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???displayArticle.abstract??? CDMP1/GDF5 has not demonstrated biological activity in Xenopus embryos when overexpressed by mRNA injection. We provide biological and biochemical evidence that to become active, the protein requires cleavage by two distinct proteolytic enzymes. We demonstrate a specific overlap in the expression patterns of CDMP1/GDF5 with the proteases required to release the mature peptide at the location of the future articular surface but not in the future joint space. Taken together, these observations provide a plausible mechanism for local action of CDMP1/GDF5 consistent with requirements imposed by current models of pattern formation in the developing limb.
FIGURE 1.
Characterization and expression analysis of Xenopus GDF5. A, dendrogram showing the phylogenetic relationship between members of the GDF5, -6, -7, and -16 subfamily. Full-length amino acid sequences were analyzed using GeneWorks® Version 2.2 (IntelliGenetics, Inc.) software. B, amino acid sequence comparison of full-length human, mouse, and Xenopus GDF5. Sequence identities are boxed; sequence similarities are highlighted in gray. C, RT-PCR analysis for Xenopus GDF5 using RNA obtained from indicated stages of Xenopus development (h = hindlimb; f = forelimb). D, whole mount hybridization in situ of Xenopus GDF5 of a stage 59 Xenopus forelimb. Arrows indicate the location of positive signal at presumptive joint interzones.
FIGURE 2. Comparison of BMP precursor cleavage sites and SPC temporal expression pattern. A, amino acid sequences surrounding the consensus RXXR cleavage sites for various BMP precursors. The residues shown in color fit the required sequence for SPCcleavage, and the boxed residues illustrate the sequence shared by CDMP1/GDF5 and Vg1. B, RT-PCR analysis of indicated Xenopus stages for RNA encoding different SPCs.
FIGURE 3. Ventralizing activity observed in stage 27 tadpoles following injection of dorsal blastomeres of Xenopus (two- and four-cell) embryos with mRNA encoding wild type or mutant GDF5 and combinations of various SPCs. A, control, sham-injected embryos. B, embryos injected with wild type GDF5 (60 pg) alone. C, embryos injected with GDF5 Lys → Arg mutant (60 pg). D, DAI distribution of embryos injected with GDF5-wild type alone (60 pg), GDF5-wild type (60 pg) + Furin (300 pg) or SPC6 (300 pg) GDF5-wild type (60 pg) + Furin (150 pg) + SPC6 (150 pg) or GDF5 Lys → Arg mutant (60 pg). Injection with Furin (150 pg) + SPC6 (150 pg) only was included as a negative control. The DAI of the embryos was scored at stage 27, and the percentage of embryos having a DAI score <5 for each treatment is shown. Numbers of embryos examined (n) are indicated above each column. Embryos with a DAI of 0 lack dorsal structures completely, and those with a DAI of 5 are normal. Similar results were obtained in three separate experiments.
FIGURE 4.
Immunoblot and RT-PCR analyses demonstrating GDF5 requires both Furin and SPC6 to produce mature protein and become biologically active. A, immunoblot analysis of secreted proteins following mRNA injection of Xenopus oocytes. Xenopus oocytes (stage VI) were isolated, defolliculated, and injected with mRNAs encoding GDF5-T7 (25 ng), GDF5-T7 (25 ng) + Furin (25 ng), GDF5-T7 (25 ng) + SPC6 (25 ng), or GDF5-T7 (25 ng) + Furin (12.5 ng) + SPC6 (12.5 ng). Injected oocytes were incubated at 18 °C for 24 h, and oocyte supernatants were prepared for analysis as described under “Materials and Methods.” Arrows indicate the locations of the pro- and mature forms of GDF5-T7. Similar results were obtained in three separate experiments. B, conjugated animal caps (as indicated), produced at stage 9 and cultured to stage 17, analyzed by RT-PCR for the mesodermal marker Brachyury and the ventral markers Xhox3, and Xvent-1. Histone H4 is included as a loading control. Xenopus embryos were injected equatorially at the four-cell stage with mRNAs for either GFP (500 pg), Furin (150 pg) + SPC6 (150 pg) + GFP (200 pg), GDF5 (100 pg) + GFP (300 pg), GDF5 (100 pg) + Furin (150 pg) + SPC6 (150 pg) or GDF5Mut Lys → Arg (100 pg) + GFP (300 pg).
Supplemental Figure 1. Co-injection of GDF5 with increased doses of SPC mRNA into Xenopus embryos at the two-cell stage does not increase the biological activity of GDF5. A. GDF5-wt (60pg) only and with Furin or SPC6 (150pg) B. GDF5-wt (60pg) only and with Furin or SPC6 (300pg). The effect of GDF5-K→R (60pg) is shown in each experiment.
Supplemental Figure 2. Dorso-Anterior Index (DAI) distribution of embryos injected with GDF5-wt alone (60pg), GDF5-wt (60pg) + Furin (300pg) or SPC4 (300pg) or SPC6 (300pg), GDF5-wt (60pg) + Furin
(150pg) + SPC4 (150pg), GDF5-wt (60pg) + Furin (150pg) + SPC6 (150pg) or GDF5-wt (60pg) + SPC4 (150pg) + SPC6 (150pg). The DAI of the embryos was scored at stage 27, and the percentage of embryos having a DAI score of < 5 for each treatment is shown. Numbers of
embryos examined (n) are indicated above each column. Embryos with a DAI of 0 lack dorsal structures completely, and those with a DAI of 5 are normal. Similar results were obtained in three separate experiments.
gdf5 (growth differentiation factor 5) gene expression in Xenopus laevis embryoforelimbautopod,, assayed via in situ hybridization, NF stage 59.
