Figure 3.

Functional analysis of Gdf5 mutants in micromass culture. (A) Chicken micromass cultures were assayed after 4 days for extracellular matrix production and analyzed after 7 days for ALP activity. Cells were infected with virus containing WT Gdf5 or mutant sequences and coinfected with Nog or not coinfected. The mutants differed drastically in their biological activity. Coinfection with Nog completely represses chondrogenesis irrespective of the Gdf5 variant expressed. Magnification, ×1 objective (AxioCam HRc camera; Zeiss). (B) Alcian blue incorporation into the extracellular matrix of micromass cultures reflecting the production of proteoglycan-rich cartilaginous matrix measured at day 4 was quantified after extraction. Biological activity of R438L mutant was comparable to that of Gdf5, whereas the L441P mutant displayed only small effects on matrix production. (C) ALP activity of micromass cultures at day 7 was quantified by a specific enzymatic assay. While Gdf5 and R438L induced ALP activity effectively, L441P caused only a small amount of induction, slightly above control activity. Contr, control.