RETROVIRAL EXPRESSION OF FGF-2 (BFGF) AFFECTS PATTERNING IN CHICK LIMB BUD

To investigate the role of fibroblast growth factor-2 (basic fibroblas t growth factor) in chick limb development, we constructed a replicati on-defective spleen necrosis virus to ectopically express fibroblast g rowth factor-2 in stage 20-22 chick limb bud. Because infecting cells in vivo proved to be inefficient, limb bud cells were dissociated, inf ected in vitro, and then grafted back into host limbs. This procedure caused duplications of anterior skeletal elements, including proximal humerus, distal radius, and digits 2 and 3. Eighty-nine percent of hos t wings receiving infected grafts at their anterior borders had duplic ations of one or more of these elements. The frequency of duplication declined dramatically when infected cells were grafted to progressivel y more posterior sites of host limb buds, and grafting to the posterio r border had no effect at all. Several techniques were used to determi ne the role of infected tissue in forming skeletal duplications. First , staining with an fibroblast growth factor-2 specific monoclonal anti body showed higher than endogenous levels of fibroblast growth factor- 2 expression associated with extra elements. Second, the host/donor co mposition of duplicated elements was determined by simultaneously infe cting donor cells with viruses encoding fibroblast growth factor-2 or beta-galactosidase; donor tissue was then visualized by X-gal staining . Patterns of ectopic fibroblast growth factor-2 expression and X-gal staining confirmed the presence of infected donor tissue near duplicat ed structures, but the duplicated skeletal elements themselves showed very little staining. Similar results were obtained in duplications ca used by infected quail wing bud cells grafted to the chick wing bud. T hese observations suggest that fibroblast growth factor-2-expressing d onor tissue induced host tissue to form normally patterned extra eleme nts. In support of this conclusion, implanting beads containing fibrob last growth factor-2 caused partial duplications of digit 2. These dat a provide the first direct evidence that fibroblast growth factor-2 pl ays a role in patterning in the limb bud.