Apoptosis in the chick embryo wing bud and the permanence of FGF-2 rescue

Two regions of programmed cell death that occur in the mesoderm of developing chick wing buds were studied in vitro. The permanence of FGF-2 rescue in the opaque patch (OP) and posterior necrotic zone (PNZ) was examined using the chromium release assay. While rescue in the OP was found to be permanent, rescue in the PNZ only delayed death while FGF-2 was present in the culture medium. Also, the OP and PNZ were examined for the presence of internucleosomal DNA degradation and for rescue by protein synthesis inhibition, two defining characteristics of apoptosis. Agarose gel electrophoresis showed that DNA from OP and PNZ tissue was cleaved into nucleosome size pieces and this cleavage was prevented by the presence of cycloheximide. Both the OP and PNZ showed rescue by protein synthesis inhibition with cycloheximide, as determined by the chromium release assay and examination of electron micrographs. This research suggests that in vitro FGF-2 rescue is permanent in the OP but is merely a delay of cell death in the PNZ, and that death in both of these regions is prevented by inhibition of protein synthesis with cycloheximide. It also shows that death in the OP and PNZ exhibits internucleosomal DNA fragmentation, biochemically characterizing this death as apoptosis.