(A) Whole-mount Alcian blue images of retrovirus GFP and DN-GNAS coinjected embryos. GFP encoding avian retrovirus vector RCAS-B was coinjected with DN-GNAS encoding retrovirus RCAS-A to allow visualization of viral expression. Thickening of the cartilaginous ribs (arrow) as well as the pelvic girdle (arrowhead) was observed. DN-GNAS virus injection alone yielded the same results as DN-GNAS coinjection with GFP virus. (B) Alcian blue and Alizarin red staining showing a cartilage nodule in the forelimb of DN-GNAS–injected side (arrow), while the contralateral side showed uniform mineralization. (C) An ectopic cartilage element was present in the hindlimb of the DN-GNAS–injected side (arrow) of a representative embryo. Note that the mineralized femur was also thicker on the injected side (asterisk) than on the contralateral side. (D) μCT analysis of GFP-injected embryos, indicating normal mineralization. (E) A DN-GNAS–injected embryo showing altered mineralization during endochondral bone formation in the ribs (arrowheads). (F) A DN-GNAS–injected embryo showing thickened mineralized ribs after endochondral bone formation (arrows). Original magnification, ×0.71 (A, low mag); ×2.0 (A, high mag); ×1.6 (B); ×1.25 (C).