Figure 3

Shhb genes carry a functional ar-C homolog enhancer with diverged sequence and tissue specificity. (a) Top panel: Vista plot comparison (AVID) between zebrafish shha intron 2 (baseline), zebrafish shhb intron 2, and tench shha intron 2. Bottom panel: comparison between zebrafish (baseline) and tench shhb intron 2. The peaks exhibiting more than 70% identity in a 50 base pair window are highlighted in orange. The schemes of zebrafish shha (top) and shhb (bottom) intron 2 mark the position of the shha ar-C (blue box), the putative shhb ar-C (red box), and exons 2 and 3 (black boxes). Dashed lines demarcate equivalent sequence regions. Panels b to d show a transgenic analysis of shhb genomic fragments for enhancer activity. Embryos injected with the plasmid constructs are shown at 24 high-power field (hpf), lateral view, onto the trunk at the level of midline. Shown are embryos injected with gfp-reporter constructs containing zebrafish (b) 2.7 kilobase (kb) shhb promoter, (c) 2.7 kb shhb promoter plus zebrafish shhb intron 1, and (d) shhb intron 2. Panels e to j show transgenic analysis of the enhancer activity of shha and shhb intron 2 fragments. Shown are embryos injected with (e) promoter-control construct, (f) plasmids containing zebrafish shha intron 2, (g) zebrafish shhb intron 2, (h) tench shhb intron 2, (i) the nonconserved part of zebrafish shhb intron 2, and (j) the conserved part (putative ar-C). Arrows and arrowheads indicate green fluorescent protein (GFP) activity in the floor plate and notochord cells, respectively. Lines on the left side indicate the level of the floor plate and notochord on the images. The quantification of the gfp expression is shown on the graphs, as described above. ar, activation region; E, exon; ect, ectopic; fp, floor plate; I, intron; nt, notochord; pr, promoter; t, tench; z, zebrafish.