Images of 18 hpf (A) and 36 hpf (B) zebrafish embryos showing abcb4 expression patterns.Abcb4 transcripts where visualized with whole mount in situ hybridization. The faint but global staining patterns indicate weak, ubiquitous constitutive expression of abcb4 in the embryos at both stages.

Fluorescence micrographs of 48 hpf zebrafish embryos that had been incubated with 0.5 μM rhodamine B or 1.0 µM calcein-am either in the presence of different ABC transporter inhibitors (A) or upon knock-down of Abcb4 or Abcb5 (B). ABC transporter inhibitor compounds were cyclosporin A (CsA), PSC833 and MK571. Morpholino oligonucelotides were used for knock-down of Abcb4 (Abcb4-SP-MO/Abcb4-ATG-MO) or Abcb5 (Abcb5-SP-MO). Exposure water of control embryos contained dye only (ctrl, ctrl-MO). The morpholino control (ctrl-MO) served to exclude unspecific morpholino effects on dye accumulation in the embryos. Brighter appearing embryos indicate accumulation of higher amounts of dye in the embryo tissue.

Quantification of rhodamine B (RhB) dye accumulation in zebrafish embryos. (A) Amounts of RhB accumulated in 1 hpf, 6 hpf, 12 hpf, 24 hpf and 48 hpf zebrafish embryos that, simultaneously to RhB, were exposed to various concentrations of transporter inhibitors cyclosporin A (CsA), PSC833 or MK571. (B) Amounts of RhB accumulated in 48 hpf zebrafish embryos upon morpholino knock-down of Abcb4 (Abcb4-SP-MO/Abcb4-ATG-MO) or Abcb5 (Abcb5-SP-MO). Controls contained dye only (ctrl, ctrl-MO), the morpholino control (ctrl-MO) served to exclude unspecific morpholino effects on dye accumulation in the embryos. Experiments with CsA (5 µM CsA, ctrl-MO + 5 µM CsA) were set up as comparisons for the effects of this pharmacologic transporter inhibitor on RhB accumulation in the embryos. Values are means +/ standard deviations from three independent experiments. Statistically significant differences from controls were determined with one-way analysis of variance (ANOVA), followed by Dunnett’s test and are indicated by asterisks (P <0.05).

Images of 120 hpf zebrafish embryos in which abcb4 mRNA transcripts where visualized with whole-mount insitu hybridization (WISH). The intestinal bulb and intestine are strongly stained at this stage showing high expression of abcb4. The images are provided here to confirm specificity of the abcb4 WISH probes we used.