Fig. 3

Generation of inducible transgenic zebrafish expressing GFP-tagged bbs6.

Gateway cloning was used to generate a GFP-bbs6 construct driven by the hsp70 promoter, flanked by tol2 recombination sites (A). Expression of the transgene can be activated and observed in live fish by a single, 30-minute heat-shock; lateral view of 48 hpf embryo (top) and higher-magnification image of the 48hpf larval head (bottom), individual cells expressing GFP-bbs6 can be observed in the developing eye (B). RT-PCR of 1dpf zebrafish detecting GFP expression in Transgenic (Tg) embryos which received a single heat-shock (HS), but not in wildtype (WT) or Non-HS Tg embryos. Actin was used as cDNA library control (C). Knockdown of bbs6 results in delayed melanosome transport. Shown are dorsal views of 5dpf zebrafish with expanded and contracted melanocytes (D). Retrograde melanosome transport rate after epinephrine treatment in WT and Tg 5dpf zebrafish. The transgene rescues the bbs6 knockdown defect. Statistics performed in GraphPad Prism 6: 1-way ANOVA with Dunnet’s multiple comparisons test, p-value ** < 0.01; plot: box and whisker plot with whiskers extending from 5–95 percentiles, outliers shown as points, orange boxes are WT zebrafish, blue boxes are Tg zebrafish (E). WT = wildtype, Tg = Tg(hsp:70:GFP-bbs6), HS = heat-shock, dpf = days post-fertilization.