Joint patterning defects caused by single and double mutations in members of the bone morphogenetic protein (BMP) family

Elaine E. Storm and David M. Kingsley* - Joint patterning defects caused by single and double mutations in members of the bone morphogenetic protein (BMP) family SUMMARY The mouse brachypodism locus encodes a bone morphogenetic protein (BMP)-like molecule called growth/differentiation factor 5 (GDF5). Here we show that Gdf5 transcripts are expressed in a striking pattern of transverse stripes within many skeletal precursors in the developing limb. The number, location and time of appearance of these stripes corresponds to the sites where joints will later form between skeletal elements. Null mutations in Gdf5 disrupt the formation of more than 30% of the synovial joints in the limb, leading to complete or partial fusions between particular skeletal elements, and changes in the patterns of repeating structures in the digits, wrists and ankles. Mice carrying null mutations in both Gdf5 and another BMP family member, Bmp5, show additional abnormalities not observed in either of the single mutants. These defects include disruption of the sternebrae within the sternum The skeletal elements of the vertebrate limb are derived during embryonic development from mesenchymal cells, which condense and initiate a differentiation program that results in cartilage and bone. The diverse shapes and patterns of skeletal elements arise from several fundamental morphogenetic behaviors of the limb mesenchyme (Shubin and Alberch, 1986; Oster et al., 1988). These behaviors include the initial de novo condensation of cells into the rough outlines of the future skeletal elements, the growth and branching of some of the condensations to produce Y-shaped bifurcations, and the segmentation of condensations into individual elements. For example, the humerus, radius and ulna of the forelimb arise initially from a single condensation that grows, branches and segments to produce the precursors of the three separate bones. Variations in the temporal and spatial patterns of condensation, branching and segmentation underlie much of the evolutionary modifications of limb structures that are seen in different vertebrate species (Shubin and Alberch, 1986; Oster et al., 1988). Despite the importance of mesenchymal condensations in skeletal patterning, relatively little is known about the factors that control their formation, branching and segmentation (Hall and Miyake, 1992). Recent studies, however, suggest that bone and abnormal formation of the fibrocartilaginous joints between the sternebrae and ribs. Previous studies have shown that members of the BMP family are required for normal development of cartilage and bone. The current studies suggest that particular BMP family members may also play an essential role in the segmentation process that cleaves skeletal precursors into separate elements. This process helps determine the number of elements in repeating series in both limbs and sternum, and is required for normal generation of the functional articulations between many adjacent structures in the vertebrate skeleton. morphogenetic proteins (BMPs) may play a crucial rule in inducing the formation of particular skeletal condensations. BMPs were originally purified from adult bones based on their remarkable ability to trigger the entire sequence of condensation, cartilage differentiation and bone formation when implanted at ectopic sites in animals (Urist, 1965; Reddi and Huggins, 1972). Biochemical purification, cloning studies and homology screens have shown that the vertebrate genome contains a large number of related BMPs, most of which belong to the transforming growth factor beta (TGF-b ) family of secreted signalling molecules (Rosen and Thies, 1992; Kingsley, 1994a,b for review). Particular members of the BMP family are expressed at times and places consistent with a role in inducing the formation of early skeletal condensations (Lyons et al., 1989; King et al., 1994). Null mutations in the Bmp5 gene have also been shown to reduce or eliminate particular skeletal condensations during normal mouse development, confirming an essential role for this signal in formation of particular anatomical features in the skeleton (Kingsley et al., 1992, 1994b; King et al., 1994). We recently reported that the mouse brachypodism locus encodes a new member of the BMP family called growth differentiation factor 5 (GDF5) (Storm et al., 1994). Mutations at this locus have been studied for forty years because of their interesting effects on the pattern of repeating elements in the vertebrate limb (Landauer, 1952; Grneberg and Lee, 1973; Elmer and Selleck, 1975; Hinchliffe and Johnson, 1980). Brachypodism mutations reduce the length of several long bones of the limb, and cause the first two bones in most digits to be replaced by a single element. Molecular studies have shown that these limb-patterning defects are due to frame-shift mutations in the Gdf5 gene (Storm et al., 1994). Although these mutations should completely inactivate Gdf5, brachypodism mice are viable and fertile, and show little defects in skeletal structures outside the limbs. In order to gain insight into how Gdf5 participates in skeletal patterning, we have undertaken a detailed analysis of RNA expression during limb development and have generated double mutants that lack two different BMPs: Gdf5 and Bmp5. These combined studies suggest that BMP family members may play important roles not only in generating early skeletal condensations, but also in controlling the segmentation events that generate the joints between skeletal structures. MATERIALS AND METHODS The bpJ and bp3J alleles occurred spontaneously on the inbred A/J and BALB/cJ strains, respectively, and were maintained on the corresponding isogenic backgrounds. The se20Zb mutation is a null allele at the Bmp5 locus and is maintained on an outbred background (Kingsley et al., 1992). Double mutants were generated by crossing bpJ or bp3J homozygotes to se2OZb homozygotes, intercrossing the F1 progeny and collecting short-eared animals with short feet and limbs. Double mutants were maintained by intercrossing. In situ hybridization The mouse Gdf5 probe was generated from a 267 bp PCR product in the 3 untranslated region (bases 1837-2103, Storm et al., 1994) cloned into pCRII (Invitrogen). The mouse Bmp5 probe was generated from a 1.03 kb PCR product of the pro-region of the cDNA (bases 671-1702, King et al., 1994) cloned into pCRII (Invitrogen). Digoxigenin (DIG) cRNA probes were prepared using the Genius 4 kit (Boehringer Mannheim). Timed matings were performed and embryonic day 0.5 was designated as noon on the day a vaginal plug was observed. Embryos were dissected in cold PBS and frozen quickly on dry ice in OCT compound (Miles). Frozen tissue was stored at - 80C until sectioned at 12 m m on Vectabond (Vector)-treated slides at - 20C. In situ hybridization was carried out using a modification of previous protocols (Schaeren-Wiemers and Gerfin-Moser, 1993). Sections were dried at (...truncated)