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Fig. 1. Morphological analyses of the bone collar. (A-D) Whole-mount skeletal staining of (A-C) the radius (R) and ulna (U) and (D) the tibia at E18.5. Alizarin Red stains the bone red and Alcian Blue stains the cartilage blue. (C) The bone collar in Cre10; Smo^n/c is separated from the cartilage (orange arrowhead) and contains cartilage in certain areas (green arrowhead). (D) No bone collar (red arrowhead) is formed in the long bones of Ihh^n/n embryos. The mineralized hypertrophic cartilage (H) is stained dark red. (E-I) Mineralization revealed by von Kossa staining (black) on sections of E18.5 tibia counterstained with Methyl Green. Genotypes for E-G correspond to A-C, respectively. (G) The bone collar in Cre10; Smo^n/c is separated from the marrow cavity (M) by several layers of cells (orange double arrowhead). (H,I) These cells assume the morphology of chondrocytes (I, a higher magnification of the boxed region in H. B, bone; C, chondrocytes; M, marrow). The asterisk in H denotes a region where the bone collar is missing. (J) In situ hybridization using ³⁵S-labeled riboprobes against Col1(II) on a section adjacent to that in H. High levels of expression (signal in red) are detected in cells separating the bone collar and the marrow cavity in Cre10; Smo^n/c embryos (between arrows). (K,L,M) Analyses of Cre activity of the Cre10 line using Rosa26 reporter (R26R) mice. Evidence of Cre activity (strong blue staining) is detected in chondrocytes (C) as well as in the perichondrium (PC) and the periosteum (PO), whereas muscles (M) and tendons (T) are negative. A higher magnification (L) of the boxed region in K shows strong Cre activity in osteoblasts of the bone collar (red arrowheads). Strong Cre activity is also evident in osteoblasts of the primary spongiosa (red arrowheads in M).

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