Nov expression and phenotypes of E16.5 skeletons. A and B. RNA in situ hybridization showing Nov expression in the hind foot at E16.5. A. Strong expression in the myotendenous junctions (arrow) and in the mesenchyme of the joints adjacent to the cartilage elements, but no expression in condensing cartilage. B. High expression in the mesenchyme overlying the cartilage elements (arrow) and in the digital tendons (arrow head). C-G. Skeletal staining with Alizarin red and Alcian blue of E16.5 Novdel3+/-, Novdel3-/- and wild type (+/+) embryos. C. Wild type forefoot showing ossification of digits (arrow). D. No ossification in Novdel3-/- digits (arrow), but intense staining with Alizarin red of the radius and ulna indicating increased bone mineralization. E-G Vertebrae and rib cages of wild type (E), Novdel3-/- (F) and Novdel3+/- (G) showing delay in ossification of the vertebrae in the mutant embryos. No ossification is present in the Novdel3-/- embryo, and whilst ossification is taking place in the Novdel3+/- embryo (G), it is not occurring in the ordered, sequential manner observed in the wild type. Thoracic vertebrae numbered 1–13. H-Q. Histological sections of E16.5 wild type and Novdel3-/- embryos. H, J, L, N, P: wild type; I, K, M, O, Q: Novdel3-/- littermate. H, I: Haematoxylin and Eosin stained sections showing expanded perichondrium and periosteum in Novdel3-/- and a thicker bone collar (arrow head) compared to the wild type. o: ossified bone; h: hypertrophic cartilage; ph: pre-hypertrophic cartilage; c: columnar chondroctyes. J, K: Haematoxylin and Eosin stained sections at higher magnification at the junction of pre-hypertrophic/hypertrophic chondrocytes, adjacent to the border of the perichondrium/periosteum, showing abnormal morphology of chondrocytes and matrix in the mutant (K). L, M: Alcian blue staining of cartilage, showing expansion of the cartilage element and blurring of its borders in the Novdel3-/- embryo (M) compared to wild type (L). N, O: PCNA staining showing a sharp demarcation (black dashed line) between proliferating chondrocytes and pre-hypertrophic chondroctes in the wild type (N), but not in the Novdel3-/- mutant (black dashed line) (O). P, Q: Von Kossa staining for mineralised bone showing a shorter and thicker bone collar (indicated by the black bar) in the pre-hypertrophic/hypertrophoic zone of the Novdel3-/- mutant (Q) compared to wild type (P). Scale bars in A B = 20 μm; H,I,N,O,P,Q = 10 μm; J-M = 5 μm.