Spatial distribution of fech in zebrafish larvae. fech expression in larvae 1 day ((a) lateral view; (b) ventral view; and (c) tail), 3 days ((d) lateral view; (e) ventral view; (f) tail; (g) head), and 5 days ((h) lateral view; (i) ventral view; (j) tail; (k) head) days post-fertilization (dpf). ICM, intermediate cell mass; PBI, posterior blood island; Li, liver; CHT, caudal hematopoietic tissue; Hr, heart; P, pronephros; In, intestine.

Tissue-specific expression of fech in healthy adult zebrafish. The relative mRNA levels of fech were assessed in various organs, with muscle tissue used as a reference. The spleen and kidneys exhibited the highest expression, followed by relatively higher levels in the gill, ovary, and liver than in other tissues. Each bar on the graph represents the mean relative mRNA level, with error bars indicating the SD (n = 3). Statistical significance was determined using a one-way ANOVA with Tukey’s post hoc test, and different letters indicate statistically significant differences (p < 0.05) between tissue types.

Generation and phenotyping of fech^−/+ fish and fech^−/− larvae. (A) Schematic representation of the organization of the zebrafish fech gene. Untranslated areas and open reading frames are depicted by white and black boxes, respectively. The target location for the sgRNA is denoted by the red arrowhead. The PAM and single-guide RNA (sgRNA) target sequences are indicated by red and blue letters, respectively. (B–D) Illustration of the insertion of 13 bp nucleotides into the target site and the introduction of a premature stop codon (*) using CRISPR/Cas9 gene editing. The inserted amino acid sequence is highlighted in yellow. (E) Genotyping of fech^−/+ and fech^−/− using PCR and agarose gel electrophoresis. (F) Confirmation of the mutation via RT-qPCR using fech target-site-specific primers in 7 dpf WT and fech^−/− larvae. (G) Detection of red fluorescence in 24 hpf fech^−/− larvae during the screening process.

PPIX accumulation in fech^−/− larvae. (A) Fluorescence images of fech^−/− zebrafish larvae across various developmental stages (PBI, posterior blood island; YCV, yolk circulation valley; CHT, caudal hematopoietic tissue; Li, liver; Hr, heart; P, pronephros). (B) PPIX accumulation in 6 dpf zebrafish larvae. Images were taken in a bright field, with the red channel used to detect PPIX fluorescence and the green channel used to detect autofluorescence. Merged images show the localization of PPIX in the liver (white dotted line), pronephros (green dotted line), and intestinal area (blue dotted line).

Reduction in PPIX accumulation in fech^−/− larvae following UDCA treatment. WT and fech^−/− larvae (3 dpf) were treated with 100 or 200 µM of UDCA, and their PPIX fluorescence intensity was compared with that of untreated WT and fech^−/− larvae. (A) Fluorescence and bright-field images of WT and fech^−/− larvae after UDCA treatment as well as the controls. (B) The relative fluorescence intensities of PPIX in the experimental larvae. The relative fluorescence intensity data are presented as the means ± SD (n = 5). The statistical significance between control and treated larvae was analyzed using the Student’s t-test (ns, non-significant; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001).

Apoptosis activation in fech^−/− larvae. (A) Acridine orange staining of WT and fech^−/− larvae at various developmental stages and (B) relative fluorescence intensity. (C) The bax/bcl2 mRNA fold induction ratios in WT and fech^−/− larvae at different developmental stages. The fold induction ratios are presented as the means ± SD (n = 3). Statistical significance between fech^−/− and WT larvae was analyzed using Student’s t-test (ns, non-significant; *, p ≤ 0.1; **, p ≤ 0.01; ****, p ≤ 0.0001).

Effect of UDCA treatment on apoptosis in fech^−/− larvae. (A) Acridine orange staining of WT and fech^−/− larvae after UDCA treatment and (B) relative fluorescence intensity. (C) The bax/bcl2 expression ratio after UDCA treatment. The bax/bcl2 fold induction ratios are presented as the means ± SD (n = 3). Statistical significance between fech^−/− and WT larvae was analyzed using Student’s t-test (ns, non-significant; *, p ≤ 0.1; ***, p ≤ 0.001; ****, p ≤ 0.0001).

Effect of UDCA treatment on the expression of bile transporters in WT and fech^−/− larvae (3 dpf) that were treated with 100 µM of UDCA and for which, after 12 and 24 h, qPCR was performed. UDCA treatment induced the expression of (A) abcb11a and (B) abcc2 to a greater extent in both WT and fech^−/− larvae compared with that in the control. RT-qPCR results are presented as the means ± SD (n = 3). Statistical significance between control and UDCA-treated fech^−/− larvae was analyzed using the Student’s t-test (ns, non-significant; **, p ≤ 0.01; ****, p ≤ 0.0001).

Temporal neutrophil production in fech^−/− larvae. (A) Changes in neutrophil production in the head, trunk, and tail of WT and fech^−/− zebrafish larvae at different developmental stages. (B) The total neutrophil counts in WT and fech^−/− larvae at various developmental stages. The highest neutrophil count in fech^−/− larvae compared to the WT was observed at 3 dpf. The total neutrophil counts are presented as the means ± SD (n = 5). Statistical significance between WT and fech^−/− larvae was analyzed using Student’s t-test (ns, non-significant; *, p ≤ 0.1; ***, p ≤ 0.001; ****, p ≤ 0.0001).

Amelioration of neutrophil accumulation by UDCA treatment in fech^−/− larvae. (A) Images of Sudan black-stained neutrophils in WT and fech^−/− larvae. (B) The total neutrophil counts in WT and fech^−/− larvae. WT and fech^−/− larvae (2 dpf) were treated with 100 µM of UDCA, and another group was treated with DMSO for 24 h. The total neutrophil counts are presented as the means ± SD (n = 5). Statistical significance between control, DMSO-treated, and UDCA-treated fech^−/− larvae was analyzed using Student’s t-test (ns, non-significant; ****, p ≤ 0.0001).

Changes in temporal macrophage production in fech^−/− larvae. (A) Alterations in macrophage production in the heads, trunks, and tails of WT and fech^−/− zebrafish larvae during different developmental stages. (B) The total number of macrophages counted in WT and fech^−/− larvae at various developmental stages. The highest macrophage counts for fech^−/− larvae, compared with those for the WT, were observed at 4 dpf. Total macrophage counts are presented as the means ± SD (n = 5). Statistical significance between WT and fech^−/− larvae was analyzed using Student’s t-test (ns, non-significant; ****, p ≤ 0.0001).

Attenuation of macrophage accumulation by UDCA treatment in fech^−/− larvae. (A) Images of neutral, red-stained macrophages in WT and fech^−/− larvae. (B) The total macrophage counts in WT and fech^−/− larvae. WT and fech^−/− larvae (3 dpf) were treated with 100 µM of UDCA, and another group was treated with DMSO for 24 h; the control group remained untreated. The total macrophage counts are presented as the means ± SD (n = 5). Statistical significance between the control, DMSO-treated, and UDCA-treated fech^−/− larvae was analyzed using Student’s t-test (ns, non-significant; ****, p ≤ 0.0001).