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Human Molecular Genetics Advance Access originally published online on July 26, 2007
Human Molecular Genetics 2007 16(19):2366-2375; doi:10.1093/hmg/ddm195
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

A novel dominant-negative mutation in Gdf5 generated by ENU mutagenesis impairs joint formation and causes osteoarthritis in mice

Hiroshi Masuya1,*, Keiichiro Nishida2, Tatsuya Furuichi4, Hideaki Toki1, Gen Nishimura5, Hidehiko Kawabata6, Haruka Yokoyama1, Aki Yoshida3, Sayaka Tominaga4, Junko Nagano1, Aya Shimizu1, Shigeharu Wakana1, Yoichi Gondo7, Tetsuo Noda1, Toshihiko Shiroishi1 and Shiro Ikegawa4

1 Mouse Functional Genomics Research Group, RIKEN GSC, 3-1-1 Kouyadai, Tsukuba, Ibaraki 305-0074, Japan, 2 Department of Human Morphology and 3 Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan, 4 Laboratory for Bone and Joint Diseases, SRC, RIKEN, 4-6-1 Shirokanedai, Minato-ku, 108-8639 Tokyo, Japan, 5 Department of Radiology, Tokyo Metropolitan Kiyose Children’s Hospital, 1-3-1 Umezono, Kiyose City, Tokyo 204-8567, Japan, 6 Department of Orthopaedic Surgery and Rehabilitation, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodou-cho, Izumi City, Osaka 594-1101, Japan and 7 Population and Quantitative Genomics Team, RIKEN GSC, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa 230-0045, Japan

* To whom correspondence should be addressed. Tel: +81 298369013; Fax: +81 298369017; Email: hmasuya{at}gsc.riken.jp

Received May 11, 2007; Accepted July 13, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Growth and differentiation factor 5 (GDF5) has been implicated in chondrogenesis and joint formation, and an association of GDF5 and osteoarthritis (OA) has been reported recently. However, the in vivo function of GDF5 remains mostly unclarified. Although various human GDF5 mutations and their phenotypic consequences have been described, only loss-of-function mutations that cause brachypodism (shortening and joint ankylosis of the digits) have been reported in mice. Here, we report a new Gdf5 allele derived from a large-scale N-ethyl-N-nitrosourea mutagenesis screen. This allele carries an amino acid substitution (W408R) in a highly conserved region of the active signaling domain of the GDF5 protein. The mutation is semi-dominant, showing brachypodism and ankylosis in heterozygotes and much more severe brachypodism, ankylosis of the knee joint and malformation with early-onset OA of the elbow joint in homozygotes. The mutant GDF5 protein is secreted and dimerizes normally, but inhibits the function of the wild-type GDF5 protein in a dominant-negative fashion. This study further highlights a critical role of GDF5 in joint formation and the development of OA, and this mouse should serve as a good model for OA.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Osteoarthritis (OA, MIM 165720 [OMIM] ) is the most common form of joint disorder and is characterized by the degeneration of articular cartilage. There are apparent genetic influences on OA (1–4): a number of genetic loci associated with OA (5–7) as well as specific associations between certain genes and OA (8–13) have been reported. The precise etiology of the disease is still unknown; however, a few animal models of OA produced by gene-targeting techniques have been created (14,15).

Growth and differentiation factor 5 (GDF5), also known as cartilage-derived morphogenetic protein 1, is a member of the bone morphogenic protein (BMP) family that are secretory signaling molecules binding to specific serine–threonine kinase receptors. Gdf5 is expressed in osteochondral progenitors of the appendicular (16–18) and axial skeleton. During early limb development, Gdf5 is expressed in the condensing mesenchyme of the cartilage primordium (16,17,19). In later development, its expression is confined to the site of joint formation termed the interzone region (18–20). GDF5 mutations in human and mice cause alterations in the appendicular skeleton. Human skeletal dysplasias associated with GDF5 mutations include the Hunter–Thompson (21), DuPan (22) and Grebe types of chondrodysplasia (22–24), angel-shaped phalangoepiphyseal dysplasia (25,26), brachdactyly type C (25,27–29), brachydactyly type A2 (30), proximal symphalangism (31) and multiple synostosis syndrome 2 (32). Various functional consequences of GDF5 mutations have been reported, including inhibition of the ligand–receptor interaction with preserved partial function (L441P), inhibition of BMP secretion (C400Y) and gain of function with acquisition of BMP2-like properties, as a result of loss of receptor-binding specificity (R438L) (23,30).

