Fig. 2. Loss of Gli3 in Ihh-deficient limbs rescues chondrocyte proliferation and the onset of hypertrophic differentiation, but not bone collar formation. (A-L) Hematoxylin and Eosin staining of E16.5 wild-type (A,D,G,H), Ihh^–/– (B,E,I,J) and Ihh^–/–;Gli3^–/– (C,F,K,L) limb sections. (A-C) Compared with Ihh^–/– mutants (B) the size of the skeletal elements is increased and hypertrophic chondrocytes are restricted to the center of the cartilage anlagen in Ihh^–/–;Gli3^–/– double mutants (C). D-F are higher magnifications of the boxed regions in A-C; G-L show the chondrocyte morphology of distal (G,I,K) and central (H,J,L) regions indicated by the boxes in D-F. In Ihh^–/– limbs no columnar chondrocytes can be detected, whereas wild-type and Ihh^–/–;Gli3^–/– cartilage anlagen have a zone of distal (D,F, red bar) and columnar (D,F, blue bar) chondrocytes. (M-O) The chondrocyte proliferation rate in Ihh^–/–;Gli3^–/– limbs (O) is similar to wild-type levels (M) and strongly increased compared to Ihh^–/– limbs (N). (P-U) Van Kossa staining detects mineralization of chondrocytes of E16.5 wild-type (P), Ihh^–/– (Q, red arrow in the humerus) and Ihh^–/–;Gli3^–/– limbs (R). S-U are higher magnifications of the boxed regions in P-R. Wild-type limbs form a bone collar adjacent to hypertrophic chondrocytes (S, red arrow), which is missing in Ihh^–/– (T) and Ihh^–/–;Gli3^–/– mutants (U, red arrow). In Ihh^–/–;Gli3 ^–/– limbs, bone collar-like structures form in restricted regions (U, black arrowhead).