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Fig. 4 CNE extension correlates with evolutionary gain of enhancer function. (a) Genomic location and conservation of CNE14 variants based on UCSC PhyloP and MultiZ alignment tools. CNE14 human–mouse conserved region (CNE-L, 1906 bp), CNE14 intermediate human–chicken region (CNE-M, 958 bp), and the core conserved human–coelacanth sequence (CNE-C, 304 bp) are compared. (b) CNE14-L and CNE14-M induce GFP expression in the developing zebrafish fish pectoral fin. (c) Transgenic LacZ reporter activities of CNE14 versions in mouse embryos at E11.5. While CNE14-L induced strong LacZ expression in the anterior limb mesenchyme, activity driven by CNE14-M and CNE14-C was progressively reduced. “n” indicates the number of embryos with reproducible transgenic reporter activity versus the total number of transgenic embryos showing any LacZ signal. (d) Pairwise sequence comparison of CNE14 by Vista plots. Blue double arrow indicates scale. FL, forelimb; HL, hindlimb.