Generation of zebrafish abcb4 knockout mutant using CRISPER/Cas9 system a chematics of the abcb4 mutant alleles generated using CRISPER/Cas9. The 4th exon of abcb4 was targeted by gRNA. The sequences of the abcb4 wild-type (WT) and 2 nucleotides deletion mutant allele (abcb4^−/−) were illustrated. Quantitative RT-PCR showed abcb4 transcript reduction in the embryos b and brain tissue c of the abcb4 mutant. Data are expressed as mean ± SD from at least three independent experiments. P-values were calculated using a two-tailed t-test or one-way ANOVA

Zebrafish Abcb4 is an efflux transporter of rhodamine123, a substrate for human Pgp. a Fluorescence micrographs indicate the accumulation rhodamine123 in the WT and abcb4 knockout embryos. The intensity of rhodamine123 dye in WT and abcb4 knockout embryos was quantified and illustrated as a bar graph b. c Protocol of drug treatment for vinblastine and doxorubicin toxicity experiments. e Representative images of embryos with defects development after vinblastine treatment. d The percentage of embryos showing developmental abnormalities for WT and abcb4 mutant in the presence and absence of 2 μM vinblastine was compared by two-way ANOVA. eabcb4 knockout embryos treated with 100 µM doxorubicin demonstrate a reduction of body length in a two-tailed t-test. Data are expressed as mean ± SD from at least three independent experiments. n numbers indicate the total number of embryos across the independent experiments

Zebrafish Abcb4 protein localizes to blood vessels in the zebrafish brain. Brain tissues of WT a and abcb4 knockout b adult zebrafish, as a negative control, were stained with anti-Pgp antibody F4 (red) as described in the Materials and Method section. Bar = 30 μm. c F4 antibody staining of whole adult zebrafish. Bar = 3 mm. Positive staining (red) was noted in the forebrain d, intestine e, and a subset of renal tubes or collecting ducts in the kidney f. g The F4 positive staining (red) in the brain colocalized with flk1:GFP positive cells (green). Fluorescence channels were interrogated individually and merged in. Nuclei were stained with DAPI (blue). Bar = 300 μm for (d, e), 200 μm for (f), and 50 μm for (g)

Efflux activity of zebrafish Abcb4 in the larval brain vasculature a Diagram of the intravascular injection of rhodamine123 experiment. Rhodamine123 (green) was injected into the cardinal vein of Tg[flk1:EGFP] and Tg[flk1:EGFP]; abcb4^−/− embryos at 3 dpf and allowed to circulate for 1.5 h before imaging. b Representative images of the dorsal view of the larval brain after rhodamine123 injection showed the level of rhodamine123 accumulation in the brain area. c Quantification of normalized rhodamine123 intensity in the brain area of Tg[flk1:EGFP] and Tg[flk1:EGFP]; abcb4^−/− embryos. Data are expressed as mean ± SD from three independent experiments (black dots). N numbers (gray dots) indicate the total number of embryos across the three independent experiments. P-values were calculated using two-way ANOVA

Age-related transcriptome profiling in abcb4 knockout zebrafish brain a Summary of significant DE genes from RNA-seq analysis of brain tissues between WT and abcb4 knockout zebrafish at 2 and 30 months. Gene Set Enrichment Analysis (GSEA) of age-associated DE genes (FDR < 0.5) in brain tissue of WT b and abcb4 mutant c (see Additional file 3 for detailed gene lists of GSEA). d Radial graph depicting the three enriched pathways more negatively regulated with age in abcb4-mutated brain than WT. e Heatmaps illustrate the relative level of transcripts in the enriched pathways from GSEA