GDF5 mutant N445T has increased prochondrogenic activity.

<p>Stimulation of chicken micromass cultures with rhGDF5, rhN445T GDF5, rhBMP2, and rhBMP9 at day one for 72 h with the indicated concentrations. (A) Alcian blue staining of micromass cultures at day four. RhN445T showed a strong induction of chondrogenesis already at low concentrations. In comparison, rhBMP2 showed a strongly reduced and rhBMP9 the highest prochondrogenic activity. (B) Quantification of the Alcian blue staining, n = 3. (C) Semi-qRT-PCR for Nog after 12 h stimulation of one day old chicken micromass cultures with 5 nM rhGDF5, rhN445T GDF5, rhBMP2, and 0.5 nM rhBMP9. Stimulation with recombinant proteins induces a strong upregulation of endogenous Nog expression. (D) Whole mount in situ hybridizations showing <i>Nog</i> expression in chicken hind limbs after implantation of heparin beads soaked with 4 mM HCl + 0.2% BSA, 0.5 mg/ml rhGDF5, 0.5 mg/ml rhN445T GDF5, 0.5 mg/ml rhBMP2, and 0.25 mg/ml rhBMP9. Beads were implanted between HH stages 25–27 and HH29/30 (BMP9) and embryos were collected 20 h later. In contrast to the control limb, all beads soaked with BMP induced <i>Nog</i> expression in the periphery.