Figure 1. atg-Gene-Deficient Zebrafish Show Larval Lethality and Defective Inflation of the Swim Bladder (A and B) Survival rate (%) of fip200+/− (n = 28), fip200−/− (n = 16) (A), atg16l1+/− (n = 23), and atg16l1−/− (n = 9) (B) zebrafish. (C) Median lethal time of atg9a−/−atg9b−/− (n = 24), vmp1−/− (n = 11), fip200−/− (n = 16), atg101−/− (n = 12), atg13−/− (n = 15), atg14−/− (n = 5), atg2a−/−atg2b−/− (n = 6), atg16l1−/− (n = 9), and atg5−/− (n = 15) zebrafish. dpf, days post-fertilization. (D) External appearance of atg-deficient zebrafish at 9 dpf (atg9 mutant zebrafish were examined at 6 dpf). Magnified images of the indicated regions are shown in the right panels. A swim bladder is indicated with a dashed line in fip200+/− zebrafish. Uninflated swim bladders are indicated by red arrowheads. Data are representative of two to four independent experiments. Scale bar, 1 mm. (E) Rate (%) of inflated (white column) and uninflated (black column) zebrafish swim bladders for each genotype at 9 dpf (atg9 mutant zebrafish were examined at 6 dpf). (F) Hematoxylin and eosin staining of sagittal sections of 9-dpf fip200+/− and fip200−/− zebrafish. G, gut; N, notochord; SB, swim bladder (indicated by dashed lines); SM, skeletal muscle. Scale bar, 20 μm. (G) Behavioral analysis of 6-dpf fip200+/− and fip200−/− zebrafish in cuvettes. The arrow indicates the upward direction of the cuvettes. The arrowheads indicate zebrafish. The video from which these stills images were captured is available as Video S1.

Figure 2. Defective Maturation of Lamellar Bodies in Swim Bladder Epithelial Cells in fip200-Deficient Zebrafish (A) Representative images of GFP-LC3B signals in swim bladders of 4-dpf fip200+/− and fip200−/− zebrafish expressing GFP-LC3B. Scale bar, 20 μm and 1 μm in the inset. (B) Representative images of GFP-LC3B and LAMP1-RFP (lamellar bodies and endolysosomes) signals in swim bladders of 4-dpf wild-type zebrafish expressing GFP-LC3B and LAMP1-RFP. Arrows indicate LC3B- and LAMP1-positive structures. Scale bar, 2 μm. (C) Representative images of LAMP1-GFP signals in swim bladders of 4-dpf fip200+/− and fip200−/− zebrafish expressing LAMP1-GFP. Arrows indicate LAMP1-positive structures. Scale bar, 20 μm and 1 μm in the inset. The average size and number of lamellar bodies were quantified and are shown in the graphs. The solid bars and boxes indicate the median and interquartile range (25th to 75th percentile), respectively. The whiskers indicate the largest and smallest values. Differences were statistically analyzed by unpaired two-tailed Mann-Whitney U tests. E, epithelial cells; L, lumen of the swim bladder.