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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).
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