Genetic System for Testing the Function of Genes in Joint Development
To generate a general system capable of specifically testing genes for functions in skeletal joint development, we engineered transgenic mice to express Cre recombinase in developing joints (Figure 1). Gdf5 is a gene strongly expressed in stripes across developing skeletal elements during embryonic joint formation. A bacterial artificial chromosome (BAC) containing the Gdf5 locus was modified by homologous recombination in bacteria to insert a cassette encoding Cre-internal ribosome entry site (IRES)-human placental alkaline phosphatase (hPLAP) into the translation start site of Gdf5 (Figure 1A). This modified BAC was then used to make lines of transgenic mice. The resulting Gdf5-Cre transgenic mice were tested for transgene expression and Cre recombinase activity by crossing them to R26R reporter mice that activate the expression of lacZ after Cre-mediated removal of transcriptional stop sequences (Soriano 1999). The resulting progeny were analyzed both for expression of the transgene by assaying HPLAP activity and for recombination of DNA by assaying LACZ activity. The progeny from all three lines showed strong LACZ expression primarily in joints, and in two of three lines HPLAP expression could also be seen in joint regions. Interestingly, HPLAP expression in the Gdf5-Cre transgenic GAC(A) line used for all subsequent breeding experiments was seen to precede LACZ expression during successive development of joints in the digits (Figure 1C) (unpublished data). These experiments clearly demonstrate that the Gdf5-Cre transgene expresses Cre recombinase and causes DNA recombination in developing joint regions.
Figure 1  A Genetic System to Drive Gene Recombination in Developing Joints
(A) A 140-kb BAC from the Gdf5 locus was modified by inserting Cre-IRES-hPLAP into the translation start site of Gdf5 and used to make transgenic mice. Not to scale. See Materials and Methods for details.
(B–E) Visualization of Gdf5-Cre driven recombination patterns based on activation of lacZ expression from the R26R Cre reporter allele. (B) LACZ activity is visible as blue staining in the ear (ea) and the joints of the shoulder (s), elbow (eb), wrist (w), knee (k), ankle (a), vertebra (vj), and phalanges (black arrowheads) of an E14.5 mouse embryo. (C) E14.5 hindlimb double-stained to show both HPLAP expression from the transgene (grey/purple staining) and LACZ expression from the rearranged R26R allele (blue staining). Note that both markers are visible in the oldest, proximal interphalangeal joint (black arrowhead), only HPLAP activity is visible in the more recently formed medial interphalangeal joint (black arrow), and neither HPLAP nor LACZ expression is visible in the youngest, most distal joint of the digit (white arrowhead). (D) Newborn (P0) forelimb with skin partially removed showing LACZ activity expressed in all phalangeal joints (red Salmon gal staining, black arrowheads) and regions of some tendons (asterisk). (E) Section through the most distal phalangeal joint of a P0 hindlimb stained with Alcian blue to mark cartilage showing LACZ expression (stained red) in all tissues of developing joints: articular cartilage (black arrowhead), precursors of ligaments and synovial membranes (black arrow), and cells where cavitation is occurring (asterisk). 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).
Figure 2  Bmpr1a Is Required for Webbing Regression and Apoptosis in Specific Regions of the Limb
(A and B) Control E14.5 forelimb (A) compared to a, E14.5 mutant forelimb (B) showing webbing between digits 1 and 2 (arrowheads) and extra tissue at the posterior of digit 5 (arrows).
(C) Gdf5-Cre induced lacZ expression from R26R in an E13.5 forelimb showing LACZ staining (blue) in metacarpal-phalangeal joints, between digits 1 and 2 (arrowhead), and in a region posterior to digit 5 (arrow).
(D and E) Sections of E14.5 hindlimbs showing apoptosis visualized by TUNEL staining (green) and proliferation visualized by staining for histone H3 phosphorylation (red). Controls show strong, uniform TUNEL staining between digits 1 and 2 (D, arrowhead) while mutants show patchy TUNEL staining interspersed with mitotic cells in similar regions (E). Scale bar = 200 μm.
(F) Quantitation of TUNEL staining and mitotic cells in the posterior region of the fifth digit shows apoptosis is reduced 30% while proliferation is increased 20% (asterisks indicate statistically significant difference).
(G and H) By E15.5, interdigital tissue has regressed in controls (G, arrowhead). In contrast, tissue remains in mutants at this location, primarily derived from cells that have undergone Gdf5-Cre-mediated recombination that inactivates Bmpr1a function and activates expression of LACZ (H). Scale bar = 75 μm. Other sites besides limb joints also have Cre-mediated lacZ expression. Starting at E13.5, LACZ activity is detected in an anterior and posterior domain of the limb bud (Figure 2C). At E14.5, LACZ activity is detectable in the developing ear pinnae, ribs, sternum, tissues in the face, and some regions of the brain and spinal cord (Figure 1B) (unpublished data). At birth, LACZ is also expressed in tendons running along the vertebral column, regions of tendons in the wrist and ankle, and some tendon insertions (Figure 1D) (unpublished data). By 5 wk of age, LACZ is also expressed in the hair follicles, ear cartilage, some cells in the growth plate of the long bones, and portions of the brain and spinal cord (unpublished data). Surprisingly, 23 of 63, or 37% of transgenic mice analyzed also show some degree of wider “ectopic” LACZ expression, which can extend throughout many different tissues in the animal. However, sustained expression of the transgene itself, as assayed by HPLAP activity, is still restricted primarily to joints in animals that show evidence of more generalized recombination based on LACZ expression (unpublished data). This suggests that in a fraction of animals, sporadic expression of Cre at some time early in development is sufficient to lead to both ectopic recombination and LACZ expression. While the fraction of animals with broader recombination patterns must be tracked and accounted for during experiments, these animals offer the potential benefit of revealing additional new functions of target genes that could be subsequently studied with additional site-specific Cre drivers.