox6^+/+) mouse. The nuclei pulposi of Sox5^+/+/Sox6^–/– and Sox5^+/–/Sox6^–/– mice are severely reduced in both width and height, and opaque instead of translucent. (E) Histological analysis of nuclei pulposi in control (Sox5^+/–/Sox6^+/+) and Sox5^+/–/Sox6^–/– newborn littermates. Mutant cells are less swollen than control cells. (F) In situ hybridization of Shh RNA (top) and histological analysis (bottom) of sagittal sections of control (Sox5^+/–/Sox6^+/+) and Sox5^+/–/Sox6^–/– newborn littermates. Pictures show a lumbar intervertebral disc and segments of flanking vertebrae. Nucleus pulposus cells (arrows) are still expressing Shh in the mutant, but not in the control. Hypertrophic chondrocytes (arrowheads) are expressing Shh in both mice. (G) In situ hybridization of Shh RNA in cross-sections of wild-type embryos. Pictures show a presumptive intervertebral disc region and the ventral part of the neural tube. Shh is expressed in E13.5 and E14.5 notochord cells (arrows), but no longer in E15.5 nucleus pulposus cells (arrow). At all three stages, Shh is expressed in the neural tube floor plate (arrowheads). (H) Picture of 1-week-old (P7) control and Sox6^–/– littermates. The Sox6^–/– mouse is developmentally delayed and exhibits a kinked tail (arrow). (I) Histological analysis of longitudinal sections through the tail of 4-day-old (P4) control and Sox6^–/– mice. Control nuclei pulposi (arrows) occupy the core region of intervertebral spaces. Mutant nuclei pulposi are small and located eccentrically, where the tail forms kinks.