In contrast to the broad spectrum of human GDF5 mutations and phenotypes, only a series of loss-of-function mutations in Gdf5 with brachypodism (bp) (Gdf5bp, Gdf5bp-H and Gdf5bp-J) have been found in mice. These animals present with a reduction in number of digits (symphalangism or fusion of digit joints) and shortening of the short tubular bones (brachydactyly) (19,32). Transgenic mice with the Gdf5 gene under the control of the type II collagen promoter and enhancer presented with chondrodysplasia (33). This phenotype is characterized by expanded cartilage, consisting of enlarged hypertrophic zones and reduced proliferating zones. These studies suggest GDF5 regulates joint formation, growth and differentiation of chondrocytes during development. However, the precise role of GDF5 signaling in the maintenance of articular chondrocytes in adults is unknown.

In this study, we report a new dominant-negative mutation of the mouse Gdf5 gene produced in a large-scale phenotype-driven screen of mice mutagenized with a chemical mutagen, N-ethyl-N-nitrosourea (ENU). This mutation produced brachydactyly and ankylosis in the distal limb skeleton in heterozygotes and ankylosis of the knee joint and OA of the elbow joint in homozygotes. The mutant protein dimerized was secreted normally and acted as a dominant-negative inhibitor of wild-type GDF5. This mouse will be a good tool for in vivo studies of the role of Gdf5 in OA development and joint formation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A novel Gdf5 mutation causes brachypodism and akylosis in heterozygote mice
ENU is a highly potent chemical mutagen, which induces multiple single base pair changes randomly on genomic DNA at very high efficiency (approximately 1 x 10–3/locus/gamete) (34–36). We screened 10 236 G1 mice (generated from crosses of ENU-mutagenized males and wild-type females) for abnormal limb phenotypes transmitted as dominant traits. We identified a mutant line, termed M100451, which exhibits a brachypodism phenotype (posted on the website: http://www.gsc.riken.jp/mouse/). The proximal and middle phalanges of this mouse were short and fused, causing an unspecified phalanx. The metacarpo-/metatarso-phalangeal joints were also fused. The lengths of the distal phalanges and metacarpal/metatarsal bones seemed not to be reduced (Fig. 1B and D). Histological analysis showed a ligament attached to the intermediate region of the unspecified phalanx (Fig. 1F). We did not find gross abnormality in the long bones, axial skeleton and large joints. The brachypodism phenotype resembles that seen in Gdf5bp-J homozygote mice (16).


Figure 1
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  		Figure 1. A hindfoot from M100451 mice at 10 weeks of age. Wild-type mice (A) showed three phalanxes (P1, P2 and P3) in the second to fifth digits. The heterozygote mutant showed a reduced number of phalanges [asterisk and P3 in (B)]. Hindlimb digit 2 of wild-type (C) and heterozygote (D) mice. In heterozygotes, the morphology of the tarsal and metatarsal joints was abnormal (black arrowhead). The proximal interphalangeal joint was missing and the matacarpo-/metatarso-phalangeal joint was fused to the sesamoid (white arrow head). Histology of the second digit (E and F) showed a ligament (pink arrowhead) attached to P2 in wild-type (E) and the unidentified phalanx in heterozygote (F). P1, phalanx 1; P2, phalanx 2; P3, phalanx 3; MT, metatarsal bone. *Fused phalanx.

