Zhang et al., 2019 - Establishment of a zebrafish hematological disease model induced by 1,4-benzoquinone. Disease models & mechanisms   12(3) Full text @ Dis. Model. Mech.

Fig. 1

Kaplan–Meier survival curve and analysis of zebrafish embryo phenotype following exposure to 1,4-benzoquinone (BQ). (A) Survival rates in zebrafish embryos following continuous exposure to BQ (n=278 per group) in the range 8–10 μM, from 2 dpf to 9 dpf (log-rank test, P<0.001). (B) Phenotypic traits (indicated by the arrows in C) observed following BQ exposure (Chi-squared test, mean±s.d., *P<0.05, **P<0.01). (C) Typical malformations were observed in zebrafish embryos exposed to BQ. Scale bars: 500 μm.

Fig. 2

Expression of lineage-specific markers in zebrafish was detected using WISH and SB staining. (A,B) Loss of βe1-globin (n=20) and rag1(control group n=37, BQ group n=49) expression in 5 dpf embryos exposed to BQ. The red boxed regions show magnified views of the caudal hematopoietic tissue (CHT). Relative rag1+ thymocyte-signal areas were analyzed using ImageJ software. The circled regions show the thymus. (C) Increase in l-plastin expression upon BQ exposure at 3 dpf (control group n=25, BQ group n=30). (D) Expression of the macrophage marker mfap4 was reduced in the BQ exposure group (control group n=52, BQ group n=76). (E,G) The neutrophil markers mpx (E) (control group n=20, BQ group n=33) and lyz (G) (control group n=39, BQ group n=35) both showed increased expression in embryos at 3 dpf following BQ exposure. (F) Increased SB+ cells were observed in the BQ exposure group compared with the control group (n=21 per group). (H–N) Quantification of WISH and SB staining for A–E (Student's t-test, mean±s.d., **P<0.01, ***P<0.001). (O) Dot plot of flow cytometry analysis of lyz:GFP+ cells from control (left) and BQ-treated fish (right). Three independent experiments (in each group, 100 embryos are pooled together) were conducted. (P) Percentage of lyz+ cells in each group [0.30% in the control group and 0.40% in the BQ group; Chi-squared test (95% c.i.), ***P<0.001].

Fig. 3

Exposure to BQ induced neutrophil proliferation in zebrafish embryos.(A,B) BrdU incorporation assay. Double staining of BrdU/lyz (A) showed BrdU incorporation of CHT lyz+ cells in BQ-exposed embryos and controls at 3 dpf. Arrows indicate lyz/BrdU double-positive cells. Scale bars: 50 μm. Percentage of the CHT-localized lyz+ myeloid cells that incorporate BrdU (B) in lyz+ myeloid cells (Student's t-test, control group n=30, BQ group n=26, mean±s.d., **P<0.01).

 
 

Fig. 4

Effect of BQ exposure on c-myb expression. (A–C) BQ exposure increased c-myb expression. Both WISH performed at 36 hpf (A,B) and RT-qPCR performed at 2 dpf (C) indicated increased expression of c-myb in BQ-exposed (right, A) and control (left, A) groups (Student's t-test, n=25 or 26, mean±s.d., *P<0.05, ***P<0.001). The red boxed regions show magnified views of the aorta-gonad-mesonephros (AGM).

Fig. 5

BQ-induced neutrophil expansion was blocked in c-myb-deficient mutants. (A–I) Expression of the neutrophil markers lyz (A,B) (sibling control group n=65, BQ group n=38; mutant control group n=25, BQ group n=35) and mpx (C,D) (sibling control group n=83, BQ group n=47; mutant control group n=31, BQ group n=16) was analyzed using WISH, and SB staining (E,F) (sibling control group n=54, BQ group n=18; mutant control group n=25, BQ group n=22) was detected, in c-mybhkz3/+ intercrossed progenies in BQ-treated groups (right column) and untreated controls (left column). Signals increased following BQ exposure at 3 dpf in siblings (A,C,E) but were unaltered in c-mybhkz3/hkz3 mutants (B,D,F). The red boxed regions show magnified views of the CHT. (G–I) Quantification of WISH and SB staining for panels A–F (Student's t-test, mean±s.d., **P<0.01, ****P<0.0001; ns, non-significant).

Fig. 6

BQ hematotoxicity to adult fish. (A) May–Grunwald/Giemsa of KM cells in BQ-exposed (right) and control (left) fish. Red arrows, erythrocytes (oval-shaped nucleus and cytoplasm); black arrows, lymphocytes (large round-shaped nucleus surrounded by minimal cytoplasm); green stars, neutrophils (segmented nucleus and cytoplasm with distinct neutral granules); yellow stars, macrophages (large irregular-shaped cytoplasm with distinct vacuoles); blue arrows, precursors (large and dense nucleus with dark cytoplasm). Scale bars: 5 μm. (B) Blood cell counts of KM in each group were calculated manually based on their morphology (Student's t-test, n=9, mean±s.d., *P<0.05, **P<0.01).

 
 

Acknowledgments:
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Dis. Model. Mech.