Ag1 x2 beads

Cyclopamine (5 μl; Sigma) dissolved in control carrier (45% 2-hydroxypropyl-β-cyclodextrin in PBS; Sigma) to a concentration of 1 μg μl⁻¹ was pipetted directly into the egg after removal of vitelline membranes using fine forceps. Retinoic acid (Sigma) and BMS-493 (Sigma and a gift from M. Torres) and colchicine (Sigma) were dissolved in DMSO (Sigma) to 5 μg μl⁻¹ (Retinoic acid and BMS-493) or 0.05μg μl⁻¹ (colchicine). Formate-derivatized AG1-X2-beads (Sigma-150 μm diameter—sieved using nylon mesh to exclude smaller beads) were soaked in these compounds for 30 min, washed twice in DMEM and implanted into chick wing buds. For 48 h Retinoic acid treatment, beads were replaced after 24 h with freshly prepared ones.

Sieved formate‐derivatized AG1‐X2 beads (150 or 200 μm in diameter, Sigma) were soaked in all‐trans‐retinoic acid (Sigma, 1 μg/μl^‐1 or 5 μg/μl^‐1 dissolved in DMSO, also Sigma) for 1 hr and then washed twice in DMEM before being grafted to the anterior margin of chick wing buds using a sharp tungsten needle. Affi‐Gel beads (Bio‐Rad) were soaked in recombinant Shh protein (10 μg/μl^‐1, a kind gift of Prof Joy Richman) for 2 hr and implanted the same way as retinoic acid beads.