Fad24^hi1019 mutants exhibit a chronic inflammatory phenotype and reduced expression of fad24. A,A′: Oblique coherent contrast (OCC) images of WT and homozygous fad24^hi1019 larvae at 3 dpf. B,B′: Zebrafish mpo in situ hybridizations of WT and fad24^hi1019 larvae at 3 dpf. Box, CHT; arrows, neutrophils in the body of fad24^hi1019 mutants. C: Genomic map of the first two exons of zebrafish fad24 showing the location of the hi1019 insertion; each section = 500 bp. D: RT-PCR analysis of WT (lanes 1,2) and fad24^hi1019 mutant (lanes 3,4) single larvae at 3 dpf; ef1α is used as a control. E, F, F′: In situ hybridization of zebrafish fad24 in WT (E,F) and fad24^hi1019 mutants (F′) at 26 hpf (E) and 3 dpf (F,F′). b, brain; e, eye; s, somite; y, yolk. Lateral view, anterior to the left. Scale bars = 200 μm. Representative results from at least three experiments with greater than 10 larvae per condition per experiment.

Injection of fad24 MO into WT embryos phenocopies the fad24^hi1019 leukocyte infiltration phenotype. Shown is the CHT and surrounding tissues of sudan black stained WT (A) and fad24^hi1019 (A′) larvae or control MO- (B), zfad24 init. MO- (C), or zfad24 ex17 MO- (D) injected larvae. Lateral view, anterior to the left at 3 dpf. Scale bar = 200 μm. E: Schematic of fad24 exon 16, 17, and 18 with the target site of the zfad24 ex17 MO labeled. Arrows, locations of primers used for RT-PCR of total RNA isolated from single embryos at 24 hr post-injection of either uninjected (lane 1), standard control (lane 2), or zfad24 ex17 (lanes 3 and 4) MO. F: Quantification of neutrophil location (either in the CHT or in the body) seen in WT and fad24^hi1019 larvae or control MO-, zfad24 init. MO-, or ex17 MO-injected larvae analyzed by one-way ANOVA at a 95% confidence level using Dunnett's multiple comparison post-test. *P < 0.05. Results reported as average ± SEM.

Injection of fad24 MOs into WT embryos phenocopies the fad24^hi1019 morphological phenotypes. OCC images of the head and yolk (A,C,E,G,I) or body (B,D,F,H,J) of WT (A,B) and fad24^hi1019 (C,D) larvae or control MO- (E,F), zfad24 init. MO- (G,H), or zfad24 ex17 MO- (I,J) injected larvae. K: Comparison of yolk sac and retina length for WT and fad24^hi1019 larvae or control MO-, zfad24 init. MO-, or zfad24 ex17 MO-injected larvae. Results reported as average ± SEM, analyzed by one-way ANOVA at a 95% confidence level using Dunnett's multiple comparison post test. *P < 0.05. Representative results from three experiments.

Zebrafish Fad24 localizes to nuclear granules. HEK cells transfected with EGFP (A) or N-terminal EGFP-tagged zebrafish Fad24 (B). DAPI staining is used to reveal the nucleus. Scale bar = 50 μm. Transient expression of EGFP (C) or N-terminal EGFP tagged zebrafish Fad24 (D) in muscle cells of 3-dpf WT larvae. Scale bar = 200 μm. Representative results from three experiments.

Fad24^hi1019 mutants exhibit defects in lipid metabolism. A, A′: Oil red O staining in a PTU-treated WT (A) and fad24^hi1019 (A′) larvae. Arrows, areas of staining in the head and heart; arrowheads, areas of staining in the vasculature that are absent in the fad24^hi1019 mutant. B, C: Oil red O staining in the body of control (B) or zfad24 init. MO- (C) injected larvae. h, heart; isv, intersegmental vessel; da, dorsal aorta; dlav, dorsal longitudinal anastomotic vessel. Lateral view, anterior to the left at 3 dpf. Scale bar = 200 μm. Representative results from three experiments with greater than 10 larvae per condition per experiment.

Leukocyte recruitment into the body of fad24^hi1019 mutants. A-C: WT larvae. A′-C′: fad24^hi1019 mutant larvae. Immunolabeling for MPO (A,A′) and L-plastin (B,B′). C,C′: Overlay of MPO and L-plastin co-immunolabeling shows that neutrophils, MPO⁺ and L-plastin⁺ (arrows), and macrophages, L-plastin⁺ and MPO^- cells (arrowhead), contribute to the inflammatory response. D: Confocal projections show the more elongated morphology of macrophages (arrowhead) compared to neutrophils (arrow). E,E′: Whole-mount in situ hybridization for c-fms, a macrophage marker. Red box, CHT; red arrows, macrophages in the body of fad24^hi1019 mutants. Black arrows point to c-fms expression in the neural crest. F,F′: XZ projections of MPO-labeled WT and fad24^hi1019 larvae. G,G′: ZY projections of MPO (green) and p63 (red, to mark the epidermis) double-immunolabeled larvae. White line, the midline of the larva. H: A 3.9-μm optical longitudinal section through the muscle of a fad24^hi1019 larvae immunolabeled for MPO. All images except F,F′G,G′ are in lateral view, anterior to the left, and all images are at 3 dpf. Scale bar = 200 μm. Representative results from three experiments.

