Fig. 4. Requirement of Cv2 for cartilage differentiation in vivo and in vitro. (A-H) E14.5 and (I-R) E12.5. (A,B) Nuclear Fast Red and Alcian Blue staining of the cross-sections of the trunk region at E14.5. The vertebral arch was lost in the mutant (B). (C,D) Hematoxylin and Eosin staining of the neighboring sections of A and B (higher magnification views of the vertebral arch region indicated by squares in A and B). The condensed cell mass corresponding to the dorsal vertebral arch cartilage (arrowheads) was replaced with the mesenchymal cells in the mutant (D). (E,F) The vertebral body was reduced and had less cartilage matrix in the mutant (F). (G,H) Sagittal sections showed that the rostrocaudal length of the vertebral body was also reduced in the mutant (bracket, compare H with G). ivd, intervertebral disc. (I,J) Immunostaining of the early dorsal sclerotomal marker Zic1 in the presumptive vertebral arch region showed no reduction in the mutant (J) at E12.5. (K,L) In situ hybridization analysis of the early sclerotomal gene Pax1 in the presumptive vertebral body region showed no reduction in the mutant (L). (M) RT-PCR analysis of the trunk tissues also showed that the sclerotomal markers Pax1 and Sox9 were expressed normally in the mutant at E12.5. The expression levels of the Bmp genes were also unchanged in the mutant. (N,O) Hematoxylin and Eosin staining of the presumptive vertebral body (surrounding the notochord) in the control (N) and mutant (O) mice. (P,Q) Ki67 immunostaining. Ki67⁺ mitotic cells were increased in the vertebral body region in the mutant (Q). Scale bar: 50 µm. (R) Statistical analysis of P and Q. The percentages of Ki67⁺ cells in the 150 µm x 150 µm square regions surrounding the notochord were presented. The averages ±s.d. (error bars) from three sets of littermates are shown. ^*P<0.05. (S) RT-PCR analysis showed that Cv2^-/- MEF cells had attenuated responses to Bmp4 for the induction of aggrecan 1 and Col2a1.