Figure 4.

Marine and freshwater Bmp6 enhancers drive more similar spatial patterns in younger fish. Ventral pharyngeal tooth plates from <20 mm (pre-tooth number divergence) fish doubly transgenic for two alleles of the Bmp6 intron 4 enhancer driving two different reporter genes (A, D): the marine enhancer driving mCh with the freshwater enhancer driving eGFP (B, C) and the marine enhancer driving eGFP with the freshwater enhancer driving mCh (E, F). Bilateral ventral tooth plates (B, E) are shown next to representative teeth from the three stages (C, F): early, middle, and late highlighted by white boxes in B, E. Early: both freshwater and marine enhancer drove expression robustly in the epithelium (black arrowheads), while both enhancers drove expression in the mesenchyme (white arrowheads), the marine enhancer drove a broader domain (yellow dotted line) compared to the freshwater enhancer (orange dotted line). Middle: both enhancers continued to drive robust, apparently similar levels of expression in the epithelium (black arrows). In the mesenchyme (white arrowheads) the domain of the freshwater enhancer was reduced compared to the marine allele. Late: marine allele continued to drive a broader domain within the mesenchyme of mineralized teeth (dashed line). The relative sizes of green and magenta hatched lines correspond to the approximate relative strength of expression in the epithelium. Overlapping mesenchyme domain is gray, and expanded marine mesenchyme is marked with white arrowhead. Scale bars=100 µm (B, E), 50 µm (C, F). n=3 fish per genotype (6 total fish), >25 teeth per fish (249 total teeth).