 
Linkage analysis using 20 backcrossed progeny localized M100451 to a 30 cM region on chromosome 2 containing Gdf5 (data not shown). Sequencing analysis of the Gdf5 coding region revealed a missense mutation, c.1222T > A in exon 2 (Fig. 2A). The mutation (p.W408R) is located in the type I BMP receptor interaction domain of the GDF5 ligand (37). This amino acid is conserved in the mouse, human and chick Gdf5 homologs and in mouse Bmp family genes, including Gdf6, Gdf7, Bmp2 and Bmp5 (Fig. 2B). We designated the new Gdf5 mutant allele as Gdf5Rgsc451. Genotypes of this allele completely correlated with the brachydactyly phenotype in 97 progeny (51 heterozygotes and 46 wild-type animals) derived from crosses of heterozygote and wild-type animals.


Figure 2
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  		Figure 2. Sequence and expression analyses of the Gdf5 mutation. (A) Direct sequence analyses of Gdf5. Genomic sequence chromatograms from wild-type (upper), heterozygote (middle) and homozygote (lower). The location of the point mutation is indicated by red arrowheads on each chromatogram. The sequence read is shown above the panel. (B) Comparison of the amino acid sequences of GDF5Rgsc451 (W408R), wild-type GDF5 of different species and Gdf5-related genes. Amino acids identical to mouse GDF5 are represented by blue letters and those not identical by red letters. The conserved cysteine residues that form disulfide bridges in all BMP family members are indicated by a yellow arrowhead. The location of the GDF5Rgsc451 mutation at position 408 of mouse GDF5 is indicated by a pink arrowhead and pink shading. (C) Expression of the mutant allele. Pyrosequencing analysis of the expression of Gdf5Rgsc451 (residue A or T) in 12-day whole embryo cDNAs. The average frequencies of Gdf5Rgsc451 transcript (residue T) are shown. The relative residue T ratios to the wild-type (N = 2) were 53.3 ± 2.5% (N = 5) in heterozygotes and 0.0 ± 0.0% (N = 2) in homozygotes. Error bars represent standard deviation.

 
We analyzed the mRNA expression of the Gdf5Rgsc451 allele in 12-day whole embryos. Direct sequencing of cDNA from heterozygotes revealed no allelic differences; pyrosequencing showed that Gdf5Rgsc451 expression in heterozygotes was half of that in homozygotes (Fig. 2C). These findings indicate that expression of the mutant allele was unaffected.

Molecular analyses of the Gdf5Rgsc451 product
GDF5 is initially synthesized as a monomeric pro-form. It then undergoes post-translational processing that includes disulfide-linked dimer formation and endopeptidase cleavage to produce a dimeric mature form (23,38). To determine whether the Gdf5Rgsc451 product (W408R) was secreted as a correctly folded mature dimer, we analyzed culture supernatants from COS-7 cells transfected with an expression vector encoding wild-type Gdf5 or Gdf5Rgsc451. Two types of fragments, corresponding to the dimeric pro-form and the mature form, were detected in supernatants from both GDF5 and W408R expressing cells (Fig. 3A). Therefore, W408R was correctly assembled into the dimer, cleaved and secreted, in a way similar to the wild-type GDF5.


