Gdf11 Is a Negative Regulator of Chondrogenesis and Myogenesis in the Developing Chick Limb

AbstractGDF11, a new member of the TGF-β gene superfamily, regulates anterior/posterior patterning in the axial skeleton during mouse embryogenesis. Gdf11 null mice display skeletal abnormalities that appear to represent anterior homeotic transformations of vertebrae consistent with high levels of Gdf11 expression in the primitive streak, presomitic mesoderm, and tail bud. However, despite strong Gdf11 expression in the limb throughout development, this structure does not appear to be affected in the knockout mice. In order to understand this dichotomy of Gdf11 expression versus Gdf11 function, we identified the chicken Gdf11 gene and studied its role during limb formation. In the early limb bud, Gdf11 transcripts are detected in the subectodermal mesoderm at the distal tip, in a region overlapping the progress zone. At these stages, Gdf11 is excluded from the central core mesenchyme where precartilaginous condensations will form. Later in development, Gdf11 continues to be expressed in the distal most mesenchyme and can also be detected more proximally, in between the forming skeletal elements. When beads incubated in GDF11 protein were implanted into the early wing bud, GDF11 caused severe truncations of the limb that affected both the cartilage elements and the muscle. Limb shortening appeared to be the result of an inhibition of chondrogenesis and myogenesis and using an in vitro micromass assay, we confirmed the negative effects of GDF11 on both myogenic and chondrogenic cell differentiation. Analysis of molecular markers of skeletal patterning revealed that GDF11 induced ectopic expression of Hoxd-11 and Hoxd-13, but not of Hoxa-11, Hoxa-13, or the Msx genes. These data suggest that GDF11 may be involved in controlling the late distal expression of the Hoxd genes during limb development and that misregulation of these Hox genes by excess GDF11 may cause some of the observed alterations in skeletal element shape. In addition, GDF11 induced the expression of its own antagonist follistatin, indicating that the activity of GFD11 may be limited by a negative feedback mechanism. The data from our studies in the chick suggest that Gdf11 plays a role in the formation and development of the avian limb skeleton