GAC(A) mice were crossed with lacZ ROSA26 Cre reporter strain (R26R) mice to analyze the pattern of Cre-mediated lacZ recombination throughout development. Joints in developing limbs begin forming in a proximal-distal pattern such that the shoulder joint forms prior to the elbow joint. In addition, three major stages of early joint development have been defined by histology as (1) interzone formation, (2) three-layer interzone formation, and (3) cavitation (Mitrovic 1978). Consistent with the proximal-distal pattern of joint development in the limbs, LACZ activity is seen at embryonic day 12.5 (E12.5) in the more proximal joints, including the shoulder and knee (unpublished data). By E14.5, LACZ expression is typically seen in all but the most distal joints of the limbs (Figure 1B and 1C), but with some variability in both strength and extent of expression from embryo to embryo. The strongest-staining embryos often have additional staining in fingertips (not seen in the E14.5 embryo in Figure 1C, but clearly detectable in the E13.5 embryo shown in Figure 2). Sections through developing joints show that LACZ is present in many cells at the interzone stage (unpublished data). However, expression of LACZ in nearly 100% of joint cells is not achieved until the three-layer interzone stage (for example, in the knee joint at E14.5 or in any of the phalangeal joints at E16.5 (unpublished data). Within the developing skeleton, Cre-mediated expression of LACZ remains strikingly specific to joints throughout development. Furthermore, it is seen in all the structures of postnatal synovial joints including the articular cartilage, joint capsule, and synovial membrane (Figure 1D and 1E) (unpublished data). These patterns are consistent with the well-established expression of Gdf5 in interzone regions during embryonic development (Storm and Kingsley 1996). Adult expression patterns of the Gdf5 gene are not as well characterized, but Gdf5 expression has previously been detected in adult articular cartilage using both RT-PCR and immunocytochemistry (Chang et al. 1994; Erlacher et al. 1998; Bobacz et al. 2002).