Fig. 2

Evolutionary functional conservation of the Bmp6 enhancer in teleosts. (A) Sequence alignments of four teleost sequences relative to the 190 bp stickleback enhancer. The perfectly conserved Smad3 dimer binding site is marked in blue, and purple arrows mark the boundaries of the 72 bp minimal enhancer (see Fig. S6). (B-D) The stickleback sequence reporter construct stably integrated into the zebrafish genome drove expression in the distal edge of the median fin at 24 hpf (arrow in B), the distal edge of the pectoral fin at 48 hpf (arrow in C), and tooth epithelium (arrow) and mesenchyme (arrowhead) at 5 dpf (D). (E-G) A 477 bp construct of zebrafish genomic sequence centered around the conserved sequence of the enhancer drove similar, but weaker expression in the median fin of a 33 hpf zebrafish (arrow in E), pectoral fins of a 48 hpf zebrafish (inset of F), and teeth of a 5 dpf zebrafish (G). (H-I) Although not detected in seven of eight stable lines, in one of eight stable lines, the zebrafish sequence drove faint expression in the distal edges of the median fin (arrow in H) and pectoral fins (arrow in I) of 5 dpf stickleback. However, no expression was detected in tooth germs in newly hatched fry in any line (J). See Table S2 for quantification of expression domains of transgenic lines. Bone is fluorescently stained with Alizarin red in (D, G, J). Scale bars=200 µm.