Fad24^hi1019 mutants exhibit muscle degeneration. A-F: WT larvae. A′-F′: fad24^hi1019 mutant larvae. A,A′: Polarized light microscopy to reveal birefringence of muscle. B,B′: OCC images of trunk muscle. C,C′: Confocal projections of trunk muscle immunolabeled with the fast-muscle myosin specific antibody EB165. Ca,C′a: Zoomed in images better showing muscle fiber striation and size. D,D′: Immunolabeling with the slow-muscle myosin specific antibody F59. E,E′: Hematoxylin and Eosin-stained longitudinal sections through trunk muscle tissue. F,F′: In situ hybridization using probe cb1045 to reveal myoseptum separating the somites of developing larvae. Lateral view, anterior to the left at 3 dpf. Scale bar = 200 μm. Representative results from three experiments with greater than 10 larvae per condition per experiment.

Onset of muscle degeneration in fad24^hi1019 mutants is not mediated by leukocytes. Tailfin views at 3 dpf. A,C: WT. B,D: fad24^hi1019 mutants. A-D: Co-immunolabeling for zMPO (green) and fast-muscle myosin (red) in uninjected (A,B) and pu.1 MO-injected (C,D) larvae. Arrows, neutrophils; white boxes, areas magnified in Aa,Ba,Ca,Da. Lateral view with anterior to the left. Scale bar = 200 μm. Representative results from three experiments.

Apoptosis or caspase activity is partially responsible for the recruitment of neutrophils to the trunk of fad24^hi1019 mutant larvae. A,A′: TUNEL staining in the trunk of WT (A) and fad24^hi1019 mutants (A′). B: TUNEL staining in fad24^hi1019 mutant coimmunolabeled with MPO. C,C′: Acridine orange staining in the trunk of WT (C) and fad24^hi1019 mutant (C′) larvae. D,D′: Acridine orange staining in fad24^hi1019 larvae can be blocked by the addition of the pan-caspase inhibitor zVD.fmk (D′) to the larvae water but not by addition of the vehicle control, DMSO (D). E,E′: MPO immunolabeling of DMSO- (E) and zVD.fmk- (E′) treated fad24^hi1019 larvae. F: Quantification of neutrophils found in the body of fad24^hi1019 larvae when treated with zVD.fmk relative to the vehicle control, DMSO (set to 100%). Average of three replicates analyzed by a paired Student's t-test at a 95% confidence level. *P < 0.05, error bar is standard error of the mean. Lateral view with anterior to the left at 3 dpf. Scale bar = 200 μm. Representative results from three experiments with greater than 10 larvae per condition per experiment.

Fad24hi1019 mutants have defects in jaw formation, heart folding, and gut morphology. A-D: WT mutants. A′-D′: fad24hi1019 mutants. A,A′: OCC images of head and jaw of larvae at 3 dpf. B,B′: Immunolabeling of fast-muscle myosin with EB165 revealing musculature of the lower jaw at 3 dpf. C,C′: Immunolabeling with muscle myosin antibody A4.1025 showing heart morphology at 4 dpf. D,D′: Gut morphology in hematoxylin-stained whole-mount larvae at 3 dpf. Lateral view, anterior to the left. Representative results from three experiments.

Neutrophils in fad24hi1019 larvae respond to wounds and have active MPO contained in cytoplasmic granules. A: A fad24hi1019;mpx:GFP mutant at 3 dpf was analyzed by time-lapse confocal microscopy. Frames are taken from Supp. Movie 3. The asterisk indicates a wound made in the dorsal fin. A single neutrophil is marked in each frame in green with a red track indicating the path it takes from the body to the fin. Indicated times are from the start of the movie. B: A blown-up image of the wound (taken from Supp. Movie 4) reveals the presence of a low-level MPO-expressing cell with a long and extended morphology (arrowhead). C: Both a low-level MPO-expressing cell with an extended morphology (arrowhead) and a high-level MPO-expressing neutrophil (arrow) can be compared at the wound. Asterisk indicates the wound. D,E: The differential subcellular localization of MPO (D) and L-plastin (E) can be seen in a confocal composite image of two neutrophils from a co-immunolabled fad24hi1019 larva. F,F′: Whole-mount endogenous MPO activity in WT (F) and fad24hi1019 (F′) larvae. Representative results from at least 3 experiments.

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