Materials and Methods

Generation of Gdf5-Cre transgenic mice
A mouse 129x1/SvJ BAC library (Invitrogen) was screened to identify a 140-kb BAC from the Gdf5 locus. This BAC was modified using a homologous recombination system in E. coli (Yang et al. 1997) to place nuclear-localized Cre recombinase (from plasmid pML78, gift of Gail Martin) followed by IRES-hPLAP (from plasmid 1726, gift of Oliver Bogler) directly behind the ATG start site of Gdf5. In the process, 583 bp of the first exon of Gdf5 was removed and no functional GDF5 protein is predicted to be produced. The 5′ homology arm was subcloned from a PCR product tailed with XhoI and Bsp120I restriction sites that contains 781 bp of 5′ genomic Gdf5 sequence ending at the ATG translation start site (forward primer 5′-CTGTCTCGAGATGAGGTGGAGGTGAAGACCCC-3′; reverse 5′-GTTTGGGCCCATCCTCTGGCCAGCCGCTG-3′). Cre was subcloned from a 1.1-kb Bsp120I/EcoRI fragment of pML78. IRES hPLAP was subcloned from a 2.1-kb PCR product tailed with EcoRI and SpeI sites that contains the hPLAP translation stop site (forward primer 5′-ATCTCTCGAGGAATTCTCCACCATATTGCCGTCTTTTG-3′; reverse 5′-AGAACTCGAGACTAGTCGGGACACTCAGGGAGTAGTGG-3′). The 3′ homology arm was subcloned from a 0.8-kb PCR product amplified from a 0.9-kb XhoI Gdf5 genomic subclone containing part of the first exon and downstream intron. The forward primer contains the 3′ end of the first exon and is tailed with a SpeI site; the reverse primer is from the T7 promoter of the vector containing the 0.9-kb subclone and flanks the intronic XhoI site (forward primer 5′-CTAAACTAGTCACCAGCTTTATTGACAAAGG-3′; reverse 5′-GATTTCTAGAGTAATACGACTCACTATAGGGC-3′). The targeting construct was built and verified in pBSSK (Stratagene, La Jolla, California, United States), then digested with XhoI and subcloned into pSV1, the vector used for homologous recombination (Yang et al. 1997). Southern blotting, PCR, and DNA sequence analysis confirmed the appropriate targeting construct and BAC modifications were made (unpublished data).
Before the modified BAC was injected to produce transgenic animals, a loxP site present in the BAC vector, pBeloBAC11, was removed to prevent the addition of undesired Cre target sites into the genome. To do this, BAC DNA was prepared by CsCl separation, digested with NotI to free the insert from the vector, and size-fractionated over a sucrose gradient. Aliquots of fractions were run on a pulse-field gel and Southern blotted using vector-specific DNA as a probe. Fractions containing unsheared insert and almost no detectable vector DNA were dialyzed in microinjection buffer (10 mM Tris [pH 7.4] with 0.15 mM EDTA [pH 8.0]) using Centriprep-30 concentrators (Millipore, Billerica, Massachusetts, United States). This purified insert DNA was adjusted to 1 ng/μl and injected into the pronucleus of fertilized eggs from FVB/N mice by the Stanford Transgenic Facility. Transgenic founder mice were identified by PCR using Cre-specific primers 5′-GCCTGCATTACCGGTCGATGCAACGA-3′ and 5′-GTGGCAGATGGCGCGGCAACACCATT-3′, which amplify a 725-bp product, and were assessed for absence of BAC vector using vector-specific primers 5′-CGGAGTCTGATGCGGTTGCGATG-3′ and 5′-AGTGCTGTTCCCTGGTGCTTCCTC-3′, which amplify a 465-bp product. Three lines of Gdf5-Cre mice were established and maintained on the FVB background. Matings with R26R Cre-inducible LACZ reporter mice (Soriano 1999) were used to test for Cre activity.
Staining for LACZ and HPLAP on whole embryos or sections of embryos was accomplished following established protocols (Lobe et al. 1999). The red LACZ substrate (see Figure 1E) is 6-chloro-3-indoxyl-beta-D-galactopyranoside (Biosynth International, Naperville, Illinois, United States).

General characterization of Bmpr1a mutant mice
Bmpr1a null and floxed alleles (Ahn et al. 2001; Mishina et al. 2002) were obtained on a mixed 129 and C57BL/6 background and maintained by random breeding. Mice carrying the null and floxed alleles were typically mated to Gdf5-Cre mice as shown in Figure 3. The resulting mice are on a mixed 129; C57Bl/6; FVB/N background, with both controls and mutant animals generated as littermates from the same matings. Whole-mount skeletal preparations were made from 34- to 36-d-old mice (Lufkin et al. 1992). Pairs of ears from euthanized 6-mo-old animals were removed, pinned, photographed, projected, and measured from the base of the curve formed between the tragus and antitragus to the farthest point at the edge of the pinnae. Grasping ability in 6-mo-old mice was measured by placing animals on a slender rod and timing how long they could remain suspended on the rod, to a maximum time allowed of 2 min. Data from five consecutive trials for each mouse were averaged. Range of motion assays were conducted on the MT/P1 and P1/P2 joints of the second hindlimb digit from euthanized 18-wk-old animals. Forceps were used to bend the joint to its natural stopping position, and the resulting angle was measured to the nearest 10° under 12.5× magnification using a 360° reticule. Analysis described in this section occurred on animals lacking R26R. Control mice included all nonmutant genotypes generated by Parent 1 being heterozygous for Gdf5-Cre and Bmpr1anull and Parent 2 being heterozygous for Bmpr1afloxP (see Figure 3). All statistical analysis used the Student's t-test or Welch's t-test, and values listed are mean ± standard error of the mean.

