Initiation of dorso-ventral axis during chick limb development

We analysed spatio-temporal expression of dorso-ventral genes - Wnt-7a, En-1, Lmx-1 and Fgf-8 - during both normal and ectopic limb formation following fibroblast growth factor (FGF) application to the flank. We confirm that Wnt-7a is the first of these genes to be expressed in dorsal ectoderm in limb-forming regions. We also noticed patterns and kinetics of gene expression specific to chick that could account for differences observed in ridge formation between chick and mouse. We find that Wnt-7a expression, in dorsal ectoderm, is rapidly and locally induced by FGF application. In contrast, ectopic induction of Lmx-1 expression, in dorsal mesoderm, is much slower, occurs first at a distance from the FGF-2 bead and seems initially independent of direct Wnt-7a signalling during FGF-2 limb induction. Finally, we show that there is no contribution to extra-limb mesoderm from normal limb mesoderm and confirm that flank cells give rise to the extra limb. Furthermore, we suggest that an inhibitor present in the flank normally prevents Lmx-1 expression in this region and restricts its expression to limb-forming regions.