Synergistic effects of FGF and non-ridge ectoderm on gene expression involved in the formation of the anteroposterior axis of the chick limb bud in cell culture

Abstract

Skeletal patterning of the vertebrate limb is controlled by the zone of polarizing activity (ZPA), apical ectodermal ridge (AER) and dorsal ectoderm. In the present study, to understand the involvement of fibroblast growth factor (FGF) and non-ridge ectoderm in anteroposterior (AP) axis formation, gene expression in chick limb bud mesenchymal cells in culture was investigated by reverse transcription-polymerase chain reaction and in situ hybridization. It was found that Shh expression was locally maintained in the mesenchymal cells underneath and near non-ridge ectoderm in coculture with the posterior mesenchymal cells and non-ridge ectoderm in the presence of FGF-4 by in situ hybridization. In Shh-expressing anterior limb bud mesenchymal cells cultured with non-ridge ectoderm, it was also discovered that Bmp-2 was activated in the presence of FGF-2, -4 and -8, while Hoxd-13 was activated in the presence of FGF-4 and that FGF-2 had a similar effect but FGF-8 did not. This result indicates that Hoxd-13 activation by SHH depends on non-ridge ectoderm and FGF-2 or FGF-4, and that there may be a difference in the effect on AP axis formation of the limb bud between FGF-2, -4 and -8. Possible roles of these genes and signal molecules in AP pattern formation are discussed.