Fig. 4. Removal of Sox5^–/–/Sox6^–/– notochord cells from intervertebral spaces. (A) Histological analysis of mid-sagittal sections of E14.5 and E15.5 control and Sox5^–/–/Sox6^–/– embryos. At E14.5, in the distal tail, the control notochord is continuous, but the mutant is interrupted in intervertebral regions (arrows in first row). In the intervertebral regions of the proximal tail, the control notochord is bulging (arrow in second row), whereas mutant notochord cells are missing. In both segments of the tail, control vertebral bodies are starting to accumulate cartilage matrix, but mutant vertebral bodies hardly are. In the E14.5 thoracic region and E15.5 lumbar region (third and fourth rows), control notochord cells have been removed from vertebral bodies, but the notochord sheath is still visible (arrow in left panel in third row). Vertebral body and inner annulus cartilage is developing, and nuclei pulposi expanding. In mutants, vertebral bodies are accumulating a deficient cartilage matrix, and notochord cells are forming trains (arrows in right panels in third and fourth rows) between vertebral and intervertebral regions. (B) In situ hybridization of Car3 RNA in an E13.5 Sox5^–/–/Sox6^–/– embryo. The RNA signal identifies notochord stretches in vertebral bodies, but not in intervertebral regions (higher cell density, arrows). Arrowheads indicate notochord cells found at ectopic positions, i.e. dorsal and ventral (mid-sagittal section) or lateral (para-sagittal section) to the normal axis of the notochord, aside intervertebral regions. (C) Histological analysis of cross-sections of E12.5 control and Sox5^–/–/Sox6^–/– presumptive intervertebral discs. The control notochord is intact in all regions. The mutant notochord is still visible in the cervical and caudal regions, but not in the lumbar region. (D) In situ hybridization of Shh RNA in cross-sections of E12.5 Sox5^+/–/Sox6^+/– and Sox5^–/–/Sox6^–/– embryos through intervertebral regions. Shh RNA is detected in the floor plate of the neural tube in both embryos (arrowheads). It is also detected in notochord cells in both lumbar and cervical control regions and in the Sox5^–/–/Sox6^–/– cervical region (arrows). By contrast, no Shh RNA signal is seen in the Sox5^–/–/Sox6^–/– lumbar region (arrow).