Limb or CNS specific enhancers show complementary regulatory potential reflecting endogenous GLI3 expression. (A) Diagrams summarizing reporter signals of two independent enhancers, CNE6 and CNE11, which regulate expression distinctly within evolutionary ancient (stylopod, zeugopod) and modern aspects (autopod) of mammalian limb. At E10.5 and E11.5, CNE6-governed reporter activity (blue color) was detected throughout the developing limb bud, with the exception of the posterior margin. At these time points no CNE11 activity was seen in the limb bud. By E12.5, CNE11 induced transgene expression specifically within precartilage condensations of mesenchyme within presumptive proximal limb elements (green). At this time point, CNE6 activity was confined to digital rays, digital inter-zones, and to the non-cartilaginous mesenchyme encasing the precartilage condensations of proximal and distal limb elements. By E13.5, when the precartilage condensations of mesenchyme are replaced by cartilage, CNE11 activity was retained in the stylopod and zeugopod, and was extended more distally up-to cartilaginous elements of digit arch (wrist/ankle). At this time point, CNE6 directed reporter expression was focused on the individual digits. A, anterior; P, posterior. (B, C) Diagrams summarizing enhancer activities of CNE1 (lavender) and CNE9 (red) along dorsal-ventral aspects of midbrain (B) and spinal cord (C). CNE1- and CNE9-directed expression during CNS development is non-overlapping except in the dorso-lateral marginal neuronal tissue of the midbrain. RP, roofplate; FP, floorplate; pMN, progenitor area of motor neurons; Dp/Vp, progenitors of dorsal/ventral interneurons; AP, alar plate; BP, basal plate; MV, mesencephalic vesicle; CC, central canal.