Fig. 7. Appropriate expression of chondrogenic regulators at early days of embryonic skeletogenesis and precocious bone formation in Pbx1-/- embryos. (A-R) In situ hybridizations were performed on sections of wild-type and Pbx1-/- rib cartilage at E13.5 and 15.5. (A-L) Digoxigenin-labeled riboprobes specific for candidate chondrogenic regulators were utilized on frozen sections. (A-D) Ihh; (E-H) Sox9; (I-L) Fgfr3. (D,H,L) Expression of these genes was physiologically lost in Pbx1-/- rib cartilage at E15.5, when many more hypertrophic chondrocytes are present, most of which are undergoing accelerated autolysis and precocious mineralization, compared to wild type (C,G,K). Rib cartilage boundaries in Pbx1-/- embryos at E15.5 are indicated by dotted lines in D,H and L. (M,P) Bright-field photos of serial sections consecutive/adjacent to those where in situ hybridization was performed (N-R). 35S-labeled riboprobes for Col1a1, a marker of bone, and Col10a1, a marker of hypertrophic chondrocytes, were utilized on wax sections. In Pbx1-/- rib the presence of hypertrophic chondrocytes and scarce proliferating chondrocytes (P), is associated with advanced expression of Col1a1 in the perichondrium of the rib (Q), indicative of the forming bone collar (green arrow). Col10a1 expression is diminished in the middle of the mutant rib (R) presaging ossification and bone deposition. By contrast, in wild-type rib, proliferating chondrocytes (black arrow in M) are still present in large numbers and Col1a1 is not expressed (N). However, Col1a1 is expressed in the developing calvarial bones of both the wild-type and Pbx1-/- mutants (M and P: insets). Col10a1 is expressed at high levels in wild-type rib (O). Representative data are shown for embryos that were extensively sectioned and analyzed.
