Fig. 6

Fig. 6 ubi:Switch is a ubiquitous lineage-tracing transgene. The asterisks indicate heart-specific cmlc2-EGFP expression marking the ubi:cre^ERt2 transgene. (A) Schematic of the ubi:cre^ERt2 transgene and Tg(–3.5ubi:loxP-GFP-loxP-mCherry) (ubi:Switch) reporter cross. Arrows indicate transcription start positions, yellow boxes are non-coding 5′ and 3′ UTR/polyA sequences, blue arrowheads indicate loxP sites. (B,B′) Imaging of live ubi:cre^ERt2; ubi:Switch double-positive zebrafish embryos at approximately 3 dpf for baseline GFP expression (B) and mCherry indicating successful loxP excision (B′). Embryos were treated at shield stage (6 hpf) with 5 μM 4-OHT, ethanol (EtOH) carrier, or a short 15 minute pulse of 5 μM 4-OHT (top to bottom). Both 4-OHT treatments induce strong mCherry expression (B′), with apparent GFP fading in the longer 4-OHT treatment (B, top), compared with ethanol control (B,B′, center). (C-F′) Approximately 30 hpf zebrafish embryos double-positive for ubi:cre^ERt2; ubi:Switch were treated with 4-OHT or ethanol control at bud stage as indicated and compound imaged for GFP fluorescence (C-F) to visualize ubi:Switch default expression or mCherry fluorescence (C′-F′) to reveal successful loxP excision (the white line indicates merge of the anterior and posterior focal planes). Whereas 5 μM 4-OHT potently induces loxP recombination (C′), and 1 μM 4-OHT causes notably reduced mosaicism (D′), 0.5 μM 4-OHT fails to induce a widespread response (E′) similar to the ethanol control (F′). (G-H) ubi:cre^ERt2; ubi:Switch embryos 5 μM 4-OHT or ethanol control treated at 24 hpf (G,G′) or 7dpf (H) reveal 4-OHT-dependent loxP recombination and de novo mCherry expression from the ubi:Switch transgene. Note the strong mCherry expression in the heart in the 4-OHT-treated larva (G′, top), suggesting promoter cross-talk between the cmlc2 marker driver and ubi:cre^ERt2. Because of their drastically increased size, the embryos induced at 7dpf (H) were treated with increased dose (10 μM) of 4-OHT or ethanol control and visualized for GFP (green) and mCherry expression (blue) channel.