Retinoic acid is required for the initiation of outgrowth in the chick limb bud

Background: Retinoic acid (RA) is present in the chick limb bud, and excess RA induces limb duplications. Here, we have investigated the role of endogenous RA during chick limb development by preventing the synthesis of RA and testing the effect on various genes expressed during limb initiation and outgrowth. Results: We demonstrate that the stage 20/21 limb bud synthesizes didehydroretinoic acid (ddRA), and that the posterior half of the limb bud synthesizes ddRA at a higher rate than the anterior half. Disulphiram inhibits this synthesis at micromolar concentrations. Administering disulphiram to embryos prior to limb bud outgrowth (stages 12-18) abolishes outgrowth, and no limb develops in the majority of cases. Disulphiram treatment also prevents the expression of Sonic hedgehog (Shh), but the expression of the fibroblast growth factor-8 gene (Fgf-8) appears as normal in the ectoderm over the prospective limb bud. The application of a bead soaked in RA can rescue Shh expression. Disulphiram treatment of later limb buds (stages 20-23) similarly down-regulates Shh, and also Fgf-4, expression, whereas the expression of Fgf-8, as at earlier stages, is initially unaffected. Again, RA can rescue the expression of Shh in these limb buds. Conclusions: RA, in conjunction with Fgf-8, may be needed for the induction of the chick limb bud and the induction of Shh and Fgf-4 expression. The expression of Shh and Fgf-4 remains dependent upon the continued synthesis of RA within the limb bud, Didehydroretinoic acid is the major active retinoid in the stage 20 chick limb bud.