Figure 3
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  		Figure 3. Molecular characterization of W408R. (A) Western blot analysis using conditioned medium from COS-7 cells transfected with expression vectors for empty (lane 2), wild-type GDF5 (lane 3), W408R (lane 4) or GDF5 and W408R (lane 5) under non-reducing conditions. About 0.5 ng of positive control (recombinant human GDF5 protein) was loaded on lane 1. Mouse monoclonal antibody aMP5, which recognizes dimeric human GDF5, was used. Two bands, which correspond to the dimeric pro-form and the mature form, were detected from W408R expressing cells, similar to wild-type GDF5 expressing cells, indicating that this mutant was correctly assembled into a dimer, cleaved and secreted. This experiment is representative of three independent experiments. (B) Reporter gene assay using an smad1/5/8-dependent reporter vector. The BRE-luc reporter vector was co-transfected with 200 ng of expression vectors for empty (—), GDF5, W408R or both (GDF5 and W408R). The ratio of the transfected expression vectors is indicated below. W408R did not activate transcription and inhibited the activity of wild-type GDF5. Values are the means ± SD of data from quintuplicate wells. P-values were determined by the Student’s t-test. *P < 0.01, **P < 0.001. (C) Expression levels of chondrocyte marker genes. ATDC5 cells grown at confluence were stimulated for 48 h with recombinant GDF5 protein (1 mg/ml), W408R (1 mg/ml) or GDF5 (1 mg/ml)+W408R (1 mg/ml). Total RNA was extracted and reverse-transcribed to generate cDNA. The mRNA expression levels of chondrocyte marker genes, Clol2a1, Agc1 and Alp, were measured by real-time PCR. W408R protein did not induce chondrocyte marker genes and had no effect on the inductive activity of wild-type GDF5. Values are the means ± SD of data from triplicate wells. P-values were determined by the Student’s t-test. *P < 0.05, **P < 0.01, # > 0.05.

 
We examined the effects of the W408R mutation on the activation of the Smad signaling pathway using an SMAD1/5/8-dependent reporter gene, BRE-luc (50). Wild-type GDF5 enhanced SMAD1/5/8-dependent transcriptional activity in HeLa and HCS-2/8 cells, whereas W408R did not (Fig. 3B). Co-transfection of GDF5 and W408R vectors inhibited downstream transcriptional activity. These results suggest that W408R is a loss-of-function mutation and acts as a dominant negative in the activation of the SMAD1/5/8 pathway.

Because GDF5 promotes chondrocyte differentiation in a dose- and time-dependent manner in the chondrogenic cell line, ATDC5 (39), we examined the function of W408R in ATDC5 cells. Treatment of the cells with purified recombinant GDF5 dimers increased the mRNA levels of Col2A1, Agc1 and Alp (Fig. 3C). In contrast, treatment with W408R dimers did not induce these chondrocyte marker genes. Co-treatment with both GDF5 and W408R showed that W408R did not affect the inductive activity of GDF5 on chondrocyte marker expression.

Gdf5Rgsc451 homozygotes show fusion of the knee joint and OA in the elbow joint
To further investigate the in vivo function of the mutation, we produced Gdf5Rgsc451 homozygotes. Radiographic examination at 8 weeks of age showed much more severe brachypodism than was observed in heterozygote animals. We measured the length of skeletal elements in distal portion of forelimbs (Table 1). The middle and proximal phalanges and metacarpals were fused and obviously shortened in digits 2 and 5 of homozygotes. The similar fusions and shortenings were observed in the distal portion of hindlimbs (data not shown). Carpal/tarsal joint fusion was observed in addition to fusion of the proximal interphalangeal joint and the metacarpo-/metatarso-phalangeal joint (Fig. 4C). In the terminal phalanx, the distal interpharlangeal joint, the wrist joint and the elbow joint, we did not find gross morphological alterations. In the forelimbs, the length of the ulna was normal (Table 1). However, complete fusion (Fig. 4I) was observed in the knee joints. The long bones of the hindlimbs (the tibia, fibula and femur) were short. Other skeletal elements, including the axial skeletons, ribs and skull, had no gross morphological alterations (data not shown).


Figure 4
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  		Figure 4. Radiographs of the autopod and elbow regions of the forelimb (A–F) and the knee joint (G–I) at 8 weeks of age. In the forelimb, heterozygotes showed mild brachypodism as shown in Figure 1(B). Homozygotes showed severe ankylosis of the digital joints and shortened digital bones (white arrowhead in C). The elbow joints of wild-type, heterozygote and homozygote animals were normal (D–F, respectively). The knee joints of wild-type and heterozygote animals were normal (G and H); however, in homozygotes, the knee joints were severely fused (white arrow in I).

