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Regulation of neutrophil maturation in zebrafish
(Figure 1E and Online Supplementary Appendix, Movies 1 and 2). These results suggest that neutrophil maturation is defective in smu350 mutants.
The alas1 mutation was responsible for neutrophil defects of smu350 mutant
Positional cloning was then performed to identify the causative gene in smu350 mutants. Initial mapping with bulk segregation analysis located the mutated site to link-
age group 11 (data not shown), then fine mapping placed the mutated gene within a region between two simple sequence length polymorphism markers, CU633745-M and CU929297-M, from the Massachusetts General Hospital panel (Figure 2A). The mutation was then mapped to a 100-kb region partly covered by two bacterial artificial chromosomes (BACs) (Figure 2A). There were 9 predicted genes in this region (Figure 2A). By sequencing, we found a T-to-A mutation in alas1 intron 7 next to the
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Figure 1. Neutrophil deficiency in smu350 mutants. (A) The Sudan black B (SB) signal was totally absent in smu350 mutants. SB staining in siblings (left) and smu350 mutants (right) at 3 days post fertilization (dpf). The boxed regions are magnified in the lower right-hand corner. (B) Whole-mount in situ hybridization (WISH) of pu.1 expression in siblings (left) and smu350 mutants (right) at 22 hours post fertilization (hpf). (C) WISH of mpx expression in siblings (left) and smu350 mutants (right) at 2 dpf. (D) DAB staining in siblings (left) and smu350 mutants (right) at 2 dpf. Signal points represent- ing neutrophil peroxidase activity (black arrowheads) were absent in smu350 mutants. Red arrowheads indicate the hemoglobin signal. The boxed regions are magnified in the lower right-hand corner. (E) Neutrophil granules were absent in smu350 mutants. In vivo imaging of neutrophils in 2-dpf Tg(lyz:DsRed);smu350 embryos by VE-DIC microscopy. Neutrophils of siblings had abundant visible and highly mobilized granules (29 of 31 embryos), while neutrophils of smu350 mutants lacked granules (12 of 12 embryos). White arrowheads indicate neutrophil granules. (Left) Bright-field DIC image; (right) an overlay of bright-field DIC and fluorescent images. See also Online Supplementary Appendix, Movies 1 and 2. (F) Quantifications of SB+ cells in the poste- rior blood island (PBI), pu.1+ cells (B), mpx+ cells in the PBI (C), and DAB+ cells in the PBI (D). Mean±Standard Deviation (SD), n>15; Student t-test: ****P<0.0001. ns: not sig- nificant; nd: not detectable. Scale bars: 200 mm (A-D) and 5 mm (E).
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