Fig. 3. Lack of notochord sheath in Sox5^–/–/Sox6^–/– embryos. (A,B) Histological analysis of mid-sagittal sections of control (left) and Sox5^–/–/Sox6^–/– (right) embryos at E11.5 (A) and E13.5 (B). Top panels show the notochord surrounded with vertebral bodies (VB) and intervertebral mesenchyme (IM). Bottom panels show the notochord at higher magnification. The control notochord is surrounded with extracellular matrix (arrows) that stains with Alcian Blue. The mutant notochord has fewer cells and lacks the matrix sheath. (C) In situ hybridization of RNAs for extracellular matrix proteins in mid-sagittal sections of control and Sox5^–/–/Sox6^–/– E13.5 embryos. The left and right panel pairs are low- and high-magnification pictures, respectively, of the same areas. Arrows indicate the notochord. Col2a1 expression is partially downregulated in mutant notochord cells and chondroblasts. Agc1 and Hspg2 expression is severely downregulated in both mutant cell types. Lamc1 expression is not significantly altered or is only slightly downregulated in mutant notochord and intervertebral cells. (D) In situ hybridization of RNAs for notochord-specific non-matrix proteins in control and Sox5^–/–/Sox6^–/– E13.5 embryos. Shh RNA was tested in cross-sections, and T and Car3 RNA in mid-sagittal sections. The Sox5^–/–/Sox6^–/– mutation does not affect expression of these RNAs.