 


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  		Table 1. Length of skeletal elements* in distal portion of the forelimbs

 
Histological examination showed that the fused knee joints were filled with bone marrow-like structures with poorly formed trabeculae without atricular cartilage (Fig. 5C and F). The epiphysial trabeculae were also poorly formed (Fig. 5I). The growth plate of the distal femur and proximal tibia showed any gross abnormality. Surprisingly, the elbow joints of all analyzed homozygotes had early degenerative changes of the articular cartilage resembling human OA (3/3:Fig. 6). The articular cartilage showed surface fibrillation, cracking, loss of the superficial zone and poor matrix staining by Safranin O because of proteoglycan loss. Chondrocytes were present at decreased number and showed pathologic hypertrophy and cluster formation. (Fig. 6F). In contrast, heterozygote animals had normal smooth articlular surfaces and proper arrangement of chondrocytes. In addition, we examined detailed morphology of peripheral (finger/toe) joints in three heterozygotes at 8 weeks of age. We found mild degenerative change in distal interphalangeal joint of one heterozygote at 8 weeks of age (Fig. 7C). However, other two heterozygotes showed any degenerative change in distal and proximal interphalangeal joints (data not shown).


Figure 5
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  		Figure 5. Histology of the knee joints at low (50 x, A–C) and high (200 x, D–F) magnification and of the epiphyses and growth plates of distal femurs (200 x, G–I) of 8-week-old animals (hematoxylin–eosin stain). Wild-type (A and D) and heterozygote (B and E) animals had normal joints with smooth articular cartilage. In homozygotes, no joint space or articular cartilage was noted, and the junction of the distal femur and proximal tibia was filled with a bone marrow-like structure with poorly formed trabecular bone (C and F). The epiphysis, growth plate and metaphysis developed normally in wild-type and heterozygote (G and H) animals. In homozygotes, the growth plate and metaphysis developed normally, but poorly formed trabecular bone was seen at the epiphysis. fe, femur; ti, tibia; epi, epiphysis; GP, growth plate; meta, metaphysis; m, meniscus of knee; AC, articular cartilage.

 


Figure 6
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  		Figure 6. OA phenotype in the elbow joints of homozygotes. Histology at low (50x, A, C and E) and high (200x, B, D and F) magnifications (Safranin O stain) at 8 weeks of age. Wild-type (A and B) and heterozygote (C and D) animals showed normal joints with smooth articular cartilage and regular cell arrangement. Articular cartilage from homozygotes showed surface irregularity (black arrow), poor matrix staining, decreased cell number and abnormal chondrocyte distribution (E and F). In homozygotes, the subchondral bone of the distal humerus and the radial head was composed of thin and poor trabecular bone (F). rad, radius; hu, humerus; CA, articular cartilage.

 


Figure 7
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  		Figure 7. Histology of a phalanx of forelimb of a Gdf5Rgsc451 heterozygote at 8 weeks of age. Low (50x, A) and high (200x, B and C) magnification (hematoxylin–eosin stain). The right and left open squares in (A) represent magnified regions in B and C, respectively. The metacarpo-phalangeal joint showed normal cartilage with smooth surface. (B). The distal inter-phalangeal joint showed mild cartilage degeneration with irregular cell arrangement and surface. P1, phalanx 1; MT, metatarsal bone; *Fused phalanx.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Through the use of phenotype-based screening in a large-scale ENU mutagenesis program that creates multiple mutants in a single locus (40–43), we isolated the M100451 mouse line, which carried a new Gdf5 allele, Gdf5Rgsc451, clearly associated with brachydactyly in heterozygotes. This allele has an amino acid substitution (W408R) at a conserved residue in the type I receptor binding domain of the ligand molecule (44). The product of the Gdf5Rgsc451 allele, W408R, was correctly secreted as a dimeric mature form, but did not provoke SMAD-mediated biological activity. Furthermore, W408R interfered with the activity of wild-type GDF5; thus, Gdf5Rgsc451 is a dominant-negative mutation. The mutant homodimer did not interfere with normal GDF5 activity, suggesting that the dominant-negative effect is elicited from heterodimerization of mutant and wild-type products.

