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Fig. 2 Zebrafish behavioral screen for nicotine response mutants. (A) Diagram of gene-breaking transposons (GBTs) deployed in this study. GBT-P9 [this Tol2-based vector is adapted from the GBT-P6 vector from (28)] harbors a transcriptional terminator cassette in cis with a strong ubiquitous promoter driving the green fluorescent protein (GFP) reporter upstream of a splice-donor sequence. Intronic insertions in the sense orientation activate the GFP reporter by providing a functional poly(A) signal from the nearby genetic locus. GBT-R15 contains a modified transcriptional terminator cassette that harbors an AUG-free monomeric red fluorescent protein (mRFP) reporter cassette. mRFP expression is only detected after an in-frame integration event. GBT-R15 also deploys the miniTol2 gene vector backbone and is flanked by loxP sites for downstream genetic causality analyses. (B) Behavioral genetic screening paradigm using GBTs as insertional mutagens. Standard transposase-mediated transgenesis protocols (20) generate a pool of mosaic F0 founder animals. These are outcrossed to generate stable transgenic F1 animals that are then outcrossed to generate mixed clutches of wild-type and mutant siblings. Mutant animals are segregated from their genetically wild-type siblings using fluorescent tags in these GBTs (GFP fluorescence is diagrammed as noted in the GBT-P9 screen; for the GBT-R15 screen, red fluorescence was used to sort). Nicotine response profile is obtained on each subpopulation (fluorescently tagged vs. wild-type). Nicotine was administered at the standard dosage of 10 μM. (C) Differential nicotine response profile between wild-type and fluorescently tagged siblings are diagrammed. Response bars are derived by subtracting the pretreated response of fluorescently tagged GBT-mutants from the pretreated response of their wild-type siblings. This analysis identified two nicotine response mutants (*, P j 0.05), bette davis (bdav) and (**, P j 0.05) humphrey bogart (hbog), out of a total of 102 lines screened: 89 GBT-P9 representing an estimated 178 expressed loci and 23 GBT-R15 representing an estimated 35 expressed loci.