Cell death and proliferation assays
Limbs from mutant and control animals at E13.5 and E14.5 were dissected and frozen in OCT (Sakura Finetek,Torrence, CA, United States). Cryosections of tissue were assayed by TUNEL using the In Situ Cell Death Detection Kit, Fluorescein (Roche, Basel, Switzerland). Following TUNEL, slides were washed in PBS, blocked with PBS + 0.05% Tween-20 + 5% goat serum, washed again, and incubated with a 1:200 dilution of a rabbit anti-phospho-histone-H3 antibody called Mitosis Marker (Upstate Biotechnology, Lake Placid, New York, United States) to identify cells in mitosis. Cy3-labeled anti-rabbit secondary antibody was used to detect the antibody. Cell nuclei were labeled with DAPI, and slides were mounted in Vectamount (Vector Laboratories, Burlingame, California, United States) and visualized at 100× magnification. The area of selected anatomical sites were measured, and the number of TUNEL-labeled nuclear fragments and the number of Cy3-labeled nuclei were counted from three 10-μm sections spanning 50 μm, from three control and three mutant animals. The number of labeled cells in the metacarpal-phalangeal and metatarsal-phalangeal joints was counted in a 290 μm × 365 μm rectangle placed around the center of the joint. The posterior region of the fifth digit was defined by drawing a line from the tip of the digit down 2.15 mm and across to the lateral edge of the tissue. For this analysis, the R26R Cre reporter was not present.

Histology and histochemistry
Tissue from animals ranging from stages E14.5 to P14 was prepared for analysis by fixing in 4% paraformaldehyde (PFA) in PBS for 45 min to 4 h depending on the stage; washing three times in PBS, once in PBS + 15% sucrose for 1 h, and once in PBS + 30% sucrose for 2 h to overnight depending on the stage; and then freezing in OCT. Tissue from animals aged 7 wk to 9 mo was processed similarly to earlier stages except that it was decalcified in 0.5 M EDTA (pH 7.4) for 4 d prior to incubating in sucrose. All solutions were prechilled and used at 4 °C with agitation, and skin from tissues of P0 or older mice was lacerated or removed prior to processing.
Tissue was then cryosectioned at 12 μm and processed. Staining of sections with Safranin O, Fast Green, and Harris' hematoxylin was carried out using standard histological procedures. Detection of LACZ activity with X-Gal was performed as described (Lobe et al. 1999) and was followed by refixing in 4% PFA, rinsing with deionized water, counterstaining with Nuclear Fast Red (Vector Labs), rinsing with water again, and then mounting in Aquamount (Lerner Labs, Pittsburgh, Pennsylvania, United States).
RNA in situ hybridization was performed as described (Storm and Kingsley 1996), with the following modifications: (1) Prior to the acetylation step, sections were incubated with 10–20 μg/ml proteinase K for 30 s to 7 min at room temperature (depending on the developmental stage), followed by refixing in 4% PFA and washing three times in PBS; (2) prehybridization step was skipped, and (3) embryonic tissue sections used a different color development mix (Thut et al. 2001). Probes for the following genes have been published previously: Bmpr1a (Mishina et al. 1995), Col2a1 (Metsaranta et al. 1991), Col10a1 (Apte et al. 1992), Gdf5 (Storm and Kingsley 1996), Osteocalcin (Celeste et al. 1986), and Sox5 and Sox6 (Lefebvre et al. 1998). The following probe templates were gifts: Agg, Dr. Vicki Rosen, Genetics Institute; Bmp2 and Bmp4, Arend Sidow, Stanford University; Col1a1, Bjorn Olsen, Harvard Medical School; Bmpr1b, Col3a1, and Mat4 probes were made from ESTs with IMAGE clone numbers 5056341, 478480, and 406027, respectively (Invitrogen, Carlsbad, California, United States).
Sections for immunohistochemistry were fixed in 4% PFA, then digested with 942–2,000 U/ml type IV-S bovine hyaluronindase (Sigma, St. Louis, Missouri, United States) in PBS (pH 5) at 37 °C for 30 min to 2 h depending on the stage. Slides were then washed in PBS, treated with 0.3% hydrogen peroxide in 100% methanol for 30 min, washed, blocked with PBS + 0.05% Tween20 + 5% goat or fetal bovine serum, washed again, and incubated with primary antibodies in PBS + 0.05% Tween 20 + 1% goat or fetal bovine serum overnight at 4 °C. Biotin-labeled secondary antibodies (Vector Labs) were tagged with HRP using the Vectastain Elite ABC kit (Vector Labs) followed by detection with DAB (Vector Labs). Primary antibodies and dilutions used were: goat anti-mouse MMP13, 1:100 (Chemicon International, Temecula, California, United States); rabbit anti-human SOX9, 1:500 (Morais da Silva et al. 1996); rabbit anti-phosphorylated-SOX9 (SOX9.P), 1:10–1:250 (Huang et al. 2000).