Fig. 5.

Western blot analyses of Smad1 phosphorylation in whole tails, MEFs derived from wild-type, Cv2^−/−, Chd^−/− and Cv2^−/−;Chd^−/− treated with BMP4 protein, and lack of skeletal genetic interactions between Cv2 and Chd mutants. (A) Western blot showing comparable amounts of endogenous Smad1 phosphorylation in tails from wild-type, Cv2^−/− and Cv2^+/− 12.5 mouse d.p.c. embryos. The tail contains multiple tissues in addition to the vertebral column which is the topic of this study. No significant differences in pSmad1 are seen. (B) Addition of purified CV2 protein to Cv2^−/− MEFs inhibits Smad1 phosphorylation induced by 30 min treatment with BMP4. (C) pSmad1 levels are higher in Cv2^−/−, Chd^−/− and Cv2^−/−;Chd^−/− MEFs compared to wild-type fibroblasts upon addition of 0.3 nM of BMP4 (compare lane 5 to lanes 6–8). (D) pSmad1 response levels are higher in Cv2^−/−;Chd^−/− MEFs compared to wild-type at low amounts of BMP4 (compare lanes 3 to 4, and 5 to 6. (E–H) Genetic interaction between Cv2 and Chd in the lumbar region of the vertebral column. Bone is stained with Alizarin Red and cartilage with Alcian Blue. Dorsal view of the skeletons of wild-type (E), Chd^−/− (F), Cv2^−/− (G) and Cv2^−/−;Chd^−/− (H) neonates. In this region the skeleton of Chd^−/− (F) is indistinguishable from wild-type (E). The posterior homeotic transformation indicated by the loss of the 13^th rib (asterisk) is present in Cv2^−/− and Cv2^−/−;Chd^−/− embryos. na, neural arches.