Limb tissue specificity of GLI3-CNE enhancers is maintained in chicken limb buds. (A) Diagram of the reporter construct employed to test the enhancer activity of CNEs in chicken embryos. The β-globin promoter was used to drive the GFP expression. The CNEs were cloned in SacII-XbaI sites. (B) Whole mount of limb buds 48 hours after co-electroporation of enhancer construct and RFP at stage HH20 analyzed simultaneously for expression of a control RFP expression vector and a GFP reporter under control of one out of five CNEs. Top row: Control bright field view of electroporated limbs; middle row: RFP fluorescence; bottom row: GFP fluorescence. Electroporated limbs showing GFP signals in addition to RFP fluorescence: CNE1 n = 0/5, CNE6 n = 3/12, CNE9 n = 0/7, CNE10 n = 0/2, CNE11 n = 4/6. Consistent with reporter expression data from mice, only CNE11 and CNE6 enhancers drove GFP reporter expression in developing chicken limbs. Arrows: GFP expressing regions at embryonic stage HH26. Anterior to the top. (C) Distribution of Gli3 transcripts shown via in situ hybridisation. At stage HH26 transcripts are seen at the distal tip (arrows) and more proximally, but absent from the distal posterior region. Note that GFP expressing cells in 4B are found in a region of the limb where Gli3 appears to be expressed. Anterior to the top.