Figure 3. The Gli3 limb-specific enhancer of S. canicula and C. milii.

(A) VISTA plots of Gli3 intron 3 from indicated animals. Blue vertical bars, exons of human Gli3; black rectangle, element 1586. Regions with >70% identity are indicated: blue, exon; pink, non-coding sequences. (B) The enhancer construct. (C) GFP expression in chick forelimb buds driven by chick element 1586 at stage 19 (top, n = 3/3), stage 23 (middle, n = 14/14), and empty vector (bottom, n = 0/7). pCAGGS-RFP (right). (D–F) GFP expression driven by element 1586 of S. canicula (D, n = 11/11), C. milii (E, n = 10/10) and Sc1586mt (F, n = 4/4). Scale bars, 100 μm. (G) Scheme of Sc1586mt, S. canicula enhancer (blue) partially replaced by chick sequence (green) and alignment. Boxes indicate tetrapod (i) and sarcopterygian (ii) specific sequences.

DOI: http://dx.doi.org/10.7554/eLife.07048.009

Figure 3—figure supplement 1. Detailed functional analyses of element 1586.

(A) Alignment of element 1586 sequences. Points where chick and S. cacnicula enhancers are recombined in (B) are indicated by arrows a and b. Box i and Box ii are sequences that are replaced with S. canicula sequence in (C), activities of element 1586 recombined at the indicated points. Note that the posterior repressive activity is not altered at arrow a, but partially destroyed at arrow b, suggesting that the repressive sequences are located between arrow a and b and after arrow b. (C) Activities of chick element 1586 that are partially recombined with S. canicula sequences at Box i and Box ii in (A). The sequences were chosen because of specific conservation among tetrapods (Box i) and sarcopterygians (tetrapod and coelacanth, Box ii). Each of replacement can slightly alter the posterior repression of GFP (n = 3/3). When the chick enhancer is replaced by both sequence (the most right panel), the distal GFP expression is significantly shifted to the posterior limb buds (n = 4/4, white arrow). And weak expressions are also detected in the posterior proximal limb buds (n = 2/4. arrow head in the bottom panel, magnified view of the above white box with enhanced contrast), suggesting that the sequences at Box i and Box ii are partially responsible for the posterior repression. Scale bars, 100 μm.

DOI: http://dx.doi.org/10.7554/eLife.07048.010