Fig. 2. Skeletal abnormalities in Wnt9a mutants. (A) Alizarin Red/Alcian Blue staining of skeletons of wild-type and Wnt9a^-/- E17.5 littermates, and of forelimbs, hyoid bones, atlas from WT (on top) and Wnt9a^-/- (below) newborn littermates. Smaller mineralized zones are found in the scapula and humerus, and, to a lesser extend, in ulna and radius in the mutant forelimbs compared with wild type. Hypoplastic hyoid bone and atlas. (B) Table showing quantification of size reduction with regard to the total length and the mineralized regions of mutant skeletal forelimb elements (Mut=Wnt9a^lacZ/lacZ) in comparison with those from wild type (wild type=Wnt9a^+/+) and heterozygous (Het=Wnt9a^+/lacZ). Average length of wild-type/heterozygous elements was set to 100%. Mutant and littermate control limbs were collected from seven litters. (C) Dorsal (a) and ventral (b) view of Alizarin Red/Alcian Blue-stained wild-type and Wnt9a^-/- heads from newborns, showing that in Wnt9a^-/- the frontal bones are further apart (arrow in a), a smaller ossification center in the supraoccipital (so) bone and an abnormally shaped basioccipital bone (bo) (arrow in b). (c) Lateral and (d) dorsal view of Alcian Blue-stained wild-type and Wnt9a^-/- newborn heads, showing expansion of the cartilaginous base in the region of the parietal bone (arrowhead in c) and presence of ectopic Alcian Blue-positive areas in the sagittal suture of the skull (arrowhead in d). (e,f) Coronal sections through skulls of wild-type and Wnt9a^-/- newborns, at the levels indicated in d. Van Kossa/Alcian Blue-stained sections (e,f), showing that the two frontal bone plates are further apart from each other in Wnt9a mutants (arrowheads in e), dorsal expansion of the cartilaginous base (arrow) and the presence of Alcian Blue-positive cells in the sagittal suture region of the Wnt9a mutant skull. (e',f') Col2a1 in situ hybridization on sections adjacent to those shown in e,f, showing Col2a1-expressing cells within the sagittal suture (asterisks), which are absent in the wild-type littermates.