Enhancers CNE6 and CNE11 govern distinct aspects of Gli3 expression in developing limbs. (A) Diagram of the lacZ reporter gene construct employed to test the enhancer activity of GLI3-CNEs in mouse embryos. (B) Whole mount views of transgenic mouse embryos at days E11.5, E12.5, and E13.5 expressing lacZ under control of CNE6 or CNE11. White and black open arrowheads indicate reporter expression within facial (VII) nerve and proximal limb elements, respectively. (C) Developing mouse forelimbs from embryonic day E9.5 to E13.5 showing domains of reporter gene activity induced by CNE6 (upper row) and CNE11 (lower row). CNE6-directed lacZ expression starts early, coinciding with the emergence of the limb bud, and is largely confined to anterior aspects. CNE11 induced reporter expression starts at E12.5 and is restricted to mesenchymal condensations of proximal elements (stylopod and zeugopod, brackets s and z). In E13.5 limbs, CNE11 governed expression extends to the proximal portion of handplate (bracket: w wrist, h handplate) but not into the digits. Longitudinal sections through the E13.5 forelimbs show CNE6-directed reporter expression within distal skeletal elements (digits, open arrowheads) and CNE11-governed X-gal staining in prospective humerus (hu), radius (ra) and ulna (ul). Asterisk symbol shows posterior margin of E9.5 and E10.5 forelimb buds devoid of reporter expression.