Although regional differences in mesenchymal cell affinity in the limb bud represent positional identity, the molecular basis for cell affinity is poorly understood. We found that treatment of the cell surface with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) could change cell affinity in culture. When PI-PLC was added to the culture medium, segregation of the progress zone (PZ) cells from different stage limb buds was inhibited. Similarly, sorting out of the cells from different positions along the proximodistal (PD) axis of the same stage limb buds was disturbed. Since PI-PLC can remove glycosylphosphatidylinositol (GPI)-anchored membrane bound proteins from the cell surface, the GPI-anchored cell surface proteins may be involved in sorting out. To define the GPI-anchored molecules that determine the segregation of limb mesenchymal cells, we examined the effect of neutralizing antibody on the EphA4 receptor that binds to GPI-anchored cell surface ligands, called ephrin-A. Sorting out of the PZ cells at different stages could be inhibited by the neutralizing antibody to EphA4. These results suggest that EphA4 and its GPI-anchored ligands are, at least in part, involved in sorting out of limb mesenchymal cells with different proximal-distal positional values, and that GPI-anchored cell surface proteins play important roles in determining cell affinity in the limb bud.
Copyright 1998 Academic Press.