The phenotype of Gdf5Rgsc451 heterozygote animals is similar to that of previously reported Gdf5 loss-of-function homozyogote mutant animals (16–19), also suggesting the dominant-negative effect of Gdf5Rgsc451. Interestingly, the phenotype of Gdf5Rgsc451 homozygotes was much more severe than that of Gdf5 loss-of-function homozygotes. Thus, the severe deformity of the proximal limbs in Gdf5Rgsc451 homozygotes cannot be explained by complete inactivation of GDF5 function. It is tempting to speculate that the W408R ligand affects signaling of some other BMP receptors in addition to inhibiting authentic GDF5 signaling. Inhibition of BMP signaling in Gdf5 expressing regions was reported for a human dominant-negative GDF5 mutation (C400Y) that creates a semi-dominant chondrodysplasia termed Grebe chondrodysplasia (23). The mutant product inhibits the processing and secretion of multiple BMP family members by selective heterodimerization and produces mild brachyphalangy in heterozygotes and severe brachyphalangy which closely resembles Gdf5Rgsc451 homozygote (normal distal phalanx and obvious shortening of proximal and middle phalanges and metacarpals) and shortening of the proximal limbs in homozygotes. Deformities of the proximal limbs have also been reported for other human GDF5 mutations associating Hunter–Thompson type chondrodysplasia with DuPan syndrome (21,22). These facts indicate that dominant-negative effect of GDF5 can induce proximal limb deformities. In addition, the amino acid substitution of W408R located at the strongly conserved residue between BMP families also supports the hypothesis that W408R product may interfere with other BMP receptors.

Ankylosis of the knee joint was a major characteristic of Gdf5Rgsc451 homozygotes, although this phenotype does not occur in homozygotes of Gdf5 null alleles. Joint formation requires GDF5, GDF6, BMP2, Noggin (an antagonist of BMP) and Wnt9a (18,45–47). W408R may interfere with some or all BMP signaling molecules. In addition, Gdf5Rgsc451 homozygotes showed abnormal trabeculae of the epiphysis. Poor trabecular bone was also observed in the ectopic bone marrow-like structure of the fused joints (Fig. 4F and I). These facts suggest that W408R not only impairs joint determination but also prevents normal proliferation, particularly epiphyseal chondrocytes.

The young adult Gdf5Rgsc451 homozygotes showed cartilage degeneration assessed as OA that progressed to an irreversible stage in elbow joint and in rare cases showed mild degenerative change in peripheral joint at 8 weeks of age. This result is quite a contrast with the case of the knockout mouse lacking collagen IX that showed no alterations at young adult stage, but a progressive degenerative joint in homozygous aged 4 months or older in the knee joints. This suggests that collagen IX chain plays a role of mechanical stability of articular cartilage (14). We preliminary investigated the progression of the OA phenotype at 25 weeks (about 3 months) of age in two Gdf5Rgsc451 homozygotes. In comparison with the phenotype at 8 weeks of age, any gross progression of the degenerative changes and hypertrophy was detected apart from one homozygote that showed slight progression of erosion in contact surface of cartilage (data not shown). These results suggest that W408R may mainly impair differentiation of the articular cartilage and induce the intrinsic early-onset OA. A human disease linked to GDF5 mutation, termed angel-shaped phalangoepiphyseal dysplasia (MIM 105835 [OMIM] ), is associated with early-onset OA of the hip and knee (25,26). Furthermore, we have found that a functional single nucleotide polymorphism (SNP) in the 5'-UTR of GDF5 is directly associated with hip and knee OA. The SNP causes decreased GDF5 expression, indicating that inhibition of GDF5-mediated signaling is causative of OA in humans (48). This strongly suggests that disruption of BMP signaling related to GDF5 could cause OA in mice and humans. Further analysis using the Gdf5Rgsc451 allele will help clarify the detailed mechanisms of joint formation. In addition, this mouse will serve as a good model for studying OA associated with GDF5/BMP signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Phenotype-based screening of ENU-induced mouse mutants
The method used for mouse ENU mutagenesis is available at http://www.gsc.riken.go.jp/Mouse/ and in previous reports (40–43). C57BL/6J and DBA/2J mice were purchased from CLEA Japan (Tokyo, Japan). C57BL/6J males administered ENU (total dosage of 150–250 mg/kg) were crossed with DBA/2J females. The F1 hybrid progeny (G1 animals) were screened for various phenotypes at 8 weeks of age. Examination for adult limb phenotypes was performed as a subtest of the modified-SHIRPA protocol, a comprehensive package of screens for morphological and behavioral phenotypes (41). A complete list of the subtests is available on the above website. The skeletal morphology of the adult G1 animals was examined using a microfocus X-ray system (DFX-100K; Pony Industry, Osaka, Japan). This system can take multiple radiographs of mice from different angles at different zoom values.

