Cellular and molecular analysis of SoxC^Prx1Cre limbs. (A) TUNEL assay (green) in hind limb sections from E13.5–14.5 control and SoxC^Prx1Cre littermates. Counterstaining is with DAPI (blue). Arrows, mesenchyme between digits 2 and 3. Arrowheads, presumptive joints. (B and C) RNA in situ hybridization of E13.5–14.5 control and SoxC^Prx1Cre hind paw sections with various probes, as indicated. Arrows, presumptive joints. Arrowheads, interdigital and perichondrium cells. (D) E14.5 hind limb sections immunostained for β-catenin (green) and counterstained with DAPI (blue). Magnified images of boxed regions are shown in side panels. j, joint; pC, perichondrium. (E) RNA in situ hybridization of E14.5 control and SoxC^Prx1Cre hind paw sections with various probes, as indicated. (F) Sox9 RNA in situ hybridization of hind limb sections from E11.5–13.5 control and SoxC^Prx1Cre embryos. (G, left) Coronal section of E14.5 tibias. (right) RNA in situ hybridization and immunofluorescence for SOX9 (red) and fibronectin (green). Pictures were taken at the level of the box shown in the left. (H and I) β-Catenin and SOX9 levels in whole-tissue extracts from control and SoxC^Prx1Cre mutant hind limb buds (H) and in membranous/cytoplasmic and nuclear extracts from primary limb bud cells (I). Cells were isolated from E11.5 SoxC^fl/fl limb buds and treated in culture with lacZ or CRE adenovirus for 24 h. Representative Western blots are shown. Fold changes in β-catenin and SOX9 levels were normalized to the levels of GAPDH and a nonspecific protein (nsp) in membrane/cytoplasm (mbr/cyt.) and nucleus fractions, respectively. *, P < 0.05; n = 3 embryo littermates (H) and experimental replicates (I). All experiments were repeated at least three times. Each panel shows the results of a representative one.