Shh, Bmp-2 and Hoxd-13 gene expression in chick limb bud cells in culture

The anteroposterior axis of the vertebrate limb bud is determined by signals from the zone of polarizing activity (ZPA). Sonic hedgehog (Shh) is expressed in the posterior mesoderm, which corresponds closely to ZPA activity. Moreover, Bmp-2 and HoxD genes are expressed in the broader posterior mesoderm, and it is thought that the ZPA signaling pathway consists of these gene products. Limb outgrowth and patterning, including expression of these genes, depend on the apical ectodermal ridge (AER). Fibroblast growth factors (FGF) have been identified as candidates for signal molecules from the AER. To further understand the ZPA signaling pathway and the participation of FGF, expressions of these genes were examined by reverse transcription-polymerase chain reaction in chick limb bud cells cultured with FGF-4. The present results indicate that FGF-4 cannot maintain Shh expression but can maintain Hoxd-13 expression in cultured posterior cells; moreover, Bmp-2 is expressed independently of FGF-4. These results suggest that Bmp-2 and Hoxd-13 expressions do not require a continuous expression of Shh. Further, it was demonstrated that posterior cells cultured with FGF-4 recovered Shh expression when grafted to the limb bud, indicating that FGF-4 maintains not Shh expression itself but competence of Shh expression.