Inheritance testing and gene mapping
M100451 was crossed with wild-type DBA/2J mice to test for phenotype transmission and genetic mapping of the causative genes. Genomic DNA was prepared from the tail tips of 20 G2 progeny using the NA-2000 automatic nucleic acid isolation system (KURABO, Osaka, Japan). We used the db-SNP website (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=snp) for SNPs and microsatellite markers listed on the MGI website (http://www.informatics.jax.org/) for simple sequence length polymorphisms. Polymorphic loci were examined using the TaqMan MGB assay (Applied Biosystems, USA) and ABI 7700 and ABI 7900 sequence detection systems.

Sequence analysis and genotyping
To search for mutations in the Gdf5 gene, we sequenced coding regions and splice sites from the M100451 line. We designed primer pairs based on the genomic sequence of Gdf5 (NT_039207 [GenBank] ). Primer sequences are 5'-CAG GCA GCA TTA CGC CAT T-3' and 5'-GTT TGG GGA GTC TCA TCC TCT-3' for the 5' portion of exon 1, 5'-CTG GAC CTG GAA CTC ATC TG-3' and 5'-ACA CAC GCT GTC CCC CAC CT-3' for the 3' portion of exon 1, 5'-AAT TGA CTT CTG GCT GTT TT-3' and 5'-TCG CTT GCC CTG GCG ATT GG-3' for the 5' portion of exon 2, 5'-AGC GAC CCA GCA AGA ACC TC-3' and 5'-GTG GGC CAG TGC TGC TAC CT-3' for the 3' portion of exon 2. The Gdf5Rgsc451 allele was genotyped by sequencing the 3' portion of exon 2 or by pyrosequencing using the following primers: 5'-GCT GCT CCG GTT CAT AGA TTA GCG ACC CAG CAA GAA CCT C-3' as the forward-specific PCR primer, 5'-GCT GCT CCG GTT CAT AGA TT-3' as the 5' biotin-labeled forward universal PCR primer, 5'-GTG GGC CAG TGC TGC TAC CT-3' as the reverse PCR primer and 5'-TGA TCC AGT CGT CCC-3' as the sequencing primer.

Analyses of Gdf5 transcript levels
Whole embryos were placed in microtubes and flash frozen in liquid nitrogen. The cold samples were crushed quickly using a multibead shocker (Yasui Kikai, Osaka, Japan). Total RNA and genomic DNA were isolated using TRIZOL reagent (Invitrogen) and treated with DNase I (Nippon Gene). Reverse transcription was performed using SuperScript II (Invitrogen) and oligo-dT primer. Gdf5Rgsc451 allele frequency was determined by pyrosequencing, as described earlier.

Cell culture
HeLa, HCS-2/8 (human chondrosarcoma cell line) (49) and COS-7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin G and 100 mg/ml streptomycin), and ATDC5 (mouse chondrogenic cell line) (reference) was maintained in a 1:1 mixture of DMEM and Ham’s F12 containing 5% FBS and antibiotics. Cells were maintained at 37°C in an atmosphere containing 5% CO2.

