Images of bilirubin-exposed larvae: Larvae were exposed to (A-A′′) carrier, (B-B′′) 3 μM, (C-C′′) 6 μM, (D-D′′) 9 μM, and (E‒E′′) 15 µM bilirubin between 2–3 dpf. Representative images recorded at the end of treatment (3 dpf, left column), 1 day after washout (4 dpf, middle column) and 2 days after washout at 5 dpf (right column) are displayed in the figure. Scale bar: 200 μm, n = 40.

Mild and severe phenotypes: The larvae exposed to 9 µM bilirubin between 2–3 dpf display (A) mild or (B) severe phenotypes at 4 dpf. Close-up images are shown next to the overview images. navy, blue and red arrows represent bilirubin, yolk edema and pericardial edema, respectively. The green arrow indicates bilirubin excreted from the GI tract. Scale bars: 200 µm. (C) Average body length of larvae treated with bilirubin at 2–3 dpf for 24 h (purple line) or at 2–4 dpf for 48 h (pink line) shows a significant shortening of body length upon 9–15 µM bilirubin exposure. (D–F) Penetrance of the severe phenotype after exposure to bilirubin between (D) 2–3 dpf, (E) 2–4 dpf and (F) 3 - 4 dpf. Larvae were treated with vehicle, 3 μM, 6 μM, 9 µM or 15 µM bilirubin. The mean ± SEM was calculated from triplicate treatments of 15 larvae in each group.

Histopathology of the liver and gut after bilirubin exposure.(A–E) Liver and (F–J) gut tissues of 6 dpf larvae are stained with H & E after paraffin sectioning. (A and F) Control larvae treated with vehicle. Larvae exposed to (B and G) 9 µM and (C and H) 15 µM bilirubin between 2 and 3 dpf. Larvae exposed to (D and I) 9 μM, (E and J) 15 µM bilirubin between 3 - 4 dpf. Arrow: enlarged vein, arrowhead: bilirubin in bile duct, scale bars: 50 µm.

Quantitative analyses of bilirubin accumulation: (A) Bilirubin concentration in larval body was measured from lysates, after exposure to 6 and 9 µM bilirubin for 24 or 48 h, mean values were plotted (n = 60 for each group, experiment was done in triplicate). (B) Relative bilirubin accumulation in the brain at 4 dpf was plotted for each group (n = 15). (C–J) Inverted images of the head that show bilirubin accumulation as bright blue spots were used for the graph in (B). Scale bar: 200 µm.

Morphologic analysis of neural tissues in bilirubin-exposed larvae: Larvae exposed to 3 μM, 6 μM, 9 μM, and 15 µM bilirubin between 2 and 3 dpf were imaged at 4 dpf. (A) Axial eye length and (B) vertical eye length decreased; (C) sclera size and (D) interocular distance increased; (E) optic tectum width decreased upon hyperbilirubinemia induction. (n = 24, n = 12, n = 10, n = 19 and n = 13 for control group, 3 μM, 6 μM, 9 μM and 15 µM).

Bilirubin-induced neurological damage (BIND) is detected in the larval brain:(A) Apoptotic cells were detected in the brains of control and hyperbilirubinemia larvae at 4 dpf. DAPI (blue) highlights the brain structure, and cleaved caspase 3 (C-Casp3) (red) labels apoptotic cells. Dorsal views of fore brain (left) and hind brain (right) are shown for control, 9 μM and 15 µM bilirubin treatment groups. (B–F) H&E-stained paraffin sections of 6 dpf larval brains. (A) Intact and healthy brain tissue in control larva; deformed brain tissue in larvae that received (B) 9 µM or (C) 15 µM bilirubin between 2–3 dpf. (D) Larva that received 9 µM bilirubin between 3 - 4 dpf had normal brain tissue. (E) Larva that was exposed to 15 µM bilirubin between 3–4 dpf had slight deformation in brain tissue. Scale bars: 100 µm.

Hyperbilirubinemia affects eye reflexes and swim behavior:(A) Turn of the eyes in response to light stimulation is displayed. Turn of the eye between t₀ and t₁ was measured as turn angle alpha. (B) Average turn angle of the eyes of larvae exposed to 3, 6, 9 and 15 µM doses of bilirubin is displayed. (C) Average swim distance of controls and larvae exposed to 9 µM bilirubin between 2 and 3 dpf at 4 and 6 dpf is displayed. Data are the mean ± SEM. (A″-C″) n = 20, 18, 22, 20, and 22 for control, 3, 6, 9, and 15 µM. (D) Directional swim behavior of the control and swirling swim behavior of the severely affected larvae are displayed.