Construction of expression vectors
Total RNA was extracted from 11.5-day wild-type or heterozygote embryos using ISOGEN (Nippon Gene) and reverse-transcribed to generate cDNA using TaqMan Multiscribe Reverse kit (ABI). Full-length wild-type Gdf5 or Gdf5Rgsc451 cDNAs were amplified by PCR and subcloned into the EcoRI/NotI sites of the pcDNA3.1(+) vector. cDNA fragments encoding the mature domains of wild-type Gdf5 or Gdf5Rgsc451 were amplified by PCR using the full-length cDNAs as templates. The amplified PCR products were subcloned into the BamHI/SalI sites of the Escherichia coli expression vector, pQE-30 (Qiagen). The inserted fragments were confirmed by sequencing.

Purification of recombinant protein from E. coli cells
E. coli Rosetta (DE3) pLysS cells (Novagen) were transformed with pQE-30 vectors containing the mature domains of GDF5 or W408R and were grown in Luria-Bertani (LB) medium in the presence of 100 µg/ml ampicillin at 37°C. Inclusion bodies harboring the expressed proteins were obtained using BugBuster protein extraction reagent (Novagen). The refolding reaction was performed as described previously (38). The refolding buffer was extensively dialyzed against 10 mM HCl and concentrated using Amicon Ultra-15 centrifugal filter devices, 5K (Millipore).

Reporter gene assays
HeLa or HCS-2/8 cells were grown to 70–80% confluence in 24-well multiplates and were transfected with plasmid DNA mixtures using Fugene 6 transfection reagent (Roche Diagnostics). The DNA mixture contained BRE-luc (100 ng) and tested pcDNA3.1(+) expression vectors (200 ng total) and a reference vector, pRL-TK (2 ng). Fourty-eight hours after transfection, cells were harvested and luciferase activities were measured using the PG-DUAL-SP reporter assay system (Toyo Ink) and a model Lumat LB 9507 luminometer (Berthold). Relative luciferase activity was calculated after normalizing the transfection efficiency by Renilla luciferase activity expressed by the reference vector.

Western blot analysis
COS-7 cells were grown to 70–80% confluence in 12-well multiplates and were transfected with 400 ng of pcDNA3.1(+) expression vectors. The cells were maintained in Opti-MEM I containing 1% FBS for 48 h after transfection. The culture supernatant was concentrated 10-fold using VIVASPIN 500, 10000 MWCO (Vivascience). Proteins were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidine (PVD) membranes. After blocking with 5% non-fat dry milk in Phosphate Buffered Saline Tween-20 (PBST), the membranes were incubated first with mouse monoclonal antibody aMP5, which recognizes dimeric human GDF5 (Biopharm, Germany), and then with goat polyclonal antibody against mouse IgG conjugated with horseradish peroxidase (Upstate).

Skeletal preparation and histology
Samples were fixed under whole mount reflux of 4% paraformaldehyde/phosphate-buffered saline. Sectioned preparations were stained with hematoxylin and eosin.


   ACKNOWLEDGEMENTS
 
We would like to thank members of the Japanese Skeletal Dyaplasia Consortium for fruitful discussions on analyses of skeletal morphology. We thank Dr Masaharu Takigawa for providing HCS-2/8 cell line and Drs Frank Ploeger (Biopharm) and Petra Seemann (Max Planck Institute for Molecular Genetics) for providing aMP5 monoclonal antibody. We thank the technical staff of the Functional Genomics Research Group of RIKEN GSC for gene-driven screening, animal care and phenotype screening. This study was supported in part by the National Bioresources Project, Grants-in-Aids from the Ministry of Education, Culture, Sports, Science and Technology and a grant for Research on Children and Families from the Ministry of Health, Labor and Welfare of Japan (contact nos 15770150, 18390423 and H18-005, respectively).

Conflict of Interest statement. None declared.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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